University of Groningen
Regulation of polarity development in hepatocytes
van der Wouden, Johanna
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CHAPTER 1
Membrane dynamics and the regulation of
epithelial cell polarity
Johanna M. van der Wouden, Olaf Maier, Sven C.D. van IJzendoorn, and Dick Hoekstra
Department of Membrane Cell Biology, Faculty of Medical Sciences,
University of Groningen, Groningen, the Netherlands
International Reviews of Cytology, 226:127-164 (2003)
Chapter 1
Abstract
Plasma membranes of epithelial cells consist of two domains, an apical
and a basolateral domain, the surfaces of which differ in composition.
The separation of these domains by a tight junction and the fact that
specific
transport
pathways
exist
for
intracellular
communication
between these domains and distinct intracellular compartments relevant
to cell polarity development, has triggered extensive research on issues
that focus on how polarity is generated and maintained. Apart from
proper assembly of tight junctions, their potential functioning as
landmark for the transport machinery, cell-cell adhesion is obviously
instrumental in barrier formation. In recent years, distinct endocytic
compartments, defined as subapical compartment or common endosome
were shown to play a prominent role in regulating membrane trafficking
to and from the polarized membrane domains. Sorting devices remain to
be determined but likely include distinct rab proteins, and evidence is
accumulating to indicate that signaling events may direct intracellular
membrane
transport,
intimately
involved
in
the
biogenesis
and
maintenance of polarized membrane domains and hence the development
of cell polarity.
8
Chapter 1
I. Introduction
Epithelial
cells
form
a
barrier
between
distinct
extracellular
environments, e.g. the blood and underlying tissues on the one hand and
a lumen on the other. For biogenesis and maintenance of such a barrier,
epithelial cells must regulate their cell shape and internal processes. A
major feature of the epithelial cell is that it exhibits a plasma membrane
polarity, i.e. the plasma membrane has two domains, an apical and
basolateral domain, which are separated by a tight junction. The
basolateral domain is faces the blood and underlying tissue and the
microvilli-covered apical domain is facing the lumen (e.g. the lumen of the
intestine, bile canaliculi and kidney tubules; figure 1).
Figure 1: Schematic representation of a polarized Hepatocyte. The apcical
bile canalicular membrane (BC) is separated by tight junctions (TJ) from the
basolateral membrane which is facing the blood and underlying tissues.
Because the molecular composition of either domain differs, consistent
with differences in functional properties and likely, mechanical stability,
intracellular transport from and towards the different membrane domains
must be highly regulated to secure that difference. Indeed, for that
purpose several transport pathways and sorting stations are operational
in epithelial cells, which are discussed in this chapter. Other important
parts of polarity biogenesis and maintenance are the extracellular
9
Chapter 1
environment and cell attachments, which are closely related to the
cytoskeleton. The cytoskeleton needs to be properly organized to provide
epithelial cell shape and to constitute tracks for vesicle movement.
Presumably, this complex composition of epithelial morphology is highly
regulated by signalling cascades to provide a proper barrier under each
condition. Disruption of the barrier function, due to malfunctioning or
disregulation of signalling cascades results often in carcinogenesis. On
the other hand, stimulation of fetal hepatocytes or metanephros with
interleukins such as Oncostatin M or Leukemia Inhibitory Factor,
respectively, result in epithelialization.
In this chapter, membrane polarity will be discussed with a focus on the
role of the cytoskeleton and tight junctions, signalling and intracellular
traffic in the development and regulation of epithelial cell polarity.
II. Structural organization of epithelial cell polarity
Because epithelial cells constitute a boundary between tissues and
extracellular interstitium, their plasma membranes face simultaneously
different environments, it is obvious that distinct functions require a
concentration at their site of action. To meet such a requirement,
appropriate barrier and transport functions of epithelia depend on the socalled polarized distribution of proteins and lipids in the plasma
membrane. To maintain such a polarized distribution, tight junctions
separate these two membrane domains. Overall cell architecture and its
organization represent fundamental aspects in the maintenance and
development of plasma membrane polarity. However, plasma membrane
polarity is often described in terms of specific intracellular transport
processes, which deliver and retrieve compounds so that the specificity of
either domain is generated and/or maintained.
10
Chapter 1
A. Cell-cell contacts
For epithelial cells to constitute a barrier, cells need to form cell-cell and
cell-substratum contacts. Important players in the formation and
maintenance of cell polarity are the cadherins, the adhesion receptors for
cell-cell adhesion, which assemble the adherens junction, present on
lateral membrane domains. They comprise a family of calcium-dependent
cell adhesion molecules. Cadherins not only function in cell-cell contact
but are also involved in the programming of cell differentiation and
polarization. For example, E-cadherin plays an important role in
maintaining the normal phenotype of epithelial cells, while N-cadherin is
needed to prevent epithelial to mesenchym conversion, as frequently
occurs in tumors with an inappropriate expression of N-cadherin (Kim et
al., 2000). The extracellular domain of E-cadherin is responsible for
calcium-dependent homophilic interactions with adjacent cells and the
cytoplasmic domain associates with proteins that belong to the catenin
family, i.e., α- catenin, β-catenin and γ-catenin (plakoglobin) are all
known to associate directly with the E-cadherin (Nathke et al., 1994). In
fact, catenins anchor E-cadherin to the cytoskeleton thereby allowing the
cells to engage in strong cell-cell interactions.
Abnormal expression of cadherins, such as in human carcinomas,
correlates with a dedifferentiated epithelial phenotype, invasion and
metastasis. More specifically, MDCK cells in which a mutated E-cadherin
is overexpressed display a reduced expression of endogenous cadherin,
which results in a decreased cell adhesion and they are more easily
dissociated from the cyst (Troxell et al., 2001). Thus, a loss of cadherin
does not induce transformation, but it promotes the development of an
invasive, non-polarized phenotype, often observed in carcinogenesis.
Further details concerning the association of E-cadherin with the
cytoskeleton will be discussed below.
11
Chapter 1
B. Tight junctional complex
The basolateral and apical plasma membrane domains are separated by a
tight junction (TJ), which is in fact a multicomponent protein complex.
TJs not only create a primary barrier to prevent paracellular transport of
solutes, but they also restrict the lateral diffusion of apical and
basolateral proteins and lipids in the outer leaflet of the plasma
membrane. Inner leaflet lipids appear to be capable of randomizing over
the entire inner leaflet, suggesting that an effective barrier exists for the
outer leaflet only. Occludin, an integral transmembrane protein, was the
first protein identified in the TJ, where it functions as the actual seal of
the junction. To localize to and function at the TJ, the protein requires to
be phosphorylated
(Wong, 1997). Detailed insight on the assembly of
occludin into the TJ complex has been obtained from a wound healingmodel, which relies on the establishment of cell polarity in mouse
epithelia. In this system, Ando-Akatsuka et al. (1999) showed that
occludin first associates with E-cadherin and ZO-1, another TJ protein,
at cell-cell contacts. Subsequently, occludin and ZO-1 separate from Ecadherin; the latter forms the adherens junction whereas ZO-1 and
occludin localize at the TJ, occludin being in its tyrosine phosphorylated
state, as a result of phosphorylation by nonreceptor tyrosine kinase c-Yes
(Chen et al., 2002).
ZO-1 is a member of the membrane-associated guanylate kinase-like
homologues (MAGUK) family, which includes ZO-2 and ZO-3, both of
which also localize to the TJ. MAGUKs contain several PDZ domains, one
SH3 domain and a guanylate kinase like domain. Not only occludin binds
to ZOs, but the actin cytoskeleton (see below) and proteins belonging to
the claudin-family also bind (via their C-terminus) to ZO-1/ZO-2/ZO-3
(Itoh, et al., 1999). Partners for the SH3 domain of ZO-1 are ZONAB-A
and -B as Y-box transcription factors, which are located at the tight
junction (Balda and Matter, 2000). ZONAB per se is also present in the
nucleus where it regulates gene expression of ErbB2 and p27kip1, which is
involved
in
cell
cycle
regulation
12
and
most
likely
in
intestinal
Chapter 1
differentiation (Tian, et al., 1999a,b). ErbB-2 is involved in alveolar
branching and morphogenesis of the mammary gland (Niemann et al.,
1998). How these functions correlate with those expressed by these
proteins in the tight junction assembly is as yet undetermined.
Given their specific localization at the boundary between the apical and
basolateral domains, the TJ could represent a landmark for the transport
machinery, acting in targeting vesicles to the correct plasma membrane
domain, thereby facilitating formation and maintenance of the polarized
epithelial phenotype (see below). In this context it is interesting to note
that TJs indeed form a microdomain in the plasma membrane, as
hyperphosphorylated occludin and ZO-1 are both enriched in the Triton
X-100-insoluble fractions, isolated from T84 epithelial cells (Nusrat et al.,
2000), suggesting an association with a detergent resistant microdomain
(DRM). Furthermore, the small GTPase rab 13, belonging to the family of
proteins thought to regulate vesicle docking and fusion, is recruited from
a cytosolic pool to the junctional complexes after cell-cell contacts have
been established. Overexpression of dominant active rab13 (Q67L) causes
a delay in the recruitment of claudin-1 to the junctional complex, which
presumably explains the delay in the formation of a sealed monolayer of
polarized MDCK cells, and an enhanced leakage of a small nonionic
tracer from the apical domain (Marzesco et al., 2002). These data suggest
that rab13 functions as a regulator in TJ assembly, and hence as a
potential parameter in governing development and maintenance of
polarity. A role of rab13 in vesicular transport has thus far not been
described, but rab13, in conjunction with rab8, is closely related to yeast
small GTPase sec4, which is involved in controlling the assembly of the
exocyst (Guo, et al., 1999), required for the polarized delivery of cargo
proteins to the plasma membrane in budding yeast.
13
Chapter 1
C. Role of the extracellular matrix and the cytoskeleton
Cell adhesion to the extracellular matrix (ECM) is important for the
determination of the axis of polarity, i.e., in defining the orientation of the
apical and basolateral membrane domains relative to the biological
environment and the direction of transport of solutes. As previously
mentioned, an important indicator for the axis of polarity is the TJ and
cell-cell contacts, but it is the cytoskeleton which provides the cell's
epithelial-like shape. We will first discuss some aspects of the
involvement of the ECM in the development of an apical plasma
membrane domain.
In early studies it could already be demonstrated that surface adhesion of
MDCK cells suffices to target a 184 kDa apical resident protein to the
non-contacting, apical domain of the cells. By contrast, at this early
phase of polarity development, a 63 kDa basolateral protein marker
randomly distributes over the entire surface of the cell (Vega-Salas et al.,
1987). In this study, the medium contained a relatively low calcium
concentration, which prevents the formation of cell-cell contacts and TJs.
Accordingly, the presence of TJs and the occurrence of cell-cell adhesion
is apparently not needed to induce recruitment of apical resident proteins
at distinct membrane domains, seemingly reflecting an initiation of
polarity. Evidently, full development of polarity requires cell-cell contacts
and the formation of TJs. Following cell attachment, MDCK cells initially
organize their microtubules radiating from an intracellular region where
also the centrosomes and Golgi complex are located. In the fully polarized
state, the microtubules are organized as an apical web, and are
longitudinally arranged in microtubule bundles in the apical-basal axis
with the minus end spread over the apical region and the plus ends
directed towards the basolateral region (Bacallao et al., 1989). Such an
organization of the microtubule network has been thought to be specific
for polarized epithelial cells, as it would strongly facilitate vesicle
transport to the correct plasma membrane domain. However, in nonpolarized fibroblasts, transport of apical and basolateral proteins in
14
Chapter 1
different vesicles along microtubules occur in a similar manner. Thus, it
is more likely that microtubules in addition to regulating overall vesicular
transport in general, are also involved in functions as maintaining cell
shape.
Apart from a microtubule network, epithelial cells also contain a highly
ordered actin network. In epithelial cells, a dense actin network is found
just underneath the apical surface. The actin network is linked to actin
filaments, which constitute the core of the microvilli, covering the apical
surface. Fath and Burgess (1993, 1994) proposed that cytoplasmic
dynein moves Golgi-derived vesicles along microtubules to the cell cortex,
where the actin-based motor protein myosin-I then mediates the local
delivery to the apical membrane across the dense actin network. Not only
apical delivery depends on actin. When actin filaments in MDCK cells are
depolymerized, following treatment with latrunculin B, both the apical
construct FcLR(5-22) and the basolaterally localized transferrin receptor
(TfR) become redistributed over both membrane domains, without
perturbation of the tight junctional complex (Sheff, el al., 2002). This
suggests that the integrity of actin filaments is also crucial in regulating
in a direct or indirect manner protein-sorting events, for example by
orchestrating protein retention in specific domains.
In this context, the involvement of additional regulators, connecting the
actin cytoskeleton with cell polarity, is of relevance, such as the Rho
family GTPases, including RhoA, Rac1 and cdc42. Mutations in these
small GTPases cause a loss or misorientation of polarity and sorting of
proteins. Expression of dominant-active cdc42 in MDCK cells slowed
endocytic and biosynthetic transport, whereas the dominant-negative
mutant slowed apical endocytosis and basolateral-to-apical transcytosis,
but stimulated biosynthetic traffic (Rojas et al., 2001). Both mutants
cause altered TJ morphology and function, as measured by a decrease in
transepithelial resistance. A downstream effector of Rho is Rho kinase
(ROCK). Inhibition of ROCK by a specific inhibitor, Y-27632, resulted in a
reorganization
of
F-actin
structures
and
enhances
paracellular
permeability, but it did not alter the distribution and detergent solubility
15
Chapter 1
of TJ-proteins (Walsh et al., 2001). Therefore it is suggested that ROCK
regulates intact TJ via its effects on the F-actin cytoskeleton. Effects on
cell-cell adhesion by the Rho family of GTPases has been shown in
keratinocytes, where Rac1 regulates adherens junctions via endocytosis
of E-cadherin (Akhtar, 2001) possibly regulated by Protein kinase C
activity (Le et al., 2002) and effects on F-actin. In conclusion, the data
suggest that regulation of actin organization by Rho GTPases is involved
in the proper functioning of cell adhesion and cell-cell contacts, and
thereby plays an important role in the development and maintenance of
epithelial cell polarity.
III. Regulation of cell polarity
A. Interleukins and epithelial cell differentiation
Several cytokines and hormones are known to be involved in epithelial
differentiation, in particular in liver development and regeneration. An
important group of cytokines in hepatic differentiation and regeneration
are the interleukin (IL)-6 type of cytokines. This family consists of IL-6,
IL-11, CNTF, CT-1, LIF and OSM, and these cytokines all share the
common signal transducer gp130 in combination with cytokine-specific
subunits. This shared use of gp130 presumably explains their almost
identical biological response. Fetal murine hepatocytes differentiate upon
stimulation with OSM in combination with glucocorticoid, and as a result
several mature hepatic markers become expressed, such as albumin
secretion (Kamiya et al, 1999). Furthermore, livers deficient in gp130
display defects in liver maturation. Not only does liver development
depend on an IL-6 type cytokine action, but mesenchymal-to-epithelial
conversion of rat metanephros is also induced by an IL-6 type of cytokine,
LIF, and following OSM treatment the same epithelialization was observed
(Barasch, et al., 1999). The classical signal transduction via gp130
involves the JAK/STAT pathway, but also Ras, PI3K and MAPK can also
be activated by OSM via a gp130-dependent mechanism (Heinrich, et
16
Chapter 1
al.1998, and Taga and Kishimoto, 1997). In the murine fetal hepatocyte
system,
overexpression
of
dominant
negative
STAT3
resulted
in
suppression of the differentiated phenotypes obtained upon OSM
stimulation (Ito et al, 2000). This indicates that the STAT3 pathway is an
essential component of OSM induced hepatic differentiation. On the
molecular level, further insight into downstream events of OSM-mediated
signalling is gradually emerging. OSM-induced formation of E-cadherinbased adherens junctions (AJ), which are fundamental for proper
organization of epithelial cells, including hepatocytes, is dependent on
direct K-Ras activation (Matsui et al., 2002). This indicates that OSM can
regulate cell adhesion in fetal hepatocytes, thereby functioning in the
organization of the cellular architecture. However the tight junctional
organization, when monitored by ZO-1 expression and localization, is not
affected by OSM.
The IL-6 type of cytokines are not only involved in epithelialization of fetal
hepatocytes. Recently, evidence was obtained that these cytokines can
also specifically induce a plasma membrane polarization in primary cell
systems. This issue was investigated in cultured hepatocytes, which
develop a bile canalicular (BC) structure, like HepG2 hepatoma cells, a
system that has been shown to represent a valuable tool to study
development of plasma membrane polarity (van IJzendoorn et al., 2000;
Maier et al., 2001; van der Wouden et al., 2002). Thus, van der Wouden
et al. showed that OSM can stimulate plasma membrane development via
a gp130-dependent mechanism. Furthermore, van IJzendoorn and
Hoekstra (2000) provided evidence that a distinct apically-directed
vesicular transport pathway is specifically activated by an intrinsic
protein kinase A-mediated mechanism during polarity development of
HepG2 cells (see below). An inhibition of this pathway by protein kinase A
(PKA) inhibitors abolishes polarity development. Similarly, stimulation of
HepG2 cells with OSM or IL-6 results in a more polarized phenotype of
HepG2 cells (see fig. 2) and, interestingly, this hyperpolarization is a
consequence of stimulation of an apically directed transport pathway,
17
Chapter 1
B. Cell cycle regulation and cell polarity
During development, coordinately regulated cell division events are
crucial
for
proper
tissue
morphogenesis.
Other
key
features
of
morphogenesis are cellular polarization and membrane dynamics. These
two intimately entwined features are well illustrated in polarized epithelia
in which, as noted above, the plasma membrane is segregated into
different domains with distinct protein and lipid compositions, and
intracellular organelles and structures such as the centrosome are
positioned in a polarized manner.
In addition to the well-defined function of TJs in maintaining a fence
between apical and basolateral membrane domains, recent evidence
suggests that TJs are also involved in basic cellular processes such as
the regulation of cell growth and differentiation (Balda and Matter, 1998;
2000), as well as polarized processes such as the orientation of the
division apparatus (Lecuit and Wieschaus, 2002). In cells that are
stimulated to proliferate, e.g. during the process of wound healing, the
localization of the prominent TJ protein zonula occludens (ZO-1) is
shifted to the nucleus (Gottardi et al., 1996). Furthermore, ZO-1, by
virtue of its interaction with ZONAB, directly participates in the control of
the expression of the ErbB-2 gene (Balda and Matter, 2000), which is
implicated
in
cell
differentiation
and
proliferation.
ZO-1
displays
homology to Discs large, a Drosophila tumor suppressor (Bilder et al.,
2000). In Drosophila epithelial cells, Discs large, together with two other
proteins, lethal giant larvae and scribble, act to properly localize apical
proteins and adherens junctions and as such contribute to epithelial
architecture. Studies with mutants of these proteins suggest that in
epithelial-derived cancers, the breakdown of the apical-basolateral
architecture may have been the primary defect, resulting in a loss of
differentiation, an increased proliferation rate (Bilder et al., 2000), and
possibly a disorganized tissue architecture. The establishment and
maintenance of epithelial cell polarity may thus be functionally related to
cell cycle control. For instance, circulating growth factors, their receptors
19
Chapter 1
typically displaying a polarized distribution in epithelial cells, may elicit
different signaling cascades that lead to changes in cell cycle control
when no longer restricted to a specific surface or membrane domain.
Many signaling events are organized in specialized PM microdomains,
called caveolae (Fielding, 2001). The main structural component of
caveolae, caveolin-1, behaves as a tumor suppressor. Thus, downregulation of caveolin-1 is sufficient to trigger cell transformation
(Galbiati et al., 1998) and its (over)expression negatively regulates cell
cycle progression (Galbiati et al., 2001). Accordingly, caveolin-1-deficient
mice display increased cell numbers and a disorganized cell architecture
(Drab et al., 2001). The molecular mechanism by which caveolin-1
controls cell growth and architecture is not clear, but may involve a
p53/p21(WAF1/CIP1)-dependent pathway (Galbiati et al., 2001) and/or
caveolin-1-mediated regulation of protein kinase A signaling (Razani et
al., 1999). In addition to its involvement in signaling, caveolin-1 also
plays a role in the delivery of certain newly synthesized proteins, many of
them
being
sequestered
in
detergent-insoluble
sphingolipid-
and
cholesterol-enriched PM microdomains (‘rafts’; Simons and Ikonen,
1997), to the apical PM (Scheiffele et al., 1998).
As noted in section II B, the TJ area appears to be a targeting patch for
polarized traffic pathways (Louvard et al., 1980; Zahraoui et al., 2000).
Moreover, the TJ area displays raft-like characteristics (Nusrat et al.,
2000) and harbors many signaling complexes (Zahraoui et al., 2000) and
integrins. Proteins that are known to direct polarized trafficking, for
example, the exocyst members sec6/sec8 (Grindstaff et al., 1998), cdc42
(Kroschewski et al., 1999; Cohen et al., 2001; Zhang et al., 2001; Rojas et
al., 2001) and the mammalian homologue of Drosophila lethal giant
larvae (Müsch et al., 2002), localize at the side of cell-cell contact in close
proximity to the TJ area. The intracellular sorting and trafficking of
proteins
and
lipids
are
fundamental
to
the
establishment
and
maintenance of cell polarity. Indeed, these processes ensure the selective
supply of molecules to the different PM domains and control the
(intra)cellular localization of signaling molecules activated at the cell
20
Chapter 1
surface, and thereby the ultimate biological response. Well-known sorting
sites are the Golgi apparatus and PM. Recently, several elements of the
endosomal system have been identified that control the polarized sorting
and targeting of both proteins and lipids, and include the early sorting
endosome and the subapical compartment (SAC) (reviewed in van
IJzendoorn and Hoekstra, 1999, van IJzendoorn et al., 2000). The latter
compartment is rich in caveolin-1 and other raft components such as
cholesterol and sphingolipids (Gagescu et al., 2000; Hoekstra and van
IJzendoorn, 2000). Early studies revealed that membrane traffic ceases
during mitosis, which suggests a functional relationship between the
regulation of cell cycle progression and intracellular transport. The
cessation of intracellular transport during the mitotic phase was reported
not to perturb the existing epithelial architecture and polarity (Reinsch
and Karsenti, 1994). However, polarity is transiently rearranged without
loss of cell-cell contact during growth factor-induced tubulogenesis
(Pollack et al., 1998).
It is not clear whether epithelial polarity can be directly influenced by
changes in cell cycle progression or by an altered expression pattern of
cell cycle-regulating molecules. However, terminal differentiation of
epithelial cells and the establishment of polarity are often associated with
withdrawal from the cell cycle following cell cycle arrest in the G1 phase.
Recent data suggest that proteins that are primarily implicated in cell
cycle
control
may
play
additional
functions
in
epithelial
cell
morphogenesis and polarity. For instance, the cyclin-dependent kinase
inhibitor p27kip1, which is involved in mediating G1 arrest, was found to
be required for mouse mammary gland morphogenesis and function
(Muraoka et al., 2001), and was also implicated in the differentiation of
kidney and intestinal epithelium (Combs et al., 1998; Deschenes et al.,
2001; Tian and Quaroni, 1999b). Furthermore, the overexpression or
activation of p53, a tumor suppressor that regulates cell cycle
progression, was reported to inhibit cdc42-dependent cell effects that
promote cell polarity, including actin cytoskeletal dynamics (Gadea et al.,
2002) and, possibly, polarized membrane transport (Kroschewski et al.,
21
Chapter 1
1999; Cohen et al., 2001; Zhang et al., 2001). Recent data indicate that
proteins involved in the regulation of the cell cycle may modulate changes
in the organization and function of the endomembranous system. Indeed,
several cyclins, e.g. cyclin A (S-phase), cyclin B (G2/mitosis) and cyclin E
(G1 phase), cdc2 and the cyclin-dependent kinase
(cdk) 2 have been
found in isolated endosomal fractions from liver (Vergés et al., 1997).
Cyclin B has also been detected in the Golgi apparatus (Jackman et al.,
1995) and both cdk2 (Gaulin et al., 2000) and the cdk2-inhibitor p27kip1
(Yaroslavskiy et al., 2001) have been found in PM fractions.
Moreover, the amount and activity of cell cycle regulatory proteins in
endosome and PM fractions is dynamically and specifically regulated, e.g.
during liver regeneration, a process that involves a highly increased
proliferation rate of hepatocytes (Vergés et al. 1997), following insulin
stimulation (Gaulin et al., 2000), or upon cellular activation (Yaroslavskiy
et al., 2001). The endosomal targets of cdk2 kinase activity are not known
but are likely governed by the cyclin type that is complexed with cdk2.
Cdk2
complexes
integrate
tyrosine
phosphorylation
and
dephosphorylation signals, and in this way may control the number of
rounds of membrane fusion at discrete domains. This leads to changes in
the intracellular location of internalized receptors and, ultimately, in their
biological response (Fiset and Faure, 2001). A target of the mitotic cyclin
B-complexed cdk2 is the monomeric small GTPase rab4, which in
epithelial cells regulates endosomal membrane trafficking and transport
to the apical PM domain (Mohrmann et al., 2002). Phosphorylation of
rab4 in mitotic cells by cyclin B/cdk2 leads to its relocalization from
endosomal membranes to the cytosol (van der Sluijs et al., 1992; Ayad et
al., 1997). Phosphorylation of rab4 by cyclin B/cdk2 inhibits both the
recruitment of rab4 effector proteins to endosomes and the docking of
rab4-containing transport vesicles. Thus in this way endocytic membrane
fusion and transport are affected during the G2 and mitotic phase. The
presence of active pools of cdk2, complexed with the G1-specific cyclin E,
at the plasma membrane and in endosomes of polarized hepatocytes
(Gaulin et al., 2000) strongly suggests that a functional relation between
22
Chapter 1
Figure 2: Oncostatin M treatment causes an increase in the number and
membrane surface area of bile canaliculi in HepG2 cells. Hep2 cells were
cultured for 72 hrs in the absence (A) or presence (B) of rhOSM (10 ng/ml). The
cells were fixed in ethanol and stained with TRITC-labeled Phalloidin to visualize
actin, which is abundantly present underneath the apical, bile canalicular
surface (van der Wouden et al., 2002). Arrows indicate BCs and the bar is 5 µm.
marked by sphingomyelin, from the subapical compartment SAC to the
bile canalicular membrane.
In epithelial development, particularly with respect to the development of
plasma membrane domain polarity, IL-6 type cytokines have thus far
been studied most extensively. Nevertheless, other interleukins could also
interfere in correct maintenance and regeneration of polarity after tissue
damage. For example, IL-15 is upregulated during inflammatory bowel
disease as well as during colitis (Liu et al., 2000), cases in which the
intestinal barrier function can be disrupted. Under these conditions, IL15 likely contributes to the reassembly of the tight junctional complex,
separating
the
basolateral
domain
from
the
apical
domain,
by
upregulation and recruitment of claudins, occludin, ZO-1 and ZO-2 into
the complex (Nishiyama et al., 2001). Although knowledge in this area is
still scanty, we anticipate that other cytokines will be identified that may,
in addition, control polarity during development, inflammation or cancer,
the latter also often being accompanied by a loss of cell polarity.
18
Chapter 1
cell cycle control and membrane dynamics may not be restricted to
mitosis.
Several factors that stimulate cell polarization also cause cells to arrest in
the G1 phase of the cell cycle, and modulate membrane traffic events. As
discussed above, the IL-6 cytokine family member OSM is required for
fetal liver maturation and, in mature hepatocytes, stimulates polarity
development (van der Wouden et al., 2002). OSM also induces G1 arrest
via p27kip1-mediated inhibition of cdk2 activity (Klausen et al., 2000). In
polarized hepatocytes, OSM-elicited signaling targets PKA-dependent
membrane trafficking events at the SAC (van der Wouden et al., 2002)
that have previously been demonstrated crucial for polarity development
(van IJzendoorn and Hoekstra, 1999; 2000). Direct stimulation of PKA
activity with cAMP analogs enhances the development of polarity in
hepatic cells (Zegers and Hoekstra, 1998; van IJzendoorn and Hoekstra,
2000). The cAMP-mediated PKA activation pathway also increases the
expression
of
the
cdk2-inhibitor
p27Kip1
by
interfering
with
its
degradation, in this manner controlling cell cycle progression (van
Oirschot et al., 2001). Functional molecular links that integrate these
events in a mechanism(s) that regulates the process of polarity
development are as yet unknown, but are likely to be revealed in the near
future.
IV. Membrane transport in epithelial cells
A. Polarized sorting in epithelial cells
As described above a major feature of polarized epithelial cells is the
formation of distinct PM domains, the apical and basolateral membrane,
which are characterized by distinct protein and lipid compositions. This
implies that sites exist in the cell where membrane components are
sorted from each other and from where they are directed specifically to
the different membrane domains. Although the polarized distribution of
PM proteins and lipids can be generated during their biosynthesis, it is
obvious that due to continuous internalization from and recycling to the
23
Chapter 1
PM, efficient sorting in the endosomal compartment(s) is required to
maintain the polarized phenotype of epithelial cells. Accordingly, two
major compartments have been implicated in polarized sorting: the transGolgi network (TGN) in the biosynthetic transport pathway and an
endosomal compartment that has been referred to as an apical recycling
endosome (ARE) or common endosome (CE) in epithelial cells such as
MDCK, and as the subapical compartment (SAC) in liver cells (Traub,
1997; van IJzendoorn et al., 1999; 2000).
The extent of sorting in the TGN is dependent on the cell type. Although
in MDCK cells most apical and basolateral proteins are directly sorted
from the TGN to the different domains (Ikonen and Simons, 1998),
hepatocytes transport most (Bastaki et al., 2002), but not all (Kipp and
Arias, 2000), apical proteins first to the basolateral membrane, where
they are internalized and transported through the endosomal membrane
system to the apical membrane, a process called transcytosis. This
transcytotic pathway is also used for the transport of molecules across
the epithelial cell layer, as, for example in case of transport of dimeric IgA
after its binding to the polymeric Ig-receptor (pIgR) at the basolateral
membrane of epithelial cells (Apodaca et al. 1991).
The differences in the sorting capacities of biosynthetic and endocytotic
pathways in MDCK cells and hepatocytes are exemplified by their distinct
sorting of sphingolipids. Sphingolipids, a class of lipids which includes
glycosphingolipids and sphingomyelin (SM), are enriched to different
extent in the apical membrane of polarized epithelial cells (Holthuis et al.,
2001), with glycosphingolipids showing a more pronounced polarity than
SM. This implies that glycosphingolipids and SM are (partly) sorted from
each other during their transport to the PM. Indeed, when fully polarized
MDCK cells were incubated with fluorescently labeled (C6NBD)-ceramide,
the newly synthesized C6NBD-SM and –glucosylceramide (GlcCer) are
transported predominantly to the basolateral and apical membrane,
respectively (van Meer et al., 1987).
In contrast, no sorting of these
sphingolipid analogues was observed in the transcytotic pathway of these
cells (van Genderen and van Meer, 1995). Careful examination of
24
Chapter 1
transcytotic lipid transport in polarized HepG2 hepatoma cells revealed,
however, that sorting in the transcytotic pathway of polarized cells can
occur. This could be demonstrated after loading the endosomal
compartment of these cells, accomplished by bulk-flow transport of
sphingolipid analogues from the basolateral to the apical membrane.
After their accumulation in the SAC, and continuing the incubation at a
transport permissive temperature, C6NBD-SM was efficiently transported
back to the basolateral membrane whereas C6NBD-GlcCer remained in
the apical pole of the cells, where it was recycling between SAC and the
apical (bile canalicular, BC) membrane (van IJzendoorn and Hoekstra,
1998; see fig. 2). HepG2 cells can transport newly synthesized
sphingolipids directly from the TGN to the BC (Zegers and Hoekstra,
1997), although direct evidence for sorting of sphingolipids in the TGN
was not provided at that time.
However, more recently indirect evidence was obtained that newly
synthesized fluorescent sphingolipid analogues are indeed sorted in the
TGN of HepG2 cells. As in MDCK cells, SM is transported predominantly
to the basolateral membrane while GlcCer is, in relative amounts, the
major lipid going to the BC (Maier and Hoekstra, 2003). Most studies on
polarized protein sorting in the endosomal system have been performed
in MDCK cells, often using TfR and pIgR as markers for recycling and
transcytosis,
respectively.
After
internalization
at
the
apical
and
basolateral membrane the endocytosed material encounters first apical
and basolateral early endosomes, respectively (Mostov et al., 2000). Here
the membrane-bound proteins are sorted from soluble components; most
of the soluble molecules are transported further into the lysosomal
degradation pathway, but a major part of the membrane-bound
components is recycled directly back to the original plasma membrane, a
mechanism which may help to restrict the intermixing of basolateral and
apical molecules. Nevertheless, in non-polarized cells approximately 50 %
of the internalized TfR is transported further into the cell to the
pericentriolar recycling compartment (PRC) from where it is efficiently
recycled back to the plasma membrane (Hao and Maxfield, 2000).
25
Chapter 1
In polarized cells, proteins internalized from the apical and the
basolateral membrane both have access to a tubular endosomal
compartment, which is localized around the centriole in the apical pole of
the cell, and has been defined as common endosome (CE; Knight et al.,
1995). From the CE, which is presumably homologous to the SAC in
hepatocytes, apical and basolateral proteins are recycled back to their
original membrane. Recently it was shown that in MDCK cells TfR does
also colocalize with the transcytosing pIgR in the CE; TfR is recycled back
from this compartment to the basolateral membrane and pIgR is
transported via the apical recycling endosome (ARE) to the apical
membrane (Brown et al., 2000). Because the ARE is a specialized
compartment involved in the apical delivery of molecules in polarized cells
it was postulated to represent the site of polarized sorting in the
endocytotic pathway. However, these data clearly indicate that it is the
CE that is responsible for polarized sorting, whereas the ARE may have
additional functions or it may act as a “back-up” sorting station, where
proteins that should be transported to the basolateral membrane and
which have escaped the sorting process in the CE are ultimately removed
from the apical pathway.
That CE and ARE are indeed distinct compartments or that they may
represent different subregions in a pleiotropic compartment, perhaps
dictated by tubular extensions; this has been verified by their distinct pH
values, which are 5.8 for the CE and 6.5 for the ARE, indicating that
intermixing of their lumenal contents is restricted (E. Wang et al., 2000).
Promising markers to further distinguish the different endosomal
(sub)compartments are small GTPases of the rab family. Interestingly
rab11, which colocalizes to the TfR-positive recycling compartment in
non-polarized cells is localized to the TfR-negative apical recycling
endosomes in MDCK cells, but not to the CE (Brown et al. 2000),
indicating that regulatory proteins can fulfill different tasks depending on
the requirements for the trafficking pathways, and indicating in this case
that ARE and recycling endosomes are not identical compartments.
Although there is evidence that rab11, as well as rab17 and rab25, may
26
Chapter 1
function in apical sorting at the level of the common endosome and
associated subcompartments (Zacchi, et al, 1998; X. Wang et al., 2000;
Prekeris et al., 2000), their exact role is not known.
Recently a direct role for another rab protein, rab3b, has been
demonstrated for the polarized trafficking of pIgR in MDCK cells (van
IJzendoorn et al., 2002). Rab3b is primarily localized at an apical
endosomal compartment, probably the ARE, and binds in its GTP-form to
the ligand-free pIgR. In MDCK cells, the unoccupied pIgR is constitutively
internalized at the basolateral membrane and the binding of rab3b-GTP
mediates its transport back to the basolateral membrane. Association of
dimeric IgA to pIgR at the basolateral membrane causes GTP-hydrolysis
and dissociation of rab3b, which in turn enables the dIgA-pIgR-complex
to reach the apical surface. Hence, in this case rab proteins provide a
mechanism that carefully regulates optimal utilization of a receptor in
polarized transcytotic transport, regulating apical-basolateral receptor
recycling.
Furthermore, binding of dIgA to pIgR induces a phosphorylation cascade,
which involves the tyrosine kinase p62yes and tyrosine phosphorylation of
PLCγ1, and which as such is important for the efficient transcytosis of
the dIgA-pIgR complex, (Luton et al., 1999). This cascade depends on an
increase of the intracellular Ca2+-concentration [via inositol 1,4,5triphosphate (IP3) signaling] and accordingly is activated by PKC
stimulation (Cardone et al., 1994). In addition, pIgR can be a substrate
for serine kinases and it was shown that phosphorylation of newly
synthesized pIgR at Ser-726 facilitates its transport from the TGN to the
basolateral PM (Orzech et al., 1999) as well as its internalization at the
basolateral membrane (Okamoto et al., 1994), whereas basolateral to
apical transcytosis is independent of pIgR phosphorylation.
In contrast to its stimulatory effect on pIgR-transcytosis, PKC activation
dramatically reduces polarity of HepG2 cells (Zegers and Hoekstra, 1997).
Indeed PKC inhibits the transport of sphingolipids from both the TGN
and the SAC to the apical membrane in these cells. In contrast,
stimulation of PKA stimulates the transport of sphingolipids to the apical
27
Chapter 1
membrane of HepG2 cells and also increases the size and number of BC
structures in these cells (Zegers, and Hoekstra, 1997). In particular, the
size increase after a short-term incubation with cAMP analogues
indicates that PKA causes a redistribution of membrane components
from an endosomal compartment to the BCs. Indeed, it was shown that
SM, which in fully matured cells is predominantly transported to the
basolateral membrane after its accumulation in the endosomal SAC, is
redirected from this compartment to the apical membrane upon PKA
activation (van IJzendoorn and Hoekstra, 1999).
Figure 3: Schematic representation of sphingolipid sorting in the SAC of
HepG2 cells. Under normal growth conditions, glucosylceramide (GC) and
sphingomyelin (SM) are laterally segregated in the SAC membrane (
). GC
is recycled back to the membrane of the bile canaliculus (BC), while SM is
transported to the basolateral plasma membrane. Activation of PKA, naturally
occurring in young developing cells or artificially-induced by exogenous addition
of cAMP analogs, causes a redirection of SM trafficking from the SAC. The lipid
now enters a nocodazole-sensitive subregion of SAC (SM*) while on its way to
the apical (BC) membrane. Moreover, this pathway is distinct from the GCrecycling pathway.
Interestingly, the apical transport of SM after PKA activation follows the
same route as the transcytosing pIgR, which is clearly distinct from the
apical recycling pathway of GlcCer (van IJzendoorn and Hoekstra, 1999),
28
Chapter 1
indicating that at least two different apical transport pathways originate
from the SAC (fig. 3)
In several studies it has been reported that PKA activation can induce the
redistribution of proteins from an internal endosomal compartment to the
apical
membrane.
Well-known
examples
are
the
water
channels
aquaporin 1 and 2 that are redistributed to the apical membrane of
kidney cells after PKA activation induced by binding of vasopressin to the
basolateral membrane (Han and Patil, 2000, Katsura et al., 1997). The
redistribution of aquaporin 2 in renal principal cells depends on the
presence of an A kinase-anchoring protein (AKAP), either at the ARE or
the apical membrane, where it is thought to sequester the RII subunit of
PKA, thereby providing the link between vasopressin signaling and
trafficking of aquaporin 2 (Klussmann and Rosenthal, 2001, Jo et al.,
2001). In hepatocytes, PKA activation induced the redistribution of
several ABC transporters, including the bile salt exit pump (BSEP), from
endosomal pools to the BC membrane (Kipp et al., 2001). Although a
similar redistribution of BSEP was found after incubating hepatocytes
with taurocholate, the effects were clearly distinct, indicating that the
increase in bile salts is not the physiological stimulus for PKA-induced
apical
transport
of
ABC
transporters.
The
extensive
membrane
redistribution in the epithelium of the urinary bladder upon stretching is
another example for PKA-mediated apical transport in the endosomal
system (Truschel et al., 2002). Most likely in these cells it is the ARE
rather than the CE or the AEE that acts as a reservoir for proteins
destined for the apical membrane. From here, they can be transported
instantly to the apical membrane upon a physiological stimulus.
Although there is no indication for the presence of such a ‘storage’
compartment in the basolateral endocytic pathway, it may exist in some
nonpolarized cells. For example, the features that are associated with the
redistribution of GLUT4 transporters in muscle cells and adipocytes from
a specialized recycling endosomal compartment upon binding of insulin
(Lampson et al., 2001), are reminiscent of events following signaling in
the apical pathway.
29
Chapter 1
Recently it was shown that PKA activation not only redirects SM in fully
polarized cells, but that intrinsic activation of PKA activity is required in
young HepG2 cells to develop apical membrane domains (van IJzendoorn
and Hoekstra, 2000). Thus, a polarized phenotype does not develop when
the cells are cultivated in the presence of the PKA inhibitor H89. At such
conditions, apical-directed transport of SM is inhibited, emphasizing once
again the close correlation between the rerouting of a SM-marked
membrane flow, exiting from SAC, and development of polarity. Whether
the rerouting of SM as such is relevant to this development remains to be
seen, for example as a necessary compound for raft-assembly (see
Hoekstra and van IJzendoorn, 2000), a sorting principle thought to be
relevant to apical membrane targeting. In this context it is interesting to
note the importance of SM for development of polarity in neurons. Here,
SM is required for axonal outgrowth (Ledesma et al., 1999), although it is
not clear whether PKA activation is involved.
B. Polarized sorting during vesicular transport
Most transport processes between membranes within eukaryotic cells are
mediated by membrane-bound vehicles that are released from the donor
compartment and fuse, after their transport, specifically with the target
membrane (Rothman, 1996). To establish and maintain the specific
protein and lipid composition of the cellular organelles it is necessary to
tightly regulate all aspects of this vesicular transport event. Originally it
was thought that transport in general is mediated by small spherical
vesicles, although in cells overexpressing GFP-tagged cargo proteins also
large irregularly shaped containers have been identified that mediate the
transport from the TGN to the apical and basolateral membrane
(Hirschberg et al., 1998; Keller, et al., 2001). Most data available deal
with the formation of spherical vesicles and it is unclear whether the
formation of large transport containers requires different mechanisms
and regulation steps, although some general features are likely to be
similar. For example: a necessary step during vesicle formation is the
30
Chapter 1
specific sequestration of proteins to be transported into a domain that
will bud into a vesicle, while resident proteins of the donor organelle
excluded. In this section we will concentrate on recent findings that may
shed light on the possible mechanisms that govern the sorting of proteins
and lipids into the budding vesicle. Because most studies so far
concentrated on the sorting within the biosynthetic pathway, the focus
will be on vesicle formation at the TGN, but we will also present some
recent mechanistic data on polarized sorting in endosomes.
1. Sorting during vesicle formation
The minimal requirements for vesicle budding and fission have been
analyzed in reconstituted systems using large liposomes. These studies
revealed that interaction of cytoplasmic coat proteins with lipids and/or
cytoplasmic tails of transmembrane proteins of the donor membrane is
sufficient for the formation of vesicles (see e.g. Matsuoka et al., 1998,
Bremser et al., 1999, Zhu et al., 1999). Consequently, the presence of
cargo proteins in the donor compartment is not required for vesicle
formation per se. In contrast, the specific sorting of transport proteins
into the forming vesicle at cellular membranes requires the interplay of
the cytoplasmic budding machinery with components of the donor
membrane as well as the cargo molecules (Schmid, 1997; Kuehn et al.,
1998, Lanoix, et al., 2001).
So
far
three
distinct
coat
protein
(COP)
complexes
have
been
characterized. The COPII coat mediates exit of newly synthesized proteins
from the ER. COPI-coated vesicles are required for the retrograde (and
possibly anterograde) transport within the Golgi and from the Golgi to the
ER, and several classes of clathrin-coated vesicles have been described
that originate from the TGN, the plasma membrane and endocytotic
organelles. These are distinguished by distinct adaptor protein complexes
(up to now AP1 to 4 have been characterized), which mediate the binding
of clathrin to the forming bud (for a review see Hirst and Robinson,
1998). These AP-complexes consist of two large subunits, one medium
31
Chapter 1
chain (µ1-4) and one small chain (σ1-4). Although one of the large
subunits (β-adaptin 1-4) is highly homologous and mediates the binding
to clathrin, the other is more specific and is used for the characterization
of the different AP complexes. These are α-adaptin (for AP2), γ-adaptin
(AP1), δ-adaptin (AP3) and ε-adaptin (AP4). Several of these AP complexes
have been localized to the TGN where they are involved in the exit of
distinct classes of proteins, indicating that they are important for sorting
in the TGN.
The “classical” AP1 complex binds to mannose 6-phosphate (M6P)
receptors (MPR) and mediates the transport of soluble lysosomal enzymes
to the endosomal system (Le Borgne and Hoflack, 1997). The small
GTPase ARF1 mediates the binding of the AP1 complex to the TGN (West
et al., 1997). Association of GTPase to the membrane is regulated by
exchange of GDP for GTP, which causes the exposure of an N-terminal
myristoyl fatty acid anchor. ARF1 also mediates the binding of COPI
complexes to the cis-Golgi (Palmer et al., 1993), indicating that additional
factors are required to recruit coat protein complexes to distinct
membranes. Indeed, although the interaction of MPRs with M6Pcontaining proteins in the Golgi lumen and AP1 components in the
cytoplasm might be sufficient for sorting, recently an additional class of
proteins, the Golgi-localizing, gamma-adaptin ear homology domain,
ARF-binding proteins (GGAs), has been described that has been
implicated in providing specificity to this pathway by binding to both
ARF1 and MRPs (Takatsu et al., 2001; Puertollano et al., 2001; Doray et
al., 2002). Although it remains to be shown whether GGAs have a role in
polarized transport, they can bind to various ARFs, including ARF6
(Takatsu et al., 2002), which is found at endosomes, indicating that they
are involved in various transport pathways originating not only from the
TGN but also from endosomal compartments.
Although it was postulated that vesicle formation requires the attachment
of a cytosplasmic coat complex to the site of vesicle formation, it was only
recently that a protein coat was identified that is involved in the transport
from the TGN to the plasma membrane. A variant of the AP1 complex
32
Chapter 1
containing a distinct medium chain, APµ1b, was described that mediates
the transport of newly synthesized proteins to the basolateral membrane
of LLC-Pk1 cells (Fölsch et al., 1999), presumably by binding tyrosinebased basolateral sorting signals, as was shown for the LDL receptor
(Fölsch et al., 2001). Nevertheless there are several indications that
APµ1b expression is not sufficient for basolateral versus apical sorting in
all epithelial cells.
First, several basolateral proteins are correctly transported and targeted,
independently of APµ1b in LLC-Pk1 cells, whereas fibroblast, which do
not express APµ1b, are able to sort at least some apical and basolateral
cargo proteins into distinct vesicles (Yoshimori et al., 1996). Second,
some polarized cells, e.g. hepatocytes, do not express APµ1b, indicating
that in these cells a different sorting mechanism must exist. AP4, which
has been implicated in basolateral transport from the TGN, may be a
candidate for mediating such an alternative pathway (Simmen et al.,
2002). But it is striking that in hepatocytes most apical proteins reported
thus far are first transported to the basolateral plasma membrane before
they are transcytosed to the canalicular membrane. This implies that
polarized sorting in hepatocytes takes place mainly at the level of plasma
membrane and/or endosomes and that other mechanisms exist in these
cells to separate apical and basolateral proteins and lipids. Whether
those mechanisms involve AP/clathrin-coated vesicles is not known, but
recently AP1/clathrin-coated vesicles budding from recycling endosomes
were described in MDCK cells, that are enriched in TfR (Futter et al.,
1998). These vesicles may therefore separate basolateral cargo from the
transcytosing pIgR, which was enriched in cup-shaped vesicles (Gibson et
al., 1998), although in another study AP1 was reported to colocalize with
pIgR at the level of the ARE (Wang et al., 2001). In addition endosomederived
ARF6-containing
vesicles
were
isolated
from
nonpolarized
HEK293 cells, which were covered with an unknown coat complex and
which contain also TfR, indicating that they may be involved in
basolateral transport in polarized cells (Peters et al., 2001). On the other
hand, because ARF6 was localized to the apical membrane in MDCK cells
33
Chapter 1
(Altschuler et al., 1999), it may have a direct function in apical transport
in polarized cells.
However, current evidence generally implies that the major function of
protein coat complexes is the active sorting of cargo molecules away from
apical cargo and resident membranes into vesicles destined for the
basolateral membrane. In contrast, so far no coat protein complex has
been described that is involved in the formation of transport vesicles
containing apical cargo. This may reflect differences in the localization of
targeting sequences in the cargo molecules. So far the only signals that
have been localized in the cytoplasmic domain of cargo proteins are those
mediating basolateral targeting, and coat components have been shown
to interact specifically with these signals. In contrast, apical sorting
signals have been shown to be localized almost exclusively in the
membrane domain or the lumenal domain of cargo proteins (Keller and
Simons, 1997). Consequently coat proteins cannot directly interact with
sorting determinants of apical proteins and would therefore require a
transmembrane receptor that recognizes both the lumenal cargo and the
cytoplasmic coat. This is actually the case for AP1/clathrin-mediated
transport of M6P-containing proteins from the TGN to endosomes (see
above), but so far no similar receptor for apical targeting has been
identified. The evidence for the polarized targeting of pIgR from AREs
indicates that in this case only basolateral transport is mediated by
AP1/clathrin-coated vesicles, because brefeldin A, which interferes with
the binding of ARF1 to the membrane, disturbs the basolateral recycling
of the TfR and results in its mistargeting to the apical membrane (Wang
et al., 2001). On the other hand special AP1/clathrin-coated vesicles
containing a distinct β-subunit, which were implicated in the transport of
H+/K+-ATPase to the apical membrane in gastric acid secretory cells
(Okamoto and Jeng, 1998), may be of a coated vesicle fraction that
mediates transport from the ARE to the apical membrane. However, if
coated vesicles would mediate only basolateral targeting, the specific
binding of adaptor proteins to the targeting signals could cause the
clustering of basolateral proteins into the forming vesicles, thereby
34
Chapter 1
resulting in their separation from apical proteins. This mechanism would
also explain the observed hierarchy of sorting signals, i.e., basolateral
targeting signals being dominant over apical ones (Ikonen and Simons,
1998).
Yet, there is good evidence that apical proteins and lipids form clusters in
the donor membrane, thus being actively sorted from basolaterally
directed
cargo.
In
particular
the
formation
of
cholesterol-
(glyco)sphingolipid-enriched membrane domains in the TGN has been
implicated in this clustering of apical cargo (Verkade and Simons, 1997).
Sphingomyelin and most glycosphingolipids are synthesized in the
lumenal membrane leaflet of the Golgi. The biophysical properties of
sphingolipids, bearing a long-chain saturated fatty acid and alkyl chain of
high
melting
temperature,
favor,
their
packaging,
together
with
cholesterol, into small membrane domains at the level of the TGN (Brown
and London, 2000). These membrane domains, often called ‘rafts’, can be
identified by their resistance to extraction with nonionic detergents and
the low buoyant density of these extracts in sucrose gradients.
Glycosylphosphatidylinositol (GPI)-anchored proteins as well as several
transmembrane proteins, which are usually expressed in the apical
membrane of epithelial cells, are associated with these rafts, whereas
basolateral proteins are excluded from these domains (Verkade and
Simons, 1997). Raft association of newly synthesized apical proteins
occurs in the TGN and disruption of rafts, e.g. by depleting the
cholesterol pool of the cells, perturbs raft association and retards apical
transport. In addition, it may cause randomization of protein transport,
indicating that rafts are important for apical targeting (Keller and Simons,
1998). Interestingly, although cholesterol depletion disrupts raft integrity
in FRT-cells, only inhibition of sphingolipid synthesis causes the
randomization of polarized transport (Lipardi et al., 2000), indicating that
sphingolipids are especially important for the polarized sorting.
Although rafts may be important for the separation of apical and
basolateral cargo, it is clear that they are not sufficient for apical
targeting. Both N- and O-glycans have been identified as apical targeting
35
Chapter 1
signals (Scheiffele et al., 1995; Alfalah et al., 1999), indicating that
lectins, e.g., VIP36 (Fiedler et al., 1994), may have a function in delivery
to the apical membrane. Other proteins that have been implicated in
targeting of vesicles to the apical membrane are the lipid-binding protein
annexin XIIIb (Lafont et al., 1998) and the transmembrane protein MAL
(Cheong et al., 1999), which were both shown to be raft-associated.
Indeed, MAL can interact with GPI-anchored proteins (Millan and Alonso,
1998) and this interaction may be important for apical sorting of these
proteins. It is still unknown whether MAL and other proteins directly
target rafts to the apical membrane or whether they constitute an
intermediate link to other targeting devices, such as SNAREs (see below).
However, so far no evidence has been provided that would support an
interaction of MAL with SNARE proteins. The observations mentioned
above indicate that interactions between the apically directed molecules
in the lumen of the vesicle are important for their clustering into rafts
and their sorting into the assembling vesicle. Consistent with this notion,
it has been shown that rafts can also mediate transport of secretory
proteins to the plasma membrane (Martin-Belmonte et al., 2001).
Intermolecular interactions, important for polarized sorting, also occur in
the lumen of the SAC, as has been shown for the sorting of fluorescently
labeled SM and GlcCer. As described above, in fully polarized HepG2
cells, C6NBD-SM and GlcCer are transported from the SAC to the
basolateral and apical membrane, respectively. Because these probes
differ only in their head group (choline versus glucose) it is evident that
head group-specific sorting is operational in this case. Moreover, C6NBDgalactosylceramide, an epimer of C6NBD-GlcCer, is transported from the
SAC to the basolateral membrane, further emphasizing head groupdependent sorting but also reflecting a very specific interaction of C6NBDGlcCer with the apical transport pathway (van IJzendoorn and Hoekstra,
1998).
Although direct extrapolation of results obtained with fluorescent lipid
analogues requires a direct comparison with their natural counter parts,
which is often hampered due to technical limitations (see Maier et al.,
36
Chapter 1
2002), these results could be taken as a first indication that membrane
domain formation also plays a role in polarized sorting in endosomal
compartments. That rafts do exist in endosomal compartments was
confirmed in a recent study that demonstrated that the CE of MDCK cells
is enriched in raft components, although these were isolated by
antibodies against the basolaterally directed TfR (Gagescu et al., 2000).
This indicates that either transport of the TfR from the CE to the
basolateral membrane is mediated by rafts or, alternatively, that these
raft components were coisolated with the TfR, although they are actually
localized in distinct parts of the CE. The first possibility seems unlikely
because cholesterol depletion has no effect on the recycling of TfR in
nonpolarized cells. In contrast, the recycling of GPI-anchored proteins,
which is much slower than TfR recycling under normal conditions, is
accelerated upon cholesterol depletion and indistinguishable from TfR
recycling (Chatterjee et al., 2001). Surprisingly, the effects of cholesterol
depletion on endocytotic trafficking appear to be different from those on
the biosynthetic pathway. Although it generally slows down the transport
from the TGN to the plasma membrane, recycling from the endosomal
system is accelerated. This indicates that rafts in the CE cause the
retention of GPI-linked proteins and such a mechanism may facilitate
their polarized sorting to the apical membrane in polarized cells. Evidence
that sphingolipid-cholesterol-enriched membrane domains in apical
endosomes may be involved in the transcytosis of pIgR comes from
studies in enterocytes (Hansen et al., 1999). In contrast, pIgR transport
seems to be raft independent in MDCK and FRT cells, although transient
interactions, e.g., in the CE or ARE, cannot be excluded (Sarnataro et al.,
2000).
Clearly, the formation of specified membrane domains may play an
important role in the clustering and sorting of proteins and lipids towards
the apical domain. Nevertheless many reports of raft-independent apical
transport exist. In this respect it should be mentioned that proteins may
display differences in resistance to detergent solubilization, a feature
used
for
raft
characterization.
One
37
example
is
prominin,
a
Chapter 1
multimembrane spanning membrane protein in the apical membrane of
epithelial cells, which is completely soluble in Triton X-100, but is
resistant to extraction with lubrol WX (Roper et al., 2000). Similar
features have been observed in the sorting of the mulitmembrane
spanning proteins MDR1 and the copper transporter ATP7B in HepG2
cells. These proteins are readily extracted with Triton X-100, but are
insoluble in Lubrol WX, implying that sorting and direct transport of
apical resident proteins in liver cells could be mediated by Lubrol instead
of Triton X-100 rafts. Remarkably, Triton-rafts are operating in the
transport of apical resident proteins that travel via the indirect
transcytotic pathway (for a further discussion, see Aït Slimane and
Hoekstra, 2002; Aït Slimane et al., 2003).
The data indicate that in a (polarized) cell type-dependent manner,
various membrane domains with distinct biophysical properties appear to
exist that mediate apical transport, and some of these may be formed
independently of sphingolipids or cholesterol. Presumably, as a rule, the
domains display a distinct degree of liquid ordering that could be
provided by sphingolipids, cholesterol and saturated lipids, (co)dictated
by the lipid and perhaps protein composition of the apposed leaflet. The
absence of distinct lipids might be compensated for by others, provided
that the overall liquid-ordered state is maintained. Also in MDCK cells,
distinct vesicles for two apically transported proteins have been
identified,
the
raft-associated
sucrase-isomaltase
and
the
raft-
independent (Triton X-100-soluble) lactase-phlorizin hydrolase (Jacob
and Naim, 2001), which were probably segregated after their exit from the
TGN. Mechanisms and players, instrumental in governing these distinct
sorting and vesicular transport pathways that lead to the same site of
destination, remain to be elucidated. In this context, it has been shown
that MAL is involved in the apical targeting of both raft-dependent and
raft-independent proteins (Martin-Belmonte et al., 2000).
In addition to their function in clustering and sorting of apical cargo,
sphingolipid-enriched
membrane
domains
may
also
facilitate
the
formation of the transport vesicles. The generation of a membrane
38
Chapter 1
curvature at the site of budding is one of the main features of vesicle
formation. As described above, coat proteins can induce the formation of
vesicles from artificial liposomes, proving that they are sufficient to
provide the driving force for vesicle budding and fission. Membrane
curvature can be induced by the lipid composition of the membrane itself,
involving the asymmetric distribution of lipids between the lumenal and
cytoplasmic leaflets of the membrane bilayer. Because of their relative
rigid, i.e., tight packing, the formation of sphingolipid-cholesterol
domains in the lumenal leaflet of the membrane would induce a
curvature of the membrane, provided that packing properties in the
cytoplasmic leaflet remain unaltered. As a consequence, the result would
be the formation of a vesicular bud at the site of raft assembly.
Association of proteins with raft lipids would therefore not only separate
these proteins from the basolaterally directed clathrin-coated vesicles,
but may also be sufficient to concentrate them into apically directed
transport carriers (for further discussion see Huttner and Zimmerberg,
2001).
For the further characterization of the molecular mechanisms that govern
and regulate polarized sorting and vesicle formation, in vitro assays using
permeabilized cells or isolated organelles are desirable. Indeed various
“budding assays” have been developed to study vesicle formation from the
TGN, and it was demonstrated that MDCK cells, after infection with
vesicular stomatitis virus (VSV) and influenza virus, release the
basolateral marker VSV glycoprotein and the apical marker influenza
hemagglutinin in distinct transport vesicles (Wandinger-Ness et al.,
1990). Also, newly synthesized sphingolipids are released in distinct
vesicles from the TGN of MDCK and HT29 cells (Kobayashi et al., 1992;
Babia et al., 1994). Although it should be possible to use these in vitro
systems to further characterize the mechanisms of apical versus
basolateral sorting during vesicle formation, so far they were mainly used
to characterize the regulation of vesicle release and to distinguish the
formation of distinct basolateral vesicles at the TGN. Thus distinct
vesicles for secretory and transmembrane proteins as well as serum
39
Chapter 1
albumin and heparansulfate proteoglycan (HSPG) have been identified in
hepatocytes (Saucan and Palade, 1994; Nickel et al., 1994).
The impact of various protein kinases on vesicle formation from the TGN
has been investigated using specific activators and inhibitors. In most
systems basolateral transport proteins, especially VSV glycoprotein, but
also HSPG, were used to monitor vesicle release. However, little is known
about the release of apical transport vesicles. In these assays it was
shown that both PKA and PKC are involved in mediating vesicle release
from the TGN, although it is not clear whether the kinase activity of PKC
is actually required (Muniz et al., 1997; Simon et al., 1996; Westermann
et al., 1996). Other regulatory proteins that have been implicated in
vesicle formation are tyrosine kinases and phosphatases as well as
trimeric G proteins (Austin and Shields, 1996; Leyte et al., 1992). These
data show that vesicle formation is a highly regulated process, although
the mode of action of these proteins is currently unknown. Possible
kinase substrates include cargo molecules (see above for pIgR),
components
of
protein
coats,
or
putative
sorting
receptors.
The
phosphorylation of MPR by a casein kinase II-like protein at the Golgi,
which facilitates its interaction with the AP1 complex, is one example for
such a mechanism (Mauxion et al., 1996). Phosphorylation of subunits of
the AP complexes may be important for their binding specificity, as was
shown for AP2 (Fingerhut et al., 2001), but so far there is no indication
that such a mechanism is involved in polarized sorting.
The analysis of polarized vesicle release from endosomes was hindered by
difficulties to exclusively label the CE with specific marker molecules.
Therefore only few in vitro assays with endosomal membranes have been
established.
Recently
the
possibility
to
accumulate
fluorescent
sphingolipid analogues in the SAC of HepG2 cells has been used to
characterize the sorting capacity of the SAC membrane and indeed
distinct vesicles enriched in either C6NBD-SM or -GlcCer were identified
after permeabilization of the cells (Maier and Hoekstra, 2003). Other
assays resulted in the discovery of ARF6-containing vesicles covered by a
new coat (Peters et al., 2001; see above) and the characterization of
40
Chapter 1
H+/K+-containing vesicles derived from apical endosomes of gastric
parietal cells (Okamoto et al., 2002). The isolation and characterization of
distinct endosomal compartments from LDL-loaded hepatocytes (Enrich
et al., 1996; Vergès et al., 1999) may provide new opportunities for the
further analysis of polarized sorting in endosomal compartments.
2. Specific targeting of vesicles
After budding from the donor membrane the vesicles have to be
transported
to
the
distinct
plasma
membrane
domains.
Efficient
transport requires an intact microtubular cytoskeleton, and disruption of
microtubules affects both apical and basolateral transport, indicating
that microtubules are important for overall targeting of the transport
vesicles (Lafont et al., 1994). In addition, microtubule disruption not only
disturbs several transport steps, but also interferes directly with the
structure of the sorting organelles. At the same time, this feature may
make it difficult to determine whether they have a direct role in polarized
sorting and targeting to the plasma membrane. Nevertheless, recent
evidence indicates that interaction of vesicles with certain kinesins,
directed to the minus end of microtubules, can be affected by the lipid
composition of the membrane so that they may associate specifically to
apically directed vesicles. Thus the raft lipids cholesterol, sphingomyelin
and GM1 enhance the binding of KIF1a to liposomes (Klopfenstein et al.,
2002), and KIFc3 has been found in detergent-resistant domains
associated with annexin XIIIb (Noda et al., 2001).
The involvement of the actin cytoskeleton in polarized transport is even
less clear, but interestingly myosin II has been localized to coated vesicles
at the TGN (Müsch, et al., 1997; Ikonen et al., 1997). Association of
myosin II to the TGN was disrupted by brefeldin A, indicating that it may
bind to AP1 complexes (Ikonen et al., 1997) and thus be involved in
basolateral transport. Indeed it was shown that myosin II is involved in
the release of VSV glycoprotein from the TGN, but the protein has no
effect on the exit of HA, indicating that attachment to actin filaments may
41
Chapter 1
be specifically involved in efficient formation of vesicles directed to the
basolateral membrane (Müsch et al., 1997).
The next step is the specific docking and subsequent fusion of the vesicle
with the target membrane. SNARE proteins have been localized to distinct
organelles and play a crucial role in the targeting process. Formation of
trans-complexes between SNAREs present at the vesicle (v-SNARE) and
target (t-SNARE) membrane are required for specific docking and possibly
also for subsequent fusion (Pelham, 1997; Pecheur et al., 2000). The tSNAREs syntaxin 3 and syntaxin 4 are localized to the apical and
basolateral membrane of polarized epithelial cells and therefore have
been implicated in the polarized targeting of transport vesicles (Gaisano
et al., 1996), whereas cellubrevin is a ubiquitous v-SNARE that may be
involved in basolateral targeting of vesicles from TGN and/or endosomes.
Indeed it was shown that cellubrevin is a component of the ARF6containing vesicles derived from endosomes (Peters et al., 2001). The
function of SNAREs in vesicle targeting was originally identified by the
sensitivity of the fusion of synaptic vesicles to botulinus and tetanus
toxin and the insensitivity of apical transport to tetanus toxin was taken
as indication that apical targeting is independent of the SNARE
machinery (Ikonen et al., 1995). Recently, a tetanus toxin-insensitive
SNARE (TI-SNARE) was identified that may be the v-SNARE that mediates
transport to the apical membrane (Galli et al., 1998).
To target vesicles to their destination v-SNAREs have to be incorporated
into the vesicle during its formation. Recent evidence as to how this may
be achieved comes from studies of ER-Golgi transport. Here, ARF1
mediates the binding of the COPI coat complex and the GTPase activity of
ARF, regulated by an ARF-GAP (ARF GTPase activating protein), is
required for efficient incorporation of cargo into the budding vesicle. In
addition ARF-GAP interacts with v-SNAREs and this interaction is
required for efficient association of COPI components with the membrane.
Thus ARF-GAP links v-SNARE incorporation with incorporation of cargo
proteins into the forming vesicle and vesicle budding (Rein et al., 2002).
Whether this is a ubiquitous mechanism that is also operational at the
42
Chapter 1
level of the TGN and of endosomes remains to be seen. In addition, the
association of SNAREs with rafts, as described for TI-SNARE and
syntaxin 3, may provide a mechanism for their specific targeting to the
apical membrane (Lafont et al., 1999).
Although SNAREs are required for specific targeting, they are not
sufficient. In budding yeast a protein complex, the exocyst, marks the
location where new membrane material has to be internalized (Guo et al.,
1999). In mammalian cells a protein complex homologous to the exocyst
has been described, which is required for basolateral transport of LDL
receptors, but not for apical transport. This complex is localized close to
the tight junctions indicating that lateral cell-cell adhesion is important
for the generation of cell surface polarity (Grindstaff et al., 1998).
Additional regulators for the assembly of SNARE complexes at the site of
the exocyst are members of the Sec1 protein family (Carr et al., 1999). In
polarized epithelial cells the mammalian sec1 homologues munc18-2 and
munc18c have been implicated in apical and basolateral transport by
inhibiting syntaxin 3 and syntaxin 4-mediated fusion of vesicles with the
respective plasma membrane domains (Riento et al., 2000; Tellam et al.,
1997). In pancreatic acinar cells zymogen granules are stored in the cell
and exocytosed after incubation with cholecystokinin (CCK). Incubation
with supramaximal CCK concentrations abolishes apical exocytosis but
has only a minor effect on overall secretion. It was shown recently that
CCK incubation caused the dissociation of munc18c from the basolateral
membrane thereby exposing fusion complexes. This in turn results in the
basolateral exocytosis of zymogen granules which may result in mild
acute pancreatitis (Gaisano et al., 2001). Interestingly the dissociation of
munc18c is also mediated by PKC activation, although it has not yet been
shown that munc18c is a PKC substrate. Such a mechanism may provide
an explanation for the decrease in polarization of HepG2 cells caused by
PKC activation (Zegers and Hoekstra 1997).
43
Chapter 1
V. Conclusions and perspectives
In the last years the investigation of polarized transport has become one
of the major subjects in cell biology. Especially the notion that certain
lipids play a crucial role in membrane domain formation and that this
domain formation is an important step for polarized sorting, opened a
new field in membrane trafficking research. Evidently, the presence of
such rafts will also have a major impact on other aspects of cell biology,
as is shown by the growing evidence that similar membrane domains are
also involved in many signal transduction cascades, including those that
might regulate polarity development. Further impact in uncovering
polarized membrane transport will come from the analysis of the various
GFP-variants, displaying distinct fluorescence spectral properties. Thus
different probes can be used to monitor simultaneously the flow of apical
and basolateral proteins. Until recently, studies on polarized transport
have been mainly restricted to the biosynthetic pathway, whereas the
involvement of endosomes has been largely ignored. However, not
surprisingly, cells exhibit a much greater complexity as originally
anticipated and the discovery of the subapical compartment or common
endosome in polarized cells will now bring the focus of research also on
the role of the endocytotic pathway in development and maintenance cell
polarity. A major challenge will be to characterize functions of these
compartments on the molecular level. Among others, their isolation and
purification would thus be highly desirable.
44
Chapter 1
Scope of this thesis
In this thesis a novel role for the IL-6 type cytokine Oncostatin M (OSM)
in the regulation of plasma membrane polarity development is described,
as investigated in polarized HepG2 cells. HepG2 cells are human-derived
hepatocellular carcinoma cells, and are well known for their ability to
adopt polarity when cultured in vitro. In this system the effect of OSM on
polarity development and sphingolipid trafficking was studied as
propagated by virtue of signaling via its receptor, present on the
basolateral membrane domain. This work, as described in chapter 2
revealed a correlation between OSM-mediated activation of PKA and
polarity development, involving a mechanism that appears to rely on a
reorganization of the intracellular distribution of PKA rather than on a
net enhancement in PKA activity. Since sphingolipids play an important
role in both cell polarization and proliferation, we analyzed the effects of
polarity stimulating-signaling cascades on sphinganine turnover, using
the mycotoxin fumonisin B1. Fumonisin B1 is known to inhibit
dihydroceramide synthase, which causes an elevation of the intracellular
levels of sphinganine, and is also associated with the development of
hepatocellular carcinoma. In chapter 3, we investigated the effects of
elevated sphinganine levels on polarized endosomal membrane traffic,
and the data indicated a correlation between polarity development on the
one hand, and the level of sphinganine and the expression of the cell
cycle regulatory protein p27kip1 on the other. Moreover, dihydroceramide
synthase turned out to be a target for the cAMP/PKA signaling cascade,
known to be involved in promoting cell polarity development. In chapter 4
we therefore analyzed in detail the effects of p27kip1, a CDK inhibitor, on
differentiation and subsequent polarization of HepG2 cells. Increased
levels of p27kip1 are associated with a block in G1-S transition, thus
keeping the cells in G1. Eventually they may leave the cell cycle and enter
G0 to undergo terminal differentiation. The data demonstrate that both
OSM and activation of PKA increased the intracellular levels of p27kip1,
which was associated with a block in proliferation and an increase of
45
Chapter 1
polarization.
Furthermore,
we
show
that
during
early
polarity
development p27kip1 is reallocated from the nucleus to the cytosol where
it
possibly
facilitates
events
that
precede
and/or
promote
the
acquirement of cell polarity.
The dynamics of the actin cytoskeleton is of importance for tissue
morphogenesis and is carefully controlled by regulatory proteins,
including the small GTPase Rho and its downstream effector ROCK. A
loss of tissue architecture is often observed in carcinogenesis. In chapter
5 it is shown that suppression of ROCK activity results in a displacement
of the adherens junction protein E-cadherin and the induction of
lamellipodia, which culminates in the formation of elongated bile
canalicular lumens, shared by multiple cells. OSM stimulation in ROCK
suppressed cells counteracted both E-cadherin junction disassembly and
complex apial lumen formation. These data indicate that OSM-controlled,
E-cadherin-based cell-cell contacts and the formation of lamellipodia are
important factors for apical lumen morphogenesis.
Taken together, the data indicate that OSM is closely involved in
controlling
plasma
membrane
polarity
development
by
regulating
membrane traffic, cell cycle control and cell-cell adhesion, as summarized
in chapter 6, in which chapter some suggestion are presented for future
work.
46