Physiol Rev
83: 871–932, 2003; 10.1152/physrev.00001.2003.
Transcytosis: Crossing Cellular Barriers
PAMELA L. TUMA AND ANN L. HUBBARD
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland
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I. Introduction
II. Documented Transcytosis In Vivo
A. Transcytosis in the vasculature
B. Transcytosis in the brain
C. Immunological protection and transcytosis
D. Role for transcytosis in the homeostasis of micronutrients
E. Additional transcytosis systems
F. Role of transcytosis in plasma membrane biogenesis in vivo
III. In Vitro Cell Models of Transcytosis
A. What constitutes a “good” transcytotic cell model?
B. Microvascular endothelial cell models
C. Epithelial cell models
D. Transcytosis outside of the epithelial world
IV. More About Two Different Transcytosis Systems
A. Caveolae-mediated transcytosis
B. Clathrin-mediated transcytosis
V. Mechanisms and Molecules Regulating Transcytosis
A. Targeting machinery
B. Cytoskeleton
C. Lipids and transcytosis
D. Perturbations of transcytosis
E. Transcytosis versus direct PM delivery
VI. Conclusion
Tuma, Pamela L., and Ann L. Hubbard. Transcytosis: Crossing Cellular Barriers. Physiol Rev 83: 871–932, 2003;
10.1152/physrev.00001.2003.—Transcytosis, the vesicular transport of macromolecules from one side of a cell to the
other, is a strategy used by multicellular organisms to selectively move material between two environments without
altering the unique compositions of those environments. In this review, we summarize our knowledge of the
different cell types using transcytosis in vivo, the variety of cargo moved, and the diverse pathways for delivering
that cargo. We evaluate in vitro models that are currently being used to study transcytosis. Caveolae-mediated
transcytosis by endothelial cells that line the microvasculature and carry circulating plasma proteins to the
interstitium is explained in more detail, as is clathrin-mediated transcytosis of IgA by epithelial cells of the digestive
tract. The molecular basis of vesicle traffic is discussed, with emphasis on the gaps and uncertainties in our
understanding of the molecules and mechanisms that regulate transcytosis. In our view there is still much to be
learned about this fundamental process.
I. INTRODUCTION
At its simplest, transcytosis is the transport of macromolecular cargo from one side of a cell to the other
within a membrane-bounded carrier(s). It is a strategy
used by multicellular organisms to selectively move material between two different environments while maintaining the distinct compositions of those environments.
Cells have other strategies not involving membrane vesiwww.prv.org
cles to selectively move smaller cargo (ions and small
solutes) across cellular barriers. Paracellular transport,
the movement between adjacent cells, is accomplished by
regulation of tight junction permeability, and transcellular
transport, the movement of ions and small molecules
through a cell, is accomplished by the differential distribution of membrane transporters/carriers on opposite
sides of a cell. Together, these three processes contribute
to the success of multicellular organisms.
0031-9333/03 $15.00 Copyright © 2003 the American Physiological Society
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PAMELA L. TUMA AND ANN L. HUBBARD
Physiol Rev • VOL
lysosomes. How? Cargo: the nature of the transcytotic
cargo also varies. Although today we might think of transcytosis as a selective process, the originally defined system, endothelial cells of the microvasculature, moves
macromolecular cargo rather nonselectively within the
fluid phase of the transport vesicle or by adsorption to the
vesicle membrane. Furthermore, transcytotic cargo is not
limited to macromolecules. Several vitamins and ions utilize endocytic mechanisms and vesicular carriers as part
of their transcellular sojourn. This brings up another unsolved mystery, that of a cell transcytosing particular
cargo for use by other cells but also using some of it for
its own metabolism. How is such apportionment made?
A major goal of this review is to summarize the
widespread occurrence of transcytosis and focus on its
many variations. First, we present documented examples
of in vivo transcytosis in mammals, using the expanded
definition given above. Next, we assess the status of in
vitro cell models currently used to study the different
types of transcytosis. We then review in more depth the
two best-studied transcytosis systems, transendothelial
transport of circulating macromolecules and transcytosis
of IgA in polarized epithelial cells, focusing on the similarities and differences of their pathways and carriers.
Finally, we present current information about the molecular mechanisms and regulation of transcytosis. Throughout, we identify gaps in our present understanding of this
process, with the hope that interested researchers will fill
in those gaps with insightful experiments and definitive
answers.
II. DOCUMENTED TRANSCYTOSIS IN VIVO
Table 1 documents that transcytosis is widespread.
As expected, epithelial cells forming barriers between the
outside world and the interstitium or between the internal
world (circulation) and the interstitium are the major
cells participating in transcytosis. However, the question
of whether transcytosis occurs in all adult epithelia (e.g.,
kidney and skin) is open. While proximal tubule cells are
endocytically active, only micronutrients seem to be
“transcytosed” by them in vivo. Other segments of the
nephron, e.g., the collecting tubule, are more difficult to
assess. Transcytosis certainly occurs in the most obvious
fetal organs, the yolk sac and placenta, and it probably
operates elsewhere in the developing fetus. Further examination of Table 1 reveals that the transport of iron,
vitamin B12, and the immunoglobulins IgA and IgG occurs
in several organs. However, the routes and fates of the
molecules are not always the same. Curiously, the routes
and mechanisms by which circulating hormones gain access to their target tissues have not been extensively
explored (181). Finally, although not yet examined in all
polarized cells, the biogenesis of apical plasma membrane
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Historically, the existence of transcytosis was first
postulated in the 1950s by Palade in his studies of capillary permeability (426). He described a prominent population of small vesicles, many of which were in continuity
with the plasma membrane, and hypothesized that these
vesicles were the morphological equivalent of the large
pore predicted by the physiologist Pappenheimer to explain the high permeability of blood microvessels to macromolecules (428). N. Simionescu was the first to coin the
term transcytosis to describe the vectorial transfer of
macromolecular cargo within the plasmalemmal vesicles
from the circulation across capillary endothelial cells to
the interstitium of tissues (538). During this same period,
another type of transcytosis was being discovered. Immunologists comparing the different types of immunoglobulins found in various secretions (e.g., serum, milk, saliva,
and the intestinal lumen) speculated that the form of IgA
found in external secretions (called secretory IgA, due to
the presence of an additional protein component) was
selectively transported across the epithelial cell barrier
(577, 578). The pathway and origin of the component
acquired during transport were actively investigated, and
in 1980 secretory component (SC) in secretory IgA was
identified as the ectoplasmic domain of the intestinal
epithelial cell membrane receptor that binds dimeric IgA
and transports it through multiple intracellular compartments to the opposite side of the cell (391, 423). These
two historic transcytotic systems are still actively investigated today.
We now know that transcytosis is a widespread
transport process; a variety of cell types use it, different
carriers and mechanisms have evolved to carry it out, and
the cargo moved by it is diverse. Cell types: we are most
familiar with transcytosis as it is expressed in epithelial
tissues, which form cellular barriers between two environments. In this polarized cell type, net movement of
material can be in either direction, apical to basolateral or
the reverse, depending on the cargo and particular cellular context of the process. However, transcytosis is not
restricted to only epithelial cells. Reports of cultured
osteoclasts (398, 490) and neurons (221) carrying vesicular cargo between two environments indicate that the
strategy of vesicular transcytosis has been used elsewhere. Mechanisms: in intestinal cells transcytosis is a
branch of the endocytic pathway, with cargo being internalized via receptor-mediated (i.e., clathrin-coated) mechanisms and progressively sorted away from internalized
material destined for other cellular destinations. However, transendothelial transport in blood capillaries does
not conform to this scenario, since different carriers and
a more direct route are used to cross the cell. Such
differences illustrate that multiple transcytotic mechanisms have evolved that depend on the particular cellular
context. Furthermore, they illustrate that cargo in the
transcytotic pathways seems able to avoid degradation in
873
TRANSCYTOSIS
proteins is an example of endogenous molecules using
transcytosis to attain their destination.
A. Transcytosis in the Vasculature
1. Structural features of continuous endothelium
The simple, squamous epithelial cells of continuous
endothelium are quite distinctive morphologically (Fig.
1B). They are remarkably thin (0.2– 0.5 ␮m) in regions not
including nuclei. A defining feature of these and all epithelial cells is a basement membrane that underlies their
basal surface (Fig. 1C). It is made collaboratively by the
endothelial and underlying interstitial cells. The most
prominent intracellular feature is a population of smoothsurfaced vesicles of 50 –70 nm diameter, some of which
are in continuity with the plasma membrane facing the
circulation (the apical or luminal surface), others in continuity with the opposite surface (the basolateral or abluminal surface), and still others apparently free in the
cytoplasm (Fig. 1C). These vesicles, which have an ⬃35nm-diameter opening with a thin diaphragm across it,
were originally termed plasmalemma vesicles but are now
called caveolae (“small caves”) because of their characteristic flask shape (Fig. 1D) (10). In continuous endothelial cells, the frequency of caveolae varies widely depending on the organ. For example, in endothelium of skeletal
muscle (the diaphragm), the estimate is ⬃1,200/␮m3,
whereas in pulmonary capillaries it is only ⬃130/␮m3
(539). This variation does not correlate with permeability,
suggesting other functions for caveolae. Caveolae are also
Physiol Rev • VOL
2. Microvascular permeability and transcytosis
Microvessels are approximately two orders of magnitude more permeable than other epithelia, making them
leaky to the passage of circulating proteins into the interstitium. Most macromolecules move across capillary endothelium by bulk-phase not receptor-mediated mechanisms. Nonetheless, there is selectivity to the process,
with the size and charge of cargo being important factors.
Furthermore, although transport is bidirectional, the concentration gradient extending from the blood (apical side)
to the interstitium (basal side) dictates that the bulk of
transport is in an apical-to-basolateral direction. Finally,
different continuous capillary beds have distinctive permselectivities, as evidenced by the varied compositions of
the lymph draining from them.
The basis for high capillary permeability has been a
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The most extensive exchange in vivo is that of
plasma constituents across the endothelium that lines the
inner surface of the blood vasculature. Of the three types
of endothelium, continuous, fenestrated, and discontinuous (sinusoidal), only the first two form selective cellular
barriers to the passage of macromolecules between the
circulation and the underlying interstitium. All continuous
and fenestrated endothelia throughout the vascular system are capable of the rapid and extensive bidirectional
exchange of small and large molecules, but those of the
capillaries and postcapillary venules are the major players
in this activity (535, 536, 539). These two parts of the
vascular tree constitute what is often called the microvascular exchange system, whose surface area is enormous
(⬃600 m2) (Fig. 1A). While fenestrated endothelia are
more permeable to small solutes and water than are continuous endothelia, their relative permeability to macromolecules, and hence participation in transcytosis, is controversial (534). Although obvious, it is nonetheless important to state that transcytosis is but one of many
important functions carried out by vascular endothelial
cells, which are dynamic and capable of rapid responses
to local changes in the environment.
found in other cell types where their functions and compositions are actively being investigated (reviewed in
Refs. 9, 546).
An important feature of endothelial cells is their
tight junctions, which represent a barrier to paracellular diffusion (219). While there is good experimental
evidence that the permeability of endothelial cell tight
junctions changes depending on local conditions (336),
the molecular basis has yet to be elucidated. The discovery of a large family of tight junction membrane
proteins, the claudins, and their capacity to form heteroligomeric complexes with distinct permeability
properties (580), will undoubtedly provide insights into
the dynamic regulation of tight junction permeability in
capillaries.
Important to an understanding of transcytosis in endothelial cells is the endocytic system, including clathrincoated vesicles, endosomes, and lysosomes. These organelles are present in all capillary endothelium but are
not abundant, and they are usually located in the thicker,
perinuclear regions of cells. The endocytic system is
clearly functional, as attested by the delivery of modified
albumins and oxidized low-density lipoproteins (LDLs) to
lysosomes (296, 506). But how do these cells distinguish
between cargo destined for transcytosis versus that for
degradation? The simplest explanation is that different
cargoes use different receptors that are localized to different entry sites in the plasma membrane (PM). However, how do endothelial cells themselves utilize the same
cargo that they transport for use by other cells; that is,
how is the apportionment of cargo for self versus others
regulated? As we shall see in section III, at least one cargo
molecule (e.g., native LDL) may use different entry ports
(i.e., caveolae versus clathrin-coated vesicles), offering
the interesting possibility that the point of entry determines the subsequent fate of a particular internalized
cargo molecule.
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TABLE
PAMELA L. TUMA AND ANN L. HUBBARD
1. Documented in vivo transcytosis
Organ System
Heart, lung,
skeletal
muscle,
adipose
tissue
Testis
Cargo
Direction
Receptor/Carrier
Molecules ⬎ 1.7
nm
Albumin
A-BL and BL-A
Fluid phase in caveolae
See text
A-BL and BL-A
gp60 in caveolae
Albumin plays multiple roles in
caveolae-mediated transcytosis
throughout the vascular tree
Orosomucoid
A-BL and BL-A
IgG
A-BL and BL-A
Unidentified in
caveolae
FcRn in unidentified
carrier
LDL cholesterol
Gonadotrophin
A-BL
A-BL
Transferrin
(Tf)-iron
Insulin
A-BL
LDL
A-BL
Iron
Tf
A-BL
BL-A
M cells in Peyer’s
patches of ileum
IgG
Membrane
associated
Antigens,
pathogens
BL-A
very little ABL; no BL-A
A-BL
Absorptive enterocytes
dIgA
BL-A
pIgA receptor via CCV
Newly
synthesized
apical PM
proteins
Vitamin B12
BL-A
Unknown
A-BL
dIgA
BL-A
Cubilin/megalin via
CCV
pIgA-receptor via CCV
BL-A
Unknown
Sinusoidal endothelium
Newly
synthesized
apical PM
proteins
Ceruloplasmin
A-BL
Unknown
Proximal tubule cells
Vitamin B12
A-BL
Vitamin D
A-BL
Vitamin A
A-BL
Maternal IgG
A-BL
TCII; megalin-mediated
in CCV
D binding protein;
megalin-mediated in
CCV
Retinol binding protein;
megalin-mediated in
CCV
FcRn via CCV
Maternal B12
A-BL
Cubilin?/megalin/TCII
Syncytiotrophoblasts
Maternal IgG
Iron
A-BL
A-BL
FcRn
Maternal Tf receptor
via unidentified
carrier
Fetal capillary
endothelial cells
Iron, maternal
IgG
BL-A
Tf receptor and FcRn
via unidentified
carriers
Continuous capillary
endothelium
Arterial endothelium
Continuous capillary
endothelium (has
markers of brain EC)
Cerebral endothelium
(tight continuous)
Choroid plexus
Adult
intestine
Liver
Absorptive enterocytes
in terminal ileum
Hepatocytes
Hepatocytes
Kidney
Neonatal
intestine
Enterocytes
Yolk sac
Placenta
Fluid-phase in caveolae
Chorionic gonadotropin
receptor in CCV
Nonspecific in caveolae
A-BL
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Insulin receptor via
unidentified carrier
LDL receptor via
unidentified carrier
Tf receptor via CCV
Tf receptor via
unidentified carrier
FcRn
?
Both phagocytic and
clathrin-mediated
uptake
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Comments
Reference No.
See text
183, 378, 503, 602
456
Contributes to IgG homeostasis
and prolonged half-life of IgG in
body
Not via LDL receptor
Target is Leydig cell
Not via Tf receptor
Rabbit, rhesus monkey, binding to
isolated human brain capillaries
In vitro study, but LDL receptor
present in vivo
Debate over Fe ⬎⬎⬎Tf
Apo-form preferred over holo
Efflux from brain
Degradation in lysosomes is
predominant fate
Lack of thick glycocalyx thought to
permit entry; ␤1-integrin on
apical PM mediates entry;
transcytosis thought to be
through prelysosome then
exocytosis
Multiple intermediate
compartments in pathway
No intracellular intermediates
identified
47
600
181, 182
236
133, 431
112
104, 476
635
501, 634
590
259, 400, 402,
404, 492
64
148, 355
See Table 2 for molecular players
128, 129, 515, 519
Multiple intermediate
compartments in pathway
Entry mechanisms unknown,
intermediate compartments may
be same as those of pIgA
238, 468
Postulated to be desialylated in
transit to hepatocytes
Degradation of TCII in lysosomes;
unknown storage site for B12
Degradation of protein carrier in
lysosomes; fate of vitamin D?
570
Same fate as above
Subsequent movement into apical
endosomes then fusion with
lateral membrane
Identification of cubilin as target of
teratogenic antibodies
Apical location of receptor
Apical location of receptor and
HFE receptor modulator;
ferreportin and endogenous
copper oxidase believed to
facilitate iron release at
basolateral PM
32, 499
311
413
347
1, 245
450, 516
543
107, 172, 432, 433
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Brain
Cell Type
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TRANSCYTOSIS
TABLE
1—Continued
Organ System
Lung
Mammary
gland
Cargo
Direction
Receptor/Carrier
Trachea
HRP, ferritin
A-BL
Unknown
Upper airways
IgA
BL-A
IgA-receptor via CCV
Bronchial epithelium
Albumin
A-BL
Unknown
A-BL
FcRn
Perfused rat lung
Bioactive-Fc
fusion
protein
Albumin
A-BL
gp60 in caveolae
Alveolar epithelium
dIgA
BL-A
pIgA-receptor
Iron
BL-A
IgG
BL-A
Thyroglobulin
A-BL
Tf receptor via
unidentified carrier
Not identified; not
FcRn
Megalin-mediated
endocytosis via CCV
Thyroid epithelial cells
Comments
Reference No.
Presence in large endosomes;
degradation also?
A-BL at low levels may be entry
point for pathogens
Degraded fragments released at BL
(Ussing chamber)
Transport of intact protein
determined by bioassay
469
Question of which alveolar cell
type involved
Presumed to be same as in
intestine and liver
Transferrin synthesized by lactating
gland; pathway of iron unknown
Neonatal rodent only
267
Separate route for Thy-T3/T4
(lysosomes)
273
269
553
497
310
245
352
A, apical; BL, basolateral; HFE, hemachromatosis gene product; PM, plasma membrane; Tf, transferrin; TCII, transcobalamin II; CCV,
clathrin-coated vesicle; Thy, thyroglobulin; T3/T4, thyroxines; pIgA, polymeric IgA; FcRn, IgG receptor; LDL, low-density lipoprotein.
FIG. 1. The ultrastructure of a capillary network, an endothelial cell, its membrane, and caveolae. A: the vascular
casts of the forestomach are shown in this scanning electron micrograph. The submucosal vessels are seen under the
two-dimensional mucosal capillary network. [From Imada et al. (254), copyright 1987 Springer-Verlag.] B: this transmission electron micrograph is representative of the ultrastructure of an endothelial cell if the capillaries indicated in A were
viewed in cross section. [From Bolender (40) by copyright permission of The Rockefeller University Press.] C: a higher
magnification of the indicated region of the endothelial cell in B. The caveolae attached to both the luminal and basal
plasma membrane (PM) domains are indicated. [From Fawcett (147), with permission from Journal of Histochemistry
& Cytochemistry.] D: a higher magnification of the indicated caveolae in C. Caveolae (vesicles; v) open to the blood or
tissue fronts or that appear to be completely closed are indicated. [From Bruns and Palade (65) by copyright permission
of The Rockefeller University Press.]
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Thyroid
Cell Type
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directions? Are clathrin-coated vesicles used, as they are
to carry maternal IgG from the gut to the interstitium
(apical to basolateral) in neonatal rodents (Table 1)? Is
excess IgG degraded when the endothelial FcRn receptor
is saturated with its ligand, absent, or dysfunctional? If so,
how? This area of endothelial cell biology deserves further study, because it might reveal the mechanism(s) used
to selectively deliver a ligand (IgG) from the interstitum
back to the circulation (basolateral to apical). We will
return to several of these issues below. Finally, one
polypeptide hormone, human luteinizing hormone/chorionic gonadotrophin (hLH/CG), is reportedly transcytosed
via clathrin-coated pits and vesicles across the continuous endothelium of the testis (Table 1). The transport is
apparently mediated by the same receptor present on
Leydig cells, the target of the hormone in the testis. This
system deserves further study, because it is one of two
documented examples in which clathrin-coated vesicles
of an endothelium transcytose cargo; the other is brain
endothelia and transferrin-Fe (see sect. IIB1C).
B. Transcytosis in the Brain
Since the 19th century dye experiments of Ehrlich,
the brain has been known as a “privileged” organ where
access is tightly regulated so that the environment remains chemically stable. The brain’s fluid is different from
either the blood or noncerebral tissue. The two principal
gatekeepers of the brain are the cerebral capillary endothelium and the epithelial cells of the choroid plexus (Fig.
2A). These cellular barriers are specialized for the passage of different nutrients from the blood (132, 552). The
capillaries move nutrients that are required rapidly and in
large quantities, such as glucose and amino acids. These
small molecules are transported by membrane carriers
using facilitated diffusion. The choroid plexus supplies
nutrients that are required less acutely and in lower quantities. These are folate and other vitamins, ascorbate, and
deoxyribonucleotides. Their transport requires energy
since the blood concentrations of these nutrients are
extremely low. Of relevance to this review is experimental evidence that transcytosis of a limited set of macromolecules occurs across brain capillaries from blood to
the interstitium (the blood-brain barrier) but does not
occur across the epithelial cells of the choroid plexus into
the cerebrospinal fluid [the blood-cerebrospinal fluid
(CSF) barrier].
1. Cerebral capillaries
Compared with other organs, the abundance of capillary endothelium in brain is very high (35). At the same time,
permeability is about two orders of magnitude lower than
that of endothelia in peripheral organs, giving rise to the
designation of this endothelium as “tight continuous” (201).
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controversy between physiologists and morphologists for
over 50 years. Numerous reviews documenting the history, experimental details, and different interpretations
have appeared in this and other journals (377, 467, 539,
571, 608, 614). Because the controversy centers on
whether tight junctions or caveolae serve as the major
(only) conduit for transported cargo, we will briefly recap
this story.
On the basis of experiments in which he compared
the compositions of the blood and lymph in the hindleg
muscle of cats injected with various size tracers, Pappenheimer et al. (428) postulated in 1951 that plasma components (ions, small solutes, and proteins) were transported
through two types of rigid pores: a small, ⬃3- to 5-nmdiameter pore present at a frequency of 100/␮m3 and a
large, ⬃20- to 40-nm-diameter pore present at 1% the
frequency of the small ones (428). When the endothelium
was seen at the ultrastructural level, no structures corresponding to the postulated pores were found. Instead,
caveolae were observed, leading Bruns and Palade (65,
66) to suggest that they performed the function ascribed
to the rigid pores. In the early years of this controversy,
differences in the cell systems, approaches, and tracers
used by researchers in the two camps often yielded conflicting results with differences in interpretations. However, both sides progressively refined their experimental
approaches and have arrived at an apparent consensus:
caveolae do play a role in transport across endothelia,
either as fused channels (physiologists) or bonafide transport vesicles (cell biologists). Our position is that caveolae contribute to the high permeability of continuous
endothelium. However, because their number exceeds
the number of functional pores predicted by Pappenheimer’s results, there must be other functions for caveolae.
In fact, they have been proposed to harbor signal transduction components in both active and inactive states
(9, 546). The recent reports of mice genetically engineered
to lack the protein caveolin-1, a major component of
caveolae, are particularly relevant and are discussed in
section IV.
Although the controversy has focused on bulk-phase
transcytosis, receptor-mediated transcytosis of specific
macromolecules also takes place across the endothelia of
the microvasculature (Table 1). Albumin and orosomucoid are both transcytosed in competable, saturable, and
temperature-dependent fashions, and caveolae mediate
their transport. A putative receptor for albumin of ⬃60
kDa that is present only on continuous capillary endothelia has been identified by several groups (Table 1), but it
has not yet been cloned and sequenced. The transcytosis
of IgG across continuous endothelial cells is particularly
interesting, in light of the expression of the neonatal Fc
receptor (FcRn) by these cells (47) and the receptor’s role
in maintaining high serum IgG in the adult (Table 1). Does
the receptor work in both the apical-to-basal and reverse
877
TRANSCYTOSIS
Two features of brain endothelia are different from endothelia in the periphery. Brain endothelial cells have the lowest frequency of caveolae (⬍100/␮m3), and the character of
their tight junctions is influenced by underlying astrocytes
through the actions of soluble factors, including cytokines
(111, 263). Claudins 1 and 5 as well as occludin appear to be
relevant players in providing a particularly tight junction
Physiol Rev • VOL
(229, 289, 319, 385). Very little macromolecular cargo is
transcytosed across the cerebral capillary endothelium. The
three best-studied ligands are insulin, LDL-cholesterol, and
iron; questions and controversy surround each.
A) INSULIN. The finding that insulin-sensitive glucose
transporters (GLUT 4) were present in the brain (312,
369) led to the search for insulin receptors on endothe-
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FIG. 2. The barriers of the blood-brain (A) and placenta (B). A: in a, a diagram indicating the location of the choroid
plexus in the human brain is shown. In b, the relationship between the blood, brain, and cerebrospinal fluid is shown.
The dashed line follows two typical open pathways connecting the ventricular cerebrospinal fluid with the basement
membrane of parenchymal blood vessels and with the basement membrane of the surface of the brain. A, astrocytic
process; C, choroid plexus epithelium; Cs, choroid plexus stroma; E, endothelium of parenchymal vessel; EC, endothelium of choroid plexus vessel; Ep, ependyma, GJ, gap junction; N, neuron; SCSF, cerebrospinal fluid of the subarachnoid
space; TJ, tight junction; VCSF cerebrospinal fluid of ventricles. [From Brightman and Reese (60) by copyright
permission of The Rockefeller University Press.] B: a “low magnification” diagram of the chorionic villus is shown (a).
The arterio-capillary-venous network (network) is indicated at the top. [From Moe (380).] In b, a cross section of a
full-term villus is shown. The placental membrane separates the maternal blood from the fetal blood. At the end of
pregnancy, this membrane becomes very thin. [From Moe (380).] In c, a more detailed version of the chorionic villus is
shown. The microvillar surfaces (MV) and basal membranes (basal) are indicated. CT, cytotrophoblast; FV, fetal vessel;
SK, syncytial knot; ST syncytiotrophoblast. [From Moore and Persaud (383) by copyright permission of Saunders.]
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PAMELA L. TUMA AND ANN L. HUBBARD
Physiol Rev • VOL
complexed to Tf. Again, there is conflicting data about the
source of this protein. Tf is synthesized and secreted by
the epithelial cells of the choroid plexus (384). However,
hypotransferrinemic mice have been shown to accumulate substantial amounts of intraperitoneally administered
human transferrin intracranially, indicating that the endogenous source could also be derived from the serum
(126).
The brain endothelial insulin and Tf-Fe transport systems have received attention from researchers working
on therapeutic drug delivery systems (96, 161, 165). An
anti-Tf-receptor antibody, OX26, is transcytosed into the
brain mass, but the amounts are extremely low, ⬍1% of
the antibody injected. Although this amount may be sufficient for drug delivery, it is not definitive evidence for
quantitative transcytosis of the receptor along with its
cargo. Nonetheless, this approach is being combined with
toxins that bind to specific claudins and transiently open
tight junctions, to deliver macromolecular drugs (100).
D) IMMUNOGLOBULIN G. It turns out that brain endothelial
cells express the FcRn and transport intracranially delivered IgG out of the brain in a receptor-mediated fashion
(Table 1). The question is how circulating IgG initially
crosses into the brain. As for the peripheral endothelium,
the vesicular carrier and molecular mechanisms responsible for IgG transport are as yet unknown.
2. Choroid plexus
The choroid plexus is composed of a highly convoluted sheet of cuboidal ependymal epithelium that sits on
a closely apposed basal lamina. Both morphological and
biochemical tracers have provided good experimental evidence that the apical tight junctions of these epithelial
cells are the blood-CSF barrier in the choroid plexus (see
Fig. 2A). To date, no obvious ultrastructural or molecular
features distinguish these junctions from neighboring
ependymal cells, but we would predict that specific claudins are responsible for this difference (323). Interestingly, the basal lamina may act as an inducer of the tight
junction specializations that make this cell type highly
impermeable to macromolecules (reviewed in Ref. 590).
The epithelial cells make much of the CSF that nourishes
and cushions the brain. The protein content of CSF (25
mg/100 ml) is low relative to that of plasma (⬃6,500
mg/100 ml), and the composition is different. Transthyretin, which binds thyroxine, and Tf are made and secreted
by the epithelial cells, while apoproteins E and A1, which
are present in lipoprotein particles in the CSF, are made
by astrocytes. The presence of these proteins in CSF
raises the obvious questions of where and how their
ligands, presumably from the blood, reach them? Furthermore, are the components of CSF fluid functionally accessible to brain tissue or part of a drainage system much
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lium and hormonal effects on the cells. Although receptors were found (431, 592), the current status of insulin’s
transport by brain capillaries is not resolved.
B) LDL. Cells in the brain require cholesterol, which is
synthesized endogenously (127), but can also be provided
by the transcytosis of plasma LDL intact across brain
endothelium (112). There is good evidence for the presence of LDL receptors on the luminal PM of cerebral
endothelium (376). Such expression is unusual, since
cells that are constantly exposed to the high LDL levels in
plasma normally downregulate their LDL receptors. Experimental evidence from in vitro studies indicates that
cholesterol levels in the underlying astrocytes play a role
in regulating LDL receptor expression levels in the overlying endothelial cells (111). The puzzle here is how a cell
distinguishes between LDL for its own needs and LDL for
use by cells behind the permeability barrier it forms. In
the periphery, this seems to have been solved by receptormediated endocytosis via clathrin-coated pits/vesicles for
internal use versus fluid-phase (non-receptor-mediated)
transcytosis via caveolae for use by interstitial cells (600).
However, in the brain, there is virtually no fluid-phase
(i.e., nonselective) transcytosis. Thus it will be important
to localize LDL receptors in brain endothelium at the
ultrastructural level; are they in caveolae or clathrincoated pits? Another important issue is how transcytosed
cholesterol is presented at the abluminal surface of endothelial cells, since apoprotein B, which is the apoproteincarrying cholesterol in the circulation, is not present in
CSF. Apoproteins A1 and E are the principal cholesterolcarrying molecules in the brain (127, 629). Underlying
pericytes of the brain endothelium have been characterized as phagocytic; perhaps they participate in the degradation of apoprotein B and release of cholesterol into the
CSF. Cholesterol dynamics in the brain have been reviewed recently (624).
C) IRON. Iron is also transported across the blood-brain
barrier, but there is conflicting data as to whether it is
delivered with or without transferrin (Tf), the principal
iron-carrying protein of plasma (52). (Iron and Tf are
discussed in more detail in sect. IID2.) While several in
vivo studies have reported that injected 125I-Tf does not
accumulate to the same extent as 59Fe administered similarly (104), others report equivalent accumulations (152,
635). Tf receptors are definitely present on brain endothelium (248), and Tf is internalized by brain endothelium in
vivo via clathrin-coated vesicles (476), leading some to
speculate that plasma Tf may be carrying Fe across and
then recycling back unloaded (52, 384, 429, 430, 545, 591,
635). Such a scenario would require a milieu on the basal
side of sufficiently low pH to effect iron’s release. Because the pH of the underlying interstitium in brain is not
known to be acidic, there must be novel dissociation
mechanisms not yet discovered. Whatever the mechanism, iron is not free in the brain interstitium but is
879
TRANSCYTOSIS
C. Immunological Protection and Transcytosis
At several stages in the intricate choreography of the
vertebrate immune response, transcytosis is used to move
antigens and protective antibodies across epithelial barriers (Table 1). “Antigen sampling” is the first step in the
mucosal immune response and entails the apical-to-basolateral delivery of soluble and particulate antigens to underlying mucosal-associated lymphoid tissue. This transcytotic event is carried out principally by M cells that
are located in lymphoid follicle-associated epithelium
throughout the gastrointestinal and urogenital tracts (175,
401, 402, 579, 621). Later in the mucosal immune response, polymeric IgA, secreted by appropriately activated plasma cells, is transcytosed along the basolateralto-apical axis by epithelial cells in the digestive tract,
liver, and mammary gland and is released as secretory IgA
into the gut lumen, bile, and milk, respectively (245, 302,
393). The third use of transcytosis occurs in a form of
systemic immune protection, termed “passive immunity,”
which is the transport of maternal IgG to the developing
fetus or neonate. Species differences dictate whether maternal blood or milk is the source of the IgG and whether
the placenta or the intestine is the site of this transfer
(178, 245). Certainly, the last two transcytotic processes
start with uptake of their cargo through clathrin-coated
pits/vesicles and may transit through parts of the endosomal system. Thus the mechanisms regulating these itineraries most likely differ from those used by endothelial
cells, where a caveolar pathway predominates.
Physiol Rev • VOL
1. Structural features of intestinal epithelial cells
Figure 3 shows the tissue organization and ultrastructural appearances of M cells and enterocytes (adsorptive columnar cells), the two epithelial cells participating in transcytosis in the intestine. These cells are very
different from one another and the capillary endothelial
cell. Depending on the species, M cells comprise a variable but small percentage of the epithelia overlying organized mucosal-associated lymphoid tissue, making them a
very minor cell population in the gastrointestinal tract.
Being epithelial cells, their basal extensions sit on a basal
lamina, but much of their basal membrane lines an extracellular “pocket” in which migrating monocytes and lymphocytes accumulate. As can be seen in Figure 3B, the
pocket is a short distance from the apical surface. Thus
these cells have evolved a short transcellular pathway
much like the endothelial cells, but in contrast they have
few to no caveolae; rather, coated pits are present on the
apical PM. Figure 3B also shows that M cells do not have
the luxuriant brush border that is present on adjacent
absorptive enterocytes. They have short microvilli, or
microfolds, hence the name M cells. In contrast, absorptive enterocytes are simple columnar cells with several
apical features in addition to their brush borders (Fig.
3C). Clathrin-coated pits are present at the base of microvilli, and a thick glycocalyx composed of integral
membrane proteins with glycosaminoglycan side chains
emanates from the microvillar membrane. This latter
structural feature as well as the rigidity of the microvilli
are thought to prohibit microorganisms from attaching
and invading enterocytes. The intracellular organization
of these columnar epithelial cells is also polarized, with
basally located nuclei, supranuclear Golgi, and an abundance of pleiomorphic membrane compartments underlying the terminal web of the brush border (Fig. 3C). The
basolateral-to-apical length of this cell is ⬃20 versus 0.2
␮m for a capillary endothelial cell, making the transcytotic route across enterocytes potentially much longer.
Furthermore, microtubules are an important structural
element of the transcytotic pathway in enterocytes, but
not in M or endothelial cells.
2. M cells, transcytosis, and antigen sampling
Quite early, researchers studying the routes of pathogen invasion discerned that specific regions of the intestine collected adherent particulate material present in the
gut lumen; when the regions were visualized at the EM
level, M cells were identified as the invasion route (72,
579, 621). The transcytotic route across M cells is thought
to be part of the mechanism by which antigens are routinely sampled along the entire mucosal surface. Not surprisingly, numerous pathogens have evolved mechanisms
to exploit the transcytotic process as a means to invade
and disseminate before a strong enough immune re-
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like the lymphatics? To our knowledge, there are no
definitive answers.
What about transcytosis in the choroid plexus? Interestingly, although endocytosis is robust at the basal surface of the epithelial cells, transcytosis across to the
apical environment is minimal to nonexistent; rather, virtually all tracers internalized from the basal side end up in
lysosomes (590). The endocytic activity may reflect the
high permeability of the fenestrated capillaries that supply the choroid plexus and hence the abundance of
plasma proteins bathing the basal side of these cells. Van
Deurs (589) examined transcytosis in the apical to basolateral direction as a possible route for elimination of
waste from the CSF. Intraventricular injection of soluble
horseradish peroxidase and cationized ferritin resulted in
their overwhelming delivery to lysosomes; very small
amounts appeared in coated pits along the lateral surface
(589). The conclusion that apical-to-basolateral transcytosis was not an active pathway has been confirmed by
others (24) using additional electron microscopic (EM)
tracers.
880
PAMELA L. TUMA AND ANN L. HUBBARD
sponse can be mounted (403, 464). In recent years, this
route of entry has been studied intensively in the hopes of
understanding the basic mechanisms of antigen sampling
and developing effective vaccine delivery systems against
stealthy invaders. Because adherence is an essential first
step in invasion, researchers have focused on identifying
the molecular basis for the selective adherence of antigens and pathogens to M cells and not adjacent enterocytes. Lectin staining in situ has been used in attempts to
identify particular glycosidic moieties that might be differentially expressed by M cells (160, 186, 187). Recently,
Physiol Rev • VOL
␤1-integrin was localized to the M cell apical surface and
proposed as the receptor for several pathogens (257–259).
This membrane protein, which is expressed on the basolateral surface of neighboring absorptive cells, has a cytoplasmic tail that could mediate the endocytosis of particles bound to its extracellular domain. In support of this
notion, M cells are avidly endocytic and their apical membrane is much more dynamic than the rigid and stable
brush border of the enterocyte. In fact, both phagocytic
and pinocytic mechanisms appear to operate at the apical
surface of these cells. Adsorbed macromolecules are en-
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FIG. 3. The small intestine (ileum) contains Peyer’s patches. A: a schematic drawing of a Peyer’s patch is shown that
illustrates the general arrangement of gut-associated lymphoid tissue. Lymphoid follicles in the submucosa are associated with dome areas that extend into the gut lumen. The domes are covered with specialized epithelium that contains
M cells. B lymphocytes mainly populate the lymphoid follicles, while T cells predominate the interfollicular areas. High
endothelial venules (HEV) in the interfollicular areas are the route through which lymphocytes enter the Peyer’s patch.
[From Gebert et al. (175) by copyright permission of Academic Press.] B: a transmission electron micrograph of an M
cell and adjacent columnar cells (CC) from the region indicated in A. The typical M cell has short, irregular microvilli
and a basolateral pocket (P) into which the lymphoid cells (here resembling plasma cells) and macrophages migrate.
Luminal antigens are endocytosed, transported across the apical cytoplasm (bracket), and delivered to the basolateral
pocket. [From Weltzin et al. (616) by copyright permission of The Rockefeller University Press.] C: a low-magnification
transmission electron micrograph of several absorptive cells and part of a goblet cell from the region indicated in A is
shown from a fasted rat. The lumen of the intestine is at the top and a small portion of the lamina propria (LP) is shown
at the bottom. A thin basement membrane separates the basal surfaces (BL) of the cells from the lamina propria. An
elongated nucleus is located in the basal region of the cell under which is a dense cluster of mitochondria and few free
ribosomes and rough endoplasmic reticulum (RER). The apical cytoplasm contains long mitochondria, a prominent
Golgi component (G), RER, and smooth endoplasmic reticulum (SER) concentrated at the terminal web (TW). The free
surface is covered by microvilli (Mv). L, lipid droplet. [From Cardell et al. (77) by copyright permission of The
Rockefeller University Press.]
881
TRANSCYTOSIS
docytosed via clathrin-coated vesicles (404) and delivered
to a prelysosomal/lysosomal compartment from which
they are released into the underlying pocket for subsequent uptake by lymphocytes and macrophages (404, 425,
616). Thus, unlike endothelial cell transcytosis, lysosomes
appear to play a role in M cell transcytosis. Whether the
cargo in this compartment is modified by acid hydrolases
present in it (6) is not currently known.
3. Transcytosis of IgA
4. Transcytosis of IgG
The transfer of maternal immunoglobulins to fetal or
neonatal offspring provides the latter with systemic immunity until their immune system matures. Several organs transport IgG-type immunoglobulins (245). As with
IgA, maternal IgG must be transcytosed across an epithelial barrier. In all mammalian species, it is transcytosed in
an apical-to-basal direction. Thus in humans, IgG in the
maternal blood is transported across the placenta (Fig.
2B), while in rodents, maternal IgG is first delivered into
milk, a basal-to-apical route, and secondarily across the
absorptive epithelial cells of the small intestine, an apicalto-basolateral route.
Physiol Rev • VOL
D. Role for Transcytosis in the Homeostasis
of Micronutrients
Most vitamins, essential minerals, and trace elements, collectively called micronutrients, come from the
diet. Thus they must cross an epithelial barrier some-
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The large amount of mucosa-associated lymphoid
tissue and its specialization for the production of IgA
make IgA the major immunoglobulin in humans (301,
389). Given that it is synthesized and secreted by plasma
cells located in the lamina propria of the digestive, respiratory, and urogenital tracts yet functions in external
secretions, IgA must be delivered across an epithelial
barrier. This requirement is accomplished by the polymeric IgA-receptor (pIgA-R), a single transmembrane protein synthesized by the epithelial cells. As discussed in
detail in section IV, this receptor has a long (⬃100 amino
acid) cytoplasmic tail that contains most of the signals
necessary to direct it through its cellular itinerary. However, unlike most other endocytic receptors that perform
repeated rounds of cargo uptake, delivery and recycling,
the extracellular domain of pIgA-R is cleaved upon delivery to the apical surface and released into the lumen with
its ligand. The presence of the added “secretory component” stabilizes IgA in the gut lumen. This unique transcytotic system is expressed in many epithelia throughout
the body, including kidney, trachea, and the digestive
tract, including the liver (Table 1). Interestingly, some
pathogens appear to have exploited the small percentage
of uncleaved pIgA-R present in the apical membrane of
nasopharyngeal epithelial cells to gain entry into the underlying interstitium (Table 1). This result suggests that
the receptor is able or can be coerced to transcytose in an
apical-to-basolateral direction. The mechanism, whether
normal or pathogen induced, may have therapeutic potential.
The receptor mediating the apical-to-basal transport
of IgG is FcRn, a distant member of the major histocompatibility complex (MHC) I family (542). As for other MHC
I proteins, the FcRn is a heterodimer, with a transmembrane heavy chain and ␤2-microglobulin light chain. The
heavy chain has a cytoplasmic tail containing an internalization motif that mediates endocytosis of maternal IgG
via clathrin-coated pits and vesicles present at the base of
the apical brush border of neonatal rodent enterocytes.
The receptor and ligand are transported through an endosomal compartment to the lateral surface of these cells
(1). Thus this transport system shares the property with M
cells of using a prelysosomal compartment to deliver its
cargo. However, the unique pH dependence of binding
allows the ligand to remain associated with the receptor
at low pH (in the gut lumen and through slightly acidic
endosomes) and be released at neutral pH (in the interstitial space) without apparent modification. The FcRn
recycles back to the apical PM in neonatal intestine.
Although the finding that ␤2-microglobulin (␤2-M)
knock-out mice lacked the apical-to-basal IgG transport
system in the neonatal intestine was expected, it was
initially surprising that circulating IgG in ␤2-M-null adults
exhibited a much shorter half-life than in wild-type mice
(177). This result suggested that FcRn played a role in IgG
homeostasis (541), confirming a long-standing hypothesis
by Brambell (54) that the prolonged circulation of IgG in
plasma was due to a receptor capable of protecting IgG
from degradation. Given the pH dependence of IgG binding to FcRn, the current view is that intracellular FcRn
binds nonspecifically endocytosed IgG within an endosome-like compartment and returns it to the circulation
(177). The tissues and cells performing the protective
function may be hepatocytes or endothelial cells, since
both express FcRn at the PM. Quantitative studies are
needed.
An important finding in the ␤2-M knock-out mouse
studies was that the levels of maternal IgG in colostrum
and milk were normal, indicating that FcRn does not play
a role in the transcytosis of IgG in the rodent mammary
gland. This is not so surprising, considering that the direction of transport is opposite to that in the placenta or
intestine, although in endothelial cells, the FcRn presumably carries IgG in the basal-to-apical direction (Table 1).
The identity of the mammary gland receptor system will
be important to determine.
882
PAMELA L. TUMA AND ANN L. HUBBARD
1. Vitamin B12
All cells require vitamin B12 as a coenzyme in onecarbon transfers. Methyl malonyl CoA mutase uses it in
the adenosyl-B12 form to convert methyl malonyl CoA to
succincyl CoA in the mitochondria; methionine synthetase uses it in the methyl-B12 form to convert homocysteine to methionine in the cytoplasm. B12’s journey to
the cytoplasm of all cells is a fascinating and curiously
TABLE
convoluted process. The players so far identified are
listed in Table 2 and placed in cellular context in Figure
4A. Several reviews cover this topic in more detail (276,
488, 514).
A) UPTAKE FROM THE INTESTINAL LUMEN. In carnivores, B12 is
present in ingested meat as the cofactors mentioned
above. Upon digestion by pancreatic enzymes in the small
intestine, free B12 is bound by intrinsic factor (IF), a
27-kDa glycoprotein secreted by parietal cells of the stomach (317). In the terminal ileum of the small intestine, the
luminal B12-IF complex binds to its receptor, cubilin, a
large membrane-associated glycoprotein that is located in
the microvillar brush border of absorptive enterocytes
(515). Cubilin’s association with megalin, a member of the
LDL receptor-related (LRP) family of endocytic receptors
(reviewed in Refs. 192, 619), leads to the internalization
via clathrin-coated vesicles of the entire cubilin-IF-B12
complex. After delivery of the IF-B12 complex to endosomes, cubilin and megalin recycle for further rounds of
endocytosis; since cubulin’s association with megalin is
stable at pH 5, it is thought to stay associated throughout.
Meanwhile, dissociated B12-IF is delivered to lysosomes,
where the protein is degraded by leupeptin-inhibitable
acid hydrolases (196) and B12 is transported out of lysosomes. This last step is mediated by a yet-to-be-discovered transporter(s). Interestingly, in this pathway there is
no avoidance of lysosomes; to the contrary, lysosomal
function is essential, since failure to degrade IF within the
intestine results in a B12 deficiency in all subsequent
tissues and cells. Some investigators are exploring the B12
entry pathway as a means to deliver drugs orally (487).
2. Vitamin B12 (cobalamin)
Molecule
Site of Synthesis/Expression
B12
Bacteria
R binders
(haptocorrins)
Salivary gland, stomach,
placenta, circulation,
and granulocytes
Parietal cells/stomach
Enterocyte in terminal
ileum
Intrinsic factor (IF)
Cubilin
Megalin
Transcobalamin
binding protein
(TCII)
TCI and III
TCII receptor
Enterocyte in terminal
ileum; also present in
kidney proximal
tubule, placenta,
thyroid
Liver and enterocyte
Granulocytes
Kidney, liver, placenta,
intestine
Biochemical Characteristics
Small secreted glycoproteins
(includes TCII, not a
glycoprotein)
27-kDa secreted glycoprotein
400-kDa peripheral membrane
glycoprotein secreted apically by
enterocyte
600-kDa transmembrane
glycoprotein, clathrin
internalization motif in
cytoplasmic tail
45.5-kDa secreted protein
Secreted glycoproteins
62-kDa transmembrane glycoprotein
Definitions are as in Table 1.
Physiol Rev • VOL
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Functions
Coenzymes
(adenosylcobalamin,
methylcobalamin)
Bind cobalamin
Binds B12 in small intestine
Binds B12-IF complex at
apical PM
Apical PM receptor that binds
cubilin-IF-B12 and TCII-B12
complexes in Ca-dependent
manner and mediates
endocytosis; recycles
Binds B12 in circulation
Bind B12; unknown functions
Basolateral PM receptor for
TCII-B12
Reference
No.
69
317
294, 515
381
448, 460,
463
49, 518
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where along the digestive tract; this occurs primarily at
the level of the intestine. However, transcytosis is the
least used route for micronutrient absorption. Lipid-soluble vitamins associate with bile acid micelles in the gut
lumen and are thought to then partition progressively and
passively across absorptive cells, associating with chylomicra somewhere before or at the basal side of the cells.
Dietary vitamin B12 (cobalamin) is an exception, because
it uses vesicle-mediated steps, in part, to cross intestinal
cells. Many minerals are assumed to be absorbed paracellularly (61). This assumption is based on calculations of
transit times and absorption rates. However, dietary iron
is transported across the intestinal epithelium via multiple
membrane transporters; once in the circulation, its delivery to the brain and fetus requires transcytosis. Additionally, Cu and Zn, as well as other heavy metals, appear to
be transported into intestinal absorptive cells via membrane transporters at the apical plasma membrane. Finally, the kidney proximal tubule cells provide an important function in vitamin homeostasis by avidly scavenging
several vitamins (Table 1) from the urine using a modified
type of transcytosis.
883
TRANSCYTOSIS
B) TRANSFER TO CIRCULATING TRANSCOBALAMIN II AND TRANSCYTO-
A puzzle is the mechanism by which cytoplasmic B12 is
subsequently transported into the basal milieu surrounding the enterocyte. Extracellular transcobalamin (TC) II, a
40-kDa protein in the interstitium/circulation, serves as
the major functional carrier of B12 (Table 2). (TCI and
TCIII are also B12 carriers, but their functions remain
unknown.) TCII is synthesized and secreted principally by
the liver (206, 495). Evidence that enterocytes express a
TCII transcript (460) suggests that B12 may be loaded onto
newly synthesized protein as it transits the secretory pathway (463). The B12-TCII complex would then be released
SIS.
Physiol Rev • VOL
at the basolateral surface of the enterocyte. Of course,
this scenario requires the presence of a B12 membrane
transporter in the secretory pathway.
Once in the circulation, how does TCII-B12 reach
cells? All cells express a TCII receptor, which mediates
the endocytosis of the complex via a clathrin-mediated
mechanism. The TCII receptor is a single transmembrane
glycoprotein of 62 kDa (49) that functions as a homodimer at the plasma membrane (Table 2). After internalization, the TCII-B12 complex is delivered to lysosomes, where TCII is degraded and B12 is again transported into the cytoplasm for subsequent use as a
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FIG. 4. The intracellular itineraries of vitamin B12 and iron in polarized enterocytes and nonpolarized peripheral
cells. A: the current model for the trafficking of vitamin B12 is shown. In the polarized enterocyte, B12 complexed with
intrinsic factor binds the cubilin-megalin complex and is internalized via a clathrin-mediated pathway. In acidic
endosomal vesicles (EV), the ligand dissociates and is sorted to the degradative pathway, whereas the cubilin-megalin
complex is sorted to dense apical tubules (DAT) and recycled back to the PM. In lysosomes, intrinsic factor is degraded
and vitamin B12 is released and then secreted coupled to transcobalamin (TC) II that is shown in a secretory vesicle. In
the peripheral cell, the TCII-B12 complex that is bound to the TCII receptor is internalized via coated pits (CP). TCII
ligand is degraded in lysosomes and B12 is released into the cytoplasm by mechanisms that are not completely
understood and delivered to other cellular destinations. B: the proposed trafficking pathways for iron (Fe) are shown.
Iron is transported into the polarized enterocyte via DMT1 present at the apical cell surface. Ferroportin present at the
basolateral PM transports iron from the cell where it binds transferrin in the circulation. The iron-transferrin complexes
bind peripheral cells via associations with the transferrin receptor, and the ligand-receptor complexes are internalized
in coated pits (CP). Iron dissociates from transferrin in an early endocytic compartment (EE) and apotransferrin, still
bound to the receptor, is delivered back to the PM via a recycling compartment (RC). It is unclear how the free iron is
released into the cytoplasm. Here we have suggested that DMT1, present on early endosomes, transports the iron. CV,
coated vesicle; lys, lysosome; mito, mitochondria; PLC, prelysosomal compartment.
884
PAMELA L. TUMA AND ANN L. HUBBARD
cofactor (249). But how does B12 reach cells behind a
selective barrier, for example, the brain or testis? Although a B12-TCII receptor on brain or testicular endothelial cells has not been reported, we predict that it must be
there. Could it be the same receptor as that found on the
basolateral PM of most epithelial cells, even though it
would be on the apical PM of these endothelia? What is
the mode of transcytosis and the subsequent fate of the
TCII protein and B12 in brain endothelial cells? Are the
fates different from those in other cells? Clearly, this
system would be interesting to explore further.
C) INVOLVEMENT OF ADDITIONAL EPITHELIA IN B12 HOMEOSTASIS.
2. Iron
An essential cofactor in the homeostasis of every
cell, iron in mammals exists in three predominant forms:
bound to the circulating plasma protein transferrin; as
heme in intracellular proteins such as mitochondrial cytochromes, hemoglobin, and myoglobin; and in ferritin,
the storage form of iron (5). Iron is avidly reutilized by
mammals, and thus the daily requirement for it is small.
Of 3– 4 g total body iron, only 1–2 mg are lost per day
through desquamation. However, because there is no
mechanism for its disposal, excess iron leads to disease
(525, 623). Furthermore, the insolubility of both valence
states at neutral pH and the toxicity of Fe2⫹ in the presence of oxygen makes control of this essential micronutrient especially vital. Studies of both iron deficiency and
overload in human disease and animal models have
helped to elucidate the mechanisms of iron homeostasis.
Physiol Rev • VOL
A) TRANSPORT ACROSS THE INTESTINE APPEARS NOT TO REQUIRE
Dietary iron is initially absorbed in the
duodenum in either the heme and nonheme (free or chelated) form; much more is known about uptake of the
latter. The current consensus is that free Fe2⫹ is transported directly across the apical membrane of absorptive
epithelial cells via a multistep process (203) involving the
divalent metal transporter DMT-1 (Fig. 4B) (202). The
protein accepts a broad range of divalent ions. Older
reports in the literature implicated a vesicular process for
iron’s uptake, consisting of a mucin at the apical surface
binding free iron, followed by its import via an integrin
and transfer to a molecule termed “mobilferrin,” which
was later identified as calreticulin, an endoplasmic reticulum lumen chaperone. With the identification of DMT-1,
the “mobilferrin” hypothesis is now open to question (12).
After transport into the epithelial cell, cytoplasmic
Fe2⫹ is subsequently directed across the intestinal basolateral membrane via another newly identified transporter, called ferroportin (372). In intestine, a membrane
form of the ferro-oxidase ceruloplasmin, called hephaestin (607), is thought to aid both in the oxidation of Fe2⫹
and the loading of Fe3⫹ onto Tf. How this occurs is still
unknown, although Caco-2 cells are reportedly capable of
transferring apically derived iron onto apo-Tf in the basal
medium (342). Interestingly, the livers of “atransferrinemic” mice become iron overloaded, implying that a nonTf-bound transport mechanism must operate to deliver
iron from the intestine into the hepatocyte. Possible carriers have recently been reported (reviewed in Ref. 277).
B) UPTAKE OF TF-BOUND IRON. Once in the circulation, iron
is carried principally by hepatocyte-derived plasma Tf.
Cells directly accessible to the circulation take up iron via
the well-studied process of Tf receptor-mediated endocytosis (Fig. 4B); that is, iron bound to Tf is internalized in
clathrin-coated vesicles, dissociated from Tf by the low
pH in endosomes, and transported across the membrane,
most likely by a DMT-like transporter. Apo-Tf bound to its
receptor recycles to the same cell surface for subsequent
rounds of uptake, delivery, and recycling.
How do cells located behind a selective barrier obtain this essential mineral? And how do cells responsible
for transcytosing it obtain sufficient iron for their own
metabolic needs? In the adult, iron reaches cells in peripheral tissues by nonselective caveolar-mediated transcytosis of Tf-bound iron across the endothelium; there is
no dissociation in transit.
C) TRANSCYTOSIS OF IRON IN THE PLACENTA AND BRAIN. As described in section IIB1 for brain, transcytosis of iron
across cerebral capillaries is receptor mediated. Also, as
described earlier, the extent to which plasma-derived Tf
moves across brain endothelial cells is controversial and
presently unresolved (52). There is a similar uncertainty
VESICULAR CARRIERS.
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There are additional aspects of B12 homeostasis that deserve comment. The kidney, yolk sac, and placenta express the protein components involved in intestinal B12
absorption (294). For example, cubilin is very abundant in
the kidney proximal tubules, where it can bind and internalize B12-IF, again via megalin (91, 517). This is strange,
since IF is not normally found in the circulation. Not
unexpectedly, given the small size of TCII and thus its
filtration by the glomerulus, there is a scavenger of B12TCII in the kidney, and it is megalin itself on the apical PM
of proximal tubule cells. Similar to its behavior in the
intestine, megalin internalizes the protein-B12 complex
and delivers it to lysosomes, where TCII is degraded and
B12 is stored in a form that is retrievable into the circulation upon demand. This last step, storage of B12, points to
possible differences between B12 dynamics in intestinal
and kidney epithelial cells and is worth following up.
Finally, antibodies to cubilin cause severe defects in developing fetuses, possibly due to a failure to deliver B12
and/or other essential components to the developing central nervous system via the yolk sac (516). As can be seen
in Table 1, the cubilin-megalin system has the capacity to
bind and move many different cargo molecules.
There are many excellent reviews covering different aspects of iron metabolism (5, 11, 203, 277, 278, 485).
885
TRANSCYTOSIS
in the transcytosis of iron across the human placenta. As
for brain endothelium, the direction across the syncytiotrophoblasts is apical to basolateral (Fig. 2A). Is maternal or fetal Tf the carrier? Finally, the fetal endothelial
capillary is an additional cellular barrier that must be
crossed and the mechanism is currently unresolved.
E. Additional Transcytosis Systems
1. Lung
2. Mammary gland
Milk is composed largely of locally synthesized nutrients that are secreted by alveolar epithelial cells in the
lactating gland (523). However, a substantial fraction of
milk proteins is thought to be derived from the serum,
meaning that transcytosis must be used to deliver these
“exogenous” molecules. To date, distinguishing between
the two sources has not been systematically done. Local
plasma cells secrete IgA, which is transcytosed by luminal
epithelial cells using the pIgA-R (Table 1). As already
described, rodents but not humans transcytose maternal
IgG into milk for several days after pups are born. The
receptor and pathway are unknown. Micronutrients in
milk are supplied from the maternal circulation, but neither their transcellular path nor the source of binding
protein (e.g., for iron, B12, or vitamin D) is clear. Iron is
transcytosed, but milk transferrin is synthesized in the
gland, potentially necessitating an intracellular transfer.
Ca2⫹ is also derived from the maternal circulation, but its
concentration in milk is ⬃100-fold higher than that in
serum. The Golgi Ca2⫹-ATPase has been proposed as the
pump that sequesters Ca⫹2 in vesicles, which are subsePhysiol Rev • VOL
3. Thyroid
The thyroid hormones triiodothyronine (T3) and thyroxine (T4) are produced from their iodinated precursor
protein, thyroglobulin, which is stored in the lumen of a
thyroid follicle. Upon stimulation by thyroid stimulating
hormone at the basolateral surface, apical endocytosis
increases and thyroglobulin is internalized. The mechanism of uptake is not yet known and may be nonspecific
(143, 226, 483). Endocytic vesicles fuse with lysosomes,
and the cathepsins act on thyroglobulin to release 20-kDa
fragments containing the hormonogenic regions, which
are further cleaved to T3 and T4 by endo- and exopeptidases. It is a mystery how the hormones reach the circulation, since T3 receptors are present in the thyroid cell,
yet net movement of the hormones is toward the basolateral surface. Once in the circulation, T3 and T4 are bound
to their protein carrier, transthyretin, which is synthesized in the liver. The complex is transcytosed across
continuous endothelium via caveolae. It is presently unclear how these hormones reach the brain. Perhaps the
choroid plexus, which synthesizes transthyretin, plays a
role.
Approximately 10% of the thyroglobulin protein internalized from the thyroid follicular lumen is not processed in lysosomes, but is transcytosed intact into the
circulation. In the 1980s, Herzog (227) found that this
amount did not represent spillover from saturation of a
putative lysosomal delivery system, since lysosomes continued to fill as increasing amounts of thyroglobulin were
internalized. More recently, researchers have identified
megalin as the apical membrane receptor mediating thyroglobulin’s apical-to-basolateral transcytosis across the
follicular cell (348, 350, 351). This finding raises several
questions. Does megalin also cross the cell and reach the
basolateral PM? How does megalin’s cargo avoid lysosomes in the thyroid, since in the intestine, its cargo,
vitamin B12 bound to intrinsic factor, is delivered to lysosomes for degradation? The in vitro cell system models
described in section III could be useful for answering
these questions.
F. Role of Transcytosis in Plasma Membrane
Biogenesis In Vivo
Of the in vivo transcytosis systems described so far,
a common feature is that the cargo is exogenous. However, in two epithelial cell types, the hepatocyte and the
absorptive enterocyte of the intestinal villus, endogenous
membrane proteins are themselves the cargo! These cells
use basolateral-to-apical transcytosis exclusively (hepatocytes) or in part (enterocytes) as a pathway for the deliv-
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Transcytosis occurs in the upper regions of the respiratory tract and involves two receptor systems already
described, pIgA-R and FcRn (Table 1). Secretory IgA is a
known constituent of the lung’s immune defense system,
with bronchial epithelial cells carrying out basolateral-toapical transport of dIgA, which is secreted by local
plasma cells in underlying lymphoid tissue (reviewed in
Refs. 444, 491). A recent study using a clever biological
read-out has documented the efficient apical-to-basolateral transcytosis of intact IgG across bronchial epithelium
via the FcRn (553). This latter pathway could possibly be
exploited to deliver genes systemically. Finally, albumin,
which is found in lung fluid, is endocytosed specifically at
the apical surface of airway epithelia but is then subsequently degraded. At the alveolar level, the question of
whether albumin is transcytosed intact is uncertain (see
Ref. 437 for review). Malik and colleagues (267) have
recently reported the presence and function in type II
epithelial cells of a gp60 membrane protein related to that
found in endothelial cells.
quently delivered to the apical membrane with release of
Ca2⫹ via exocytosis (523).
886
PAMELA L. TUMA AND ANN L. HUBBARD
III. IN VITRO CELL MODELS
OF TRANSCYTOSIS
The use of in vitro cell models to study transcytosis
has many advantages over in vivo systems. First, variation
among animals is eliminated, as is the confounding issue
of cargo possibly being modified or endocytosed by cell
types other than the one under study. Moreover, in vitro
systems can be manipulated in ways not possible in vivo,
allowing investigators to measure the effects of different
variables (e.g., temperatures, pharmacological agents,
Physiol Rev • VOL
etc.) with greater precision and to explore the molecular
mechanisms of transcytosis. However, these advantages
are offset by the loss of the in vivo context (e.g., cues
from extracellular matrix, other cell types), which undoubtedly provides levels of regulation that are missing in
vitro. For this reason, it is important to be cautious in
extrapolating in vitro results to the in vivo situation and to
compare results obtained in the two systems whenever
possible.
A. What Constitutes a “Good” Transcytotic
Cell Model?
An ideal cell model would faithfully recapitulate the
in vivo transcytotic system in the types, amounts, and
kinetics of cargo transported across the cellular barrier. If
these criteria are met (not a simple feat), we can assume
that other parameters, such as cellular organization, relevant machinery, tight junction permeabilities, etc., are in
place. What experimental factors should be considered
and assessed in establishing a good (i.e., less than ideal)
in vitro cell model? Based on a review of the literature and
our own experience, we have come up with six issues that
pertain to simple (bipolar) epithelia.
An important factor is the choice of substratum.
Quite early, physiologists recognized that simple epithelial cells achieved a higher degree of polarity and manifested a more differentiated phenotype when they were
grown on porous surfaces with media bathing them on
both sides. This three-dimensional arrangement simulates
the in vivo condition. Filters of various chemistries and
coatings are now commercially available (Table 3). To
date, cell growth, morphology, and polarity on different
filters have been compared systematically in only a few
cases (71, 601).
The issue of pore size is relevant for two reasons.
First, cells can migrate through larger pores and grow on
the underside of filters (581). Obviously, this situation
would make interpretation of transcytosis measurements
difficult. Not surprisingly, different cell types have varying
tendencies for this behavior. However, pore diameters of
1 ␮m or less seem to prohibit migration of even the most
aggressive cells. Second, the pore size can affect access of
transcytotic tracers in the basal medium to the basal
surface of cells. Of course, the larger the transcytotic
cargo (e.g., bacteria), the more relevant this issue. However, the extent to which even small reagents such as the
surface-labeling reagent NHS-biotin (molecular mass 557
Da) have access to the basolateral surface of filter-grown
cells has been debated in interpretation of experimental
results (241).
A third factor to consider in the quest for a good in
vitro cell system is the cell seeding density. The goal is to
obtain a confluent monolayer of homogeneous cells. For
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ery of specific classes of newly synthesized apical plasma
membrane proteins. In hepatocytes we determined some
years ago that three single transmembrane domain (TMD)
apical proteins and one glycosyl-phosphatidylinositol
(GPI)-anchored apical protein followed this route (32,
499). Similar observations were made by Maroux and
colleagues (148, 355) in the rabbit intestine for aminopeptidase N. Thus the question is whether such an “indirect”
pathway of apical PM biogenesis exists in other epithelia
in vivo. Unfortunately, we may never have a definitive
answer, largely because other epithelial tissues are not
sufficiently homogeneous nor as amenable as the liver
and intestine to the biochemical approaches that were
used. In these studies, animals were first administered
radiolabeled amino acids in a “pulse-chase” fashion, then
enriched populations of apical and basolateral membranes were isolated, the specific membrane proteins immunoprecipitated and their radioactive content determined. Because hepatocytes constitute ⬎70% of the total
cells in the liver, subcellular fractionation methods could
be used that yielded preparations of highly enriched hepatocyte organelles. Similarly, intestinal mucosa could be
scraped from the surface of everted intestines, thus enriching for epithelial cells and allowing subsequent subcellular fractionation of membranes into apical- and basolateral-enriched fractions.
Why are these in vivo studies important? First, they
represent “reality,” that is, the physiological situation.
Second, the results were different from those reported
earlier using in vitro polarized cell models, specifically
MDCK cells derived from dog kidney (360, 479). In the
latter cells, newly synthesized apical PM proteins were
shown to be delivered directly from the trans-Golgi network (TGN) to the apical surface. Gradually, as different
epithelial cells and more membrane proteins have been
studied, the plasticity in the routes and mechanisms for
the delivery and retention of PM proteins in epithelial
cells has become apparent. Such a realization reinforces
the importance of studying a variety of epithelial cells to
learn the full repertoire of mechanisms. It also points to
the possibility that transcytosis is an “ancient” route,
since all epithelial cells express this transport system.
887
TRANSCYTOSIS
TABLE
3. Commercially available filters
Company
Name
Material
Pore Size, ␮m
Comments
Opaque
Transparent
Coated with bovine collagen
(I and III)
Transparent, translucent,
high and low pore density
Coated with bovine collagen
(I or IV), laminin,
fibronectin, or Matrigel
Transparent, needs coating
Costar
Transwell
Transwell-Clear
Transwell-COL
Polycarbonate
Polyester
Polytetrafluorethylene (PTFE)
0.1–12
0.4–3
0.4–3
Becton-Dickinson
Falcon
Polyethylene-terepthalate
(PET) or fluoropolymer (FP)
PET or FP
0.4–8
Biocoat
Millipore
Millicell-CM
(Biopore-CM)
Millicell-HA
Millicell-PCF
Polystyrene
0.4–8
0.4
Mixed cellulose esters
Polycarbonate
0.45
0.4–12
Coating not needed
Coating not needed
example, sparse seeding of some cell types can lead to
confluence being achieved at different times across the
filter. This situation could have a profound influence on
the differentiation state of the cells. Caco-2 cells, a wellstudied model of intestinal absorptive enterocytes, progressively differentiate over a period of ⬃15–20 days after
achieving confluence. Therefore, they are seeded at high
density so that confluence is achieved across the entire
filter synchronously. The disadvantage of seeding at high
density is the possibility of selecting for more rapidly
attaching cells. Likewise, some cell lines alter their phenotype depending on the seeding density. This appears to
be the case for placental BeWo cells, which form multiple
layers if seeded too high (327). Such a situation could lead
to erroneous results and interpretations regarding transcytosis. BeWo cells seeded at low density (subconfluence) will grow into a confluent monolayer (Table 4).
The integrity of the monolayer is obviously vital to
every study of transcytosis, and there are different methods for assessing it. Transepithelial electrical resistance
(TER) measurements are commonly used as an indication
of tight junction integrity in a monolayer, and commercial
instruments are available for these measurements (241).
For meaningful interpretation of results, especially when
studying brain endothelium permeability, it is important
to compare the in vivo and in vitro TERs (201). Radiolabeled inulin (molecular mass 5,200 Da), an inert, uncharged molecule that reports paracellular leak, is often
used, because it can be added to the apical medium and
measured in the opposite bath after various times of
incubation. Here, a positive control consisting of parallel
measurements made to a filter with no cells is important,
since there is not immediate equilibration of a tracer
between the two chambers in the absence of cells. A
simple method for assessing monolayer integrity is to fill
the upper chamber to the top, then leave the cells overnight and measure leak by a fall in the fluid level in the top
chamber. The ideal would be to measure the integrity of
Physiol Rev • VOL
each filter before using it experimentally, but this is rarely
done.
Knowing the extent to which a cell’s surface is polarized at a molecular level is crucial to a meaningful
interpretation of the results of transcytotic studies. We
have termed this factor the “polarity index,” which is the
relative distribution of a membrane component in the two
PM domains, apical and basolateral. Since the polarity
index of most PM proteins is lower in vitro than in vivo, a
larger fraction of a particular transcytotic receptor (and
its associated intracellular machinery) may be in the “incorrect” PM domain in vitro. This condition would lead to
an apparent higher transcytotic activity in the “wrong”
direction. A possible solution would be to determine the
in vivo and in vitro polarity indices for the receptor under
study and then correct back to the in vivo index.
Finally, we assert that complete “balance sheets” are
essential when studying transcytosis in vitro. By this we
mean accounting for all of the tracer over the time course
of the experiment. Unfortunately, such bookkeeping is
not usually done. Of course, two measurements are routinely made: 1) tracer uptake from one side of the monolayer and 2) its appearance on the opposite side. But
these two measurements alone are not sufficient for a
definitive demonstration of transcytosis. It is also important to determine 3) the intracellular accumulation of the
cargo, 4) any possible degradation/metabolism, and 5) the
integrity of the cargo that crossed the monolayer. These
additional measurements give clues as to whether protein
cargo avoided lysosomal proteolysis, a feature of some
transcytotic pathways.
It is important here to raise a cautionary note about
the nature of tracers used to detect and measure transcytosis. Radiolabeled tracers have often been used in biochemical studies, while macromolecules adsorbed to colloidal gold or coupled to the cytochemical agent, horseradish peroxidase (HRP), have been used in microscopic
studies. Because chemical or physical modifications to a
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[Adapted from Hughson and Hirt (241).]
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Human primary
colon
carcinoma
Caco-2
Similar to fetal colon and adult
small intestine absorptive
enterocytes; brush-border
ectoenzymes high but
glycocalyx low to absent
Positive for factor VIII, acetylLDL uptake; used after 5 days
TER of 125 (rat) and 200
(human)
Rat and human
brain
microvessel
endothelial cells
immortalized
with retrovirus
Collagenasedissociated
human brain
cerebral cortex
microvessels
EC219 and
human
endothelial
cells
637
560
271, 407
113–116, 376
97
118
118, 530, 531
Reference No.
Transwell filters; cells used 15–
20 days after confluency
Intestine
Collagen-fibronectin-coated
Transwell filter
Collagen-coated Transwell filter
Astrocytes from newborn rat
cerebral cortex cocultured
with endothelial cells on
opposite sides of 0.4-␮m
pore rat tail collagen-coated
Millicell CM filter
Brain endothelial cells
3-␮m pore gelatin and
fibronectin-coated PET
filters; cells used 5–6 days
after seeding
0.4-␮m gelatin-coated
polycarbonate filter
0.8-␮m pore gelatin-coated
polycarbonate filter
Peripheral endothelial cells
Culture conditions
I-albumin and HRP
151
473
343
307, 308, 375
A-BL of intact cargo is mediated by
lipoprotein-related receptor
A-BL of only transparent forms mediated
in part by PAF receptor; slow (4–6 h)
A-BL preferred; RAGE-mediated in part;
no degradation
Single TMD apical proteins transcytosed,
but GPI did not
Bidirectional transcytosis without
degradation
A-BL transcytosis preferred
Lactoferrin
BL population of apical
PM proteins
Endogenous p137
membrane protein
Endogenous TCII
receptor and 125I-TCIIB12
Alzheimer’s amyloid beta
1–40 peptide
49, 451
141
122
A-BL transcytosis of 125I-transferrin⫹59Fe
⬃7-fold greater than BL-A
Transferrin and iron
Strep pneumococci
112
414
268, 576, 602
511
Reference No.
From A-125I-LDL and LDL-Au into
multivesicular bodies, but 125I-LDL not
degraded; from A-acetylated LDL taken
up by another receptor and degraded;
concludes that lysosomal pathway is
functional in confluent cells.
Anti-gp60 antibody stimulated A-BL
transcytosis with no change in
monolayer integrity
Intact and active lipase moved BL-A via
VLDL receptor/heparin sulfate
proteoglycan-specific process
Lung microvessel endothelium less
permeable than endothelium of larger
vessels in comparative study; 2- to 5fold higher density of plasmalemmal
vesicles in microvessel endothelium
Comments
LDL
Lipoprotein lipase
125
11- to 150-kDa proteins
Cargo
Transcytosis Studies
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
Physiol Rev • VOL
Endothelial
cells
TER of 660 after coculture for 7
days; TER maintained
⬎ 10 days; expression of
␥-glutamyltranspeptidase,
P-glycoprotein and alkaline
phosphatase; permeability to
sucrose, inulin, and
propranolol showed good
correlation with in vivo;
[3H]dextran (70 kDa) used to
test integrity of monolayer
Bovine aorta
microvessels
Characteristics
Coculture of
Bovine brain
primary
endothelial
cells (stable
up to 50
generations)
with rat
astrocytes
Primary
endothelial
Bovine pulmonary
microvessels
Primary
endothelial
Bovine pulmonary
artery
Bovine pulmonary
vein
Bovine pulmonary
microvessels
Origin
4. Useful in vitro transcytotic systems
Cell
TABLE
888
PAMELA L. TUMA AND ANN L. HUBBARD
Physiol Rev • VOL
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Human colon
carcinoma
Rat liver
Rat liver
Same
Human
hepatocellular
carcinoma
Rat hepatoma
(Fao)-human
fibroblast (WI38) hybrid
Primary
hepatocytes
Primary
hepatocyte
couplets
Same
HEP-G2
WIF-B
Human colon
carcinoma
Origin
HT29
T84
Caco-2
Cell
4—Continued
Up to 90% polarized cells, apical
cysts form, not canaliculi
Variable fraction of polarized
cells
Consistently polarized cells
after 3–4 days; canaliculi
formed
Short-term (0–24 h) culture of
groups of cells with common
bile canaliculus; visual assays
used due to small fraction
(30%) of couplets
Pluripotent intestinal cell line;
useful for studies of intestinal
development (⫹/⫺glucose)
In the presence of murine
lymphocytes of Peyer’s patch
Caco-2 cells differentiate into
M-like cells
Crypt cell features (ion
secretion)
83, 521
3
51
135
484
124
E. coli K1 strains
3-␮m polycarbonate filter;
Caco-2 also studied; day 11
best for Caco-2 and day 14
for T84
No filter needed; cells used 10–
12 days after seeding
No filter needed; cells used 3
days after seeding
No filter needed
Collagenase-dissociated cells
cultured in collagen gel
sandwich; no filter needed
No filter needed
Liver
Not used for transcytosis
studies
Botulinum neurotoxin,
types A, B, and tetanus
0.4-␮m pore, type I collagencoated filter; [3H]inulin
NBD-phosphatidylcholine
and ergosterol
pIgA
Endogenous and
exogenous apical PM
proteins
Short-chain fluorescent
lipid analogs
Horseradish peroxidase
HDL cholesterol
pIgA
Cholera toxin (84 kDa)
endogenous FcRn
Vibrio cholerae and latex
beads
Insulin-transferrin
conjugate
S. aureus toxins,
enterotoxins A, B and
toxic shock syndrome
toxin 1 (TSST-1)
Oligopeptides, bradykinin
(9 aa)
HIV
Cargo
Transwell filter
Type I collagen coated Millicell
HA filter
TER ⬎ 200 14–15 days after
seeding cells
Culture conditions
Millicell-HA filter
284
Reference No.
TER ⬎ 150
Characteristics
Comments
222
532
489
Perinuclear accumulation before delivery
to apical
Scavenger receptor-B1-mediated uptake
at B surface and selective A
transcytosis of lipid
BL-A transcytosis via tubulovesicular
intermediates
BL-A and A-BL transcytosis of C6-NBDsphingomyelin; BL-A of C6-NBDglucosyl-ceramide
Vesicular and nonvesicular BL-A
transport
MAL-2 required for BL-A transcytosis
Use of antibodies to measure BL-A
transcytosis
120
34, 250
213, 626, 627
593, 594, 596
313
70
344
125
285
520, 628
526
232, but see 8,
400
207
Reference No.
1–3% of apically applied toxin appeared
on BL side after 2 h
BL-A 3.6-fold over A-BL, bafilomycin
sensitive (vacuolar ATPase inhibitor)
Intact A toxin A-BL 1.5-fold over BL-A;
toxin B not bound; Caco-2 also
studied; MDCK totally inactive
Active bacteria transcytosed A-BL; good
controls for cytopathic effect and
paracellular movement
A-BL 4- to 10-fold over BL-A,
hydrophobicity index a factor,
transport concluded to be adsorptive,
not receptor mediated
3-Fold greater A-BL than BL-A of
conjugate; ligand not degraded after
movement (binding to anti-insulin);
transcytosis of free insulin 1/15 of
conjugate in A-BL direction; possible
oral route for insulin; caution: polarity
index of Tfn-R not measured
A-BL transport
Facilitated A-BL movement of toxin B
and TSST-1, not toxin A
Transcytosis Studies
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TABLE
TRANSCYTOSIS
889
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Human bronchial
cell
Human
pharyngeal
carcinoma
Calu-3
Detroit 562
cells
Human type IIlike cells
TER of 160
Serous cell phenotype
Substrate and cell density
important for maintaining
type II phenotype (microvilli,
lecithin synthesis, lamellar
bodies); transdifferentiation
into type I cells (with
caveolin 1)
Rat
A549
Criteria for monolayer integrity:
lack of leak into A over 24 h
Guinea pig
Primary
tracheal
cells
Primary
alveolar
type II cells
TER of ⬃50
ts-SV40 transformed; proximal
tubule proteins expressed
(glut2, megalin); TER of 140;
[3H]mannitol permeability of
1.4/40% (⫹/⫺cells)
Proximal tubule cells; lack ␮1B
adapter subunit
MDCK II, low resistance (TER
of 150), some proximal tubule
features, but no megalin,
limited brush border
MDCK I, high resistance TER
5–10,000; collecting duct
features
Oppossum
Male pig
Rat
Normal dog
Characteristics
441
153, 522
188
0.4-␮m pore Transwell-Clear
filter; 700–2500 TER
0.4-␮m pore Transwell filter
Used after 48 h; not filter
grown
267
76
Transwell-COL filter; cells used
⬎4 days after seeding; airliquid interface
Tissue culture-treated
polycarbonate filter
110
Lung
Millicell-HA filter; cells used 14
days after seeding
Co-B12-IF
Strep pneumococci
Peptides, proteins and
dextrans 1,000–150,000
mol wt
IgA
Albumin
Tf-HRP
Albumin, 70-kDa dextran
57
Vitamin B12-TCII
0.4-␮m pore Transwell filter
243
293
Retinol-binding proteinvitamin A
3-␮m pore high density PET
filter
Human FcRn
Human pIgA-R
3-␮m pore collagen-coated PET
and 0.4-␮m pore
polycarbonate filters
0.4-␮m pore Transwell filters;
cells used 3–5 days after
seeding
Various fluid-phase
markers [e.g., Lucifer
yellow, HRP, Texas
red-dextran (10 kDa)]
Exogenous rabbit pIgA-R
and ligands (anti-pIgAR IgG; dIgA)
Cargo
0.4-␮m pore Transwell filters;
cells used 3–4 days after
seeding; ⬎2,000 TER
Liver
Culture conditions
569
209, 470
Reference No.
Comments
123
⬃50% of apically internalized conjugate
moved A-BL; conjugate transported ⬎⬎
HRP alone
Saturable endocytosis; overlap with
caveolin not lysosome markers; which
alveolar cell type studying? in vivo
clearance also
Permeability same at 4 and 37°C,
indicating paracellular transport
330
549
BL-A upregulated by IFN-␥
A-BL via hpIgA-receptor; in vivo
correlation and MDCK cell also studied
291
267
110
462
405
347
454, 556
397
390
44,605
Reference No.
Albumin transported A-BL 10-fold greater
than BL-A; dextran not transported
TCII degraded ⬃60% B12 stays inside; 20–
25% B12 complexed to newly
synthesized TCII or haptocorrin goes
A; 8–12% B12 goes BL
100% A-BL of intact cargo via cubilin;
LLC-PK and MDCK have much less to
no cubilin
IgM and IgA bound, translocated and
released equally; therefore, preference
for IgA in vivo thought to be ECM
barrier to IgM pentamers’ diffusion
Synthesis and tracking of endogenous
FcRn; nonvectorial targeting of newly
synthesized with repeated rounds of
A-BL and reverse; cytoplasmic
dileucine important for transcytosis
Megalin-mediated; 20-fold greater A-BL
than BL-A transcytosis of vitamin A
Comprehensive biochemical and
morphological analysis of endocytosis,
recycling and transcytosis from both A
and BL
Multiple studies; BL-A transcytosis of
dIgA rapid (30 min)
Transcytosis Studies
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Physiol Rev • VOL
OK
Immortalized
rat
proximal
tubule cells
(IRPT)
LLC-PK
Madin-Darby
canine
kidney
(MDCK)
Origin
4—Continued
Cell
TABLE
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Physiol Rev • VOL
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Human
Fischer rat
thyroid
Clone of FRT
Rabbit
midpregnant
gland
immortalized
with SV40
Retinal pigment
epithelial cells
immortalized
with ts-SV40
Same
FRT
FRTL-5
Mammary
alveolar
epithelial
cells
RPE-J
Adult pig
Primary
culture
Human placenta
Human chorionic
carcinoma
BeWo
Primary
endothelial
cells
Normal human
term placenta
Origin
Primary
placental
syncytiotrophoblasts
Cell
4—Continued
174
396
TER of ⬃300
38, 395
406
405
363
266
436
39, 283, 288
Reference No.
Lost expression of tissuespecific genes (e.g., whey
protein)
Polarized but no thyroid-specific
gene expression; TER of
10,000
Question about polarity
TER of 1,500
Expression of thyroid-specific
genes
Cytotrophoblasts isolated and
cultured; keratinocyte growth
medium induces synctia
formation; alkaline
phosphatase expressed on
apical surface
Formation of synctial cells that
produce placental hormones
and have apical brush border;
TER of 60
Characteristics
FITC-IgG and FITCdextran (70 kDa)
0.4-␮m pore low-density filter;
cells used 7 days after
seeding at subconfluence
IgA
Newly synthesized
exogenous apical PM
protein
0.4-␮m pore Matrigel-coated
polycarbonate filters; plus
retinoic acid for 7 days at
permissive then 36 h at
nonpermissive
Thyroglobulin
Apical PM proteins
Cationized ferritin
None
Thyroglobulin
IgG
Tf-iron; endogenous Tf-R
pIgA-R-transfected cells seeded
on 0.45-␮m pore Transwell
filters, coated with types I,
III, or IV collagen
Additional systems
Transwell
Transwell filter ⫹ TSH; inulin
permeability low
Transwell filter; cells used 1–7
days after confluency
Grown on floating collagen gels
Thyroid
0.4-␮m pore Transwell filter;
cells used 4 days after
seeding; TER of 600
0.4-␮m pore Transwell COL
filter; TER of 20–50
LDL and IgG
0.45-␮m pore Millicell-HA filter
IgG
IgG
Cargo
Millicell filter
Placenta
Culture conditions
Comments
497
45
Indirect route from TGN-BL-A of
hemagglutinin in infected RPE-J cells
348, 349, 352
636
227, 228
405
351
14
587, 588
557
139, 140
550
550
Reference No.
Two cell layers with phenotypes similar
to alveolar and myoepithelial cells;
TER of 200–10,000; ⬃6-fold BL-A over
A-BL transcytosis
Megalin-mediated transcytosis
Biotinylation
A-BL movement of intact protein via
megalin-mediated endocytosis and
avoidance of lysosome
Injected into colloidal space
BL-A via FcRn
A-BL of hIgG mediated by FcRn and 3.5fold greater than BL-A; dextran both
directions, much less
compared A-BL of IgG in MDCK ⫹/⫺
placental alkaline phosphatase
expression to BeWo; A-BL IgG in
MDCK low ⫹/⫺ PLAP; A-BL IgG much
higher in BeWo; conclusion, not PLAPmediated
Bidirectional transcytosis of receptor and
Tf
LDL-Au to lysosomes and IgG-Au to basal
tubular elements
10% of apically applied transcytosed in 60
min; A-BL ⬎⬎ BL-A ⬃10-fold; HRP
transcytosis ⫽ in both directions
Transcytosis Studies
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TABLE
TRANSCYTOSIS
891
892
Tf
28
Human, rat, and
rabbit
Osteoclasts
Hippocampal
cells
Newborn rat skin
Primary
epidermal
cells
A, apical; BL, basolateral; TSH, thyroid stimulating hormone; RAGE, receptor for advanced glycation end products; SR, scavenger receptor; TER, transepithelial electrical resistance;
TMD, transmembrane domain; PAF, platelet activating factor.
398, 490
Late endosome to “functional secretory
domain,” which was identified as
“apical” based on delivery of
hemagglutinin to it
Quantitatively little transported from
dendrite to axon
Degraded bone products;
Ca2⫹
221
418
Must have been apical to basolateral—
need to look in Ohkura, 1990;
avoidance of lysosomes
LDL
417
0.4-␮m pore type IV bovine
collagen-coated Millicell-CM
filter; cells used 20 h after
seeding
Cargo
Culture conditions
Reference No.
Origin
Cell
TABLE
4—Continued
Characteristics
Physiol Rev • VOL
molecule can change its biological properties, we assert
that one must determine if those changes have altered the
molecule’s qualitative or quantitative behavior. Unfortunately, many researchers assume that the modified molecule is normal, which can lead to erroneous or, at the
least, uncertain results.
In the remainder of this section we highlight in vitro
cell systems currently used for studies of transcytosis.
Table 4 presents a partial list. We particularly focus on the
extent to which a particular cell line being used and the
cargo being followed represent good physiological models. In several cases, there is the need for development
and/or improvement of cell models.
B. Microvascular Endothelial Cell Models
Cultured endothelial cells originating from various
tissues and vessels abound (155), but few have been used
to study transcytosis in vitro. Why? There seem to be two
reasons. First, primary endothelial cells show multiple
changes after isolation, including a decreased number of
caveolar profiles, increased permeability (101), and even
transformation from a continuous to fenestrated phenotype (477). These phenomena point to the plasticity of the
cells and reinforce the notion that local context dictates
the physiology of the vascular bed (538). A second reason
is that physical forces (osmotic pressure, flow) in vitro
are totally different from those in vivo. Nonetheless, selected aspects of endothelial cell transcytosis have been
successfully studied in vitro (Table 4). For example,
mechanistic aspects of the albumin receptor, which stimulates fluid-phase transcytosis, have been studied in vitro
by Malik and colleagues (13, 89, 379, 576, 602 and discussed below). Interestingly, although the binding/uptake
of modified LDLs is commonly used to characterize endothelial cells, and even has been used to purify them
(606), to our knowledge there has not been a comprehensive in vitro analysis of the dynamics of the different
scavenger receptors (558) or their cargoes. Are the diverse modified LDLs degraded via the lysosomal system
and/or transcytosed? Are there regional differences
among endothelial cell responses to modified LDLs in
vivo? A fruitful area for future research might be to map
the surface distributions of the endothelial scavenger receptors, LOX and CD36 (caveolae or clathrin-coated
pits?), and determine the intracellular/transcellular fates
of the cargoes. Considerable work has been done (2, 36,
108, 296, 326, 399, 461, 496, 606), but we still lack an
understanding of the role played by the endothelium in
handling atherogenic particles.
Several reasonable in vitro models of brain endothelial cells have been reported (105, 113, 201). Kim (286) has
used primary cells and secondary lines from rat and human cerebral cortex to study bacterial adherence and
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Transcytosis Studies
Comments
Reference No.
PAMELA L. TUMA AND ANN L. HUBBARD
893
TRANSCYTOSIS
C. Epithelial Cell Models
1. Intestine
Three cell models of the intestinal epithelium have
been used to study the transcytosis of macromolecules
ranging from endogenous apical PM proteins and toxins
to bacteria (Table 4). Caco-2 cells are the most commonly
used because they differentiate furthest along the cryptto-villus axis and are the easiest to transfect. As mentioned above, the intestinal differentiation program takes
up to 20 days in vitro. However, even the most differentiated Caco-2 cells lack the thick glycocalyx expressed by
Physiol Rev • VOL
mature enterocytes in vivo; this apical surface feature is
considered by some to represent a functional barrier to
the adherence and invasion of the intestinal mucosa by
many pathogens (160). Therefore, its absence in Caco-2
cells needs to be considered when extrapolating from in
vitro results to in vivo possibilities (8). Furthermore, the
apparent bidirectional transcytosis of some ligands (e.g.,
of a transferrin-insulin conjugate, Table 4) may represent
reduced polarity in vitro. Interestingly, by coculturing
lymphocytes in the basal medium with Caco-2 cells on a
filter, the latter have been induced to express M cell-like
morphology and activity (284). This achievement has exciting implications for the study of pathogen invasion in
vitro (513). Let’s hope that the model is further developed.
HT29 cells can also differentiate along the enterocyte
pathway to a mid-mature state, and they additionally have
the capacity to differentiate into goblet cells (637). However, HT29 cells are not as easily transfected as Caco-2
cells, nor do they form as tight a monolayer; thus the
latter cells are preferred for studies of transcytosis in
vitro. The third intestinal cell model, T84, is arrested at an
immature enterocyte stage, similar to a crypt cell in vivo.
They have been used recently to study IgG transcytosis
(Table 4). Additional in vitro models are under development (e.g., Ref. 425a).
2. Liver
The dissociation of liver tissue into isolated cells that
were suitable for culture was first described over 30 years
ago (37). However, the loss of structural polarity and
mixing of membrane domains made them poor models for
studies of polarity. The development of isolated couplets
was a significant advance for short-term studies of polarized hepatocyte functions, such as transcytosis and bile
formation (50). As the importance of the extracellular
matrix in maintaining gene expression and promoting cell
repolarization became apparent, investigators began culturing hepatocytes on different matrix components in
various geometries and physical states. The most successful reconstitution of polarity has come from the use of
collagen gels or Matrigel in a sandwich configuration (194,
328). Despite these advances, the limited lifespan of primary hepatocytes and the difficulties in reproducibly obtaining such polarized cultures prompted the use of secondary hepatocyte cell lines.
A) WIF-B CELLS. Cassio and colleagues (83, 84) have generated many somatic cell hybrid lines in their studies of
the genetic basis of liver-specific gene expression. The
WIF-B cells that we currently use were generated by
fusion of the differentiated (i.e., expressing liver-specific
genes) but nonpolarized rat hepatoma cells (Fao) with
human skin fibroblasts (WI-38 cells). Clonal selections
ultimately yielded a polarized hepatic phenotype, the
WIF12–1 cells, which exhibited a maximum apical polar-
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penetration. One example is in Reference 407. Dehouck
and colleagues (85, 114 –116) developed a coculture system that appears to represent a good in vitro model of
brain capillary endothelium. In brief, freshly isolated astrocytes from newborn rat cerebral cortex are plated on
the bottom side of commercial filters (or the bottom of a
dish) coated with rat tail collagen, cultured for 3 wk, at
which time a subconfluent monolayer of adult bovine
brain endothelial cells is plated on the upper side of the
filters. After 8 days, confluent endothelial monolayers
have formed, and only those exhibiting ⬎500 ⍀䡠cm2 are
used. In a series of articles, these researchers have characterized the coculture system and validated its use for in
vitro studies of brain capillary permeability. The endothelial cells express all of the standard markers of brain
endothelial cells, such as ␥-glutamyl transpeptidase, Pglycoprotein, and glucose transporters. Furthermore, a
comparison of drug transfer in vivo to that in this in vitro
system comes out favorably.
Several studies from this group deserve further comment. Earlier in this review, we referred to the conflicting
evidence for and against the quantitative transcytosis of
plasma Tf when iron is delivered to the brain interstitium.
Results from the in vitro brain endothelial system showed
that Tf is transcytosed with iron to the abluminal (basal)
side of the monolayer (122). However, the subsequent
fates of both components still remain a mystery. The story
of the brain endothelial cell LDL receptor is equally puzzling. In confluent endothelial cells (i.e., with underlying
astrocytes), LDL is transcytosed in an apical to basolateral direction via LDL receptor residing in caveolae. In
contrast, in growing cells, LDL is endocytosed via receptors in clathrin-coated pits, the protein components are
degraded, and cholesterol is subsequently used by the
endothelial cells themselves (111, 112). The molecular
basis for this switch, from receptor-mediated endocytosis
of LDL for intracellular utilization to receptor-mediated
transcytosis of LDL for use by interstitial cells, is currently unknown. Its elucidation should yield important
insights into the machinery required for each type of
vesicle traffic.
894
PAMELA L. TUMA AND ANN L. HUBBARD
Physiol Rev • VOL
tain to increase in the next few years. However, it is
important to remember that results from studies using in
vitro cell models and artificial lipids as surrogates need to
be confirmed by in vivo studies.
One obvious feature of polarized hepatocytes is that
they sequester their apical surface away from the substrate or bathing medium. This structural organization
prohibits direct access to the apical PM, which is confined
between adjacent cells and bounded by tight junctions.
Hence, quantitative determinations of the polarity index,
TER, or rates and extents of PM protein transport to or
between the two PM domains using accepted methods
(e.g., surface biotinylation as in simple epithelial cells) are
not feasible. However, Hoekstra and colleagues (596)
have reported methods for differentially measuring fluorescent lipid analogs present at the two PM domains of
Hep-G2 cells. We have developed methods in WIF-B cells
for detection of trafficked antibodies at the apical PM
(250) and proteins secreted into the apical lumen (34).
3. Kidney
As discussed in section II, there is little evidence for
in vivo transcytosis of macromolecular cargo in kidney.
Nonetheless, MDCK cells, which are derived from dog
kidney, are the most-studied epithelial cell model and
have been used extensively to study transcytosis (Table
4). These cells were originally developed by nephrologists
for permeability and electrical studies. Their subsequent
use by cell biologists (86) for studies of the formation of
tight junctions, establishment of polarity, and vesicle traffic have popularized MDCK cells to the point that they are
now the “NIH-3T3 fibroblast” of the epithelial field. An
advantage is that MDCK cells are easily cultured, easily
transfected, and become polarized 3–5 days after seeding.
They were used in the now classical studies showing that
enveloped viruses bud in a polarized fashion and that the
newly synthesized viral membrane glycoproteins are targeted directly from the TGN to the appropriate PM domain (480). Furthermore, much of our current understanding of the IgA transcytotic pathway and the sorting
signals in the pIgA-R comes from the elegant studies
performed in MDCK cells by Mostov and colleagues (393,
394). We expand on this latter topic in section IV.
Two MDCK strains with very different features were
identified some time ago (Table 4). The MDCK I cell has a
high TER and characteristics reminiscent of the renal
collecting duct, whereas the more commonly used MDCK
II strain, whose TER is one order of magnitude lower than
that of MDCK I cells, has phenotypic features closer to
those of the renal proximal tubule. Our own anecdotal
experience with MDCK II cells indicates to us that there
are many cell variants, some well-documented (367) and
others not. Tsukita and colleagues (166) recently reported
that stable expression of exogenous claudin 2 in MDCK I
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ity of 40%; that is, a maximum of 40% of cells in a mature
culture formed apical poles (or cysts) with adjacent cells.
In collaboration with Cassio, we performed further selections and generated the WIF-B cells, whose apical polarity
index is 80% (521).
Before using these cells for membrane traffic studies,
we characterized their mature phenotype, especially with
regard to cell polarity (251). Most proteins show the same
distribution as in vivo, as does microtubule organization,
at least near the apical PM (374). Microtubules radiate
from the apical membrane, with actin and foci of ␥-tubulin also concentrated in this region. The presence of ␥-tubulin leads us to conclude that the minus ends of polarized microtubules are closest to the underlying apical PM.
We also explored the structural and functional properties
of the tight junction boundary (251). ZO-1, the tight junction-associated protein, marks the boundary between the
two PM domains. Using short chain lipid analogs in living
cells, we established that the tight junctions were an
effective “fence” prohibiting the lateral mixing of outer
leaflet PM lipids. Surface-labeling of living cells with
sNHS-LC-biotin (557 Da) indicated that small molecules
had access to the entire PM, while streptavidin (60 kDa)
was restricted to only the basolateral domain, establishing that the tight junctions also provided an effective
barrier to the diffusion of large molecules into the apical
space.
B) HEP-G2 CELLS. The human hepatoma Hep-G2 is currently being used for studies of polarized membrane traffic (Table 4). This line, which expresses many liver-specific genes, was generated in the late 1970s from liver
tumor biopsies in which the histology presented as “a
well-differentiated hepatocellular carcinoma with a trabecular pattern” (3). The existence of bile canalicular-like
cysts within and between cultured HepG2 cells was reported (90); EM observations and one apical PM marker
were used in this study. Unfortunately, neither a systematic characterization of these cells nor the culture conditions giving maximum polarity have been performed.
In addition to membrane protein trafficking, lipid
trafficking has been actively studied in polarized HepG2
and to limited extent in WIF-B cells and hepatocyte couplets (102, 198, 213, 233–235, 251, 364, 626, 627). This
focus stems from the fact that the major exocrine function of hepatic cells is secretion of bile, whose principal
nonprotein components are phosphatidylcholine (PC),
cholesterol, and bile salts. To date, lipid analogs have
been used in these studies. Given the recent identification
of transporters and carriers implicated in cholesterol and
phospholipid transport (reviewed in Refs. 46, 138, 150a,
256) and the interest in sphingolipids as possible organizers of lipid microdomains (reviewed in Ref. 237), our
understanding of the molecular bases of the vesicular and
nonvesicular transcytotic pathways between the basolateral and apical domains of polarized hepatocytes is cer-
895
TRANSCYTOSIS
4. Additional epithelial cell systems
tiotrophoblasts. However, the variable TER values reported suggest that different culture conditions are being
used that could affect the monolayer integrity of the
BeWo cells (Table 4); furthermore, the cell line’s fidelity
to syncytiotrophoblasts in vivo is still not totally characterized. Nonetheless, two predominant in vivo cargoes,
IgG and iron, are being successfully studied and the molecules involved in their transcytosis identified. B12 and
cholesterol are also supplied maternally, yet have not
been studied in the isolated placenta. Clearly, there is
fertile ground for future work in this system.
C) THYROID. Culture of primary thyroid follicular cells
has been reasonably straightforward (Table 4). Additionally, a rat thyroid cell line, FRT, was generated and several sublines exist, with varying degrees of thyroid-specific expression. Both endogenous PM protein and thyroglobulin pathways have been studied with interesting
results; that is, Zurzolo et al. (636) reported that immature
FRT cells use transcytosis to deliver apical PM proteins,
while mature cells use the direct TGN-to-apical route. The
mechanistic differences in the two pathways could be
identified using such model.
D) MAMMARY. Although it is important to explore the
mechanism by which plasma constituents reach the milk,
the culture of primary mammary epithelium from lactating glands for such studies has not been reported (382).
Because these cells contain and secrete fat globules, they
are apparently very difficult to establish in culture and
maintain in a differentiated state.
D. Transcytosis Outside of the Epithelial World
Both primary cells and cell lines, alone and in
coculture with endothelial cells, are being used to study
transcytosis (Table 4). Calu-3 cells, derived from the upper airways, form tight monolayers and have been found
to express and secrete IgA. It would be interesting to
know if they also express FcRn and transport IgG in a
polarized fashion. Could they be useful for therapeutic
drug delivery studies? Likewise, the fact that alveolar type
II cells are capable of trans-differentiating into type I cells
under defined conditions may make it possible to sort out
the confusion over which cell type, if either, is able to
transcytose albumin from the thin epithelial fluid layer in
the alveolar lumen to the interstitium. Although type I
cells contain a huge number of caveolae, Malik and colleagues (267) have recently reported that it is the type II
cell that transcytoses albumin via the gp60 membrane
protein and caveolae.
B) PLACENTA. Both primary cells originating from protease dissociation of human term placenta and a human
placental cell line, BeWo, have been used in in vitro
studies of placental transcytosis (Table 4). Under appropriate culture conditions, these preparations differentiate
from single cell cytotrophoblasts to multicellular syncyA) LUNG.
Physiol Rev • VOL
1. Bone-resorbing osteoclasts
In the final part of this section, we present a fascinating in vitro transcytotic system that probably operates
in vivo but has not yet been confirmed. Bone is a dynamic
tissue that it is constantly being laid down and resorbed.
Bone degrading cells, osteoclasts, become polarized under appropriate hormonal stimulation and set up an elaborate, sealed structure, called a lacuna, against a segment
of bone (572, 585). The cells then secrete hydrolytic enzymes and acid into this extracellular lacuna, which is
lined by a ruffled border with membrane markers of lysosomes/late endosomes. Inside the lacuna, the bone mineral content (principally Ca and phosphate) is solubilized
and collagen I is partially degraded. These components
are then endocytosed at the ruffled membrane front, transcytosed across the cell to a secretory zone, an apical PM
domain in the middle of an otherwise basolateral PM (398,
490), and secreted into the marrow cavity. Subsequently,
the products are carried into the circulation. Since these
cells lack tight junctions, a morphological hallmark in
epithelial cells, the system is ideal for studying the establishment of a dynamic polarity in the absence of junc-
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cells lowers its TER to that of MDCK II cells, which
express endogenous claudin 2. Does the expression of
this tight junction family member in MDCK II cells induce
morphological and biochemical changes beyond those
contributing to the change in TER?
The first comprehensive and quantitative analysis of
apical and basal endocytosis in polarized epithelial cells
was performed by Simons and colleagues on MDCK I cells
(44, 605). In that study, fluid-phase endocytosis from the
apical PM was much less robust than from the basolateral
PM. However, apically endocytosed material was transcytosed basally to a relatively greater extent than the converse (material taken up from the basal surface transcytosed to apically). Without knowing the equivalent in vivo
endocytic and transcytotic extents at the two poles, this
information is hard to interpret.
The apical-to-basolateral “transcytosis” of micronutrients by the proximal tubules of the kidney in vivo
makes this topic appropriate for study in kidney cell lines.
An important requirement is that the cells express the
apical scavenger receptors cubilin and megalin, which
have been implicated in the retrieval mechanism (Table
4). Unfortunately, MDCK cells appear not to express these
receptors while OK and LLC-PK cells do. The latter cells
have been used for studies of vesicle traffic (109, 334). We
favor the rat proximal tubule line (IRPT, Table 4), because
it expresses functional levels of megalin, and useful reagents are available to rat proteins.
896
PAMELA L. TUMA AND ANN L. HUBBARD
tional elements. Furthermore, the transcytotic pathway in
these cells differs from that of intestinal epithelial cells,
because the cargo is carried from a late endosome (the
ruffled border) to the apical PM.
include most plasma proteins, were excluded from this
pathway. Instead, they were included in the fluid internalized by apical PM caveolae, shuttled to the basal side, and
released into the interstitium.
2. Neurons
2. Caveolae and caveolin
IV. MORE ABOUT TWO DIFFERENT
TRANSCYTOSIS SYSTEMS
A. Caveolae-Mediated Transcytosis
Transendothelial transport differs in several ways
from other types of transcytosis. The transport is rapid
(⬃30 s), the cargo is predominantly fluid not receptorbound, and a unique vesicle acts as the shuttle between
apical and basal surfaces. Despite these differences, the
targeting and fusion machinery are reported to be similar
to those used elsewhere (see sect. V). In this section, we
briefly summarize early work then focus on recent developments that raise new questions about caveolae-mediated transcytosis in endothelium.
1. The endothelial cell surface
The blood-endothelial cell interface is complex in
composition and dynamic in its functions (Fig. 5). The
apical PM outside of caveolae is negatively charged and
thus capable of repelling negatively charged blood cells.
Early in vivo ultrastructural studies of endothelium
throughout the vascular tree established that sialo-glycoconjugates and glycosaminoglycans (principally heparan
sulfate proteoglycans) provided the negative charge barrier. Combinations of enzymatic treatments, lectins, and
tracers of different pI values were used (537, 540). Clathrin-coated membranes and pits along the apical and basal
surfaces of endothelial cells were also negatively charged.
Thus cationic molecules were found to preferentially bind
at these sites with subsequent delivery to and degradation
in lysosomes (536). In contrast, anionic molecules, which
Physiol Rev • VOL
As stated earlier and shown in Figure 1, caveolae are
flask-shaped pits present on and continuous with the
apical and basal PMs of all endothelial cells. Although
nearly ubiquitous, they are most abundant in terminally
differentiated cells, such as adipocytes, smooth muscle
cells, type I pneumocytes and, of course, endothelial cells
throughout the vascular tree. Even in the early 1980s,
endothelial cell caveolae were recognized as chemically
distinct “microdomains” of the PM (Fig. 5). Numerous
excellent reviews cover caveolar structure and function
in greater detail than we can (9, 546, 553a). Our focus is
on their role in transcytosis.
As stated in section II, there is still considerable
debate over whether caveolae mediate transcytosis in
endothelial cells. Perhaps much of this conflict can be put
to rest now that caveolin-1-deficient mice have been generated (130, 465). In animals lacking caveolin-1, the major
structural component of caveolae in many tissues including endothelia, no identifiable PM-associated caveolae
were observed. In cultured fibroblasts from transgenic
embryos, internalization of albumin was inhibited
whereas transferrin uptake by clathrin-coated vesicles
was not changed (465). Furthermore, and perhaps most
significantly, albumin delivery from blood vessel lumens
to the underlying interstitium was completely inhibited in
perfused transgenic mice (512). Morphologically, goldlabeled BSA was found only in the lumen of caveolin-1deficient blood vessels, whereas in control endothelial
cells, the marker was concentrated at caveolar PM invaginations and in internalized caveolae that were detached
and closed from the PM. In aortic ring segments isolated
from control or caveolin-1-deficient mice, iodinated albumin transcytosis was measured. Robust transport was
seen in control mice, whereas transcytosis was virtually
eliminated in the knock-out mice. Interestingly, no accumulations of the tracer during longer incubations were
observed in the transgenic mice, suggesting that albumin
was not being internalized via clathrin-coated vesicles.
However, altered extravascular oncotic pressure normally associated with albumin transcytosis defects was
not observed in these animals (130), nor was there any
change in cerebrospinal fluid albumin concentrations, a
transport pathway thought to be caveolae mediated.
These results along with the surprising result that these
animals are viable and fertile suggest other compensatory
transport processes must be present. Nonetheless, the
data strongly indicate that caveolae do indeed mediate
transcytosis in endothelial cells.
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It is relevant in this section to discuss the possible
existence of transcytosis in nerve. Early studies in
cultured neurons suggested that the dendritic and axonal PM domains in this polarized cell are functionally
equivalent to the respective basolateral and apical PM
domains of epithelial cells (544). Carrying this analogy
a step further, Dotti and co-workers (221) asked if
transcytosis also occurred. Using hippocampal cultures, the researchers found evidence for limited transcytosis of Tf and its receptor from dendrites to axons
(221). Given the subsequent work on targeting of native
and foreign PM proteins (68, 264, 533), it seems that
transcytosis is a minor activity in vitro; its occurrence
in vivo is questionable.
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TRANSCYTOSIS
Despite these major advances, there are still many
unresolved issues. Although caveolae are the likely transcytotic carriers, it is not clear how they transport molecules across the endothelial cell layer. Do caveolae, like
clathrin-coated vesicles, pinch off from the PM, travel the
short distance across the cell, and fuse with the opposite
surface? N-ethylmaleimide (NEM) sensitivity of transport
and the identification of caveolae-associated dynamin and
specific members of the vesicle docking and fusion machinery (see sect. V) are consistent with this idea. In
particular, Schnitzer and colleagues (416) have reported
that dynamin activity leads to the scission of caveolae
from endothelial PM. Also, EM of albumin-containing free
Physiol Rev • VOL
caveolae and caveolae associated with both the basolateral and apical surfaces of the cell are consistent with
caveolae as vesicular shuttles (183–185, 378). Alternatively, caveolae on opposing membrane surfaces may
transiently (?) fuse with each other producing channels
through which ligands pass (Fig. 4). Such structures that
contain transcytosing albumin have been occasionally observed at the ultrastructural level (183). One possibility is
that both mechanisms operate but mediate the transport
of specific ligands or ligands differentially modified. Since
more caveolae are observed than the predicted number of
pores, the question as to whether all caveolae are transcytosis-competent is also raised. Are some caveolae or
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FIG. 5. Transendothelial transport pathways. A: the four transport pathways present in endothelial cells are
depicted. Also shown is the preferential distribution of anionic sites on the endothelial cell surface and basal lamina. B:
the flask-shaped plasma membrane invagination, the caveola, contains the oligomeric coat protein caveolin. C: an
enlarged version of the indicated region in B is shown. Caveolin forms a hairpin structure in the bilayer with both its
carboxy- and amino-terminal ends facing the cytoplasm. Three palmitoyl chains attached to the caveolin carboxyterminal tail insert into the bilayer. Caveolin is present in membranes enriched for glycosphingolipids and cholesterol.
The glycosphingolipids are asymmetrically distributed to the outer leaflet while cholesterol is present in both leaflets
where it is thought to fill spaces around the glycosphingolipid headgroups. Although the phospholipids present on the
cytoplasmic face have not yet been identified, their fatty acid chains may be saturated to optimize packing. cp, Coated
pit; CV, clathrin-coated vesicle; cav, caveolae; ch, channel.
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PAMELA L. TUMA AND ANN L. HUBBARD
channels reserved for transport while others serve as
signaling centers? If so, how are they different?
3. Albumin and orosomucoid transcytosis
B. Clathrin-Mediated Transcytosis
As discussed in section IIC3, one of the best-studied examples of transcytosis emerged from efforts
aimed at understanding the molecular basis of mucosal
immunity. Here we will discuss in detail the transcellular itinerary of the pIgA-R and its ligand, pIgA. Also,
Physiol Rev • VOL
1. The pIgA-R transcytotic pathway
The intracellular itinerary that pIgA-R follows during
its life cycle is complex and may vary among epithelial
cell types examined and among species (388, 439). Nonetheless, from studies performed in hepatocytes, enterocytes, and their respective in vitro model systems as well
as from MDCK cells that stably express exogenous
pIgA-R, a general picture is emerging that is summarized
in Figure 6. From work performed in rat hepatocytes
(565), it was determined that pIgA-R is synthesized in the
ER as a 105-kDa polypeptide and travels to the Golgi
where it is terminally glycosylated giving rise to an 116kDa glycoprotein. This form of the receptor is delivered
from the TGN to the basolateral surface where it is further
modified (probably phosphorylated, see below) reaching
its mature form of 120 kDa. Early in vivo experiments,
also performed in hepatocytes, further determined that
the basolaterally located receptor is subsequently internalized (in the presence or absence of bound pIgA) by
clathrin-coated pits and delivered to early endosomes
(176, 238). Other receptor-mediated ligand systems (e.g.,
ASGP-R) were also present in these early endosomes, but
only the pIgA-R and its ligand were delivered to a subapical compartment before their release into the bile. The
other internalized receptor proteins moved along one of
two other arms of the endocytic pathway, to lysosomes or
back to the basolateral PM.
The transcytotic pathway taken by pIgA-R in
MDCK cells has been studied extensively with the goal
of identifying and characterizing all of the intracellular
intermediates. Debate over nomenclature and different
experimental approaches led to conflicting views that
now appear to be resolved. Recent reports (316, 609)
have established that at least three compartments comprise the basolateral-to-apical transcytotic pathway of
pIgA-R in MDCK cells: basolateral early endosomes, a
“common” endosome, and an apical recycling endosome (Fig. 6). In hepatocytes and enterocytes, there are
only two identified transcytotic intermediates: basolateral early endosomes found in both cell types and a
“common” endosome described in enterocytes or the
subapical compartment (SAC) in hepatocytes (29, 250,
290) (Fig. 6). The basolateral early endosomes in the
different cell types appear to be rather analogous structures. They are the first entry points for molecules
internalized from the basolateral surface, they are biochemically distinct from apical early endosomes (that
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Although most transcytosed cargo in endothelial
cells is fluid, two of the best-studied molecules, albumin
and orosomucoid, are transported predominantly via receptor-mediated mechanisms. The first clues that albumin
was internalized via receptors came from morphological
studies where albumin association with endothelial cells
was concentrated in caveolae open to blood vessel lumens (183, 184, 378). Three major albumin binding proteins were subsequently identified (p18, p31, and p58 – 60)
that were present on endothelial luminal cell surfaces
(179, 180, 507, 508). Further work revealed that p18 and
p31 were likely scavenger receptors and that gp60 was the
bona fide albumin receptor (506, 575). Albumin binding to
gp60 and gp60 antibody cross-linking activates the transendothelial transport of albumin in vitro and in vivo (602).
In both cases, the tyrosine phosphorylation of gp60 itself,
caveolin-1, and the tyrosine kinases pp60 src and Fyn
were induced upon activation. Addition of the specific
tyrosine kinase inhibitors herbimycin and genistein
blocked albumin internalization, indicating that transcytosis is a tyrosine kinase-dependent process (576). More
recently, this activation has been further correlated with a
Gi-coupled src kinase signaling pathway that is discussed
below (see sect. VD1).
Like albumin, when examined ultrastructurally,
transport of orosomucoid is rapid and occurs via caveolae
(456). Previous studies showed that orosomucoid binding
to cultured endothelial cells was saturable, specific, and
high affinity, implying the presence of specific receptors
(504). Ligand blotting of endothelial cell lysates with 125Iorosomucoid identified three receptor candidates of 14,
20, and 7 kDa that have not been examined further (456).
Like albumin, orosomucoid transcytosis is also NEM sensitive, consistent with the identification of caveolae as
vesicle shuttles between the two domains during transcytosis (456). Because none of these possible receptors or
gp60 has been cloned, the structural signals that direct
transcytosis of these molecules and their associated ligands have not yet been identified. Will there be any
similarities to those identified in pIgA-R (see below)?
What dictates their placement in caveolae over clathrincoated pits?
we will describe what is understood about the signals
encoded in the cytoplasmic tail of the receptor that
direct the complex’s circuitous journey. Finally, we will
compare the pIgA-R transcytotic pathway with that of
newly synthesized hepatic and intestinal apical PM residents.
899
TRANSCYTOSIS
are first to receive apically internalized molecules), and
morphologically, they are located at the basolateral
pole of the cell. The MDCK apical recycling endosome
is most similar to the hepatic SAC. Both have a neutral
pH, only apically destined membrane components traverse them, and they are located closest to the apical
PM. The major difference between them is the absence
of recycling apical PM proteins in hepatic SAC and their
presence in the MDCK apical recycling endosome. Why
such a similar structure is not present (or not yet
identified) in enterocytes is a puzzle. Also, why do
hepatocytes lack a common endosome (that receives
cargo from both cell surfaces)? These differences may
be pointing to unique and important differences in
membrane transport among epithelial cell types.
Once the receptor has reached the apical cell surface,
it is cleaved and its ectodomain is released into the luminal spaces. The receptor is constitutively synthesized and
transcytosed whether ligand is present or not such that
both the unoccupied SC and secretory IgA are recovered
from apical secretions. In hepatocytes, the pIgA-R is
Physiol Rev • VOL
cleaved efficiently at the apical surface such that the
pathway is “one-way”; all receptor molecules that reach
the apical cell are clipped. However, in MDCK cells,
pIgA-R proteolysis is less efficient such that a small proportion of intact pIgA-R at the apical cell surface can be
recycled, and even a smaller portion can be transcytosed
back to the basolateral surface (Fig. 6). Whether this is a
physiologically meaningful process is not clear. These
MDCK cells are overexpressing the rabbit form of pIgA-R,
and it is possible that in the canine context, this receptor
has altered dynamics. Examination of the dynamics of the
very low levels of the endogenous MDCK pIgA-R may help
clarify this point. Work performed in vitro in nasopharyngeal cells has shown that uncleaved apical pIgA-R mediates endocytic entry of Streptococcus pneumoniae, suggesting that apical internalization of pIgA-R may be part of
its life cycle in certain cell types (273). Identifying the cell
surface protease and understanding its proteolytic activity, specificity, and tissue distribution will help us clarify
the posttranscytotic fate of the receptor in these other cell
types.
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FIG. 6. The intracellular itinerary of polymeric IgA receptor (pIgA-R) in hepatocytes, MDCK cells, and enterocytes.
In general, pIgA-R is synthesized in the endoplasmic reticulum and transported to the trans-Golgi network, where it is
sorted for delivery to the basolateral surface. At the basolateral surface, the receptor binds its ligand and is transcytosed
through several intracellular intermediates to the apical surface. The receptor is cleaved by a cell surface protease
releasing the pIgA-R ectodomain and bound ligand into the lumen.
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PAMELA L. TUMA AND ANN L. HUBBARD
2. Signals and regulation of pIgA-R transcytosis
FIG. 7. The targeting signals encoded by the cytoplasmic carboxy-terminal tail of pIgA-R. A schematic drawing is
shown summarizing the mutations made in pIgA-R’s cytoplasmic domain and their effects on receptor trafficking. From
these studies, the indicated trafficking signals were identified. The more detailed versions of the basolateral targeting and
internalization signals were identified in Refs. 19, 20, 421, 422. aa, Amino acid; BL, basolateral; TMD, transmembrane
domain.
Physiol Rev • VOL
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The 103-amino acid cytoplasmic domain of pIgA-R
contains multiple signals that mediate its circuitous journey. From work performed mainly in MDCK cells, the
signals were found to fall into three categories: targeting
to the basolateral PM, promoting rapid endocytosis, or
preventing degradation (Fig. 7). With the use of deletion
analysis, the pIgA-R basolateral targeting signal was identified in a sequence located just adjacent to the TMD, and
this amino acid stretch was sufficient to direct chimeric
reporter proteins to the basolateral PM (81). Site-directed
mutagenesis of the receptor tail revealed that internalization from the basolateral surface required two cytoplasmic tyrosine residues, both of which are found in sequences similar to previously identified internalization
motifs (421). Phosphorylation of serine-726 has also been
shown to mediate rapid pIgA-R basolateral internaliza-
tion, and it has been suggested this modification is reflected in the increased molecular mass (120 kDa) of the
basolaterally located receptor (231, 422). Once internalized, the receptor is rapidly transcytosed across epithelial
cells, and phosphorylation of serine-664 may facilitate this
process. Also, a region of the tail has been shown to
prevent the receptor from being degraded further, ensuring its efficient transcytosis once internalized (55). From
these studies, it has been proposed that newly synthesized pIgA-R leaves the TGN in a nonphosphorylated
form. Upon delivery to the basolateral surface, the receptor is phosphorylated (serine-726), which exposes the
tyrosine-based internalization motifs allowing rapid internalization. Subsequent phosphorylation at serine-664 promotes transcytosis, thus sorting the receptor away from
the degradative or recycling pathways (388).
Transcytosis of pIgA-R is also regulated upon ligand
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TRANSCYTOSIS
3. Transcytosis of pIgA-R versus newly synthesized
apical PM residents
As described in section IIF, hepatocytes and enterocytes use the transcytotic pathway to deliver newly synthesized resident proteins to the apical surface. This “indirect” pathway includes transport of the newly synthesized apical proteins from the TGN to the basolateral PM
where they are then selectively endocytosed and transcytosed to the apical PM (33, 499). Are the transcytotic
pathways of the pIgA-R and apical residents shared? We
tested this hypothesis by immunolocalizing newly synthesized apical PM proteins and pIgA-R in hepatocytes from
bile-duct ligated rats, a condition reported to perturb
hepatic IgA transport (29, 306). We found that both the
residents and pIgA-R accumulated in the SAC. We next
applied pulse-chase metabolic labeling combined with
subcellular fractionation, vesicle immunoisolation, and
immunoprecipitation of apical PM proteins to obtain
more quantitative information about this intracellular
compartment (30). We found newly synthesized dipeptidyl peptidase IV (DPPIV), a single TMD apical PM protein,
first in immunoisolated early endosomes and subsequently in isolated SAC. The high specific radioactivity of
DPPIV (i.e., 35S-DPPIV/immunoreactive DPPIV) in this
latter vesicle fraction indicated that very little preexisting
(unlabeled) DPPIV was present. This meant that recycling
of resident apical PM proteins was either very limited or
did not involve SAC.
The transcytosing molecules, although ultimately directed to the apical cell surface, must first traverse the
Physiol Rev • VOL
basolateral cell surface. Besides the basolateral targeting
signal identified for pIgA-R, at least two other such signals
have been identified. They either contain a tyrosine in the
context of a short degenerate sequence or a dileucine
motif (21), and in some cases, they overlap with those
used at the basolateral PM in receptor-mediated endocytosis. In general, the tyrosine-based motifs are thought to
be recognized by the ␮-subunit of the Golgi tetrameric
adaptor protein-1 (AP-1), whereas the leucine-based signals are thought to interact with the ␤-subunit (361, 392).
Identification of a new epithelial-specific ␮1-subunit, ␮1B,
(157) has added a new twist to basolateral sorting. Its role
in TGN to basolateral delivery was proposed when it was
observed that introduction of ␮1B into LLC-PK1 kidney
cells, which lack endogenous ␮1B, redirected its missorted LDL receptor (LDL-R) and Tf-R with high fidelity to
the basolateral PM (157). Since these cells express the
“original” ␮1-isoform, ␮1A, the ␮1B targeting appeared to
be dominant (157). However, the specific transport step at
which the ␮1B-subunit functions was not identified in this
study. More recently, Gan et al. (173) reported that in the
cells lacking the ␮1B-subunit, TGN to basolateral targeting of LDL-R and Tf-R occurred, but basolateral recycling
was impaired, thus ␮1B sorts basolateral proteins postendocytically. This is consistent with the observation that
basolateral targeting signals function both to deliver molecules to the basolateral domain, but also regulate basolateral internalization and recycling (for example, see Ref.
20). Despite where ␮1B functions, it is still a paradox that
in liver that lacks ␮1B expression, the same basolateral
receptors as in LLC-PK1 cells are delivered directly and
with high fidelity to the basolateral PM. How do hepatocytes, without functional ␮1B/AP-1 complexes, sort proteins to the basolateral surface and maintain them there?
What interprets the nontyrosine, nonleucine-based pIgA-R
basolateral targeting signal? Also, how are the apical residents targeted to the basolateral surface in hepatocytes?
Unlike pIgA-R, the cytoplasmic tails on several apical
ectoenzymes that we have studied are very short (6 – 8
amino acids), with no identified or nonexistent (the GPIanchored proteins) signals suggesting that the mechanisms regulating transport must be quite different.
A related puzzle is the mechanism(s) regulating internalization of the apical residents from the basolateral
domain. Both newly synthesized pIgA-R and apical residents rapidly traverse the endoplasmic reticulum and
Golgi and reach the basolateral PM within 45 min (32,
499). However, the rates of their redistribution to the
apical PM vary greatly, which might reflect differences in
their modes of basolateral internalization. The different
cytoplasmic tails may also reflect different internalization
routes, and in the case of the apical residents, these may
likely be non-clathrin-mediated mechanisms. Alternatively, the proteins are internalized at the same rates and
to the same extents. The variable trafficking kinetics in-
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binding, which significantly enhances its transport from
the apical recycling endosome to the apical PM in MDCK
cells (548). Ligand binding rapidly causes the tyrosine
phosphorylation of many proteins in MDCK cells including phospholipase C-␥1, from which a signal transduction
cascade has been proposed (388). Similarly, pIgA-R transcytosis in intact hepatocytes is stimulated by intravenous
injection of pIgA into rats (189). Although the accelerated
transport step was not identified, clues to the activated
tyrosine kinase came from studies performed in mouse
hepatocytes (341). Coimmunoprecipitations showed that
p62yes interacts with the pIgA-R tail in rat hepatocytes,
and in p62yes knock-out mice, both basolateral to apical
constitutive and activated transcytosis of pIgA were impaired (341). However, this mechanism may not be universal, since dIgA does not activate transcytosis in FRT
cells that express exogenous pIgA-R (493). Also, the human forms of pIgA-R do not respond to ligand binding in
polarized MDCK cells (all other studies were performed
using the rabbit form), further suggesting that mechanisms regulating transcytosis differ among organisms.
Careful comparison of cytoplasmic tail sequences may
help pinpoint the reasons for the differential regulation.
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PAMELA L. TUMA AND ANN L. HUBBARD
stead may reflect differences in sorting or transport mechanisms elsewhere in the transcytotic pathway (e.g., basolateral early endosomes).
V. MECHANISMS AND MOLECULES
REGULATING TRANSCYTOSIS
A. Targeting Machinery
1. The SNARE hypothesis
How do cargo-bearing vesicles deliver their contents
to the correct target domain? Morphological, biochemical, and genetic approaches have successfully identified
many molecules involved in “vesicle targeting,” a complex
series of molecular events that minimally includes docking and fusion. These approaches indicate that the basic
mechanisms are conserved among the membrane compartments throughout the biosynthetic and endocytic
pathways and in organisms ranging from yeast to humans.
The players so far identified fall into two broad categories: some are used repeatedly throughout the pathways,
others belong to discrete protein families, where one or a
few members act at one or a few transport sites. Members
of three protein families are central players in vesicle
targeting and fusion (216, 262, 335, 443). They are as
follows: 1) the SNAREs which, in general, are a group of
cytoplasmically oriented integral membrane proteins that
are present on vesicles (v-SNAREs) or target membranes
(t-SNAREs) (also referred to as Q- and R-SNAREs; see
Refs. 613 and 144); 2) the Sec1/Munc18 proteins; and 3)
small-molecular-weight GTP-binding proteins, the rabs.
Physiol Rev • VOL
2. NSF and ␣-SNAP
NSF was initially implicated as a regulator of polarized PM targeting from studies that examined the effects
of NEM on protein transport. Addition of NEM reduced
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In this section, we discuss what is known about the
mechanisms and molecules that regulate transcytosis in
polarized epithelial cells. Are there unique mechanisms/
molecules required? Do different cell types require specific molecules or combinations of molecules to perform
their specialized transcytotic functions? How does the
cell discriminate between transcytotic delivery and self
use? Surprisingly, there is little direct evidence for the
involvement of specific molecules in transcytosis, but
many promising candidates are emerging. We will describe some of these candidates, emphasizing those involved in vesicle transport, targeting, and consumption
along the transcytotic route. However, a cautionary flag
must be raised. Much of what is discussed is based on
studies performed only in tissue culture models of polarized cells (especially MDCK cells) and has not yet been
tested in a physiological context. Furthermore, many candidates have been named solely upon their subcellular
location; the functional studies have not been performed.
Nonetheless, there are exciting possibilities and the
groundwork is clearly set to direct future investigations.
The “SNARE hypothesis” emerged to describe the
mechanism by which the SNAREs promote membrane
docking and fusion through interactions with an ATPase,
N-ethylmaleimide sensitive factor (NSF), and its “receptor,” ␣-SNAP (547), but has undergone considerable revision as more is learned about these and other essential
proteins and the cyclical nature of the process is better
appreciated (262). For vesicle trafficking to continue past
one round, some of the machinery must continually cycle
between vesicle and target membranes, making the order
of events difficult to define. Also, the t- and v-SNAREs,
whose pairwise coupling was originally thought to confer
targeting specificity, show promiscuity in their binding in
vitro, suggesting that other factors are required for the
high fidelity of membrane targeting observed in vivo.
Furthermore, the ATPase, NSF, which was originally postulated to function in the fusion reaction itself, most likely
functions as a chaperone to disassemble the SNARE pairs
(204, 216). Whether this occurs when the pairs are in
“trans” (on the cognate membranes) or in “cis” (on the
same membrane after vesicle consumption) may differ
depending on the cell type or transport step examined.
It is also important to note that the “SNARE hypothesis” developed mainly from studies examining vesicle
docking and fusion at the PM where SNAP-25 family
members (t-SNARE isoforms) are required. Because these
molecules are predominantly PM associated, the mechanisms regulating intracellular vesicle transport are likely
different. Consistent with this is the finding that two
t-SNAREs (both syntaxins) and two v-SNAREs are required during late endosome fusion, whereas at the PM,
two different t-SNAREs (a syntaxin and SNAP-25 protein)
and one v-SNARE participate (15). Interestingly, in both
cases the molecules form core complexes that are structurally similar, indicating that the mechanisms, albeit different, are conserved. Newer models have integrated the
enormous and confusing body of information and have
identified common steps in membrane targeting which
minimally include 1) SNARE activation (which likely includes NSF chaperone activity) and rab recruitment to
proper organelle sites; 2) cognate membrane attachment;
and 3) membrane fusion and bilayer mixing (262). The
order of events and the mechanistic details are not clear,
yet there is considerable evidence that the SNAREs, rabs,
and Sec1/Munc18 proteins are key players in the process.
In the following sections, we discuss the possible roles of
selected members of the transport machinery in regulating transcytosis in polarized epithelial cells. Table 5 summarizes what we currently know.
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TRANSCYTOSIS
3. t-SNARES and v-SNAREs
There are two families of t-SNARE proteins: the syntaxins and the SNAP-25 family. To date, at least 18 syntaxin family members have been identified in mammalian
cells of which 5 (syntaxins 1A, 1B, 2– 4) are PM-specific
Physiol Rev • VOL
isoforms (262). The SNAP-25 family is much smaller, with
only three identified members: SNAP-25, SNAP-23, and
SNAP-29 (262). Like the syntaxins, these proteins are
relatively small (23–29 kDa), cytoplasmically oriented
molecules. Although not integral membrane proteins as
are the PM syntaxins, SNAP-25 proteins associate with
the bilayer through cysteine-linked palmitoyl chains located near the middle of the protein. Biochemically,
SNAP-25 proteins bind PM-associated syntaxins and vSNAREs in vitro to form the four-stranded ␣-helical ternary complexes required for vesicle docking and fusion
(262).
The distributions of syntaxins 2, 3, and 4 at the PM
have been examined in different epithelial cells. In particular, we found that rat hepatocytes express three endogenous PM-associated syntaxin isoforms (syntaxins 2,
3, and 4) and SNAP-23 (163). Quantitative immunoblotting
revealed that all four t-SNAREs are relatively abundant in
liver (⬃11– 668 nM corresponding to 0.5–28 ⫻ 105 molecules/cell). Biochemically, each of the t-SNAREs was observed predominantly in hepatocyte PM sheets with overlapping but distinct expression patterns among the PM
domains. Both syntaxin 4 and SNAP-23 are restricted to
the basolateral PM while syntaxins 2 and 3 are more
apically distributed, with greater enrichment of syntaxin 3
in this domain. Despite the biochemical abundance of the
molecules, we were able to detect only syntaxins 2 and 4
in rat liver sections in situ. However, the distributions did
not fit with our biochemical data; we found syntaxin 4 in
both domains. Similar discrepancies were observed in
WIF-B cells. Like in liver hepatocytes, syntaxin 3 was at
the apical domain, but unlike liver, syntaxin 2 was restricted to the apical domain. Also, syntaxin 4 and
SNAP-23 were found in both domains. These varied distributions likely reflect important differences in regulation of PM dynamics between the in vivo and in vitro
systems and may point to interesting features of the cellular itineraries and functions of the t-SNAREs. Little is
known how these putative targeting molecules are themselves targeted to the correct PM domain, and once delivered, how and if they are retained there.
Interestingly, the PM syntaxins display remarkable
variability in their domain distributions in other polarized
cells (see Table 5). Only syntaxin 3 appears to be consistent with apical distributions observed in all polarized
cells examined (117, 163, 170, 331, 438). SNAP-23 distributes to both PM domains in all but two epithelial cell
types (see Table 5). Since it is thought to be required for
vesicle docking and fusion, SNAP-23’s uniform PM distribution fits with the current model of its ubiquitous involvement in t- and v-SNARE ternary complex formation.
However, its absence at the apical PM in hepatocytes and
pancreatic cells is somewhat paradoxical. Either these
cells have unique mechanisms for regulating transport at
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IgA transcytosis by ⬃70% and its basolateral targeting by
⬃90% in permeabilized MDCK cells (17, 332). It also inhibited fusion (as assayed by pIgA-R processing) of transcytotic carrier vesicles with the apical PM in a hepatic
cell-free system reconstituting the final step of transcytosis (564). Because NSF ATPase activity is inhibited by
alkylating (617), it was the likely target for NEM. In
support of that, addition of recombinant NSF restored
much of the activity lost in MDCK cells or hepatic cell-free
systems treated with NEM or anti-NSF antibodies (17,
253, 332, 564). Interestingly, direct apical PM targeting
was insensitive to NEM (253, 332), indicating that it requires an unidentified NSF homolog or does not require
NSF-like activity at all. Other key factors, the SNAP family
of NSF receptors (␣-, ␤-, and ␥-isoforms), recruit NSF to
organelles and activate its ATPase activity (617). ␣-SNAP
has been identified in polarized epithelial cells and its role
in TGN to PM targeting examined in MDCK cells. Addition
of ␣-SNAP antibodies and treatment of cells with botulinum E (an ␣-SNAP-specific toxin) inhibited direct transport to both domains (17, 253, 300). Unfortunately, its role
in transcytosis was not examined.
NSF has also been implicated as an important regulator of transcytosis in endothelial cells. Treatment of
myocardial or rat lung endothelium with NEM significantly inhibited transcytosis in situ (80 and 50%, respectively) (455, 505). NSF and its receptor ␣-SNAP were also
found associated with caveolae, supporting the role of
this vesicle type as the transcytotic carrier in endothelial
cells (509). Furthermore, immunoprecipitation of exogenously added recombinant myc-tagged NSF from rat
lung endothelial cell extracts revealed the presence of
large, ATP-dependent, NEM-sensitive complexes that
contained many members of the transport machinery including ␣- and ␥-SNAP, cellubrevin, syntaxin, and rab5 as
well as caveolin and dynamin (457). The complexes were
also found to contain cholesterol, the ganglioside GM1,
and other unidentified lipids. As predicted, these complexes were immunoprecipitated from membrane extracts, but they were also surprisingly recovered from
cytosol preparations. How these supramolecular proteinlipid complexes function in membrane transport is not yet
known but supports a role for both caveolae (the complexes contained caveolin) and the molecules of the
SNARE hypothesis in endothelial transcytosis. Do the
complexes present in the cytosol or on membranes serve
the same function? Are similar complexes present in
other polarized epithelial cell types?
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Tight junctions (M)
Basolateral PM (M), apical vesicles and tubules
(M), transcytotic vesicles (F), apical recycling
endosome (M)
Basolateral PM (M), subapical tubules (M)
Subapical tubules (M)
Subapical structures (F, M), apical recycling
endosome (M)
Rab13
Rab17
Rab18
Rab20
Rab25
Rab6
Rab8
Rab11
Apical PM (M)
Apical and basolateral early endosomes, apical
and basolateral PM (M, F)
Golgi
Golgi, basolateral PM (M, F)
Subapical structures (M, F), apical recycling
endosome (M)
Rab3D
Rab4 and 5
␣-SNAP
Golgi (F), transcytotic vesicles (F)
Tight junctions (M)
N/D
Munc 18-2
Kidney tubule epithelia
Kidney tubule epithelia
Gastric parietal cells, MDCK cells
Hepatocytes
Intestinal and kidney epithelia,
hepatocytes, HT-29, and T84
cells
Hepatocytes
Kidney epithelia, hepatocytes,
MDCK and WIF-B cells
Hepatocytes
MDCK
Gastric parietal, pancreatic acinar
and prostate epithelial cells,
enterocytes, hepatocytes, and
MDCK cells
Small intestinal and kidney
epithelia, hepatocytes, Caco-2
and LLC-PK1 cells
Hepatocytes, enterocytes, kidney
epithelia, MDCK, and Eph4 cells
Rab proteins
Murine intestinal, lung, kidney,
testis and spleen epithelia,
MDCK cells
MDCK cells
Placenta, pinealocytes, MDCK cells
Others
Gastric parietal, MDCK and IMCD
cells
Hepatocytes
Caco-2 cells
Hepatocytes, MDCK cells, and
kidney epithelium
v-SNAREs
Hepatocytes, pancreatic acinar and
intestinal epithelial cells, kidney
collecting duct, Caco-2, MDCK
and WIF-B cells
Hepatocytes, WIF-B cells
N/D
Hepatocytes, MDCK, WIF-B, and
IMCD cells
t-SNAREs
Epithelial Cells Examined
Location, epithelial-specific expression; mutational
analysis and overexpression in MDCK and Eph4
cells
Location
Location
Location; mutational analysis in MDCK cells
Location
Location; TGN budding assay
Location; sensitivity to rab8 inhibitory peptides
Location; change in distribution upon parietal cell
activation; mutational analysis in gastric parietal
and MDCK cells
Location
Location; overexpression in MDCK
Copurification with transcytotic vesicles
Location
Anti-␣-SNAP sensitivity in MDCK
NEM and anti-NSF sensitivity and mutational
analysis in MDCK
Location; epithelial-specific expression
Location
Location
Location
Location; toxin sensitivity in MDCK and IMCD cells
Location; toxin sensitivity in MDCK and IMCD cells
and anti-SNAP23 sensitivity in MDCK cells
Location
Location; syntaxin 3 overexpression in MDCK and
Caco-2 cells and antibody sensitivity in MDCK
cells
Evidence for Role in Transcytosis
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Physiol Rev • VOL
Rab1 and 2
Rab3B
Golgi (M, F), endosomes (F), coated vesicles
(F)
Apical PM (M)
NSF
Endosomes (F)
VAMP 2
Basolateral endosomes (M, F)
Subapical structures (M), apical PM (M)
Basolateral early endosomes (F, M), apical PM
(M), subapical endosomes (M)
BL early endosomes (F, M)
TGN
Apical and/or basolateral PM (M, F), endosomes
(M, F)
Syntaxin 8, 13
Syntaxin 6, 10
SNAP 23
VAMP 3/cellubrevin
VAMP 7/Ti-VAMP
VAMP 8/endobrevin
Apical and/or basolateral PM (M, F)
Syntaxin 2, 3, 4
Subcellular Location in Epithelial Cells
[Morphology (M)/Fractionation (F)]
5. Putative regulators of transcytosis in polarized epithelial cells
Protein
TABLE
338
338
74, 82
165, 210, 247, 337, 631
632
354
240
82, 134
305
67, 272, 583
265
595, 611
253, 509
17, 253, 332, 359, 457,
509
472
75
171
554, 622
27, 74, 253, 371
562, 583
262
26, 88, 169, 255, 314, 333
59, 117, 163, 168, 331,
345, 438
Reference No.
904
PAMELA L. TUMA AND ANN L. HUBBARD
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Golgi (F), endosomes (F)
Zymogen granules (M, F), Golgi (F), apical PM
(M, F)
Apical PM (M, F), apical recycling endosome
(M)
Apical PM (M, F)
Basolateral endosomes (M)
Apical recycling endosomes (?)
Cytoplasmic dynein
Myosin I
Myosin V
Motor proteins
Intestinal epithelia, hepatocytes
Pancreatic acinar cells, intestinal
epithelia, hepatocytes, Caco-2
cells
Intestinal epithelia, MDCK and
WIF-B cells
Intestinal epithelia
MDCK cells
MDCK cells
MDCK cells
Endothelial and MDCK cells
Dynamins
Intestinal epithelia
Enterocytes and MDCK cells
MDCK cells
218, 304, 321
Location, actin dependence, mutational analysis in
MDCK cells
Location, actin dependence
Location, mutational analysis
Location, mutational analysis
215, 218
315
270
146, 415
25, 94, 136, 145, 440, 452
7
7, 416
357
618
150
260, 424, 509, 566, 615
142, 149, 357, 509, 615,
620
274, 292, 356, 357, 365
200
Reference No.
MT dependence and orientation; NEM sensitivity
Location, actin dependence, mutational analysis in
Caco-2 cells
Location; GTP dependence, overexpression and
mutational analysis in MDCK cells, anti-dynamin
sensitivity in endothelial cells
Location, GTP dependence, overexpression and
mutational analysis in MDCK cells
Intestinal-specific expression; location
Location; intestinal-specific expression
Location; kidney-specific expression
Location
Location; change in distribution upon activation of
transcytosis with bile acids in hepatocytes
Location
Location
Evidence for Role in Transcytosis
IMCD, inner medullary collecting duct; MT, microtubule; N/D, not determined; TGN, trans-Golgi network; PM, plasma membrane.
Myosin VI
RhoA
Rac1
Apical and BL PM (M, F)
Dynamin-1
Annexins
The exocyst
Intestinal and mammary epithelia,
hepatocytes, MDCK cells
Kidney, intestinal, uterine, tracheal
and fallopian tube epithelia, T84
cells
Hepatocytes
MDCK cells
Epithelial Cells Examined
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Dynamin-2
Caveolae, apical PM (M, F)
Annexin VIII
Annexin XIIIa
Annexin XIIIb
Apical and/or basolateral PM (M), early
endosomes (F), subapical structures (M, F)
Apical and/or basolateral PM (M, F), subapical
structures (M)
Apical and/or basolateral PM (M, F), subapical
structures (M, F)
Apical PM (M)
Apical PM ⬎⬎ basolateral PM (M)
Apical PM, subapical structures (M, F)
Annexin VI
Annexin IV
Annexin II
rSec6 and rSec8
Subcellular Location in Epithelial Cells
[Morphology (M)/Fractionation (F)]
Tight junction (M)
5—Continued
Protein
TABLE
TRANSCYTOSIS
905
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PAMELA L. TUMA AND ANN L. HUBBARD
Physiol Rev • VOL
plicated in regulating apical vesicle delivery (150). This
sequence in annexin XIIIb encodes a lipid-binding domain, but whether VAMP 7/TI-VAMP shares this biochemical property is not yet known.
A possible role for VAMP 2 in transcytosis has been
suggested from studies in SLO-permeabilized rat lung endothelial cells using VAMP-specific neurotoxins (371). In
cells treated with botulinum D and F, VAMP 2 cleavage
occurred concomitant with the impairment of caveolaemediated cholera toxin B endocytosis. At the ultrastructural level, large, aberrant subplasmalemmal organelles
were observed in treated cells, indicating that delivery of
cholera toxin to intracellular intermediates (endosomes?)
was impaired. Unfortunately, transcytosis was not examined in toxin-treated cells to determine whether VAMP 2
is a general regulator of caveolae-mediated internalization
in endothelial cells. These findings also expand the function of VAMP 2 to include regulation of endocytic transport, whereas previously, this v-SNARE was thought to
function in exocytic membrane docking and fusion.
Whether VAMP 2 functions in PM vesicle docking and
fusion in endothelial cells has not yet been tested. Likewise, a possible role for VAMP 2 in endocytosis in other
cell types may warrant further investigation.
4. Munc18
Munc18 homologs have been identified in systems
from yeast to neurons and are thought to participate in
multiple vesicle transport steps (262, 442). The 68-kDa
mammalian Munc18 proteins peripherally associate with
the PM through interactions with syntaxins; in vitro, they
bind syntaxins 1, 2, and 3 with nanomolar affinity. Interestingly, Munc18 binding to syntaxins cannot occur when
the syntaxins are bound to SNAP-25 proteins, suggesting
that the different complexes form reciprocally. However,
it is presently not known whether Munc18 isoforms play
a positive or negative regulatory role in PM targeting.
Mutational analysis of related proteins in yeast, Drosophila, and Caenorhabditis elegans all implicate Munc18
species as positive regulators whereas in vitro assays
suggest the opposite (262, 442). As for most of the SNARE
molecules, no direct evidence for the involvement of
Munc18 proteins in polarized PM targeting exists. However, the Munc18 –2 isoform is primarily limited to polarized epithelial cells (472). Furthermore, its expression
seems restricted to the apical PM where it forms complexes with syntaxin 3 (471), a characteristic that suggests a unique function for Munc18 –2 in vesicle delivery
to the apical PM.
5. The rab proteins
The rab proteins belong to the largest family of small
molecular mass (20 –30 kDa) GTP binding proteins. There
are 11 known yeast isoforms and at least 60 rabs in
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their apical surfaces, or other SNAP-25 isoforms are yet to
be identified.
To date, the roles of the t-SNAREs in polarized PM
targeting have been tested directly mainly in MDCK cells
that were stably expressing pIgA-R and overexpressing
wild-type syntaxins 2, 3, or 4 (332). Neither transcytosis
nor basolateral transport of pIgA-R was affected in these
cells. Similarly, in Caco-2 cells overexpressing syntaxin 3,
no changes in basolateral protein targeting were observed
(59). Together, these results suggest that the PM-associated syntaxins do not regulate these transport pathways
or that other yet-to-be isoforms are involved. Alternatively, overexpression was not high enough to be inhibitory or does not negatively regulate these processes.
However, overexpression of syntaxin 3 in MDCK and
Caco-2 cells did lead to alterations in apical PM dynamics.
In MDCK cells, a slight impairment (⬃20%) of direct TGN
to apical PM delivery of a chimeric pIgA-R molecule and
IgA was observed as well as apical recycling (also ⬃20%).
Likewise in Caco-2 syntaxin 3 overexpressors, the direct
apical targeting of sucrase-isomaltase and the apical secretion of ␣-glucosidase was significantly impaired. Furthermore, anti-syntaxin 3 antibodies inhibited direct targeting of hemagglutinin (HA) in MDCK cells, confirming a
role for this syntaxin in apical delivery (300). Unfortunately, the effects on transcytosis of anti-syntaxin 3 or
syntaxin 3 overexpression in Caco-2 cells were not examined. However, the role of SNAP-23 in transcytosis has
been examined in MDCK cells by treating with SNAP-23/
25-specific neurotoxins. In these cells, basolateral to apical transport of pIgA was inhibited by 30% (17), as was
basolateral targeting of the receptor, but the TGN to
apical targeting of HA was not affected (17, 253). More
recent studies confirmed this result and found that toxin
activity also impairs transferrin recycling (314).
The direct involvement of v-SNARES in transcytosis
has not been explored, but the obvious prediction is that
they are required. VAMP 1, 2 and VAMP 3/cellubrevin are
ubiquitously expressed, and the presence of VAMP 3/cellubrevin on endosomal structures in hepatocytes and
VAMP 2 on endothelial caveolae has been reported (75,
371). VAMP 8/endobrevin is enriched in epithelial tissues
and has been localized to the apical pole in kidney epithelium (622). Interestingly, in hepatocytes, this VAMP
species was found to be enriched in basolateral early
endosomal fractions, whereas in MDCK cells, it was localized to both apical endosomes and the apical PM (554).
Whether this protein functions in basolateral or apical PM
targeting (or both) is not yet known. In Caco-2 cells,
another VAMP isoform, VAMP 7/TI-VAMP, is also localized to both the apical PM and in subapically located
structures where it has been proposed to function in the
later steps of apical PM delivery (171). Interestingly, this
VAMP species has a long NH2-terminal extension that
resembles a region in annexin XIIIb, another protein im-
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TRANSCYTOSIS
Physiol Rev • VOL
transcytosis in a number of different epithelial cells, first
because of its restricted expression pattern and subcellular location, and more recently from functional studies. In
kidney, rab17 expression was induced only upon mesenchymal differentiation to polarized cells (337). In intestinal tissue sections, it was detected only in polarized cells
and not in surrounding, nonpolarized cells. In enterocytes, rab17 was detected in the basolateral domain,
whereas in kidney cells it was found both at the basolateral PM and in apical tubules underlying the brush border
(337). In MDCK and polarized Eph 4 cells, it was detected
in subapically located vesicular structures (247, 631), and
in hepatocytes, rab17 copurified with transcytotic vesicles
(210). Functionally, the overexpression of wild-type rab17
in MDCK cells impaired the basolateral to apical transcytosis of dIgA (247). Conversely, in Eph 4 cells expressing
GTPase-deficient mutants, the basolateral to apical transcytosis of Tf-R and a chimeric receptor was enhanced as
was apical recycling of the chimeric receptor (631). Rab17
has also been copurified with a population of transcytotic
vesicles from rat liver, suggesting it is an important regulator of hepatic basolateral to apical transcytosis, too.
Surprisingly, both rab1 and -2 also copurified with the
vesicles, implicating them as additional regulators of
transcytosis (265). The next steps will be pinpointing the
site of function in the transcytotic pathway, examining
whether the role of rab17 is universal among epithelial
cells and to identify the cellular effectors that rab17 activity regulates.
Rabs 11 and 25 have also been identified as important
regulators of basolateral to apical transcytosis from functional studies performed in gastric parietal and MDCK
cells (82, 134, 610). Both of these rabs have been localized
to the apical recycling endosome in MDCK cells (82), and
when the respective GTP-binding mutants were overexpressed, basolateral to apical transcytosing IgA accumulated in these structures (610). Interestingly, the activated
form of rab25 inhibited transport more than the rab11
mutant. Also interesting is the observation that unlike for
nonpolarized cells, rab11 is not required for Tf recycling
(610). The puzzle is why these rabs apparently regulate
the same transport steps at the apical PM. Do the differential responses suggest separable roles in transport?
Also, why is rab11 required for Tf recycling in nonpolarized cells, but not polarized cells? Although rab25 expression is enriched in epithelial tissues, it is surprisingly
absent in liver (193), pointing out yet another important
difference in apical PM targeting in polarized hepatocytes.
The recent identification of two rab11 effectors, rip11
and myosin Vb, has further confirmed a role for this rab in
regulating late steps in basolateral to apical transcytosis
(304, 458). In both cases, dominant negative mutations
significantly impaired IgA apical PM delivery, and corresponding accumulations of IgA in the apical recycling
endosome were observed in MDCK cells. Like rab11, my-
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mammalian cells (195, 354, 500). Examination of transfected cells either overexpressing wild-type or dominant
negative mutant (usually the GDP-bound conformer)
forms of various rabs either stimulate or inhibit protein
transport and in some cases alter organelle morphology.
Although their precise roles are not known, they have
been proposed to function in one of three ways: 1) facilitating vectorial traffic via associations with the cytoskeleton; 2) regulating vesicle docking by recruiting effector
molecules, thereby promoting the formation of “molecular tethers”; and 3) “priming” docking and fusion by activating SNARE molecules (195, 500).
Given the large number of mammalian rabs and their
varied distributions, it is likely that transcytosis in epithelial cells is regulated by multiple isoforms, but which
ones? Rabs 3B, 13, 17, 18, 20, and 25 are preferentially
expressed in epithelial cells (see Table 5), suggesting a
unique function in polarized membrane transport. Although also expressed in nonpolarized cells, rabs 1, 2, 3D,
4, 5, 6, and 11 have also been implicated in regulating
polarized PM transport. Of these 13 rabs, 9 have been
localized to the apical pole: at the apical PM (rab3D), the
tight junction (rabs 3B and 13), or in subapical structures
(rabs 5, 11, 17, 18, 20, and 25). The multiple rab proteins
in the apical region may point to the complexity of membrane transport events at this PM domain both in terms of
specific transport steps as well as organellar intermediates.
Rab5 is the most extensively studied isoform, and
much is known about the relationship between its catalytic activity and function in membrane transport (478). In
all nonpolarized cells examined to date, rab5 is localized
to the PM, clathrin-coated vesicles, and/or early endosomes. Overexpression of rab5 increased endocytic transport and stimulated early endosome fusion in vitro,
whereas inhibition of rab5 led to the opposite effects.
Recently, it was proposed that rab5 recruits EEA1 (one of
its many effectors) to sites of endosome fusion along with
NSF and syntaxin 13 that together drive formation of a
large multimeric complex which then coordinates fusion
pore assembly (368). This general role of rab5 in endocytosis strongly suggests an important role in the early steps
of the transcytotic pathway in most polarized cells. Consistent with this is the presence of rab5 on early apical
and basolateral endosomes in hepatocytes, hepatic WIF-B
cells, in mouse kidney epithelia and MDCK cells (67, 272,
583). Furthermore, when overexpressed in MDCK cells,
increased rates of fluid-phase internalization from the
apical and basolateral PM were observed, suggesting a
role for rab5a in endocytosis from both domains (67). In
endothelial cells, rab5 is also localized to the PM and early
endosomes, suggesting a role in endocytosis. However, it
is not found associated with caveolae, suggesting it is not
required for endothelial transcytosis (509).
Rab17 has been strongly implicated in regulating
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PAMELA L. TUMA AND ANN L. HUBBARD
Physiol Rev • VOL
though these studies provided insight into rab function,
careful enumeration and examination of the endogenous
rab isoforms in a single cell type is required to clearly
understand their role in vivo. Nonetheless, the concentration of rab proteins in the subapical regions of epithelial
cells is striking and may point to the complexity of intracellular compartments and membrane transport events at
this PM domain. It remains to be determined whether
other putative transport machinery molecules or rab effectors are also concentrated at the apical regions of cells.
6. The exocyst
Over a dozen genes have been identified in yeast that
are required for TGN to PM transport (275, 408, 409), and
of these, more than two-thirds of the gene products form
a multimeric complex referred to as the exocyst. The
exocyst subunits were localized to the yeast PM, but only
to sites of rapid cell growth at small bud tips (573).
Interestingly, this expression pattern differs from the
yeast t-SNARE molecules, which are evenly distributed at
the PM. From these data, it was suggested that the exocyst mediates vesicle delivery to restricted regions of the
cell surface such that it additionally discriminates (beyond SNARE function) whether and/or where a vesicle
docks (573).
Mammalian homologs to the exocyst subunits are
ubiquitously expressed and also form a multimeric complex that is mainly peripherally associated with the PM
(239, 282, 574). The mammalian counterparts of the yeast
Sec6, Sec8, and Sec10 exocyst have been localized to tight
junctions in MDCK cells, and their discrete staining patterns were dependent on intact junctional complex formation (200, 324). Anti-rSec6 antibodies blocked transport of LDL-R from the TGN to the basolateral PM in
permeabilized cells, whereas direct transport of an apical
PM antigen, p75, was not changed (200). Conversely, overexpression of mammalian Sec10 enhanced PM transport
of E-cadherin and basolateral secretion, whereas apical
delivery of the integral PM protein gp135 was unchanged
(324). Surprisingly, Sec10 overexpression also enhanced
apical secretion. Taken together, these results implicate
the exocyst as an important regulator of vesicle targeting
to both PM domains, but delivery to the apical domain
may be restricted to vesicles carrying secreted cargo. The
placement of the complex further implicates the tight
junction as an important site for vesicle delivery to either
domain. Whether transcytotic vesicles are specifically recruited to these or other sites on the PM inhabited by the
exocyst has not been rigorously examined, but remains an
interesting possibility.
7. Annexins
Annexins are a large family of proteins grouped together due to shared amino acid sequence similarity and
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osin Vb was found to regulate Tf recycling only in nonpolarized cells (304) and rip11 mutants did not impair
basolateral transferrin recycling (458). Are different rab11
effectors required for transferrin recycling in nonpolarized cells? How are these two effectors both regulated by
rab11 at the same transport step? Careful dissection of the
molecular events required for vesicle budding from the
apical recycling endosome, transport to the apical PM,
and subsequent docking and fusion are required to specifically identify how these molecules function.
The expression patterns of rabs13 and 3B are also
highly dependent on the polarized state of a cell (611,
632). In nonpolarized cells, they were found in cytoplasmic vesicles, whereas in polarized cells, they were recruited to tight junctions. Rab13 has so far been detected
at tight junctions of Caco-2 cells, mouse intestinal cells,
kidney, and liver (632) while rab3B has been observed in
tight junctions of colonic epithelia, kidney, and liver
(611). In both cases, their localizations were dependent
on the integrity of the tight junction. When junctions were
disassembled by Ca2⫹ withdrawal, staining of rabs13 and
3B at the cell surface was lost, suggesting these rabs
function in vesicle delivery to the tight junction and, in
particular, regulate vesicular delivery of junctional components. At present whether any transcytotic vesicles are
also specifically delivered to the cell surface at sites of
cell-cell contact, and by extension, under the control of
these rab isoforms, is not yet known. Immunoadsorption
and examination of the vesicles with which these rab
isoforms are associating will provide important clues to
their function.
Interestingly, in a recent report (595) overexpressed,
myc-tagged rab3B was localized not to tight junctions in
MDCK cells, but to apically located structures that also
contained unoccupied pIgA-R. At present, there are no
explanations for this different staining pattern. Nonetheless, in the presence of dIgA, rab3B dissociated from
pIgA-R and the GTP-bound mutant rab3B impaired dIgAactivated transcytosis to approximately control (-dIgA)
levels. Together these results suggest that rab3B is a
negative regulator of ligand-stimulated transcytosis in
MDCK cells. It will be interesting to see if rab3B also
regulates other forms of ligand-activated transcytosis in
other cell types and whether the sites of delivery are at or
near the tight junction.
Because of the congestion of different rab isoforms
in the apical and subapical region of the polarized epithelial cell, it is important to identify the specific intermediates participating in basolateral-to-apical transcytosis.
How many different subapical compartments exist, and of
those, which receive transcytosing molecules? How do
the rabs distribute among them? Furthermore, much of
the morphological and functional analysis on the different
rab isoforms has been performed in transfected cells
overexpressing either wild-type or mutated proteins. Al-
909
TRANSCYTOSIS
8. Dynamin
Dynamins are a family of high molecular mass (100
kDa), peripheral membrane-associated GTPases that
function in the early stages of endocytosis (502, 586).
Examination of dynamin function in transfected or microinjected mammalian cells has indicated its participation
in clathrin-mediated endocytosis (106, 568) and suggested
a role for dynamin in mediating fission of caveolae from
the PM (223, 416). A role for dynamin in transcytosis has
best been established in endothelial cells. Not only was
dynamin found associated with caveolae in these cells,
but it was also identified as a required factor for the
release of caveolae from endothelial PM preparations in
vitro (416). Caveolae-mediated internalization of cholera
toxin B was also impaired in cultured endothelial cells
expressing dynamin dominant negative constructs (416).
Although these results imply that dynamin is a general
regulator of caveolae-mediated internalization in endothelial cells, the effects of the dominant negative dynamins
on transcytosing proteins were not analyzed.
Examination of dynamin isoforms in other polarized
mammalian epithelial cells has been limited to cultured
MDCK cells. Dynamin-2 was found at both PM domains
Physiol Rev • VOL
and corresponding defects in IgA and Tf internalization
from both domains were observed in cells expressing
dominant negative dynamin mutants (7). Interestingly,
dynamin-1 (the brain-specific isoform) localized only to
the apical PM, and when the dominant negative dynamin-1 was expressed, internalization was inhibited only
from that domain (7). Whether this is a physiologically
relevant finding is not yet clear but may point to similarities in PM dynamics at the apical domain in epithelial
cells and at the synapse in neurons. Nonetheless, many
transcytosing receptors (including pIgA-R) are internalized via clathrin-coated vesicles, a process requiring dynamin activity. The prediction is that dynamin is required
for the internalization of transcytosing molecules in polarized epithelial cells. Does dynamin regulate internalization of all transcytosing molecules, through caveolae,
clathrin-coated, or noncoated endocytic vesicles, and at
both PM domains?
B. Cytoskeleton
1. Microtubules and microtubule-based motors
In addition to the asymmetric distribution of PM
proteins, the polarity of epithelial cells is also reflected in
the organization of the cytoskeleton. In nonpolarized
cells, microtubules emanate from a juxtanuclear microtubule organizing center (MTOC). In polarized cells, there is
accumulating evidence that microtubules are instead (or
additionally?) organized from sites at or near the apical
PM such that the emanating microtubules are oriented
with their minus ends at the apical PM and their plus ends
attached to or near the basolateral PM (22, 131, 374, 475).
Such an arrangement also dictates the placement of organelles within the interior of the epithelial cell (410, 434).
In particular, in many epithelial cells, the compartments
of the transcytotic pathway are linearly situated along the
parallel microtubules. However, this arrangement is not
universal to all polarized cells. For example, the microtubules in endothelial cells are arranged parallel, perpendicular, and obliquely to the long axis and in some cases
even form criss-crossed helical arrays (53, 481).
In general, disruption of microtubules does not inhibit internalization of molecules (either soluble of membrane-associated) into early endosomal compartments,
but does impair their movement to other compartments.
This is true for molecules destined for transcytosis and
internalized from the basolateral side in epithelial cells
where the transcellular path is long, such as in colchicineor nocodazole-treated MDCK cells, isolated hepatocytes
or Caco-2 cells (56, 110, 137, 190, 222, 246, 346, 362).
Interestingly, addition of colchicine impaired transcytotic
delivery of albumin in endothelial cells in situ despite the
comparatively short distance between the apical and basolateral surfaces (13). However, addition of microtubule
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their biochemical properties (103, 387, 563). One distinguishing feature of most annexins is their ability to aggregate membrane vesicles in the presence of Ca2⫹ in vitro
(103). This activity led to the idea that annexins initiate
membrane-membrane contact that results in fusion. Of
the numerous annexin isoforms, a few have been identified as playing roles in polarized membrane targeting, but
many of those were implicated based only on their subcellular location (see Table 5). However, more direct
evidence has been demonstrated for annexins XIIIa and
XIIIb, the former being an intestinal-specific isoform and
the latter, a kidney-specific isoform (150, 618). In SLOpermeabilized MDCK cells, addition of either of these
annexin isoforms enhanced direct apical PM delivery of
HA (150, 309). Only the addition of recombinant XIIIa
inhibited basolateral delivery of VSV-G. Accordingly, the
addition of anti-annexin XIIIb antibodies specifically
blocked transport of TGN-derived vesicles to the apical
cell surface while transport to the basolateral surface was
not changed (299). Unfortunately, the roles of annexin
XIIIa and XIIIb in transcytosis were not tested in these
studies. Some evidence for a role for annexin II in transcytosis comes from studies performed in isolated hepatocyte couplets (620). Upon induction of transcytosis by the
addition of bile salts, annexin II immunofluorescence increased dramatically and was redistributed sequentially
from beneath the basolateral PM to perinuclear structures
and finally to the apical pole (620). Whether the annexin
was simply a passenger on these transcytotic intermediates or was mediating transport activity is not known.
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PAMELA L. TUMA AND ANN L. HUBBARD
Physiol Rev • VOL
regulates transcytotic vesicle delivery, a recent study examining the trafficking of transfected rhodopsin in MDCK
cells implicates this motor in direct apical vesicle targeting. The 14-kDa endogenous dynein light chain Tctex-1
directly binds transfected rhodopsin in MDCK cells. When
RP3, a non-rhodopsin-binding Tctex-1 homolog, was overexpressed in MDCK cells, it displaced the endogenous
Tctex-1 in the dynein complex and disrupted apical delivery of rhodopsin (567). Interestingly, the apical distributions of HA and gp135 were not changed in these cells,
nor were any basolateral antigens, suggesting that distinct
light chains might regulate vesicle translocation. In permeabilized MDCK cells, when cytoplasmic dynein activity
was abolished by either ultraviolet/vanadate photocleavage or immunodepletion from cytosolic extracts, direct
apical delivery was impaired (298). On the other hand,
immunodepletion of kinesin, a plus-end directed microtubule motor, inhibited transport to both the basolateral (to
the plus ends as expected) and the apical (to the minus
ends which was unexpected) domains. One possible explanation for this last result is that kinesin may be required to translocate vesicles through the microtubule
meshwork that is postulated to exist between the Golgi
and apical PM in MDCK cells (22). Finally, a surprising
finding is that apical-organized microtubules were not
required for transport from the TGN to either cell surface
domain in MDCK cells (199). This implies that microtubules of both polarities, and by extension, both motor
proteins facilitate transport to either PM domain, a report
that contradicts numerous reports. Such continued confusion highlights the need for more information from
multiple systems before we can confidently assign specific and/or generalizable roles for microtubules and their
motors in transcytosis.
2. Actin and actin-based motors
The actin cytoskeleton also has a unique organization
in many polarized cells. In general, actin microfilaments
extend to the basolateral PM and form attachments
through interactions with proteins of zonulae adherens,
tight junctions, and focal adhesions. At the apical surface,
actin is found as the core filament of microvilli and also as
a dense subcortical web (58, 146, 366). At the basolateral
domain, the actin-associated proteins fodrin and ankyrin
form a scaffold that restricts the movements of certain
integral PM proteins, including the Na⫹-K⫹-ATPase,
thereby stabilizing the basolateral population (208, 366,
386). At the apical surface, actin was shown to be an
important factor in maintaining the apical distribution of
gp135, a membrane glycoprotein in MDCK cells, even in
cells not in contact with their neighbors (419).
In general, actin is thought to be an important regulator of endocytosis from the apical, but not basolateral,
PM (16). In MDCK, Caco-2, and pancreatic acinar cells,
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disrupting agents does not impair transcytosis of molecules internalized from the apical surface in MDCK or
Caco-2 cells (246, 362). The reasons for this differential
dependence on microtubules to the two domains are not
yet understood. One possibility is that basolaterally destined vesicles have a shorter path to the lateral surface
and thus a higher probability of encountering their target
by simple diffusion. Alternatively, the machinery associated with basolaterally destined vesicles may promote
more efficient and specific binding interactions with their
target.
Microtubules are probably not a direct requirement
for transcytosis; they likely facilitate delivery by providing
the tracks upon which vesicles are translocated (43).
Thus, when microtubules are disrupted, the kinetics of
delivery are slowed, i.e., it takes a vesicle longer to encounter its appropriate target membrane by diffusion
than when tracked along microtubules. However, this
passive role for microtubules does not account for the
mistargeting observed for some transcytosing molecules
upon microtubule disruption. For example, the apical PM
proteins, aminopeptidase N, DPP IV, and alkaline phosphatase, lost their polarized expression patterns in Caco-2
cells treated with nocodazole or colchicine (56, 137, 190).
One explanation for the mistargeting is that different
vesicle populations require the activities of distinct assemblies of docking and fusion molecules that differ in
their binding specificities. Those vesicles with more promiscuous docking capabilities are able to associate with
the improper domain upon microtubule disruption (e.g.,
apically targeted vesicles) resulting in apparent missorting. Alternatively, the mistargeting may reflect normal
basolateral delivery of these PM proteins and the subsequent need for microtubules to facilitate transport to the
apical PM domain.
The microtubule-based motor molecule that has received the most attention as a possible regulator of transcytosis is cytoplasmic dynein. In vitro analysis indicated
that this megadalton, multisubunit molecule translocates
vesicles in an ATP-dependent manner toward microtubule minus ends and that its activity is enhanced by
another megadalton, multisubunit molecule, dynactin
(279, 373). Since the microtubule minus ends are anchored in the apical PM in many epithelial cells, it is
thought that dynein mediates delivery of vesicles from the
basolateral to the apical cell surface. Although this is an
attractive proposal, the evidence confirming it is only
circumstantial and largely stems from the effects of microtubule disruption on transcytosis. Additional evidence
comes from the observation that transport in this direction is NEM sensitive. Although NEM sensitivity is a litmus test for the involvement of NSF in transport, this
alkylating agent is also a potent inhibitor of dynein ATPase activity at similar concentrations (93).
Despite the absence of direct evidence that dynein
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TRANSCYTOSIS
Physiol Rev • VOL
ing at the basolateral surface and rac1 at the apical PM
(270, 315). In particular, basolateral-to-apical transcytosing IgA accumulated in basolateral early endosomes in
MDCK cells expressing inactivated forms of rhoA,
whereas it accumulated in apical early endosomes in cells
expressing dominant negative forms of myosin Vb or a
constitutively activated form of rac1 (270, 304, 315). The
mechanisms by which these motors, GTPases, and actin
regulate membrane transport in polarized epithelial cells
are not yet known but are the subject of a recent review (16).
C. Lipids and Transcytosis
The identification of proteins as important regulators
of membrane transport is widely accepted and nearly
indisputable. In the last several years, it has become more
accepted that lipids also play significant roles in membrane transport. In particular, phosphoinositides, PC,
cholesterol, and glycosphingolipids have been shown to
be important players. Here we will focus on the roles of
phosphoinositide 3-phosphate [PI(3)P], cholesterol, and
glycosphingolipids in polarized membrane transport.
Other lipids and their modifying enzymes have been the
subject of many recent reviews (99, 242, 353).
1. PI(3)P
In the past several years, the role PI(3)P lipids play in
regulating membrane transport has received considerable
attention. This interest arose from early studies examining the effects of the selective phosphoinositide 3-kinase
(PI 3-kinase) inhibitors wortmannin and LY294002 on
membrane trafficking (98, 524, 584). To date, many transport pathways, including transcytosis, have been shown
to be wortmannin and/or LY294002 sensitive. In MDCK
cells, wortmannin treatment impaired basolateral to apical dIgA transcytosis (78, 212). Likewise, both wortmannin and LY294002 disrupted transcytosis of pIgA-R and
three newly synthesized resident apical PM proteins to
the WIF-B apical domain (582) while wortmannin perfusion in isolated rat livers decreased the biliary release of
basolaterally internalized HRP (156). Both agents were
also observed to impair transcytosis in both directions of
ricin in FRT cells and of neonatal FcR in IMCD cells (212,
370). Although the transcytosing proteins in treated
WIF-B cells eventually reached their final destination (the
apical PM), they transiently accumulated in basolateral
early endosomes, indicating a block early in the transcytotic pathway (583).
Mammalian cells encode at least three different
classes of PI 3-kinase isoforms (23, 162). Class I includes
the p85/p110 heterodimeric kinases which consist of an
110-kDa catalytic subunit associated with a regulatory
85-kDa subunit. The other member is PI 3-kinase-␥, whose
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addition of the actin disrupting agent cytochalasin D impaired internalization only from the apical domain (197,
261, 528). Clathrin-coated pits accumulated at the apical
PM in each cell type, suggesting a block in clathrin-mediated internalization. In treated Caco-2 cells, apical internalization of folate was also decreased, implying that
caveolae-mediated internalization was altered, and in
MDCK cells, apical uptake of Lucifer yellow, which is
internalized via noncoated vesicles, was decreased (197,
261). Thus actin may be important in many modes of
apical internalization. However, there are exceptions to
this generalization, as it was observed that the receptormediated internalization of ligand from the basolateral
domain of hepatocytes was impaired by cytochalasin B
treatment (280). Furthermore, treatment of Caco-2 cells
with latrunculin B, another actin-disrupting agent, misdirected basolaterally internalized Tf-R and LDL-R into the
apically directed transcytotic pathway (136). Limited evidence from studies in MDCK cells suggests that actin is
also involved in facilitating steps in postendocytic transcytotic trafficking. In the presence of cytochalasin D, the
delivery of transcytosing dIgA from the basolateral early
endosome to the apical recycling endosome was impaired
45% (346). Interestingly, dIgA transcytosis was completely
blocked when both cytochalasin D and nocodazole were
added, suggesting that microfilaments and microtubules
work in concert to facilitate transport to the apical cell
surface (16, 346).
In support of the evidence indicating a role for actin
in regulating membrane dynamics, recent studies have
also implicated specific actin-based motors as important
players in polarized vesicle trafficking. In particular, many
classes of myosin motors have been localized to the subcortical actin network in many different epithelial cell
types. The single-headed, short-tailed myosin I isoform
has been localized to the apical brush border of intestinal
(440) and kidney cells (94). They have also been found in
association with zymogen granules at the apical aspect of
pancreatic acinar cells (452) and in hepatocytes (25, 95).
Also present at the intestinal brush border, although
much less abundant, are the related myosin isoforms V
and VI (218). Myosin VI has also been placed at the apical
brush border of the proximal tubule cell line LLC-PK1
(215), whereas myosin Va has been also localized to subapical structures in polarized hepatic WIF-B cells (321)
and myosin Vb at the apical recycling endosome in MDCK
cells.
Functional studies on two different myosin isoforms
have also placed them as potential regulators of transcytosis. Brush-border myosin 1 (BBM1) plays a role in postendocytic traffic at the basolateral pole, whereas myosin
Vb is functioning at the apical pole (136, 304, 315). RhoA
and rac1, two small GTPases that regulate actin cytoskeletal dynamics, also have also been implicated as regulators of polarized membrane transport with rhoA function-
912
PAMELA L. TUMA AND ANN L. HUBBARD
Physiol Rev • VOL
cytosis as did anti-Vps34p injection (583). However, increased basolateral surface staining of the transcytosing
markers was observed with no corresponding intracellular accumulations, suggesting that Vps34p and p110␣ act
at separate steps of the pathway, with p110␣ possibly
acting at internalization. Together, these results lead to a
number of questions. Are the apical and basolateral pathways in other polarized epithelial cells also differentially
regulated by PI(3P)? Are the functions of p110␣ and
Vps34p in basolateral-to-apical transcytosis conserved
among other epithelial cells? What PI(3)P-binding proteins are required for membrane transport at the distinct
steps?
2. Cholesterol and glycosphingolipids
Glycosphingolipids and cholesterol are enriched in
cell surface membranes in all eukaryotic cells. In polarized epithelial cells, the apical surface is even further
enriched for these lipid species (158, 281, 295, 597). One
possible function of cholesterol and glycosphingolipids is
to impart structural rigidity and decreased permeability to
the apical domain, which in turn protects the cell against
the harsh external environment it faces (e.g., the detergent-like bile or high acidity) (295). The differences in
these environments would therefore dictate the lipid compositions required for appropriate protection and function in different epithelial cell types. The intrinsic properties of glycosphingolipids and cholesterol promote their
assembly into specialized membrane domains called
“rafts” (63, 214). Within these cell surface domains are
selected proteins that are recruited based on their biophysical properties. In particular, GPI-anchored proteins
that are predominantly expressed at the apical surfaces of
epithelial cells localize to rafts.
These observations in combination with studies performed mainly in MDCK cells have led to the “raft” hypothesis for protein sorting. According to this hypothesis,
rafts form in the biosynthetic pathway where they recruit
apically destined proteins (especially GPI-linked proteins); the rafts with their recruited cargo are then transported directly to the apical domain in vesicles. There is
considerable experimental evidence to support this hypothesis. For example, GPI anchors have been shown to
be sufficient to target proteins to the apical domain (62,
325). Furthermore, rafts have been isolated based on their
insolubility in nonionic detergents (especially Triton
X-100) at 4°C, GPI-anchored proteins copurify with them
(297), and cholesterol-depleting drugs and sphingolipid
synthesis inhibitors disrupt delivery of apical PM residents (252, 297). Recent work has also suggested that
caveolins and caveolae-like vesicles are important for PM
delivery of GPI-anchored proteins. In caveolin-1 or caveolin-3 knock-out mice, GPI-anchored proteins were re-
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catalytic activity is regulated by the ␤␥-subunits of heterotrimeric G proteins (80, 162, 591a). Class II PI 3-kinases include higher molecular weight kinases that contain C2 domains and class III kinases share the highest
sequence similarity with the sole isoform identified in
yeast, Vps34p (224, 604). The last kinase is also under the
control of a regulatory subunit, p150 (in mammalian cells)
or Vps15p (in yeast) (225, 427). All mammalian PI 3-kinase
isoforms are sensitive to wortmannin and LY294002 (class
II kinases at higher concentrations), which has led to
ambiguity in distinguishing the roles that specific PI 3-kinases play in membrane transport. However, more recent
studies that examined the effects of microinjection of
specific inhibitory reagents on membrane transport have
helped to begin assigning specific roles to specific kinase
(529).
Using these inhibitory reagents in polarized hepatic
cells, we found that specific inhibition of the class III PI
3-kinase Vps34p led to the formation of prelysosomal
vacuoles containing endocytosed resident apical PM proteins and to the transient accumulation of transcytosing
apical proteins in basolateral early endosomes (583).
These results indicate that the lipid product of Vps34p,
PI(3)P, regulates the two endocytic pathways differentially, at an early endosomal stage from the basolateral
surface and from prelysosomes to lysosomes from the
apical surface. The current model of PI(3)P’s role in endocytosis invokes recruitment of Vps34p/p150 by activated rab5 to the sites of endosome-endosome fusion and
local production of PI(3)P (98, 559, 625). The PI(3)Pbinding protein early endosomal antigen 1 (EEA1) is recruited to these sites where PI(3)P and rab5 binding
stabilize its membrane association. The stabilized EEA1
molecules then form oligomers that coordinate the formation of a large vesicle docking site also containing NSF
and syntaxin13, all of which drive endosome fusion. Thus,
in nonpolarized cells, when PI(3)P lipids were depleted by
wortmannin, EEA1 association with early endosomes was
lost and the subsequent events were affected. Likewise,
we found that EEA1 dissociated from basolateral early
endosomes in treated WIF-B cells; concomitantly, we observed delayed basolateral to apical transcytosis. All of
these results are consistent with the current model. In
contrast, the block we observed at a late endocytic step in
the apical pathway is not consistent with the existing
model (583). Disruption of EEA1 function in the fusion of
early endosomes arising from the apical surface should
have blocked an earlier step in the pathway. However, a
subpopulation of EEA1 near the apical surface remained
membrane-bound under PI(3)P-depleting conditions, suggesting that the protein remained active and allowed progression of endocytosed proteins along the apical route
thereby revealing a block downstream.
Injection of p110␣ inhibitory antibodies into WIF-B
cells was also found to impair basolateral to apical trans-
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TRANSCYTOSIS
Physiol Rev • VOL
lipids at the apical domain, caveolae have only been observed at the basolateral domain of MDCK cells and in
kidney epithelial cells in situ (57, 435, 498). Like endothelial cells, MDCK caveolae formation is dependent on cholesterol levels (205, 267, 511). In both cases, no morphologically definable caveolae were observed in cells treated
with cholesterol-depleting drugs such as filipin or cyclodextrin. In endothelial cells, the cholesterol-dependent
loss of caveolae corresponded to decreases in albumin
transcytosis both in vitro and in vivo (267, 511). When the
drugs were withdrawn and cholesterol synthesis stimulated by the addition of mevalonate in MDCK cells, or the
addition of 10 –20% serum in endothelial cells, the caveolae reformed (205, 511). FRT and Caco-2 cells express
little to no detectable caveolin, and no caveolae have been
observed (322, 603). However, when caveolin-1 was overexpressed in these cells, caveolae were observed in both
domains in FRT cells and only at the basolateral domain
of Caco-2 cells (322, 603). Interestingly, the formation of
caveolae in FRT cells did not promote apical sorting of
GPI-anchored proteins or their sorting into rafts (322),
suggesting that rafts are not responsible for apical targeting in these cells. Alternatively, other components may be
lacking in FRT cells that are necessary for sorting into
rafts and/or subsequent apical delivery.
Our challenge is to begin carefully examining the
detergent solubility properties of multiple endogenous
apical PM protein types (e.g., GPI-linked, single transmembrane, polytopic) during their entire life cycles
within one cell type to determine the role of rafts in
transcytosis. The detergents used should not be limited to
Triton X-100, as it has been recently reported that in PC12
cells, subpopulations of rafts exist with different solubility properties (482). The effects of both cholesterol and
glycosphingolipid disrupters on transcytosis should be
directly tested. Also, examination of purified organellar
intermediates for raft components and protein insolubility properties may help clarify at which step(s) rafts are
required. Such a strategy has shown that recycling endosomes (immunoisolated with anti-Tf-R antibodies) are enriched for raft components including glycosphingolipids,
cholesterol, caveolin, and another raft-associated protein,
flotillin (167). Such careful and consistent experimentation will more clearly determine the role of rafts in apical
vesicle targeting. Recent evidence also suggests that the
PM-associated t-SNAREs are organized in cholesterol-dependent surface domains in nonpolarized cells (87, 303).
Are these domains required for vesicle delivery to the
apical PM?
D. Perturbations of Transcytosis
The use of perturbants has long been a way to begin
dissecting the mechanisms and molecules involved in
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tained at the TGN in mouse embryo fibroblasts or muscle
tissue, respectively (551).
However, there are many observations that are inconsistent with the raft hypothesis of sorting (612). In
FRT cells, both glycosphingolipids and GPI-anchored proteins are sorted to the basolateral domain, whereas in
certain MDCK strains, they are evenly distributed between the two domains. However, in both cases, other
apical PM proteins are sorted properly to the apical surface. In hepatocytes, GPI-anchored proteins, such as 5⬘nucleotidase, are first transported to the basolateral domain before apical delivery (499). Furthermore, many
nonapical proteins have also been detected in purified raft
fractions.
Despite the debate, the question still remains. Do
rafts sort apically destined proteins in the transcytotic
pathway? If so, are rafts present at the basolateral domain
or in other transcytotic intermediates? Two approaches
have been taken to begin answering these questions. First
is examining whether transcytosing proteins are present
in detergent-insoluble fractions and second is whether
they are internalized from the cell surface via caveolae,
specialized raft domains. So far, the first approach has
yielded conflicting results from studies performed in enterocytes as well as FRT and MDCK cells (210, 493). In
polarized enterocytes from mouse intestinal explants, a
significant proportion of basolateral to apical transcytosing IgA (secreted from neighboring mucosal plasma cells)
was found in detergent-insoluble rafts (210). Because IgA
is internalized and delivered to the apical surface via the
pIgA-R, the data imply that the receptor must also be raft
associated. Accordingly, ⬃50% of the pIgA-R was recovered in detergent-insoluble fractions (210). This result
contradicts that reported for pIgA-R in MDCK and FRT
cells; pIgA-R was not found in Triton X-100-insoluble
fractions at any point during its life cycle (493). The
reasons for these opposing observations are likely not due
to differences in raft preparation, since the methods used
were very similar. Instead, the differences may be related
to the intrinsic differences between cell types, between in
vitro versus in vivo systems, or between endogenously (in
enterocytes) or exogenously (in FRT and MDCK cells)
expressed molecules. None of these possibilities has yet
been well explored.
The second approach to determine whether rafts sort
apically destined proteins in the transcytotic pathway has
focused on examining interactions of transcytosing proteins with caveolae. Because not all rafts are associated
with caveolin, caveolae have more recently been classified as specialized rafts. Much of what we know about
caveolae in polarized epithelial cells comes from studies
in endothelial cells where these structures are highly
abundant (see sect. IV). Otherwise, caveolae have been
examined in only a limited number of polarized cells.
Despite the enrichment of cholesterol and glycosphingo-
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PAMELA L. TUMA AND ANN L. HUBBARD
regulating complex cellular processes. Chemical perturbants are commonly used and have provided insight into
understanding vesicle transport in mammalian cells. Of
particular note are alkylating agents (e.g., NEM), drugs
that specifically disrupt the cytoskeletal systems of the
cell and specific lipid kinase inhibitors which were discussed in section V, A–C. In this section, we focus on the
perturbation of acute regulation of vesicle transport by
agents that alter the functions of heterotrimeric G proteins, intracellular calcium (Cai) homeostasis, protein
kinase A (PKA), or protein kinase C (PKC) activity
(Table 6).
Accumulating evidence supports a role for heterotrimeric G proteins in regulating vesicle transport in both
the endocytic and exocytic pathways (4, 41, 42, 220, 412,
445, 561). Examination of the effects of various agents on
epithelial cells, especially MDCK cells, have indicated that
transcytosis is also regulated by G proteins. The first clues
came from treating cells with nonselective G protein activators, guanosine 5⬘-O-(3-thiotriphosphate) (GTP␥S)
and AlF. In both cases, transcytosis to the apical, but not
basolateral, domain was slightly enhanced (42). Addition
of specific G protein ADP-ribosylating toxins (cholera or
pertussis toxins) further indicated that transport to the
apical domain (from both the TGN and via transcytosis) is
regulated by the Gs ␣-subunit (31, 42, 211). Mastoparan, (a
Gi-activating peptide) had no effect on pIgA-R apical delivery while anti-Gs ␣-antibodies were slightly inhibitory,
confirming a role for Gs in transcytosis to the apical
domain (42). In intact cells, the overexpression of the
wild-type or constitutively active Gs ␣-subunit led to a
modest increase in transcytosis (211). Interestingly, the
addition of either the Gs ␣- or ␤␥-subunits to an in vitro
system led to a small increase in the formation of transcytotic vesicles (42). However, it is not known whether
the ␤␥-subunits were acting to inhibit another G protein
that negatively regulates transcytosis or in concert with
the ␣-subunit.
One well-studied result of Gs activation is the activation of adenylyl cyclase and the subsequent increased
production of cAMP. PKA is then activated by the increased cAMP levels, which puts in motion numerous
(but not well-defined) cellular processes. To determine
whether this cascade of events is involved in vesicle
transport, another handful of chemical perturbants has
been useful. In particular, forskolin (a direct activator of
adenylyl cyclase) has been shown to enhance transcytosis
to the apical, but not basolateral, domain in MDCK cells
(211). This effect was also seen by the addition of exogenous cAMP to both MDCK cells and intact rat hepatocytes (211, 217). A PKA inhibitor, H-89, produced the
opposite effect, consistent with a role for PKA in regulatPhysiol Rev • VOL
2. Calcium, calmodulin, and PKC
The use of another set of pharmacological agents has
indicated that vesicle trafficking is also acutely regulated
by changes in Cai levels. Thapsigargin, a selective inhibitor of sarco/endoplasmic reticulum Ca2⫹-ATPases, enhanced transcytosis to the apical domain in MDCK cells,
whereas BAPTA, a Ca2⫹-chelating compound, inhibited it
(79). Neither agent altered transcytosis in the opposite
direction. The mechanism whereby Cai fluxes are manifested in changes in vesicle transport is not known, but
likely includes alterations in Ca2⫹-dependent enzyme activities. This has been substantiated by studies examining
the effects of the calmodulin antagonists W-7, W-13, and
trifluoperazine in MDCK cells (18, 244, 329). All three
agents significantly impaired basolateral to apical transcytosis of dIgA while transcytosis in the same direction of
the fluid-phase marker ricin was enhanced. In both cases,
endocytosis from the basolateral surface was unchanged,
suggesting that the agents were acting later in the pathway. Consistent with this is the finding that dIgA accumulated in large endosomal structures located in the apical
region of cells treated with W-13 (18). Interestingly, apical
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1. Heterotrimeric G proteins and PKA
ing transcytosis (211). Some evidence suggests that G
protein/PKA regulates transcytosis from apical endosomal compartments to the apical PM (31). Interestingly,
this is also the site(s) at which at least nine small-molecular-weight GTP-binding proteins have been proposed to
function, further exposing the complexity of vesicle transport events in this region of a polarized epithelial cell.
Trimeric G proteins are also important regulators of
endothelial transcytosis, but in this case, Gi has been
examined most extensively. In polarized endothelial cells
in vitro, addition of pertussis toxin (a Gi inhibitor) or
expression of a dominant negative Gi␣ peptide inhibited
apical to basolateral albumin transcytosis mediated by
activated gp60 (379). Previously, treatment of endothelial
cells with tyrosine kinase inhibitors suggested that a srcmediated signaling pathway regulated transcytosis (Ref.
576 and discussion in sect. IVA3); thus Minshall and colleagues examined whether Gi and src signaling were coupled. Consistent with this hypothesis, overexpression of
dominant negative src prevented the association of Gi␣
with caveolin (caveolae?) in caveolin-1-overexpressing
cells. Furthermore, dominant negative src also inhibited
albumin transcytosis. From these results, the authors suggested that gp60 activation recruits Gi to caveolae that in
turn sets off a src-mediated signaling cascade that activates transcytosis (379). This situation is somewhat analogous to the activation of pIgA-R transcytosis by ligand
binding where another tyrosine kinase, p62yes, is involved
(341). Thus tyrosine phosphorylation may be a common
mechanism for regulating activated transcytotic pathways.
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TRANSCYTOSIS
TABLE
6. Effects of pharmacological agents on transcytosis
Agent
Mode of Action
BL-A
A-BL
Epithelial Cell Types
Examined
Comments
Reference No.
SNAREs
NEM
Alkylates sulfhydryls
N/D
MDCK and endothelial
cells, hepatocytes
MDCK cells
Bacterial
toxins
Proteolytically cleaves v- and
t-SNAREs
2
N/D
Wortmannin
PI 3-kinase inhibitor
2
⫺/2
LY294002
PI 3-kinase inhibitor
2
2
Filipin
Binds and removes cholesterol
from PM
2
N/D
Endothelial cells
Nocodazole
Microtubule depolymerizer
2
⫺/2
Colchicine
UV/vanadate
Inhibits tubulin polymerization
ATPase inhibitor
2
2
Cytochalasin D
Actin depolymerizer
2
Very nonselective and will inhibit multiple
ATPases
Each toxin is highly selective for its target
17, 253, 505, 564
17, 26, 27, 314
Lipids
MDCK, FRT, IMCD and
WIF-B cells,
hepatocytes
IMCD and WIF-B cells
Class I and III kinases inhibited at low
concentrations (nM), all classes inhibited at
high concentrations (␮M), irreversible
All classes inhibited at high concentrations
(␮M), reversible
Specific for unesterified cholesterol, reversible
78, 156, 212, 370, 582
Many cell types
Specific, cell permeant, reversible
⫺
⫺
Many cell types
MDCK cells
⫺
Many cell types
Specific, irreversible
Not readily cell permeant, potent inhibitor of
many ATPases, especially ion pump ATPases
Specific, cell permeant, reversible
56, 110, 137, 222, 246,
346, 362, 370, 453,
548
13, 390
298
370, 582
509
197, 261, 346, 528
G proteins and PKA
GTP␥S
AIF
Cholera toxin
G protein activator
G protein activator
ADP-ribosylates and activates
Gs ␣-subunit
1
1
1/2
N/D
N/D
N/D
MDCK cells
MDCK cells
MDCK cells
Pertussis toxin
ADP-ribosylates and activates
Gi, Go, and Gt ␣-subunits
⫺
N/D
MDCK cells
Mastoparan
Gi, Go, and Gt activator
⫺
N/D
MDCK cells
Forskolin
cAMP
Adenylyl cyclase activator
PKA activator
⫺/1
⫺/1
N/D
N/D
H-89
PKA inhibitor
2
⫺
MDCK cells
MDCK cells and
hepatocytes
MDCK cells
Activates all GTPases
Activates all heterotrimeric G proteins
Holotoxin must bind GM1 to be internalized, A
subunit (with ADP ribosylation activity) is not
cell permeant
Holotoxin must bind cell surface to be
internalized, A protomer (with ADP
ribosylation activity) is not cell permeant
Cell permeant, also inhibits calmodulin and
activates phospholipase A2
Specific
Not readily cell permeant
Potent, selective
41
41
31, 41, 211, 379
31, 42, 211
42
211
211, 217
211
Calcium, CaM, and PKC
Thapsigargin
Ca2⫹-ATPase inhibitor
1
⫺
MDCK cells
BAPTA
Calcium chelator
2
⫺
MDCK cells
W-7
W-13
Trifluoperazine
CaM antagonist
CaM antagonist
CaM antagonist
1/2
2
1
2/⫺
N/D
2/⫺
FRT and MDCK cells
MDCK cells
FRT and MDCK cells
KN-62
PMA
H-7
CaM kinase II inhibitor
PKC activator
Serine/threonine kinase
inhibitor
⫺
1
⫺
⫺
N/D
N/D
MDCK cells
MDCK cells
MDCK cells
Herbimycin
Tyrosine kinase inhibitor
2
N/D
Genestein
Tyrosine kinase inhibitor
2
N/D
PP1
Tyrosine kinase inhibitor
2
N/D
Endothelial and MDCK
cells
MDCK and endothelial
cells
MDCK cells
Potent and specific for sarco endoplasmic
reticulum Ca2⫹-ATPases
105-fold greater affinity for Ca2⫹ than Mg2⫹;
available in a cell permeant form
Cell permeant
Cell permeant
Elevates intracellular Ca levels at low
concentrations, antagonizes CaM at higher
concentrations, cell permeant
Potent, selective
Cell permeant, potent
Nonselective, cell permeant
79
79
244, 329, 349
18, 349
329
329
79
18
Tyrosine kinases
Highly selective for tyrosine kinases, cell
permeant, irreversible
Nonselective
Selective for Src tyrosine kinases
340, 576
340, 576
340
1, Increases; 2, decreases; ⫺, no change; CaM, calmodulin; IMCD, inner medullary collecting duct; N/D, not determined; NEM, Nethylmaleimide; GTP␥S, guanosine 5⬘-O-(3-thiotriphosphate); PKC, protein kinase C; PKA, protein kinase A; PI, phosphatidylinositol.
to basolateral transcytosis of ricin was not altered by
these agents, yet an increase in its endocytosis from the
apical domain was observed (329). However, megalinmediated transcytosis of thyroglobulin in the same direction was inhibited in thyroid cells (FRTL-5) treated with
W-7 and trifluoperazine (349). Thus CaM regulation is
Physiol Rev • VOL
important for many steps in transcytosis. The challenge is
to pinpoint the specific calmodulin-dependent enzymes
that are functioning at these transport steps to understand
the differential effects of these agents.
PKC has also received attention as a possible regulator
of transcytosis based both on the effects of changing Cai
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Cytoskeleton
916
PAMELA L. TUMA AND ANN L. HUBBARD
3. Possible mechanisms
In all the studies cited above, it is important to point
out that in many cases, the inhibitory and stimulatory
effects of transfection or addition of pharmacological
agents on PM targeting were small. Does the size of the
response in vitro reflect loss of normal regulation that
would be observed in vivo or does it represent fine-tuning
that may be critical for proper organ function? If it is the
latter, what appears to be a minimal change in vitro may
have a large impact on the overall homeostatic balance of
the organism. Thus physiological studies are needed. Specifically, our challenge is to identify the molecules in
membrane transport that are regulated by phosphorylation or calmodulin/Ca2⫹ binding. Few if any direct links
have yet been established, but there are some examples of
where the modifications of molecules implicated in transcytosis correlate with their proposed functions.
Many of the SNARE molecules are phosphorylated in
vitro by purified kinases, and the modification alters their
binding properties. In particular, ␣-SNAP is a substrate for
PKA and when phosphorylated, its ability to bind the core
docking and fusion complex was decreased 10-fold (230).
In vitro, syntaxin 4 was shown to be phosphorylated by
PKA, casein kinase II (CKII), and PKC, and this phosphorylation disrupted its binding to SNAP23 or SNAP25 (92,
159, 474). When syntaxin 4 was used as bait in a yeast
two-hybrid screen, a novel SNARE kinase (SNAK) was
identified, but surprisingly, SNAP-23 was overwhelmingly
its preferred substrate in vitro and in vivo (73). SNAKphosphorylated SNAP-23 was not associated with the ternary complex, whereas phosphorylation of syntaxin 1 by
CKII enhanced t-SNARE’s association with SNAP-25
(154). Interestingly, by using phosphospecific antibodies
to stain neurons, it was found that the phosphorylated
Physiol Rev • VOL
form of syntaxin 1 was localized to discrete regions along
the axonal PM that did not colocalize with synaptic vesicles (154). Another SNARE hypothesis molecule,
Munc18 –1, is a substrate for PKC and cyclin-dependent
kinase 5, and phosphorylation in this case inhibited binding to syntaxin 1 (123a, 164, 527).
Several rabs and rab effector proteins have also been
shown to be phosphorylated. In vitro, GDI phosphorylation is mediated by PKA (555). In vivo, the phosphorylated
GDI associated to the cytosolic form of rab5, while the
unphosphorylated GDI was bound to the membrane-associated rab5. These data suggest that the cycling of rab
proteins between donor and acceptor membranes is also
a regulated process. The rab effector protein rabphilin 3A
is phosphorylated by PKA in vitro (411). Another rab
effector, rab8ip, is a serine/threonine protein kinase itself
(GC kinase) that is activated by the stress response in
lymphocytes (466). The phosphorylation state of rip11, a
rab11 effector, may regulate its membrane binding properties. In polarized MDCK cells, conditions that decrease
rip11 phosphorylation (e.g., staurosporine treatment) enhanced its binding to membranes (458). Caveolin, the
major structural protein of caveolae, has been shown to
be tyrosine phosphorylated in endothelial cells under conditions where transcytosis was stimulated (191, 318, 358).
Both kinesin and cytoplasmic dynein are phosphorylated
in vitro and in vivo, and this modification has been correlated with their ability to transport vesicles (including
transcytotic vesicles?) along microtubules (320, 486, 494).
The light chain of the actin based myosin I motor proteins
is calmodulin, which is thought to regulate motor activity
(449). Annexin binding to membrane lipids, and by extension, ability to promote intermembrane associations, is
dependent on Ca2⫹ (103).
E. Transcytosis Versus Direct PM Delivery
Transcytosis is only one pathway that molecules take
to a specific PM domain. Both newly synthesized PM
proteins and secreted molecules can also be delivered
directly from the TGN to either PM domain. How different
are the mechanisms regulating vesicle transport along
these pathways? As expected, transport directly from the
TGN to either the apical or basolateral domains is regulated differently. In particular, differences in the involvement of SNARE molecules were observed in permeabilized MDCK cells (17, 253, 332). Addition of anti-NSF
antibodies, NEM, mutant NSF, rab-GDI, or tetanus and
botulinum F neurotoxins all inhibited basolateral targeting of VSV-G protein, whereas targeting from the TGN to
the apical domain of HA was not changed (17, 253). This
implies that basolateral targeting requires NSF, rab proteins, and VAMP 2. Conversely, syntaxin 3 overexpression
or anti-syntaxin 3 antibodies inhibited only apical delivery
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levels and on the use of phorbol esters, potent PKC activators. When one such phorbol ester, phorbol 12-myristate
13-acetate (PMA), was applied to MDCK cells, both apical
recycling and basolateral to apical transcytosis of dIgA and
transferrin were enhanced, suggesting PKC was acting at an
apical recycling compartment (79). PMA treatment also led
to the membrane recruitment of ␣ and ⑀ PKC isoforms (79).
Interestingly, dIgA binding to its receptor (conditions that
activate its own transcytosis) also activated PKC-⑀, which
led to increased levels of inositol 1,4,5-trisphosphate and
Cai, the latter of which stimulated transcytosis (79, 548). The
rise in Cai is likely mediated via inositol 1,4,5-trisphosphatesensitive intracellular stores; thus ligand binding initiates a
signal that is propagated across the cell independent of the
ligand-receptor complex itself (340). Paradoxically, treatment of MDCK cells with H-7, a specific PKC inhibitor, did
not inhibit dIgA or ricin transcytosis in MDCK cells (18, 329).
At present, there is no good explanation for these disparate
results.
917
TRANSCYTOSIS
Physiol Rev • VOL
cific protein sorting. This is further suggested by the
finding that yet another MAL family member, BENE, is
expressed in endothelial cells where it is associated with
caveolae (119).
VI. CONCLUSION
We have learned a lot about transcytosis since its
existence was first postulated over 50 years ago. We have
identified cargo, uncovered pathways, and determined
possible mechanisms. Nonetheless, many things remain
mysterious. How does the cell discriminate between
cargo destined for transcytosis versus degradation in lysosomes? In peripheral endothelial cells, it appears that
separate entry points dictate different fates; internalization via coated pits sends cargo to the endocytic pathway,
whereas caveolae-mediated internalization ensures a
transcytotic fate. However, we need further study to make
a definitive conclusion on this point. In other epithelial
cells, the transcytotic pathway is a branch of the endocytic pathway. Where and how is transcytotic cargo
sorted in these cells? Signals have been identified in the
pIgA-R cytoplasmic tail that prevent its degradation. How
are they recognized? Are they universal? In enterocytes,
megalin recycles to the PM from endosomes after internalization and dissociation from its transcytotic cargo,
whereas in thyroid, the same membrane receptor escorts
thyroglobulin across the cell. What signals navigate megalin along these different itineraries and how do cells discriminate between the possible fates? Another unsolved
problem is understanding how the cell determines what
should be transcytosed versus that which should be diverted for its own use. Are the acute regulatory mechanisms described in section VD3 important here?
How might transcytosis dysfunction or corruption
contribute to human disease? For example, do mistakes
in self-apportionment lead to vitamin B12 deficiency? Do
mistakes in cargo selection cause disease? In enterocytes,
the transcytosis of undigested food antigens to underlying
interstitial cells has been linked to food hypersensitivity
and allergies (630). The antigenic epitopes stimulate the
immune system leading to the production of cytokines
that ultimately result in the loss of enterocyte barrier
function and diarrhea. Similarly, certain pathogens co-opt
the transcytotic pathway of M cells for infection. Recent
evidence suggests that Streptococcus pneumoniae binds
unoccupied, uncleaved pIgA-R that has been transcytosed
to the apical surfaces of nasopharyngeal epithelial cells.
The receptor-pneumonia complex is thought to be apically internalized and transcytosed to the basolateral surface where the pathogen is released.
Finally, how is transcytosis used or adapted in development? This is a huge unexplored area where there will
undoubtedly be surprises. Let us hope that the next 50
years will provide us with some exciting answers.
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(300, 332). Addition of ␣-SNAP antibodies and treatment
of cells with botulinum E inhibited transport to both
domains (17, 253), whereas TI-VAMP antibodies inhibited
apical transport (the effects of theses antibodies on basolateral targeting were not examined) (300). Taken together, targeting to both domains requires SNARE molecules, but in different combinations. Surprisingly, apical
targeting is NSF independent, suggesting the involvement
of an as yet unidentified homolog. Microtubule and actin
filament disruption also has differential effects on direct
delivery to either domain in MDCK cells; apical delivery is
inhibited, whereas, in most cases, basolateral is not (56,
137, 190, 362, 420, 598). In addition, PKA, PKC, and calmodulin-mediated mechanisms appear to acutely regulate
delivery mainly to the apical domain (79, 329, 447).
How different are the targeting mechanisms regulating transcytosis? This question has so far been best addressed in studies performed in MDCK cells. Unlike TGN
to apical delivery of HA-containing vesicles, pIgA-R-mediated transcytosis appears to require NSF activity (17,
253). Furthermore, syntaxin 3 (required for direct apical
targeting) is not involved in mediating transcytosis of IgA
(332). SNAP-23, on the other hand, is involved in both
basolateral to apical transcytosis, and direct apical and
basolateral targeting (332). Also, both direct and transcytotic delivery to the basolateral domain does not require
microtubules (see sect. VB). Based on the effects of pertussis toxin on MDCK cells, transport from the TGN to the
basolateral PM was found to involve the Gi␣ subunit of
heterotrimeric G proteins, whereas transcytosis to this
domain was not (31, 211, 446). From these results (and
others) it is clear that the mechanisms cells use to regulate cell surface delivery are complex and are dependent
on factors that are not yet understood.
Another interesting twist to apical PM sorting has
come from studies looking at the raft-associated protein
referred to as MAL. This 17-kDa tetra-spanning TMD protein, first identified in myelin and lymphocytes (hence
MAL), is also expressed in many epithelial cell types
where it is concentrated at the TGN (287). With the use of
an antisense approach, it was shown that MDCK cells
lacking MAL showed decreased specific apical delivery of
a single TMD apical protein surrogate, the influenza HA
the ectopic expression of human MAL rescued the defect
(459). Thus MAL has been implicated as an important
player in apical sorting. Interestingly, liver does not express this isoform of MAL, a finding consistent with the
absence of a direct apical delivery mechanism for the
single-TMD class of apical PM proteins in hepatocytes.
Recently, another MAL family member has been identified, MAL2, that is enriched in hepatic cells (120). In
HepG2 cells treated with antisense MAL2 oligonucleotides, transcytosis of pIgA was impaired from early endosomes to a juxta-apical compartment. Thus different MAL
isoforms may be responsible for specialized domain-spe-
918
PAMELA L. TUMA AND ANN L. HUBBARD
We thank R. Fuchs, L. Ghitescu, M. Lisanti, M. Molliver, M.
Neutra, S. C. Silverstein, and M. Wessling-Resnick for sharing
their expertise in areas of transcytosis less familiar to us.
Thanks also to the Editorial Board (Susan Hamilton) for their
patience during the long gestation period before seeing this
review.
Our research has been supported by National Institutes of
Health Grants P01-DK-44375, R01-GM-29185, NRSA-DK-09620,
and T32-DK-07632.
Present address of P. L. Tuma: Biology Department, The
Catholic University of America, Washington, DC 20064.
Address for reprint requests and other correspondence:
A. L. Hubbard, Hunterian 119, Dept. of Cell Biology, 725 N. Wolfe
St., Baltimore, MD 21205 (E-mail: [email protected]).
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