Biochem. J. (2008) 412, 415–423 (Printed in Great Britain)
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doi:10.1042/BJ20080474
REVIEW ARTICLE
Focusing on clathrin-mediated endocytosis
Joshua Z. RAPPOPORT1
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Investigations into the mechanisms which regulate entry of
integral membrane proteins, and associated ligands, into the cell
through vesicular carriers (endocytosis) have greatly benefited
from the application of live-cell imaging. Several excellent recent
reviews have detailed specific aspects of endocytosis, such as
entry of particular cargo, or the different routes of internalization.
The aim of the present review is to highlight how advances in livecell fluorescence microscopy have affected the study of clathrinmediated endocytosis. The last decade has seen a tremendous
increase in the development and dissemination of methods for
imaging endocytosis in live cells, and this has been followed by a
dramatic shift in the way this critical cellular pathway is studied
and understood. The present review begins with a description
of the technical advances which have permitted new types of
experiment to be performed, as well as potential pitfalls of these
new technologies. Subsequently, advances in the understanding of
three key endocytic proteins will be addressed: clathrin, dynamin
and AP-2 (adaptor protein 2). Although great strides have clearly
been made in these areas in recent years, as is often the case,
each answer has bred numerous questions. Furthermore, several
examples are highlighted where, because of seemingly minor
differences in experimental systems, what appear at first to be
very similar studies have, at times, yielded vastly differing results
and conclusions. Thus this is an exceedingly exciting time to
study endocytosis, and this area serves as a clear demonstration
of the power of applying live-cell imaging to answer fundamental
biological questions.
INTRODUCTION
PtdIns(4,5)P2 , a phospholipid concentrated at sites of clathrinmediated endocytosis [23,24,34,35]. Important roles for actin
have also been demonstrated in the process of clathrin-mediated
endocytosis (see the Other proteins section below). Finally, the
nascent coated vesicle is severed from the plasma membrane
through a fission reaction thought to be mediated by dynamin
[18].
Until James Keen’s group was able to image fluorescent clathrin
in live cells in 1999 [36], nearly all previous investigations
of clathrin-mediated endocytosis had been performed in fixed
cells (e.g. using electron microscopy or immunocytochemistry),
through quantification of radiolabelled cargo uptake, or with
isolated coated vesicles [37]. Subsequently, there have been
numerous investigations employing live-cell imaging. These
studies have come out of nearly all of the above-mentioned
endocytosis laboratories, as well as from investigators for whom
live-cell fluorescence imaging has always been the tool of choice
(e.g. Christien Merrifield) [38,39]. The focus of the present review
is to specifically detail the entirely new perspective on questions
of clathrin-mediated endocytosis that has been permitted by
developments in high-resolution live-cell fluorescence imaging.
Finally, although several recent studies have elegantly employed
similar techniques to study endocytosis in Saccharomyces
cerevisiae, for the sake of clarity and brevity, this review focuses
upon investigations in mammalian systems [40–43].
‘Bristle-coated vesicles’ were first observed using electron
microscopy in 1964 by Roth and Porter [1]. However, it would
be over a decade before Barbara Pearse devised a method for
isolating coated vesicles, and employed it in the purification
of clathrin [2,3]. Along the way, there have been numerous
groundbreaking contributions in this area. These include the
exquisite electron microscopy of John Heuser, the structural
studies of Tomas Kirchhausen, as well as the identification and
analysis of cargo adaptors by people such as Margaret Robinson
and Linton Traub, including the heterotetrameric AP (adaptor
protein)-2 complex, in addition to a series of ‘alternative adaptors’
[4–10]. Work from researchers such as Sandra Schmid and Mark
McNiven has provided great insight into the understanding of
dynamin, a large regulatory GTPase involved in vesicle fission
[11–14]. Furthermore, endocytosis at the synapse has been a
focus of people such as Pietro De Camilli and Timothy Ryan
[15–18]. Finally, contributions from researchers including Harvey
McMahon, Peter McPherson, Alexander Sorkin, Francis Brodsky,
Lois Greene and Alexandre Benmerah, as well as many others,
have added to the understanding of clathrin-mediated endocytosis
[7,19–31].
The general progression of clathrin-mediated endocytosis has
been explicitly detailed in numerous reviews, and will only
be summarized here (Figure 1) [32,33]. Integral membrane
proteins (e.g. activated receptors) bind to cytosolic adaptors
which form a link to the clathrin lattice. Accessory proteins,
such as the GTPase dynamin, have the ability to affect the process of endocytosis, e.g. through promoting clathrin polymerization or inducing membrane curvature. Furthermore, both
adaptors and accessory proteins can have the capacity to bind
Key words: adaptor protein 2 (AP-2), clathrin, dynamin, endocytosis, fluorescent protein, live-cell imaging.
LIVE-CELL FLUORESCENCE IMAGING
In many types of live-cell studies, the use of chemical fluorophores
can be hindered by the barrier imposed by the plasma membrane.
However, in the case of endocytosis, the process being studied is
Abbreviations used: AP, adaptor protein; FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; GAK, cyclin
G-associated kinase; GFP, green fluorescent protein; siRNA, small interfering RNA; TIRFM, total internal reflection fluorescence microscopy.
1
email [email protected]
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J. Z. Rappoport
Figure 2
Imaging fluorescent clathrin
Elimination of out-of-focus emission in TIRFM (right) compared with epifluorescence imaging
of the same focal plane with the same objective lens (middle). A bright-field image of HeLa cells
expressing clathrin–dsRed is shown on the left. Images acquired in the laboratory of Sanford
Simon.
Figure 1
Schematic diagram for clathrin-mediated endocytosis
At sites of local PtdIns(4,5)P 2 enrichment, cargo clustering and membrane deformation
occur, aided by adaptor and accessory proteins respectively. Adaptor proteins also bind to
clathrin triskelia. The accessory protein dynamin is believed to facilitate fission of the nascent
clathrin-coated vesicle. Proteins and lipids are not drawn to scale. PE, phosphatidylethanolamine; PI4,5P2 , PtdIns(4,5)P 2 .
that of cargo entry. Consequently, fluorescent endocytic ligands
have provided a valuable resource in the study of clathrinmediated endocytosis [44,45]. Furthermore, as in many other
areas of biology, the use of endogenously fluorescent proteins
[e.g. GFP (green fluorescent protein)], has revolutionized the field
[46,47]. Thus usage of fluorescently tagged proteins of interest
(e.g. ligands, receptors, adaptors, and accessory and coat proteins)
can be extremely powerful, assuming that the appropriate imaging
technology is employed.
The initial report utilizing GFP-tagged clathrin light chain
for live-cell imaging employed epifluorescence microscopy [36].
Even with a high NA (numerical aperture) objective lens,
which provides efficient light collection, as well as excellent
lateral and axial resolution, this configuration has the drawback
that out-of-focus light can contaminate the observed region of
interest (Figure 2). Thus, when imaging fluorescent clathrin
by epifluorescence microscopy, puncta can only generally be
resolved towards the cell periphery. This is due to the tremendous
contribution of intracellular signal outside of the plane of focus
resulting from clathrin at the trans-Golgi network, as well
as certain populations of endosomes [48,49]. Furthermore, if
intracellular structures can be imaged, how can one be sure that
plasma-membrane-associated clathrin is being tracked?
c The Authors Journal compilation c 2008 Biochemical Society
Although post-acquisition methodologies such as deconvolution can be applied to these types of dataset to assign
emitted light to the appropriate focal planes, drawbacks to these
approaches exist [50]. Generally speaking, the most useful form
of deconvolution involves reassignment of out-of-focus emission
based upon point-spread functions either determined empirically
or calculated. However, this requires the acquisition of images at
multiple focal places, with precise z-stepping. This can greatly
increase the time between acquisition of successive images at
the focal plane of interest, which can make deconvolution timeprohibitive in studies of events occurring in live cells. Finally,
the reassignment of out-of-focus light in ‘nearest neighbour’
deconvolution algorithms precludes many forms of quantitative
data analysis, as pixel value read-out no longer corresponds
directly to emission from the sample [50].
Confocal microscopy has also been applied in certain studies
of clathrin-mediated endocytosis [30,31,51]. This technique has
the benefit of optical sectioning: pinhole optics prevent the
collection of out-of-focus light originating outside of the ∼ 1μm-thick plane of interest, generally speaking. Thus it is easy
to see how this could avoid unintended imaging of intracellular
structures. However, confocal scanning of a whole-cell field with
high resolution can easily take 1 s or more. Thus, as with ‘threedimensional’ deconvolution, rapid events such as internalization
of nascent clathrin-coated vesicles cannot be readily resolved.
Because of these issues, application of confocal microscopy to
studies of individual events of clathrin-mediated endocytosis
has been limited. One place where confocal imaging has had
a major impact is the analysis of clathrin subunit exchange (see
the Imaging clathrin section below) [30,31]. However, even in
that area, other imaging methodologies have been shown more
recently to be potentially more appropriate [52].
It should be noted that several microscope companies
are currently producing ‘rapid-scan’ or ‘swept-field’ confocal
microscopes, which can increase acquisition rates by orders of
magnitude. One such configuration involves the use of a linescanning (rather than point-scanning) laser which is coupled to
a linear pixel-array-type camera. Images are then constructed
rapidly from the acquired series of lines. However, although
these types of microscope have been available for some time,
this technology has not been widely employed in cell biological
studies, and has yet to be applied to the analysis of clathrinmediated endocytosis [53]. One place where this type of imaging
could be very useful would be in the tracking of individual coated
vesicles from formation, to fission and subsequent internalization
and entry into the endocytic system.
Spinning (or Nipkow) -disk microscopy uses an array of
pinholes arranged on a disk that rapidly rotates [54]. This imaging
Clathrin-mediated endocytosis
Figure 3
Schematic diagram of total internal reflection
When the incident beam (arrow) hits the interface between medium 1 (high refractive index) and
medium 2 (low refractive index) at a super-critical angle, total internal reflection occurs. This
results in the production of the evanescent field (in yellow).
modality permits increased sampling rates relative to traditional
confocal microscopy. Furthermore, spinning-disk microscopes
employ cameras for image collection, while confocals use a
photomultiplier tube. Thus the experimenter has the added benefit
of being able to select an appropriate camera depending on
the particular characteristics of the experimental configuration
(e.g. sensitivity, pixel size and sampling rate). However, the gain
in speed requires a trade-off in confocality, and images from
spinning-disk microscopes generally suffer from some unwanted
collection of out-of-focus light, due in part to larger pinhole sizes.
Thus, as with epifluoresence and confocal microscopy, spinningdisk microscopy has been employed only in a few studies of
clathrin-mediated endocytosis in live cells [55].
TIRFM (total internal reflection fluorescence microscopy) has
proven to be very useful for studying events occurring at or near
the plasma membrane [56,57]. When incident light encounters an
interface with a medium of lower refractive index (n) at an angle
greater than the ‘critical angle’ (θ c ) defined by Snell’s law (sin
θ c = n2 /n1 ; n1 >n2 ), total internal reflection occurs (Figure 3). This
results in production of the ‘evanescent field’, a standing wave that
decays exponentially with distance from the interface between the
two media. The penetration depth (d) is defined as the distance
at which the intensity of the evanescent field decreases to 1/e. In
TIRFM, this is usually less than ∼ 100 nm, and depends upon the
refractive indices, angle of incidence and wavelength [57].
As fluorophores deeper into the cell are not excited, the images
that are collected have a very high signal-to-noise ratio due to
Figure 4
417
the elimination of out-of-focus light. Thus fluorophores entering the cell will be observed to disappear. In this way, events of
endocytosis can be observed directly (Figure 4). However, several
criteria have had to be satisfied in order to verify that clathrinmediated endocytosis was indeed imaged. (i) In order to ensure
that photobleaching is not interpreted as vesicle internalization,
neighbouring spots should be analysed alongside putative events
of endocytosis (e.g. Figure 4, spot B). (ii) Disappearance must
be observed to be progressive over successive frames, and
not be the result of lateral spot motility out of the field of view
[36,44,58]. (iii) To ensure that the dynamics of other clathrinpositive compartments (e.g. endosomes) are not being observed,
other markers for clathrin-mediated endocytosis (e.g. dynamin)
should be coincident with sites of clathrin internalization [38,59].
(iv) Cargo for clathrin-mediated endocytosis (e.g. ligands and
receptors) have to be present as well [39,45]. Thus, given the
sensitivity, relatively high frame rates (e.g. 5–10 frames/s), ability
to image multiple fluorophores simultaneously and quantitative
read-out, TIRFM has emerged as the methodology of choice
for imaging events of clathrin-mediated endocytosis in live cells
[38,59].
Furthermore, some studies have combined TIRFM with alternating images acquired via epifluoresence [13,26,38,39,60,61].
This powerful approach has the added benefit of permitting
imaging of events at the cell surface and deeper into the cytosol.
However, it should be noted that, as the epifluoresence and TIRFM
images have not been acquired simultaneously, care must be
taken in subsequent analysis. One area where this technique
has been particularly useful is the confirmation that clathrin
disappearing from the evanescent field, while still present in the
epifluorescence images, has not been lost due to photobleaching or
coat depolymerization before endocytosis [38,39]. However, once
lost from the evanescent field, all information collected will be
subject to the limitations of epifluorescence microscopy described
above.
Finally, one area that is developing particularly rapidly is that
of automated data analysis [39,55]. This provides the ability to
identify and quantify many more events than could be evaluated
manually. Thus greater statistical power can be applied to specific
questions still being addressed at the level of individual events.
However, it should be noted that automated data analysis carries
potential caveats which must be taken very seriously. Although
specific bias can be removed with regards to inclusion or exclusion
Disappearance of clathrin from the evanescent field
Spot A undergoes progressive internalization, while Spot B remains in place without loss of fluorescence intensity. Line-scan analysis depicts fluorescence across a single-pixel-thick line on the first
image over time. Region size = 2.7 μm × 2.8 μm. Data acquired in the laboratory of Sanford Simon.
c The Authors Journal compilation c 2008 Biochemical Society
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J. Z. Rappoport
Figure 6 Models for the formation of nascent clathrin-coated vesicles at
the plasma membrane
Figure 5 Separation and subsequent disappearance of a portion of a static
clathrin spot
Line-scan analysis depicts fluorescence across a single-pixel-thick line on the first image over
time. Region size = ∼ 2 μm2 . Data acquired in the laboratory of Sanford Simon.
of events on a case-by-case basis, this has now been replaced
by systematic decisions which can affect all data analysed. A
very interesting example of the complexity of automated data
analysis came with the publication of two papers analysing
similar questions, published in the same journal, less than 1
year apart [39,55]. Although it cannot be known to what extent
the vast differences in conclusions stemmed from the use of
different cell lines, microscopy modalities or automated data
analysis paradigms, this does provide evidence that all aspects of
methodology, including analysis regimes, can potentially affect
experimental outcomes.
IMAGING CLATHRIN
Numerous observations were made in the initial report by
Gaidarov et al. in 1999 [36]. These included the determination
that GFP-tagged clathrin light chain incorporates into clathrin
triskelia, localizes properly and does not perturb endocytosis.
Furthermore, it was determined that individual clathrin spots
could be seen to increase in intensity (forming coated pits),
decrease to background (internalization) and move laterally in the
plane of the plasma membrane (vesicle motility). Additionally,
the striking observation of a population of clathrin spots which
did not seem to disappear, but seemed to be sites from
which nascent clathrin-coated vesicles could bud and internalize,
was quite surprising. This result has been validated by numerous
subsequent studies employing TIRFM (Figure 5) [44,45,61].
However, the determination that specific sites on the plasma
membrane serve as hotspots for repetitive de novo coated-vesicle
formation has been more elusive.
Two general paradigms exist for the formation of nascent
clathrin-coated vesicles (Figure 6). The ‘iterative budding’ model
suggests that flat clathrin lattices are present on the cytosolic
leaflet of the plasma membrane from which coated vesicles
form repeatedly. This model draws support from both classical
electron microscopy studies, as well as more recent TIRFM data
(Figure 5) [33,39,44,45,61–64]. Interestingly, one report from the
group of Sandra Schmid clearly demonstrated that these types of
separation events are spatially and temporally correlated with
transient flashes of actin [61]. Thus localized increases in actin
concentration seem to occur when and where the nascent vesicle
c The Authors Journal compilation c 2008 Biochemical Society
The ‘iterative budding’ model depicts production of vesicles from long-lived static sorting
centres. The ‘de novo formation’ model illustrates sites of endocytosis as membrane patches
devoid of clathrin both before and after coated-vesicle production. Adapted from image generated
by Dr Marina Fix (Laboratory of Cellular Biophysics, Rockefeller University, New York, NY,
U.S.A.).
separates from the static spot [61]. One tantalizing hypothesis
arising from these types of observation is that cargo could be
sorted from flat clathrin lattices into adjacent forming vesicles.
However, direct evidence regarding this aspect of the model
is lacking. It is possible that these issues will be more clearly
elucidated by the application of new imaging techniques (see the
Future directions section below).
Evidence for the apparent production of clathrin-coated vesicles
from bare membrane initially, and subsequently, devoid of
clathrin (‘de novo formation’) has also been reported [38,55,59].
Ultimately, both mechanisms for coated-vesicle production most
likely occur. However, whether some cargo enters preferentially
through one pathway or the other has not been adequately
investigated. Finally, although it might seem logical that receptors
which undergo stimulated endocytosis following ligand binding
internalize through clathrin-coated vesicles formed de novo, this
has not been observed. Both G-protein-coupled receptors and the
epidermal growth factor receptor seem to cluster at pre-formed
clathrin spots following activation ([51,65] and J. Z. Rappoport
and S. M. Simon, unpublished work). Thus this would suggest
that, at least in these systems, receptor activation does not induce
the formation of new clathrin-coated pits.
Another area of active investigation involves studies of FRAP
(fluorescence recovery after photobleaching). Led by the group
of Lois Greene, a series of studies have yielded very interesting
results potentially relevant to the formation and uncoating of
clathrin-coated vesicles [27,30,31,66]. FRAP of clathrin puncta
at the plasma membrane (with a t1/2 for recovery of ∼ 16 s)
demonstrated that the clathrin coat is not a fixed entity [30].
Instead, polymerized subunits are constantly exchanging with
those that are freely diffusible in the cytosol. Furthermore, clathrin
subunit exchange proceeded in the face of perturbations which can
inhibit vesicle internalization (e.g. dominant-negative dynamin or
cholesterol depletion), but not those shown previously to elicit
global structural changes in the clathrin coat (e.g. hypertonic
sucrose treatment or potassium depletion) [30].
More recently, observations from both the Greene group, as
well as that of Tomas Kirchhausen, have shown that the uncoating
factor GAK (cyclin G-associated kinase) and the neuronal
homologue auxillin are recruited to clathrin spots immediately
Clathrin-mediated endocytosis
before internalization [26,67]. This was demonstrated further
to occur subsequently to dynamin recruitment, suggesting that
uncoating might begin immediately after vesicle fission. However,
this understanding seems to be inconsistent with previous
suggestions that clathrin exchange is catalysed by these same
factors, in concert with Hsc70 (heat-shock cognate 70), and might
function to convert flat lattices into curved coated pits and vesicles
[68]. If auxillin/GAK mediates clathrin subunit exchange, but
recruitment of these factors occurs only after that of dynamin,
it might not fit that subunit exchange reflects the induction of
curvature before vesicle fission.
Finally, FRAP of clathrin has also been observed in two
studies employing TIRFM [52,69]. In the first, it was very clearly
demonstrated that subunit exchange occurs more rapidly than
suggested by previous studies [52]. As confocal imaging will
bleach ∼ 1 μm of cytosol, whereas TIRFM only bleaches approx.
1/10 of this, it is not hard to imagine that the pool of free
clathrin in the earlier studies was effectively depleted during
the photobleaching step. Furthermore, this study demonstrated
FRAP for the clathrin heavy chain, and very interestingly, that it
occurred with different kinetics from that of clathrin light chain A,
but not light chain B. Finally, the second study employing FRAP
through TIRFM specifically focused upon the ‘static’ population
of clathrin spots [69]. The results of these experiments provided
further evidence that, as subunit exchange is observed, static
spots do not reflect non-functional clathrin aggregates. Overall,
these live-cell imaging studies have provided a more complex
and potentially heterogeneous view of clathrin dynamics than
envisioned previously, as well as many more exciting questions
to answer in future experiments.
DYNAMIN
In the nearly 20 years since being identified as a mechanochemical
enzyme suggested to mediate microtubule sliding, dynamin has
continually shown a tendency to confound reductionist models
[70]. Implicated in processes ranging from regulation of nitric
oxide synthesis to degradation of extracellular matrix, dynamin is
clearly a complex molecule with numerous cellular roles [71,72].
Confusing things further, dynamin exists in three isoforms, each
with numerous splice variants [11,73–75].
Dynamin-1 and dynamin-2 have been shown to play roles in
clathrin-mediated endocytosis [76–78]. Although dynamin-1 is
expressed only in cells from neuronal lineage, dynamin-2 is found
ubiquitously [11,77]. Before the application of live-cell imaging
to the study of endocytosis, the groups of both Sandra Schmid
and Mark McNiven employed tagged dynamin constructs to
permit specific analysis of individual isoforms and splice variants
[11,76]. More recently, several studies have utilized GFP-tagged
dynamins in studies of clathrin-mediated endocytosis by live-cell
imaging [13,26,38,55,59,67].
The first such study came out of Wolf Almers’s laboratory,
and clathrin, dynamin-1 and actin were imaged in various
combinations [38]. This provided, for the first time, a functional
molecular time course analysing the relative recruitment of
endocytic proteins in real time. This paradigm of comparing
spatiotemporal kinetics has been applied in numerous subsequent
studies. One observation from the 2002 study of Merrifield et al.
[38] was that dynamin-1 fluorescence intensity seemed to increase
rapidly just before clathrin internalization. Although such data
have been interpreted as the self-assembly of dynamin at the neck
of the budding vesicle immediately before fission, it has yet to
be determined whether this reflects dynamin recruitment from
the cytosol, or the redistribution of dynamin from the vesicle
419
to the neck. Importantly, there is electron microscopy evidence
suggesting that dynamin can indeed be found at all stages of
coated-vesicle production, from flat lattice onwards [12,79].
Also, consistent with a potential role for dynamin before
vesicle fission are two reports which demonstrated a more gradual
increase of clathrin and dynamin-2 together before endocytosis
[55,59]. However, there are also reports which have shown
a rapid increase in dynamin-2 relative to clathrin just before
internalization, more like the dynamin-1 paradigm [26,67]. As
each study was performed in a different cell line, it might
be that the behaviour of dynamin-2 depends upon the cell
type in which it is expressed. Interestingly, however, the kinetics
of dynamin-1 have been relatively consistent across several
studies [13,26,38,59]. One important caveat is that, to date, no
published study has investigated the behaviour of dynamin-1
at sites of clathrin-mediated endocytosis in cells in which it
is endogenously expressed. This is of particular importance as
there are dynamin-1-binding partners which share the neuronal
specific distribution [28,80]. However, it should be noted that
we have recently observed that, in dynamin-1-positive PC12
(phaeochromocytoma) cells, the kinetics of recruitment are
identical with those reported in other cells [80a].
Similarly to the question of whether dynamin functions in
clathrin-coated vesicle production before fission is the issue of
when and how dynamin signal drops. The study by Merrifield
et al. [38] claimed that a proportion of dynamin seemed in fact
to gradually internalize along with clathrin, while the majority
disappeared more rapidly. The later might reflect diffusion
following the dissolution of self-assembled dynamin subsequent
to GTP hydrolysis. Our recent studies in PC12 cells have
demonstrated that both dynamin-1 and dynamin-2 demonstrate a
biphasic drop in fluorescence: ∼ 30 % of the signal drops away at
a much higher rate than the remaining ∼ 70 % which internalizes
along with the nascent clathrin-coated vesicle [80a].
Thus it does seem as though dynamin might have a role beyond
the vesicle neck. However, clearly more experiments are required
to determine exactly what, if anything, dynamin does apart from
mediating fission. It will be very interesting to see whether studies
employing new technologies and data analysis strategies (see the
Future directions section below) will be capable of determining
the nature of any dynamin function before fission, as well as the
origin of the rapidly brightening population of dynamin. Finally,
whether dynamin truly plays a functional role in the nascent
vesicle is not currently clear.
AP-2
The first clathrin adaptor identified, the AP-2 complex, consists
of α, β2, μ2 and σ 2 subunits, and is able to link clathrin to cargo
[81]. Furthermore, AP-2 serves as a ‘hub’ for protein–protein
interactions, essentially clustering numerous proteins at locations
where clathrin-coated vesicles form [82]. AP-2 can also bind to
PtdIns(4,5)P2 , which is required for the proper localization of
clathrin, as well as progression of endocytosis [34,35].
As AP-2 can be found within a clathrin-coated vesicle
preparation, it seemed reasonable to predict that it would be
internalized as an inherent part of the nascent vesicle [9,81].
However, it is possible that production of these types of
preparations also results in vesicularization of the plasma
membrane. Interestingly, two quantitative proteomic studies have
suggested that clathrin-coated vesicles derived from non-neuronal
sources contain, as a whole, much more AP-1 than AP-2
[83,84]. Thus this would suggest, among other possibilities,
that either most of these vesicles derived from compartments
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J. Z. Rappoport
other than the plasma membrane, or that vesicles produced via
clathrin-mediated endocytosis are depleted of AP-2. Interestingly,
using an in vitro assay, removal of ∼ 90 % of AP-2 did not
significantly decrease clathrin-coated vesicle formation [85].
Finally, biochemical evidence has been obtained suggesting
that AP-2 can be released independently, leaving membraneassociated clathrin coats behind [86].
Although the heterotetrameric nature of AP-2 provides
numerous sites where fluorescent proteins could be appended,
not all GFP-fusions have appeared to be reliable markers. GFPtagged β2-adaptin was observed to mislocalize to endosomal AP1 complexes when overexpressed [44]. Similarly, the distribution
of σ 2-adaptin–GFP appeared to include partial localization to
the nucleus [55]. Although mislocalization does not preclude
targeting to the proper sites as well, and does not necessarily
prevent acquisition of useful data, it should be avoided if
possible. To this end, GFP-tagged α-adaptin constructs have been
demonstrated to properly localize to clathrin-coated pits [31,87].
However, even in that case, longer expression times (e.g. 48 h)
can be required to minimize gross cytosolic signals [45].
In recent years, numerous studies have been performed imaging
clathrin and AP-2 in live cells [33,35,44,45,55,60,69,87]. There
are currently a number of differing perspectives on whether AP-2
serves a functional role as a cargo adaptor within the nascent
vesicle or whether it is mainly involved in cargo sorting at
the cell surface. Our work employing TIRFM has repeatedly
demonstrated that AP-2 does not seem to internalize along
with clathrin, although the two do demonstrate significant colocalization in static images [33,45,69,87]. Two hypotheses were
put forth in our initial report on this subject: (i) AP-2 is excluded
from the forming vesicle before endocytosis; and (ii) AP-2 enters
the coated vesicle, but localizes to the cytosolic pole, a sufficient
distance away from the origin of the evanescent field as to preclude
excitation [87]. However, as was noted, the penetration depth in
this study nearly matched the diameter of a clathrin-coated vesicle.
More recently, it has been shown in two studies that AP-2
exits the evanescent field significantly before clathrin [60,69].
Furthermore, when imaged by both TIRFM and epifluorescence
microscopy, AP-2 could be seen to exit the evanescent field
before loss of the wide-field signal [60]. Thus it was concluded
that the second hypothesis was in fact occurring. Importantly, a
previous study from the same group which employed spinningdisk confocal microscopy suggested that AP-2 and clathrin
disappeared simultaneously [55]. This determination seems to
have arisen from the relatively deep acquisition volume of
spinning-disk microscopy relative to TIRFM. Thus it would
seem that combining the shallow penetration of TIRFM with
conventional epifluoresence microscopy has provided a more
accurate view of these events.
If the localization of AP-2 during clathrin-mediated endocytosis
is becoming clearer, this cannot be said of the underlying
mechanisms. Numerous questions still remain which might be
addressed through the application of new technologies (see the
Future directions section below). What proportions of AP-2 exit
the forming vesicle, are left behind at the plasma membrane or
polarize to the cytosolic side of the coated vesicle? Is this the same
for all events? What about vesicles formed de novo compared
with those produced via iterative budding? Finally, what about
comparing neuronal and non-neuronal cells, or vesicles containing
cargo which is internalized following ligand binding, relative to
constitutive entry?
Finally, a recent report from the laboratory of Margaret
Robinson has cast doubt on all studies employing GFP-tagged
α-adaptins [88]. In this study, the authors were unable to rescue
the phenotype of endogenous α-adaptin knockdown with a version
c The Authors Journal compilation c 2008 Biochemical Society
that had been mutated to become ‘siRNA (small interfering
RNA)-resistant’, and was linked at the C-terminus to GFP (αGFP). These results led the authors of this study, and of several
subsequent review articles, to conclude that all studies of αadaptin dynamics might be severely flawed [88–90]. However, it
must be noted that, although some studies of AP-2 FRAP and AP2 and clathrin dynamics have employed α-GFP, all of our studies
imaging clathrin and AP-2 have involved the use of α-adaptin
linked at the N-terminus to GFP (GFP-α) [31,33,44,45,69,87].
This is a very important point, as there are examples of tags to
one terminus of a protein perturbing function, whereas tags to the
opposing end do not [91]. Finally, in order to test this directly,
we have recently attempted rescue experiments with GFP-α, and
can report that it does indeed complement the phenotype resulting
from siRNA silencing of endogenous α-adaptin (J.Z. Rappoport
and S.M. Simon, unpublished work).
OTHER PROTEINS
In addition to other adaptors and accessory proteins, the actin
cytoskeleton has also been an active area of research. The paper
by Merrifield et al. [38] included data from imaging clathrin,
dynamin and actin in various combinations and found that a local
increase in actin signal was detected at the site of endocytosis,
beginning between the rise in dynamin and decrease in clathrin
[38]. Subsequent work from the Merrifield group showed that this
rise in actin fluorescence was also marked by similar increases
in the actin associated proteins N-WASP (neuronal Wiskott–
Aldrich syndrome protein), Arp2/3 (actin-related protein 2/3)
complex and cortactin [39,92]. Furthermore, results from Sandra
Schmid’s laboratory extended these observations implicating
actin at multiple steps during clathrin-coated vesicle formation
[61].
Other clathrin adaptors [e.g. ARH (autosomal recessive
hypercholesterolaemia) and Dab2] and accessory proteins (e.g.
Snx9 and eps15) have also been shown to localize to sites
of clathrin-mediated endocytosis in live cells [13,44,51]. These
observations include the determination that Snx9 recruitment
is spatially and temporally coincident with dynamin, and that
Snx9 promotes dynamin function [13]. Furthermore, TIRFM
imaging has been employed to demonstrate that the clathrin
adaptor/accessory protein epsin enters the cell as part of the
nascent vesicle [35,69].
Epsin has been shown to play a role in inducing membrane
curvature, and also functions as an adaptor for ubiquitinated
cargo [23,93]. Previous biochemical studies were unable to
resolve whether epsin represents an integral component of the
clathrin-coated vesicle, and it has been suggested that epsin might
be displaced before internalization [20,94–96]. However, livecell TIRFM was able to unambiguously demonstrate that epsin
internalizes along with clathrin [35,69]. This demonstrates the
importance of direct observation of the machinery implicated in
particular biological processes.
FUTURE DIRECTIONS
Technical advances in the area of live-cell imaging are continuing
to provide ever new options for conducting experiments. The
application of automated data analysis to the study of clathrinmediated endocytosis will certainly continue to permit questions
to be addressed with an ever increasing degree of statistical power.
The use of newly developed microscopy apparatus will hopefully
provide data sets more amenable to techniques such as particle
tracking. Increasing signal-to-noise ratios, and reducing exposure
Clathrin-mediated endocytosis
times, thus minimizing blur associated with motile structures, are
both areas where advancements would be useful.
Improvements in imaging technology are already driving
analysis to greater spatial, temporal and spectral degrees. So called
‘super-resolution imaging’ has recently been applied, albeit in
fixed cells, to analyse the morphology of clathrin-coated pits at
nearly one order of magnitude higher lateral resolution than can be
offered by traditional light microscopy [97,98]. Other advances
are being made in stride with the development or combination
of advanced imaging instrumentation. TIRFM imaging of FRET
(fluorescence resonance energy transfer) will certainly permit
the analysis of protein–protein interactions relevant to clathrinmediated endocytosis. This could be particularly applicable
to studies of signal-transduction events following receptor
activation.
Analysis of polarized emission from fluorophores illuminated
via TIRFM can provide the ability to measure membrane
curvature [99]. Thus this could enable observation of the progressive invagination of coated pits in living cells. Furthermore, it
could be employed to unequivocally demonstrate that static
clathrin spots reflect flat clathrin lattices from which curved
vesicles can form and bud. When put together with recently
developed methods to identify the moment of vesicle fission, all
steps in the endocytosis cycle could be independently identified
in real time [39].
Although four-colour emission splitters have been marketed,
alignment issues have hindered use in high-resolution studies
such as imaging clathrin-mediated endocytosis. However, use
of such technology could provide the simultaneous analysis of
cargo, adaptor and accessory proteins. This could be employed to
determine whether cargo is transferred from AP-2 to alternative
adaptors as nascent coated vesicles form from flat clathrin lattices.
Another potentially relevant advance would be the simultaneous
acquisition of donor, acceptor and FRET emissions, if polarized
imaging is employed [100]. Thus the stage is set for another wave
of technologically driven advances in this area.
CONCLUSIONS AND PERSPECTIVES
Clathrin-mediated endocytosis is an important topic which represents a synthesis of biochemical, biophysical and cell biological
issues. The application of live-cell fluorescence imaging has
created somewhat of a revolution in this area, both permitting
new types of experiments and the re-evaluation of old hypotheses.
Numerous examples of seemingly inconsistent data have arisen,
and these can, in some cases, be attributed to differences in
imaging methodology, cell type and/or marker protein. However,
the gains in our understanding of clathrin-mediated endocytosis
in the last decade have been phenomenal and have been driven in
large part through the power of microscopy.
Great thanks are due to Sanford Simon (Laboratory of Cellular Biophysics, Rockefeller
University, New York, NY, U.S.A.) for permitting the inclusion of unpublished data acquired
in his laboratory. Furthermore, Alexandre Benmerah and Laura Machesky provided a critical
reading of this manuscript. Finally, the support of the following people has been greatly
appreciated: Sandra Schmid, Harvey McMahon, John Heuser, Mark McNiven, Alexander
Sorkin, Barbara Pearse and Christien Merrifield.
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Received 28 February 2008/20 March 2008; accepted 27 March 2008
Published on the Internet 28 May 2008, doi:10.1042/BJ20080474
c The Authors Journal compilation c 2008 Biochemical Society