Journal of Cell Science, 103, 1-8 (1992)
Printed in Great Britain © The Company of Biologists Limited, 1992
COMMENTARY
Endocytosis: what goes in and how?
COLIN WATTS
Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, UK
and MARK MARSH
MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK
Introduction
During the last decade the term 'endocytosis' has
become virtually synonymous with the activity of
clathrin-coated vesicles. These vesicles, which are
derived from cell surface clathrin-coated pits, are
transport vehicles responsible for the transfer of plasma
membrane receptors and their ligands, between the first
two stations of the endocytic pathway: namely, the
plasma membrane and early endosomes (Goldstein et
al., 1985; van Deurs et al., 1989; Griffiths and
Gruenberg, 1991). Despite the irrefutable evidence that
clathrin-coated vesicles mediate endocytosis, their
contribution to the total endocytic activity of the cell
and the composition of the membrane they internalise
remains controversial. Here we discuss: (1) the evidence that non-clathrin-mediated endocytic mechanisms operate alongside the clathrin-mediated pathway;
(2) the evidence that endocytosis occurs for surface
molecules that are not enriched in clathrin-coated pits
and; (3) the sorting activities of cell surface clathrincoated pits and the notion that plasma membrane
proteins that show particularly slow rates of uptake are
actively excluded from the endocytic pathway.
Clathrin and non-clathrin-mediated endocytosis
Endocytosis is usually considered to occur either
constitutively, by the continuous fluid-phase (pinocytic)
pathway, or by phagocytosis, a ligand-induced process
responsible for the uptake of large particles and not
discussed further here. The constitutive pathway is
mediated, at least in part, by clathrin-coated pits and
vesicles (herein referred to as coated pits and coated
vesicles), which bud continuously from the plasma
membrane and bring both the membrane and the fluid
content of the vesicle into the cell. The 0-COP
containing coats found on transport vesicles in the
exocytic pathway (Duden et al. 1991) are not known to
function in endocytosis and are not discussed in this
article.
The coated vesicle membrane can be enriched in
plasma membrane proteins, such as the receptors for
transferrin (Tf-R) and low density lipoprotein (LDLR), that interact with adaptor proteins of the coated pit
(Pearse and Robinson, 1990; Trowbridge, 1991). In
contrast, the fluid content of the vesicles has the same
composition as the extracellular medium. Thus constitutive coated vesicle formation can be responsible for
both receptor-mediated and fluid-phase endocytosis.
Experiments with Semliki Forest virus indicated that
Baby Hamster Kidney (BHK) cells can internalise,
1500-3000 coated vesicles/min (Marsh and Helenius,
1980). Significantly, the membrane area and fluid
volume contained by this number of vesicles could
account for most, if not all, of the membrane and fluidphase endocytosis measured in these and other cells (cf.
Steinman et al. 1976; Thilo and Vogel, 1980; Marsh and
Helenius, 1980) and suggested that coated vesicles are
the major vehicles of constitutive endocytosis. Subsequent experiments supported these conclusions.
Firstly, acidification of the cytosol, which blocks
clathrin-mediated endocytosis (see below), inhibited
most of the fluid-phase endocytosis in BHK cells
(Davoust et al. 1987; Cosson et al. 1989). Secondly,
morphometric quantification of the number of coated
pits at the plasma membrane, coupled with estimates of
the life time of coated pits (1-2 min), indicated that in
several cell lines the predicted rate of coated vesicle
formation could account for the bulk of constitutive
endocytosis (Griffiths et al. 1989; Pelchen-Matthews et
al. 1991). Thirdly, plasma membrane proteins lacking
endocytosis signals can be non-selectively included in
coated pits, and are internalised by bulk membrane
flow at rates which can be fully accounted for by coated
vesicle formation (Pelchen-Matthews et al. 1991; Miettinen and Mellman, 1989). Finally, in rat hepatocytes
the activation energies for fluid-phase and receptormediated endocytosis are essentially the same, suggesting that both forms of endocytosis are mediated by the
same vesicle population (Oka and Weigel, 1989).
Prior to the discovery of coated vesicles, fluid-phase
endocytosis was believed to occur through flask-shaped
Key words: endocytosis, clathrin, vesicles.
C. Watts and M. Marsh
invaginations of the plasma membrane and non-coated
vesicles (see, for example, Steinman et al. 1976; Huet et
al. 1980; Montesano et al. 1982; Hopkins et al. 1985).
Several recent reports have once again suggested that
clathrin-independent endocytic mechanisms make a
significant contribution to a cell's total constitutive
endocytic activity. In the first case, anti-clathrin
antibodies delivered to the cytoplasm of monkey
kidney-derived CV-1 cells inhibited receptor-mediated
and fluid-phase endocytosis by 40-50% (Doxsey et al.
1987). The failure to block all fluid-phase endocytosis
could reflect the technical problem of getting sufficient
antibody into all of the target cells; alternatively, it
could indicate the existence of clathrin-independent
endocytic mechanisms. Secondly, exposing various cells
to hypertonic medium, potassium depletion or mild
cytosolic acidification removes or paralyses the clathrin
lattice and almost completely inhibits the uptake of TfR, for example (Larkin et al. 1983; Daukas and
Zigmond, 1985; Sandvig et al. 1987; Heuser and
Anderson, 1989; Heuser, 1989). However, these treatments only partially inhibited fluid-phase endocytosis
and the uptake of ligands such as ricin in, for example,
human epidermoid carcinoma (HEp-2) cells, isolated
rat hepatocytes and NIH3T3 fibroblasts, in contrast to
BHK cells (reviewed by van Deurs et al. 1989; Oka et
al. 1989; Fuhrer et al. 1991). Thirdly, it has been known
for a number of years that some cells undergo a
dramatic stimulation of fluid-phase endocytosis following treatment with growth factors or phorbol esters
(Haigler et al. 1979; Phaire-Washington et al. 1980;
Swanson et al. 1985), or following microinjection of
p21 raj proteins (Bar-Sagi and Feramisco, 1986). Significantly, inhibitors of Na + /H + exchange, such as amiloride, block EGF-stimulated pinocytosis in A431 cells
without affecting the endocytosis of either EGF itself or
Tf (West et al. 1989; L. Hewlett and C. Watts,
unpublished), and clathrin-mediated uptake of Tf-R is
essentially unaffected during EGF-stimulated pinocytosis in A431 cells (Wiley, 1988; West et al. 1989;
Sandvig and van Deurs, 1990), indicating that the
increase in fluid-phase uptake is not due to enhanced
coated pit activity but to the induction of a distinct
endocytic pathway. Taken together these results
suggest that in certain cell types clathrin-independent
pinocytic mechanisms operate in addition to the coated
vesicle pathway.
What is known about these clathrin-independent
mechanisms? Firstly, Hansen et al. (1991) have
attempted to visualise the vesicles responsible (Fig. 1).
They made their observations in HEp-2 cells that were
incubated at 4°C then briefly (30-60 s) warmed to 37°C
prior to fixation. Their tracer molecules could be seen in
coated vesicles and in small (95 nm) non-coated 'preendosomal' vesicles (Fig. 1). The authors argue that the
vesicles arise from distinct non-coated invaginations of
the plasma membrane, that they are responsible for the
uptake of membrane and fluid in cells whose coated pit
activity has been arrested, and that they occur constitutively in the absence of any perturbation. Given the brief
lifetime of endocytic coated vesicles, it is also possible
that these profiles are coated vesicles that have become
uncoated, or recycling vesicles. Furthermore, the EM
analysis alone does not give an indication of the
prevalence of these non-coated vesicles or their turnover times.
The nature of the vesicles involved in growth factorand phorbol ester-induced pinocytosis have also been
analysed morphologically (Fig. 2). These appear as
large (up to several fim in diameter) vacuoles that arise
adjacent to areas of the plasma membrane showing
pronounced growth factor-stimulated ruffling activity
(West et al. 1989; Racoosin and Swanson. 1989), and
similar to previously described macropinocytic vesicles
(Lewis, 1931; Abercrombie and Ambrose, 1958; Brunk
et al. 1976; Chinkers et al. 1979). These macropinosomes have a greater volume/surface area ratio than
coated vesicles or the vesicles observed by Hansen et al.
(1991). This may explain why, relative to the basal
uptake rates, volume uptake is stimulated 6- to 10-fold
by growth factors (Haigler et al. 1979; West et al. 1989)
whereas the uptake of membrane-bound markers such
as ricin is increased only 1.5- to 2-fold (Sandvig and van
Deurs, 1990). If the constitutive endocytosis observed
under conditions of cytosolic acidification or increased
tonicity can be attributed to the structures seen by
Hansen et al. (1991), then on the basis of vesicle size,
site of formation and sensitivity to amiloride, the
growth factor-induced macropinosomes should be considered a distinct endocytic pathway.
In contrast to the morphological studies, experiments
with various drugs suggest some mechanistic similarities
between clathrin-independent pinocytosis and macropinocytosis. Both of these processes, and phagocytosis as
well, are inhibited by similar concentrations of cytochalasin D (CCD) and colchicine (Racoosin and
Swanson, 1989; Sandvig and van Deurs, 1990; but see
also Haigler et al. 1979), drugs which suggest a role for
microfilaments and microtubules but which have no
significant effect on receptor-mediated endocytosis
(Marsh and Helenius, 1980; Sandvig and van Deurs,
1990). Since phagocytosis and macropinocytosis are
inducible processes (by particles and growth factors,
respectively), it is possible that the CCD/colchicinesensitive process responsible for clathrin-indepedent
pinocytosis might also be induced by the treatments
which inhibit coated vesicle formation.
One difficulty in assessing the recent work is that the
data have been obtained from different cell types using
various experimental systems, and perturbations are
frequently used to observe specific forms of endocytosis. These perturbations are notoriously tricky; they
are not necessarily specific for the clathrin-mediated
processes (see Cosson et al. 1989), the effects may be
variable in different cell types and, depending on the
specific tracer, may alter the properties of endocytic
markers. Acidification of the cytosol is counteracted by
most cells and the treatments can have significant
effects on cell morphology and viability. Aside from
ligand-induced macropinocytosis and phagocytosis, it is
unclear why cells require additional endocytic mechanisms.- If the clathrin-dependent and -independent
Endocytosis
8
Fig. 1. Non-coated endocytic vesicles in HEp-2 cells. HEp-2 cells were chilled to 4°C and incubated for 120 min with 5 nm
colloidal gold-labelled Concanavalin A (Con A). After washing with PBS at 4°C, the cells were rapidly wanned to 37°C for
30 s and then immediately treated with ice-cold fixative. After rinsing, the cells were further incubated with rabbit-anti-Con
A antibodies conjugated to horseradish peroxidase (HRP) followed by diaminobenzidine (DAB) cytochemistry and finally
processed for electron microscopy. With the protocol, non-internlaized Con A-gold particles are tagged by the HRPconjugated anti-Con A antibodies and are thus surrounded by a cloud of DAB reaction product. In contrast, endocytosed
Con A/gold is inaccessible to the HRP-conjugated anti-Con A antibodies and therefore remains unlabelled. The figure
shows sections 1, 3, 6 and 8 from a series of 8 serial sections of a non-coated endocytic vesicle. A statistical analysis
revealed that the mean diameter of non-coated endocytic vesicles was significantly smaller than that of clathrin-coated
vesicles (see Hansen et al. 1991, for further details).
pathways transfer their membrane and content to the
same endosomes Tran et al. (1987), why should the cell
sort membrane components into different endocytic
vesicles only to remix them at the next endocytic
station? The evidence that the two pathways might
mediate the uptake of different subsets of plasma
membrane components is not established and with the
growing evidence that coated vesicles may be able to
internalise a wider range of plasma membrane components than previously thought (see below), it appears
that the clathrin pathway alone could cope with
constitutive endocytic requirements. One situation in
which the cell may need additional endocytic capacity
could occur following periods of stress, in which case
the conditions used to block the clathrin pathway might
be sufficient to induce an alternative mechanism.
The evidence as it stands suggests that tissue culture
cells and ex vivo cells such as macrophages (clathrin-
independent endocytosis has yet to be demonstrated in
situ) may contain multiple mechanisms of pinocytosis.
It is clear that the coated vesicle pathway operates
constitutively. However, it remains unclear whether the
other pathways have constitutive activity or occur only
following induction. Macropinosomes can form occasionally in the absence of EGF in A431 cells and,
given their large size, may make a significant contribution to clathrin-independent pinocytosis (L. Hewlett,
A. Prescott and C. Watts, unpublished), but it is not
clear that this is a general property of all cells. Whether
each of the routes represents a biochemically distinct
mechanism is also unclear - it could be argued that the
different pathways are driven by one or the other of two
basic mechanisms; the first clathrin, and the second an
actin/tubulin-based mechanism. The latter system
would mediate phagocytosis and, when cells are
stimulated with growth factors or stressed by having the
C. Watts and M. Marsh
understanding of how endocytic activity is regulated,
especially during periods of stress, how the sizes of
endocytic vesicles are controlled and the extent to
which clathrin-independent mechanisms contribute to
total endocytic activity, still needs to be established.
Recently, Rothberg et al. (1990a,b) have reported
that folate receptors, which mediate the accumulation
of 5-methyltetrahydrofolic acid and are anchored to the
plasma membrane by a glycosylphosphatidyl inositol
(GPI) linkage, are clustered into cholesterol-rich
domains of the plasma membrane. By EM these
domains appear as pits which are coated on their
cytoplasmic aspects with protein complexes containing
a 22 kDa ppoXT0 substrate (Rothberg et al. 1992) and
which resemble the plasmalemmal vesicles or caveoli
observed in capillary endothelial and smooth muscle
cells (Palade, 1953; Fawcett, 1965; Severs, 1988).
Intriguingly, a fraction of the receptors appears to be
cryptic. They can be detected at 37°C but not at 4°C,
and are seemingly in equilibrium with the cell surface.
However they cannot be detected morphologically in
intracellular vesicles. To explain these results Rothberg
et al. (1990a,b) suggested that the caveoli can transiently seal without scission from the plasma membrane, to allow the transfer of bound folate across the
membrane of the invagination. If the caveoli do not
leave the cell surface their role in constitutive endocytosis is limited. Clearly, more work will be required to
establish whether caveoli exist on all cell types, whether
they have functions in addition to the uptake of folate
and how they are related to other endocytic vesicles.
Inasmuch as the proposed sealing/unsealing mechanism
would not contribute to net fluid-phase and membrane
endocytosis, the caveoli should be considered distinct
from the clathrin-dependent and -independent endocytic pathways discussed above.
Beyond the parameters of the transferrin
receptor cycle
Fig. 2. Macropinocytic vesicles in EGF-stimulated A431
cells. A431 cells were stimulated with 100 ng/ml EGF in
the presence of 5 mg/ml FITC-dextran for 6 min.
(A) Phase-contrast. (B) Same cells viewed by
epifluorescence microscopy. Note formation of varioussized macropinosomes especially at the cell margins (photo
by L. Hewlett).
clathrin-mediated pathway shut down, could also
mediate macro- and other forms of pinocytosis. Despite
a growing interest in clathrin-independent endocytosis,
the coated vesicle system still remains the bestunderstood endocytic mechanism. Nevertheless, an
The well-characterised receptors which mediate the
uptake of ligands such as transferrin (Tf), LDL and
various growth factors comprise a relatively small
proportion (10%) of the protein content of the plasma
membrane (Hubbard, 1989), and much of what we
know about constitutive endocytosis is based on studies
of these receptors. For receptors such as the Tf-R, the
cell surface pool of receptor rapidly exchanges with an
intracellular population which may comprise 50-70% of
the total receptor complement. As a consequence,
endocytosis of these molecules is relatively easy to
demonstrate because a large fraction can be labelled on
the cell surface and subsequently found within the cell
at steady state (Bleil and Bretscher, 1982). However,
endocytosis is also involved in turning over, redistributing and regulating the cell surface expression of the
90% of surface proteins that are not represented by the
Tf-R. Furthermore, the internalisation of these molecules can influence, for example, cell-cell interactions,
cell adhesion and cell movement. Measuring the
Endocytosis
endocytosis of these molecules can, however, be
difficult. We have often encountered the misconception
that when only a small percentage of a specific cell
surface protein can be detected intracellularly at steady
state, endocytosis of these molecules is considered to be
either insignificant or limited to a minor proportion of
the surface pool. This might be true in some cases, but it
does not follow from the small intracellular pool size.
As in a metabolic pathway the level of a particular
intermediate says nothing about the rate of flux through
that step, or about the fraction of molecules taking part
in the flux. This point is underlined by the LDL-R,
which usually has a small intracellular pool size (Basu et
al. 1981; Bretscher and Lutter, 1988), but which is
internalised and recycled efficiently (Goldstein et al.
1985). Recently there has been an increase in the range
of reagents that can be used for measuring the
endocytosis of cell surface molecules, so that now
intemalisation can be detected for molecules which
have hitherto been regarded as non-endocytic.
Many cell surface molecules do not have readily
available soluble ligands and must be labelled by other
means. Antibodies can be used as surrogate soluble
ligands, though care should be taken to ensure that the
multivalency of immunoglobulins does not modify the
trafficking properties of the relevant antigen (see
Mellman et al. 1984). As an alternative to soluble
ligands, methods of covalently modifying cell surface
glycoproteins have been improved. The range of
techniques now includes multiple means of radioiodination (see, for example, Bleil and Bretscher, 1982;
Watts, 1985; Bretscher and Lutter, 1988), labelling of
carbohydrate groups (Thilo and Vogel, 1980), and
biotinylation (Le Bivic et al. 1990; Pelchen-Matthews et
al. 1991). These can be combined with enzymic
modifications, such as neuraminidase or protease
treatment, to distinguish between cell surface and
intracellular antigens (Krangel, 1987; Davis and Cresswell, 1990).
The usefulness of these labelling procedures is
determined by the efficiency with which the internalised
marker (if any) can be distinguished from what remains
on the cell surface (i.e. the label remaining on the
surface should be quantitatively removable) and the
background of non-removable label will limit how
accurately the internal pool size can be measured. In
addition, if one wants to analyse recycling after
stripping the surface label, the means of removal should
be as mild as possible. Cells subjected to proteolysis or
acid-stripping are often compromised, though conditions can be found to limit the adverse effects of these
treatments (see Pelchen-Matthews et al. 1989). One
recent innovation which allows mild removal of the cell
surface label uses iodinated succinimide ester-based
reagents. A disulphide bond separates the iodinated
phenolic ring from the reactive ester so that the label
can be removed by mild reduction (Bretscher and
Lutter, 1988; Bretscher, 1992). These reagents have
proved useful in the analysis of endocytosis and
recycling of the major histocompatibility (MHC) glycoproteins on human lymphoblastoid cells (Reid and
Watts, 1990) and members of the integrin family
(Bretscher, 1992). For class II MHC molecules on
human lymphoblastoid cells 7% of the labelled cell
surface molecules equilibrated with an intracellular
pool as judged by protection from the reducing agent
(Reid and Watts, 1990). Variable pool sizes (8-20%)
and rates of uptake (1-4%/min) were observed for
members of the integrin family though, interestingly,
intemalisation of some integrins, for example LFA-1,
was undetectable (Bretscher, 1992).
Taken together these studies have shown that class II
MHC, members of the integrin family and other cell
surface molecules do endocytose and recycle, and that
endocytosis is not restricted to the subset of cell surface
receptors typified by Tf-R. However, their rates of
uptake from the cell surface are significantly slower
than those of the Tf-R and efficient recycling prevents
the accumulation of large intracellular pools.
Positive, neutral and negative selection in
endocytosis
The ability to measure accurately the intemalisation of
slowly internalised molecules has increased the spectrum of cell surface components known to undergo
endocytosis. Although the possibility exists that in
certain circumstances some of this uptake may occur by
the clathrin-independent mechanisms discussed above,
it is clear in several cases that slowly internalised
antigens are taken up through coated pits (see below).
This finding raises the question of how selective a filter
coated pits are.
Early analyses of receptor-mediated endocytosis
indicated that the concentration of receptors, such as
the LDL-R, was manyfold higher in coated pits than
over the rest of the plasma membrane (see Goldstein et
al. 1985, for review). Subsequently, the cytoplasmic
domains of these receptors have been found to contain
sequences which enable them to interact with components of the clathrin lattice (reviewed by Trowbridge,
1991). The concentration of certain molecules into
coated pits suggested that others might be excluded,
thereby providing coated pits with the properties of a
molecular filter. Evidence in support of this notion was
provided by an analysis of the GPI-linked Thy-1
antigen, which was found to be largely excluded from
cell surface coated pits (Bretscher et al. 1980).
Does this exclusion occur because coated pits are
filled with receptors containing intemalisation motifs?
Morphological analyses have indicated that a number of
different cell surface receptors can cluster into coated
pits. However, quantitative data on the degree of
clustering and the correlation of these estimates with
measured endocytosis rates are limited. Estimates for
the LDL-R suggest that up to 70% of the receptor may
cluster into pits, but recent analyses of Tf-R in
transfected chicken fibroblasts, HEp-2, HeLa, T47D
and A431 cells, and the HI chain of the asialoglycoprotein receptor (ASGP-R), indicate that only 6-15% of
the cell surface molecules are in coated pits at a given
C. Watts and M. Marsh
time (Miller etal. 1991;Fuhreretal. 1991; Hansenet al.
1992). As coated pits account for 1.2-2% of the cell
surface (Anderson et al. 1977; Griffiths et al. 1989;
Pelchen-Matthews et al. 1991; Hansen et al. 1992),
these data suggest that Tf-R is concentrated only 3- to
12-fold in coated pits. Furthermore, as the life time of a
coated pit at the cell surface is estimated to be 1-2 min
(see above), a 3- to 12-fold concentration in pits fits well
with the rates of Tf internalisation measured in
different cell types (~15% of the surface pool/min).
Thus the density of positively selected cell surface
receptors in coated pits may not be as high as had
previously been thought.
Is the density of receptors in coated pits sufficient to
exclude proteins lacking internalisation signals? When
the T cell differentiation antigen CD4 is expressed in
non-lymphoid cells, the molecules are clustered 3- to 6fold into coated pits and internalised relatively efficiently in coated vesicles (2.5-6% of the cell surface
pool/min; Pelchen-Matthews et al. 1989, 1991, 1992).
There is no tyrosine in the cytoplasmic domain of CD4
and the one phenylalanine does not influence the rate of
uptake (Pelchen-Matthews, A. and Marsh. M., unpublished), indicating that the internalisation signal is
different to the tyrosine-containing signals found in TfR and LDL-R (see also Miettinen et al. 1992). In T
lymphocytes, in contrast, CD4 does not undergo
efficient endocytosis; for a range of T cell lines the rates
are <0.5% of the surface pool/min and electron
microscopy indicates that the CD4 molecules are
excluded from coated pits (Pelchen-Matthews et al.
1991). However, when mutant forms of CD4, lacking
the majority of the cytoplasmic domain, are expressed
in both lymphoid and non-lymphoid cells the molecules
are internalised at similar rates (approx. 1.2% of the
surface pool/min) and to similar extents. EM analysis
shows that the tail-minus molecules are present on
coated and non-coated membrane at the same density,
i.e. they are neither concentrated into, nor excluded
from, coated pits. Furthermore, at steady state, when
endocytosis is balanced by recycling and the intracellular and cell surface pools are equilibrated, the endosome to cell surface ratio of tail-minus CD4 is about 1:5;
very similar to the ratio of the surface areas of the early
endosomes and the plasma membrane (Griffiths et al.
1989; Pelchen-Matthews et al. 1991).
These data illustrate several important points.
Firstly, they indicate that the clustering of endocytic
receptors into coated pits is not sufficient in all cases to
exclude proteins lacking internalisation signals. This
observation is not unique to CD4. Experiments with the
Tf-R, the HI chain of the ASGP-R, the LDL-R, the
murine FcRII-B2 and the cation-dependent mannose 6phosphate receptor, for example, show that when the
cytoplasmic domain is removed, or the internalisation
signal disrupted, basal levels of endocytosis similar to
those measured for tail-minus CD4 molecules are
observed (Fuhrer et al. 1991; Jing et al. 1990; Miettinen
and Mellman, 1989; Johnson et al. 1990; Chen et al.
1990). This uptake appears to occur by non-specific,
bulk flow endocytosis and, at least in the case of tail-
minus forms of CD4 and FcRII, occurs through coated
pits (Pelchen-Matthews et al. 1991; Miettinen and
Mellman, 1989).
Secondly, molecules which fail to undergo endocytosis may require active mechanisms to keep them
out of coated pits. How is CD4 endocytosis prevented
in T cells? The protein tyrosine kinase p56 , which is
expressed specifically in T lymphocytes and which
interacts with the cytoplasmic domain of CD4, prevents
the entry of CD4 into coated pits (hence the ability of
tail-minus CD4 to internalise in T cells; PelchenMatthews et al. 1992). Furthermore, expression of
p56/c* in CD4 positive non-lymphoid cells inhibits CD4
association with coated pits and internalisation.
Whether the association of p56/cAr with CD4 hides the
endocytosis signal is unclear. Such an association
should reduce CD4 uptake to a rate equivalent to that
of the cytoplasmic domain-deleted form of CD4. In fact
p56'c* reduces CD4 uptake below the level of tail-less
mutants of CD4 and appears to act as a retention
mechanism to prevent CD4 entry into coated pits. How
this occurs is unclear, but preliminary data indicate that
p56/c* may directly, or indirectly, interact with elements
of the cortical cytoskeleton, possibly by means of the
conserved src-homology domains (Pawson, 1988). Significantly, the murine FcRII has two isoforms, the
endocytic form (FcRII-B2) that is found on macrophages, and a second form (FcRII-Bl) found on B cells.
This second form contains a 47 amino acid insertion into
the cytoplasmic domain, which reduces the endocytosis
of the receptor to levels less than that of a tail-minus
mutant (Miettinen and Mellman, 1989). As with
CD4/p56/c*, the insertion appears to prevent the FcR
molecules from entering into coated pits and evidence
again suggests that this form of the receptor may
interact with cytoskeletal elements (Miettinen et al.
1992).
Thirdly, the cytoplasmic domains of cell surface
molecules can have both positive and negative signals
controlling endocytosis, and these signals can be
interpreted differently in different cellular environments. For CD4 one signal, for clustering into coated
pits, is seen in non-lymphoid cells or following
dissociation of p56/c*, and a second, for exclusion from
coated pits, is seen in T cells in association with p56'c*.
Removal of the cytoplasmic domain removes both
signals so that the molecules are internalised by bulk
flow. The LDL-R and the FcRII-Bl molecule also
appear to contain both positive and negative endocytosis signals (Pathak et al. 1990; Miettinen and
Mellman, 1992).
How do these observations fit with GPI-linked
proteins that lack cytoplasmic sequences, undergo very
little endocytosis and appear to be excluded from
coated pits (Bretscher et al. 1980; Lemansky et al.
1990)? Solubility studies and diffusion measurements
suggest that significant amounts of various GPI-linked
molecules are not free in the bilayer but are tethered in
some way. Whether this occurs through sequestration
of the molecules into, for example, lipid-rich domains
in the plasma membrane, as reported for the folate
Endocytosis
receptor and other GPI-linked proteins (Rothberg et al.
1990b; Brown and Rose, 1992), or the GPI-linked
proteins interact with other transmembrane proteins, is
unclear (Robinson, 1991). However, one recent, and
yet to be repeated result, suggests that in T cells GPIlinked membrane proteins may indirectly interact with
p56'c* or related tyrosine kinases (Stefanovd et al.
1991).
The idea that certain plasma membrane proteins
associate with the cortical cytoskeleton is well established (see, for example, Hammerton et al. 1991).
Nevertheless, the concept that these interactions regulate the endocytic properties of cell surface molecules is
often forgotten. If molecules that are not endocytosed
are actively excluded from coated pits, how selective is
the clathrin system? It could be argued that the function
of endocytic coated vesicles is to select a subset of cell
surface receptors that require rapid and efficient
endocytosis, and that they do not play a significant role
in preventing the movement of cell surface proteins out
of the plasma membrane. The retention of proteins in
the plasma membrane being mediated by other mechanisms such as association with the cortical cytoskeleton. These arguments are of course compromised by
the possibility that there is more than one pathway of
constitutive endocytosis, and that clathrin-independent
endocytosis makes a significant contribution to the turnover of certain subsets of cell surface molecules. It is
clear that a number of important and fundamental
questions need to be answered before we have a full
understanding of how endocytosis operates.
We thank our colleagues Colin Hopkins, Dan Cutler,
Annegret Pelchen-Matthews, Pamela Reid, Bo van Deurs,
Steen Hansen, Mark Bretscher and Barbara Pearse for critical
comments on the manuscript. We thank Bo van Deurs, Steen
Hansen and Lindsay Hewlett for providing the Figs. M.M. is
supported by grants from the Medical Research Council and
the Leukaemia Research Fund, and C.W. by the Medical
Research Council and the Wellcome Trust.
References
Abercrombie, M. and Ambrose, E. J. (1958). Interference microscope
studies of cell contacts in tissue culture. Exp. Cell Res. 15,332-345.
Anderson, R. G. W., Brown, M. S. and Goldstein, J. L. (1977). Role
of the coated endocytic vesicle in the uptake of receptor bound low
density lipoprotein in human fibroblasts. Cell 10, 351-364.
Bar-Sagi, D. and Feramisco, J. R. (1986). Induction of membrane
ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras
proteins. Science 233, 1061-1068.
Basu, S. K., Goldstein, J. L., Anderson, R. G. W. and Brown, M. S.
(1981). Monensin interrupts the recycling of low density lipoprotein
receptors in human fibroblasts. Cell 24, 493-502.
Bleil, J. D. and Bretscher, M. S. (1982). Transferin receptor and its
recycling in H e U cells. EMBO J. 1, 351-355.
Bretscher, M. S. (1992). Circulating integrins: a5/Jl, o6/J4 and Mac-1,
but not o3£l, o4/Sl or LFA-1. EM BO J. 11, 405-410.
Bretscher, M. S. and Lutter, R. (1988). A new method for detecting
endocytosed proteins. EMBO J. 7, 4087-4092.
Bretscher, M. S., Thomson, J. N. and Pearse, B. M. F. (1980). Coated
pits act as molecular filters. Proc. Nat. Acad. Sci. USA 77, 41564159.
Brown, D. A. and Rose, J. K. (1992). Sorting of GPI-anchored
proteins to glycolipid-enriched membrane subdomains during
transport to the apical cell surface. Cell 68, 533-544.
Bronk, U., Schellens, J. and Westermark, B. (1976). Influence of
epidermal growth factor (EGF) on ruffling activity, pinocytosis and
proliferation of cultivated human glia cells. Exp. Cell Res. 103,295302.
Chen, W.-J., Goldstein, J. L. and Brown, M. S. (1990). NPXY, a
sequence often found in cytoplasmic tails is required for coated pitmediated internalisation of the low density lipoprotein receptor. /.
Biol. Chem. 265,3116-3123.
Clunkers, M., McKanna, J. A. and Cohen, S. (1979). Rapid induction
of morphological changes in human carinoma cells A-431 by
epidermal growth factor. J. Cell Biol. 83, 260-265.
Cosson, P., de Curtis, I., Pouyssegur, J., Griffiths, G. and Davoust, J.
(1989). Low cytoplasmic pH inhibits endocytosis and transport
from the trans-Golgi network to the cell surface. J. Cell Biol. 108,
377-387.
Daukas, G. and Zigmond, S. H. (1985). Inhibition of receptormediated but not fluid phase endocytosis in polymorphonuclear
leukocytes. J. Cell Biol. 101, 1673-1679.
Davis, J. and Cresswell, P. (1990). Lack of detectable endocytosis of
B lymphocyte MHC class II anigens using an antibody independent
technique. J. Immunol. 144, 990-997.
Davoust, J., Gruenberg, J. and Howell, K. (1987). Two threshold
values of low pH block endocytosis at different stages. EMBO J. 6,
3601-3609.
Doxsey, S. J., Brodsky, F. M., Blank, G. S. and Helenius, A. (1987).
Inhibition of endocytosis by anti-clathrin antibodies. Cell 50, 453463.
Duden, R., Griffiths, G., Frank, R., Argos, P. and Krels, T. E. (1991).
Beta-COP, a 1l0kD protein associated with non-clathrin-coated
vesicles and the Golgi complex, shows homology to Beta-adaptin.
Cell 64, 649-665.
Fawcett, D. W. (1965). Surface specializations of absorbing cells. J.
. Histochem. Cytochem. 13, 75-90.
Fuhrer, C , GefTen, I. and Speiss, M. (1991). Endocytosis of the
ASGP receptor HI is reduced by mutation of tyrosine-5 but still
occurs via coated pits. J. Cell Biol. 114, 423-431.
Goldstein, J. L., Brown, M. S,, Anderson, R. G. W., Russell, D. and
Schneider, W. (1985). Receptor mediated endocytosis: concepts
emerging from the LDL receptor system. Annu. Rev. Cell Biol. 1,
1-39.
Griffiths, G., Back, R. and Marsh, M. (1989). A quantitative analysis
of the endocytic pathway in Baby Hamster Kidney cells. J. Cell
Biol. 109, 2703-2720.
Griffiths, G. and Gruenberg, J. (1991). The arguments for preexisting early and late endosomes. Trends Cell Biol. 1, 5-9.
Haigler, H. T., McKanna, J. A. and Cohen, S. (1979). Rapid
stimulation of pinocytosis in human carcinoma cells A-431 by
epidermal growth factor. J. Cell Biol. 83, 82-90.
Hammerton, R. W., Krzeminskl, K. A., Mays, R. W., Ryan, R. A.,
Wollner, D. A. and Nelson, W. J. (1991). Mechanisms for
regulating cell surface distribution of Na + , K + -ATPase in polarized
epithelial cells. Science 254, 847-850.
Hansen, S. E., Sandvig, K. and van Deurs, B. (1991). The
preendosomal compartment comprises distinct coated and
noncoated endocytic vesicle populations. J. Cell Biol. 113, 731-741.
Hansen, S. E., Sandvig, K. and van Deurs, B. (1992). Internalisation
efficiency of the transferrin receptor. Exp. Cell Res. 199, 19-28.
Heuser, J. (1989). Effects of cytoplasmic acidification on clathrin
lattice morphology. J. Cell Biol 108. 401-411.
Heuser, J. E. and Anderson, R. G. W. (1989). Hypertonic media
inhibit receptor-mediated endocytosis by blocking clathrin-coated
pit formation. J. Cell Biol. 108, 389-400.
Hopkins, C. R., Miller, K. and Beardmore, J. M. (1985). Receptormediated endocytosis of transferrin and epidermal growth factor
receptors: A comparison of constitutive and ligand-induced
uptake. J. Cell Sci. Suppl. 3, 173-186.
Hubbard, A. L. (1989). Endocytosis. Curr. Opin. Cell Biol. 1, 675683.
Huet, C , Ash, J. F. and Singer, S. J. (1980). The antibody-induced
clustering and endocytosis of HLA antigens on cultured human
fibroblasts. Cell 21, 429-438.
Jing, S., Spencer, T., Miller, K. M., Hopkins, C. R. and Trowbridge,
I. S. (1990). Role of human transferrin receptor cytoplasmic
C. Watts and M. Marsh
domain in endocytosis; localization of a specific signal sequence for
internalisation. J. Cell Biol. 110, 283-294.
Johnson, K. F., Chan, W. and Kornfeld, S. (1990). Cation-dependent
mannose 6-phophate receptor contains two internalisation signals
in its cytoplasmic domain. Proc. Nat. Acad. Sci. USA 87, 1001010014.
Krangel, M. S. (1987). Endocytosis and recycling of the T3-T cell
receptor complex. The role of T3 phosphorylation. J. Exp. Med.
165, 1141-1159.
Larkin, J. M., Brown, M. S., Goldstein, J. L. and Anderson, R. G.
W. (1983). Depletion of intracellular potassium arrests coated pit
formation and receptor-mediated endocytosis in fibroblasts. Cell
33, 273-285.
Le Bivic, A., Sambuy, Y., Mostov, K. and Rodriguez-Boulan, E.
(1990). Vectorial transport of an endogenous apical membrane
sialoglycoprotein and uvomorulin in MDCK cells. J. Cell Biol. 110,
1533-1539.
Lemansky, P., Fateml, S. H., Gorican, B., Meyale, S., Rossero, R.
and Tartakoff, A. M. (1990). Dynamics and longevity of the
glycoplipid-anchored membrane protein, Thy-1. /. Cell Biol. 110,
1525-1531.
Lewis, W. H. (1931). Pinocytosis. Bull. Johns Hopkins Hosp. 49, 1727.
Marsh, M. and Helenius, A. (1980). Adsorptive endocytosis of semliki
forest virus. /. Mot. Biol. 142, 439^*54.
Mellman, I., Plutner, H. and Ukkonen, P. (1984). Internalisation and
rapid recycling of macrophage Fc receptors tagged with
monovalent antireceptor antibody: possible role of a prelysosomal
compartment. J. Cell Biol. 98, 1163-1169.
Miettinen, H., Matter, K., Hunziker, W., Rose, J. K. and Mellman, I.
(1992). Fc receptor endocytosis is controlled by a cytoplasmic
domain determinant that actively prevents coated pit localization.
J. Cell Biol. 116, 875-888.
Miettinen, H. and Mellman, I. S. (1989). Fc receptor isoforms exhibit
distinct abilities for coated pit localization as a result of cytosplamic
domain heterogeneity. Cell 58, 317-327.
Miller, K., Shipman, M., Trowbridge, I. S. and Hopkins, C. (1991).
Transferrin receptors promote the formation of clathrin lattices.
Cell 65, 621-632.
Montesano, R., Roth, J., Robert, A. and Orci, L. (1982). Non-coated
membrane invaginations are involved in binding and
intermalization of cholera and tetanus toxins. Nature 296, 651-653.
Oka, J. A., Christensen, M. D. and Weigel, P. H. (1989).
Hyperosmolarity inhibits galactosyl receptor-mediated but not
fluid phase endocytosis in isolated rat hepatocytes. /. Biol. Chem.
264, 12016-12024.
Oka, J. A. and Weigel, P. H. (1989). The pathways for fluid phase and
receptor-mediated endocytosis in rat hepatocytes are different but
thermodynamically equivalent. Biochem. Biophys. Res. Commun.
159, 488-494.
Palade, G. E. (1953). The fine structure of blood capillaries. J. Appl.
Phys. 24, 1424.
Pathak, R. K., Yokode, M., Hammer, R. E., Hofmann, S. L., Brown,
M. S., Goldstein, J. L. and Anderson, R. G. W. (1990). Tissuespecific sorting of the human LDL receptor in polarized epithelia of
transgenic mice. J. Cell Biol. I l l , 347-359.
Pawson, T. (1988). Non-catalytic domains of cytoplasmic proteintyrosine kinases: regulatory elements in signal transduction.
Oncogene 3, 491-495.
Pearse, B. M. F. and Robinson, M. S. (1990). Clathrin, adaptors, and
sorting. Annu. Rev. Cell Biol. 6, 151-171.
Pelchen-Matthews, A., Armes, J. E., Griffiths, G. and Marsh, M.
(1991). Differential endocytosis of CD4 in lymphocytic and nonlymphocytic cells. J. Exp. Med. 173, 575-587.
Petcnen-Matthews, A., Armes, J. E. and Marsh, M. (1989).
Internalisation and recycling of CD4 transfected into HeLa and
NIH-3T3 cells. EMBO J. 8, 3641-3649.
Pdchen-Matthews, A., Boulet, I., Fagard, R., Littman, D. and
Marsh, M. (1992). CD4-p561ck interaction inhibits CD4
endocytosis. J. Cell Biol. 279-290.
Phaire-Washington, L., Silverstein, S. C. and Wang, E. (1980).
Phorbol myristate acetate stimulates microtubule and 10-nm
filament extension and lysosome redistribution in mouse
macrophages. /. Cell Biol. 86, 641-655.
Racoosin, E. L. and Swanson, J. A. (1989). Macrophage colonystimulating factor (rM-CSF) stimulates pinoytosis in bone marrow
derived macrophages. /. Exp. Med. 170, 1635-1648.
Reid, P. A. and Watts, C. (1990). Cycling of cell surface MHC
glycoproteins
through
primaquine-sensitive
intracellular
compartments. Nature 346, 655-657.
Robinson, P. J. (1991). Phosphatidylinositol membrane ahchors and
T-cell activation. Immunol. Today 12, 35-41.
Rothberg, K. G., Heuser, J. E., Donzell, W. C , Ying, Y.-S., Glenney,
J. R. and Anderson, R. G. W. (1992). Caveolin, a protein
component of caveolae membrane coats. Cell 68, 673-682.
Rothberg, K. G., Ying, Y.-S., Kamen, B. A. and Anderson, R. G. W.
(1990b). Clustering of the folate receptor and the presence of
caveolae are dependent on the presence of membrane cholesterol.
J. Cell Biol. I l l , 2931-2938.
Rothberg, K. G., Ying, Y., Kolhouse, J. F., Kamen, B. A. and
Anderson, R. G. W. (1990a). The glycophospholipid-linked folate
receptor internalizes folate without entering the clathrin-coated pit
endocytic pathway. /. Cell Biol. 110, 637-649.
Sandvig, K., Olsnes, S., Petersen, O. W. and van Deurs, B. (1987).
Acidification of the cytosol inhibits endocytosis from coated pits. J.
Cell Biol. 105, 679-689.
Sandvig, K. and van Deurs, B. (1990). Selective modulation and
endocytic uptake or ricin and fluid phase markers without
alteration in transferrin endocytosis. J. Biol. Chem. 265,6382-6388.
Severs, N. J. (1988). Caveolae: static inpocketings of the plasma
membrane, dynamic vesicles or plain artifact? J. Cell Sci. 90, 341348.
Stefanovii, I., Horejsf, V., Ansotegui, 1. J., Knapp, W. and
Stockinger, H. (1991). GPI-anchored cell surface molecules
complexed to protein tyrosine kinases. Science 254, 1016-1019.
Stelnman, R. M., Brodie, S. E. and Cohn, Z. A. (1976). Membrane
flow during pinocytosis. /. Cell Biol. 68, 665-687.
Swanson, J. A., Yirinec, B. D. and Silverstein, S. C. (1985). Phorbol
esters and horse radish peroxidase stimulate pinocytosis and
redirect the flow of pinocytosed fluid in macrophages. J. Cell Biol.
100, 851-859.
Thilo, L. and Vogel, G. (1980). Kinetics of membrane internalisation
and recycling during pinocytosis in Dictyostelium discoidcum.
Proc. Nat. Acad. Sci. USA 77, 1015-1019.
Tran, D., Carpentier, J-L., Sawano, F., Gorden, P. and Orci, L.
(1987). Ligands internalized through coated or noncoated
invaginations follow a common intracellular pathway. Proc. Nat.
Acad. Sci. USA 84, 7957-7961.
Trowbridge, I. S. (1991). Endocytosis and signals for internalisation.
Curr. Opin. Cell Biol. 3, 634-641.
van Deurs, B., Peterson, O. W., Olsnes, S. and Sandvig, K. (1989).
The ways of endocytosis. Int. Rev. Cytol. 117, 131-177.
Watts, C. (1985). Rapid endocytosis of the transferrin receptor in the
absence of bound transferrin. J. Cell Biol. 100, 633-637.
West, M. A., Bretscher, M. S. and Watts, C. (1989). Distinct
endocytotic pathways in EGF stimulated human carcinama A431
cells. J. Cell Biol. 109, 2731-2739.
Wiley, H. S. (1988). Anomalous binding of epidermal growth factor
to A431 cells is due to the effect of high receptor densities and a
saturable endocytic system. J. Cell Biol. 107, 801-810.
{Received II May 1992 - Accepted 29 May 1992)