Distinct dynamics of endocytic clathrin-coated pits and coated plaques.

Abstract

Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then pinch off to become traffic carriers. These carriers are the principal vehicles of receptor-mediated endocytosis and are the major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of endocytic clathrin coat formation, which we designate as "coated pits" and "coated plaques." Coated pits are small, rapidly forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane--classical coated pits and coated plaques--and that these two assemblies interact quite differently with other intracellular structures.

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Distinct Dynamics of Endocytic Clathrin-Coated Pits and
Coated Plaques
Saveez Saffarian, Emanuele Cocucci, Tomas Kirchhausen*
Department of Cell Biology, Harvard Medical School, Children’s Hospital and Immune Disease Institute, Boston, Massachusetts, United States of America
Abstract
Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then
pinch off to become traffic carriers. These carriers are the principal vehicles of receptor-mediated endocytosis and are the
major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from
spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of
endocytic clathrin coat formation, which we designate as ‘‘coated pits’’ and ‘‘coated plaques.’’ Coated pits are small, rapidly
forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other
regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived
structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the
cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward
movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we
have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane—
classical coated pits and coated plaques—and that these two assemblies interact quite differently with other intracellular
structures.
Citation: Saffarian S, Cocucci E, Kirchhausen T (2009) Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques. PLoS Biol 7(9): e1000191.
doi:10.1371/journal.pbio.1000191
Academic Editor: Fred Hughson, Princeton University, United States of America
Received March 4, 2009; Accepted July 30, 2009; Published September 8, 2009
Copyright: ? 2009 Saffarian et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been supported by a National Institutes of Health grant GM075252 (TK), a NERCE grant U54 A1057159 (TK), and by an American Heart
Association postdoctoral fellowship (SS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: DiNa, differential evanescence nanometry; LCa, light chain a; LCb, light chain b; RNAi, RNA interference; sRNAi, short RNA interference; TIR, total
internal reflection; WF, wide-field.
* E-mail: kirchhausen@crystal.harvard.edu
Introduction
Clathrin-mediated endocytosis is a mechanism for selective
retrieval and internalization of membrane lipids and membrane-
bound proteins. Clathrin-coated pits capture their molecular cargo as
they invaginate from the cell surface and bud inward to form coated
vesicles, in a process that involves a complex sequence of interactions
among structural and regulatory proteins and lipids [1–3].
The assembly of clathrin-coated pits can be followed in vivo by
contemporary live-cell imaging methods, including laser scanning
confocal fluorescence microscopy, spinning disk confocal micros-
copy, and a combination of wide-field (WF) and total internal
reflection (TIR) fluorescence microscopy [4–16]. These studies
have yielded an array of data and models, in part depending on
the cell type or imaging method uses, and a range of different
views of how coats form, of potential roles for actin and the
cytoskeleton, and of possible mechanistic similarities between
clathrin-based structures in yeast and mammalian cells (reviewed
in [1,17,18]).
A combination of high- and moderate-resolution molecular
structures from X-ray crystallography and electron cryomicro-
scopy (cryoEM) and dynamic data from live-cell fluorescence
imaging of BSC1 cells [5,14,19–22] has led to the following picture
for coated pit formation. At widely distributed locations on the
plasma membrane, sequential recruitment of clathrin and its
adaptors progressively bends the underlying bilayer as a clathrin
lattice assembles; the adaptors selectively capture membrane-
bound proteins destined for endocytosis; membrane pinching
separates the fully formed coated vesicle from its parent
membrane; and an ATP-dependent chaperone uncoats the
released vesicle. Important features of this process in BSC1 cells
are that most of the endocytic structures are smaller than the
diffraction limit of the optical microscope (,250 nm) and that the
coated pits form progressively, over a time between nucleation and
pinching of 35–65 s [5].
In other types of cells—HeLa and COS cells in particular—
there are, in addition to the rapidly forming coated pits with
characteristics similar to those in BSC1 cells, larger and more
stable clathrin structures. The latter are present only on the
‘‘bottom’’ (adherent, coverslip proximal) surface of the cell, and we
initially thought that they might represent clathrin reservoirs [5].
Relatively flat, extended clathrin arrays, referred to as ‘‘clathrin
plaques’’ [23], have been observed by conventional electron
microscopy on the bottom surface of HeLa cells and osteoclasts.
These plaques probably correspond to the extended hexagonal
lattices of clathrin seen in these and other cell types (e.g., Swiss
3T3 cells) when imaged by rapid-freeze, deep-etch electron
microscopy [24–27]. We believe that the plaques also correspond
to the relatively long-lived clathrin structures (lifetimes .3 min) at
the substrate-facing surfaces of primary adipose cells imaged in
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real time by TIR [28], of Swiss 3T3 cells imaged in real time by
TIR and WF fluorescence illumination [9,10], and of HeLa and
COS cells as described above [5]. Many of these long-lived
clathrin structures display an abrupt inward movement at the end
of their lifetime—a process associated with membrane internali-
zation and transferrin uptake [9,10]. Because no firm distinction
has previously been made between the dynamics of clathrin-coated
pits and plaques, the observations just summarized have spawned
a model in which preformed clathrin lattices on the plasma
membrane undergo an extensive structural reorganization, leading
to coat curvature, membrane invagination, and vesicle budding
[18,29].
Different results and interpretations on the potential role of
actin in clathrin-based endocytosis have accompanied these
fundamentally different models for the mechanics of coat
assembly. A connection between actin and clathrin-driven
membrane uptake was first described in yeast [30,31]. Acute
depolymerization of actin by latrunculin A inhibits clathrin-based
endocytosis in some metazoan cells, but not in others [32].
Latrunculin treatment has no detectable effect on the dynamics of
endocytic coated pits in BSC1 cells [33], but it strongly inhibits
endocytosis in Swiss 3T3 cells, with a marked reduction in the
number of clathrin-based endocytic events [15]. Similarly,
perturbation of the actin cytoskeleton by genetic means, such as
depletion of Hip1R [34], perturbations in Hip1R association with
cortactin [8], and depletion of N-WASP [35], has variable effects
that depend on cell type. Some clathrin endocytic structures
recruit actin or cortactin at the time they bud and disappear, but
others, in the same cells, do not [9,10,15,36–38].
In an effort to differentiate among distinct modes of clathrin-
mediated endocytosis, we have measured the properties of clathrin
assemblies on both free (top) and adherent (bottom) lower surfaces
of several mammalian cell types. Recently introduced methods in
live-cell imaging enable us to make distinctions more readily than
has previously been possible. We find that a coated pit grows
continuously as an invaginating shell, which pinches off immedi-
ately upon completion, as in the usual pictures. The entire process
typically takes 30–60 s. A coated plaque grows initially at about
the same rate as a coated pit, but without displacement from the
cell surface. Its growth reaches a fluctuating plateau, generally two
to three times that of a typical pit, remaining in place for up to
several minutes before moving uniformly inward a few seconds
before membrane pinching. The actin cytoskeleton is not required
for normal formation and budding of the rapidly growing pits, but
it is essential for the formation, inward movement, and dissolution
of plaques. We have thus distinguished two modes of clathrin-coat
formation at the plasma membrane, with quite different
mechanisms for coat internalization. That is, clathrin is a scaffold
for at least two distinct, membrane-associated processes. The
distinction allows us to resolve apparent contradictions in the
previous literature of clathrin-mediated endocytosis.
Results
Comparison of Clathrin Dynamics on the Upper and
Lower Surfaces of Swiss 3T3 Cells
We compared the dynamics of clathrin-containing assemblies
on the free surface of Swiss 3T3 cells with their dynamics at the
surface in contact with the coverslip (Figure 1 and Videos S1 and
S2). For these experiments, we used a previously described cell line
in which clathrin is labeled by stable expression of LCa-dsRed [9],
and we recorded images using spinning disk confocal imaging. We
restricted our analysis to objects that were relatively stationary in x
and y, as most of the more motile ones correspond to endosomes
[5,7]. By using exposure times of 100 ms per frame and imaging
every 2 to 10 s, we could record for periods of up to 60 min
without obvious signs of phototoxicity. All experiments reported
here were carried out under conditions in which internalization of
transferrin, used as a probe of clathrin-dependent uptake, was not
affected by expression of the fluorescent chimeric proteins.
Nearly all the fluorescent spots on the free surface of the Swiss
3T3 cells belonged to a single class of diffraction-limited objects, as
expected for coated pits or vesicles ,200 nm in diameter
(Figure 1A). These spots displayed the growth and abrupt
disappearanceof fluorescence, characteristic of assembling
clathrin-coated pits [5,14]. In general, newly forming pits
appeared at positions uncorrelated with previous events—that is,
‘‘hotspots’’ were rare or absent (Figure 1C and 1D). The mean
lifetime of these structures was 62616 s, with an average
maximum intensity of approximately 800 fluorescence units,
recorded just prior to their disappearance (Figure 2A). These
values are similar to those determined for conventional clathrin
pits that form on the top or bottom surfaces of astrocytes, BSC1,
COS, or HeLa cells, stably or transiently expressing light chain a
(LCa) fused to mRFP, YFP, EGFP, Tomato, Cherry, or DsRed
[5,14,22,33]. Clathrin-coated pits, visualized in human U373
astrocytes by stable expression of s2-EGFP (the AP-2 small chain),
also have the same characteristics (Figure 2B); the s2-EGFP
colocalizes completely with clathrin tagged with Tomato-LCa
(unpublished data). An advantage of marking endocytic coated pits
with AP-2 is that this adaptor complex is not directed to any
intracellular membranes. Its use in our previous work with BSC1
cells therefore eliminated confusion from recorded events due to
endosomes or other intracellular structures [5,14,22,33,39].
Clathrin-containing structures at the adherent surface of the
Swiss 3T3 cells have a more complex pattern of formation and
dissolution. They can be divided into three categories, each with
distinct dynamic properties (Figure 1B–1D). Those in the first
category have the same time course as described above for
conventional coated pits and vesicles (Figure 1C, pit, and 1D, dark
gray tracing; Figure 2D, gray dots). The intensity profiles of
structures in the second category are similar to those in the first,
but they form sequentially at the same positions (hotspots)
(Figure 1D). A new spot starts to appear before full disappearance
Author Summary
Here, we identify and characterize two distinct modes of
clathrin-mediated uptake at the plasma membrane. The
‘‘canonical’’ coated pit is where assembly of a curved
clathrin lattice, linked to deformation of the underlying
membrane, gives rise to coated vesicles. Clathrin coated
‘‘plaques’’ are extended clathrin lattices of low curvature
(enriched in hexagonal arrays) that have been observed by
electron microscopy at the bottom of cells, but their
relationship to the canonical, curved pit assembly has
been obscure. Recognition of the difference between two
distinguishable classes of events detected by fluorescence
microscopy has resolved a number of conflicts and
misconceptions in the literature. In particular, a large
fraction of the clathrin endocytic processes studied at the
adherent surface of HeLa, Swiss 3T3, and astrocyte cells are
the long-lived coated plaques, not canonical coated pits,
whereas most, if not all, of the clathrin endocytic processes
at the free surface of these cells are coated pits. Conflation
of data from the two distinct processes has previously led
to misleading mechanistic conclusions, which are now
resolved.
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Figure 1. Formation of clathrin-coated structures on the plasma membrane of cells. (A–E) Time-series images of clathrin assemblies at the
top or bottom of Swiss-3T3 cells acquired with a spinning disk confocal microscope (Videos S1 and S2). The fluorescent assemblies were tagged with
clathrin LCa-DsRed stably expressed in Swiss-3T3 [9] in the absence (A–D) or presence (E) of transiently expressed auxilin1-EGFP. (A) Representative
kymograph obtained from a time series recorded every 5 s (100-ms exposure) for 15 min from the free (top) surface of the cell, showing that the
majority of the structures are relatively short-lived. (B) Representative kymograph obtained from a time series recorded every 5 s (100-ms exposure)
for 15 min from the adherent (bottom) surface of the cell. All structures are dynamic, but many have longer average lifetimes than those in (A). (C)
Selected snapshots from the time series shown in (B) to illustrate examples of clathrin structures displaying the dynamic behavior characteristic of
canonical coated pits, of a group of canonical coated pits forming consecutively at a single location (hot spot), and of a plaque. The peak fluorescence
intensity of pits assembling in isolation is similar to those forming as part of a hot spot; the overall intensity of the plaque is significantly higher. The
acquisition time of each snapshot (in seconds) is indicated. (D) Fluorescence intensity profiles of the clathrin assemblies shown in (C). (E) Fluorescence
intensity profiles of clathrin coats tagged with LCa-DsRed and Auxilin1-EGFP to illustrate the dynamics of canonical pits forming in a hot spot and of
plaques. A burst of auxilin recruitment coincides with the uncoating step of a canonical pit [22,43], or with each of the uncoating steps observed
within a hot spot; in contrast, variable amounts of auxilin are recruited during the entire life of the plaque, ending with a larger burst. (F) Electron
microscopy of clathrin-coated structures on the adherent surface of unroofed BSC1 and HeLa cells. Representative electron micrographs illustrate in
(a) the exclusive presence of clathrin-coated pits with various degree of invagination in the plasma membrane of unroofed BSC1 cells; 79 pits were
captured in 49 pictures (156 mm2). Representative electron micrographs illustrate in (b and c) the coexistence of clathrin-coated pits and clathrin flat
arrays in the plasma membrane of unroofed HeLa cells. 69 pits and 48 sheets were captured in 59 pictures (188 mm2); Bar indicates 100 nm.
doi:10.1371/journal.pbio.1000191.g001
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of the previous one. The number of sequential events is variable,
and the positions of their sites of initiation cannot be resolved (i.e.,
are less than 500 nm apart) (Figure 1B–1E). This hotspot behavior
has been described by others for structures on the adherent surface
of Swiss 3T3 [10,15] and COS cells [5,6].
Clathrin-containing structures in the third category, which we
term clathrin-coated plaques (borrowing a phrase from Maupin and
Pollard [23]), have substantially longer lifetimes (typically 2–
16 min) (Figures 1B–1E, 2D, and 2E, black dots and circles).
Quantitative determination of their lifetimes was possible, because
of the longer imaging periods we used for this work. The clathrin
fluorescence associated with coated plaques is, on average,
stronger than the fluorescence of conventional endocytic coated
pits, whether the pits arise at random positions or at hotspots on
the coverslip proximal surface (Figure 1D). The intensity of coated
plaques often undergoes prominent fluctuations (see Figure 1D
and 1E), in agreement with similar observations from large
clathrin ensembles located at membrane in contact with the
coverslip [9,10,28]. The rate of clathrin accumulation at the
beginning of assembly is roughly the same for pits and plaques,
and the uncoating phase of 6–10 s is likewise similar in the two
cases, but the rate at which clathrin accumulates during
fluctuations at later times in the life of a plaque is often greater.
We also detected clathrin-coated plaques, visualized with tagged
AP-2, on the adherent surfaces of U373 astrocytes stably
expressing s2-EGFP (Figure 2E, black dots; see the comparison
in the dynamics of coat formation on the adherent surface of
BSC1 and U3T73 cells in Video S4), and long-lived clathrin
Figure 2. Sizes and lifetimes of clathrin-coated structures forming at the cell surfaces. In these double logarithmic plots, each dot
corresponds to the duration (lifetime) and the corresponding size (maximum fluorescence intensity prior to dissolution) of a clathrin or AP-2
fluorescent spot recorded at the top or bottom plasma membrane of adherent cells. The data derive from time series (100-ms exposures) of at least
15-min duration recorded every 5 s (full circles) or 60-min duration recorded every 10 s (empty circles) using a spinning disk confocal fluorescence
microscope. The dots are color coded to distinguish clathrin coats displaying dynamics characteristics of canonical pits (gray) from those behaving as
plaques (black). The time series were acquired from Swiss 3T3 stably expressing clathrin LCa-DsRed, or from U373 astrocytes and BSC1 cells stably
expressing AP-2 s2-EGFP. Similar results were obtained from data acquired in two additional Swiss, two additional astrocytes, and two additional
BSC1 cells (Figure S4). (A and D) The average lifetime of canonical pits tagged with LCa-DsRed is 62616 s (n=19) on the top of the Swiss 3T3 cells
and 64624 s (n=71) on the bottom; the average lifetime of plaques (bottom only) is longer and more variable (2716141 s; n=87). The
corresponding maximum fluorescence intensities are 1,3576698, 1,1306564, and 2,54261,784, respectively. The differences in average lifetime or of
maximum fluorescence intensity between pits and plaques in Swiss 3T3 cells are statistically significant (p,0.0001) (B, C, and E) The average lifetimes
of canonical pits tagged with s2-EGFP of AP-2 is 60626 s (n=34) and 56619 s (n=221) on the top and on the bottom of U373 astrocytes, and
56626 s (n=105) on the bottom of BSC1 cells. The lifetime of the AP-2 containing plaques on the bottom of astrocytes is 2196112 s (n=67). The
corresponding maximum fluorescence intensities are 9326431, 1,1096428, and 2,54261,784, respectively. The differences in average lifetime or of
maximum fluorescence intensity between pits and plaques in astrocytes are statistically significant (p,0.0001). (F) Effect on clathrin-coat dynamics of
actin cytoskeleton depolymerization by 15-min treatment of Swiss 3T3 cells expressing LCa-DsRed with 5 mM latrunculin A. The average lifetime
(84618 sec; n=12) and maximum fluorescence intensity (1,1786775; n=41) of the canonical coated pits remains constant, while the number of
newly formed pits decreases. In contrast, the plaques became stationary, with average lifetimes in excess of 20 min (p,0.0001), and there is a
decrease in the number of newly formed plaques.
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structures are present on the adherent surfaces of COS and HeLa
cells (unpublished data). The absence of detectable AP-2 hot spots
or plaques on the free surface of astrocytes (Figure 2B) agrees with
earlier observations showing that similar structures are absent
from the nonadherent surfaces of BSC1, HeLa cells, or astrocytes
[5,22,33]. We estimate that the relative amounts of clathrin
engaged in coated plaques and coated pits, respectively, at the
attached surface of a cell after overnight plating are 70% and 30%
for Swiss 3T3, 65% and 35% for U373, and 80% and 20% for
HeLa cells. As described below, the endocytic traffic capacity of
plaques is relatively small, because they are only present on the
adherent surface and because the frequency of plaque-mediated
endocytic events (pinching off) is significantly lower than that of
coated-pit mediated.
To correlate the coated pits and coated plaques, as defined
above by their properties when observed by live-cell imaging, with
morphologies seen by electron microscopy, we visualized the
inward-facing surface of adherent plasma membranes from
‘‘unroofed’’ BSC1 and HeLa cells by the well-established methods
of rapid freezing, freeze etching, and rotary shadowing [40].
Adherent surfaces of BSC1 cells contain coated pits but essentially
no plaques, whereas the corresponding surfaces of HeLa cells
contain both. We detect only curved clathrin coats of various sizes
and degrees of invagination in BSC1 cells that by live-cell imaging
have only coated pits (Figure 1Fa: 70 pits in a total surface area of
156 mm2; 49 electron microscopy images). In contrast, we find
both invaginated pits (Figure 1Fb: 67 pits in a total surface area of
188 mm2; 59 electron microscopy images) and relatively shallow
sheets (Figure 1Fc: 48 sheets in the same area) on the adherent
surface of HeLa cells. (Because of limitations in the way cells are
treated to reveal the inward-facing surface of the adherent
membrane, these micrographs correspond to views from the
thinnest, most peripheral regions of the attached cell surface,
where the relative number of plaques seen by optical microscopy is
substantially lower than in the center; the count of 48 sheets and
67 pits, therefore, considerably underestimates the plaque:pit ratio
across the full adherent surface.) We conclude that clathrin-coated
pits and coated plaques as defined by live-cell imaging criteria
correspond respectively to the canonical coated pits and extended
sheets observed by classical electron microscopy.
To probe whether AP-2 is essential for plaque formation, we
depleted AP-2 in HeLa cells stably expressing s2-EGFP (to
monitor AP-2) and transiently expressing Tomato-LCa (to
monitor clathrin). We used RNA interference (RNAi) specific for
m2 [41] for a period of approximately 5 d, with a double
transfection protocol (Materials and Methods) that gave full
ablation of fluorescent transferrin uptake in more than 90% of the
cells and a 10-fold reduction in the number of clathrin structures,
as described before [41]. Under these conditions, clathrin and AP-
2 were completely absent from the top surface of the cells
(unpublished data).Immobile,
(.500 s) clathrin structures were present on the adherent surface,
however (Video S5). These spots contained a very small amount of
AP-2, with a fluorescent signal barely above background—less
than 2% of the signal from plaques in HeLa cells not depleted of
AP-2. As there were no conventional coated pits on the top or
bottom surface of the cells, we interpret the immobile structures on
the bottom surface of AP-2–depleted cells as nascent plaques that
were unable to grow, because they had exhausted the extremely
limited amount of residual AP-2 present in those cells. We also
detected motile clathrin-coated structures in the cytosol and
perinuclear regions, with the usual characteristics of endosome- or
TGN-associated coats containing AP-1, AP-3, GGAs, or Hrs. We
conclude that AP-2 is essential for the formation of conventional
diffraction-limited, long-lived
coated pits and probably also for the initiation and growth of
coated plaques.
Recruitment of Auxilin
Auxilins 1 and 2 are J-domain cofactors for Hsc70, the
uncoating ATPase [42]. Auxilins appear in coated vesicles in a
characteristic burst that lasts several seconds, just at the onset of
disassembly of the clathrin/AP-2 coat [22,43]. We find similar
bursts of transiently expressed auxilin-1-EGFP in coated vesicles of
the LCa-DsRed–expressing Swiss 3T3 cells (Figure 1E), and we
can record auxilin bursts at the onset of each of the serial
dissolutions of the clathrin signals observed during the lifetime of a
hotspot (Figure 1E). Small, variable levels of auxilin are also
present in association with clathrin during the lifetime of
conventional coated pits and hotspots. These common features
of isolated pits and hotspots support the interpretation that the
latter correspond to sequential assembly and independent budding
of conventional coated pits and vesicles, at positions that all lie
within the resolution limit of the microscope. In contrast, coated
plaques recruit substantially greater quantities of auxilin during
their lifetime and at the onset of their dissolution than do the
conventional structures (Figure 1E). We could not detect any
direct correlation between variations in clathrin and auxilin
fluorescence during the lifetime of the plaques (Figure 1E and
unpublished data). We conclude that sequential formation of
conventional pits and vesicles and their detachment from a plaque
is not an adequate explanation for the fluctuations in clathrin and
AP-2 seen during the lifetime of the plaques.
Role of Actin
Coated pits and plaques differ particularly strikingly in the
relationship between their properties and the dynamic state of
actin. Treatment of Swiss 3T3 cells with latrunculin A, which
depolymerizes the actin cytoskeleton, prevents both formation of
new plaques and dissolution of old ones but has no effect on the
dynamics of assembly and dissolution of conventional clathrin-
coated pits (Figure 2F, Figure S1, and Video S6). These
observations confirm our earlier work on clathrin dynamics in
BSC1 cells (which lack plaques) [33]. We showed that formation of
conventional clathrin-coated pits is not sensitive to loss of actin
dynamics after treatment with either latrunculin A or cytochalasin
D. Other groups have reported that treatment with latrunculin A
of Swiss 3T3 cells (which contain both pits and plaques) prevents
coat dissolution at the adhered surface [15]—presumably because
the observations were weighted heavily toward the coated plaques.
There are two known connections between actin and clathrin
assemblies in cells. One is huntingtin-interacting protein 1-related
(Hip1R), which has binding sites for F-actin, cortactin, and
clathrin light chains [44–47]. Interference with the interaction
between Hip1R and clathrin light chains by overexpression of a
light-chain mutant unable to associate with Hip1R retains the
cation-independent mannose-6-phosphate receptor (CI-MPR) in
the TGN but has no detectable effect on transferrin or EGF
uptake [47]. The other connection between clathrin coats and
actin dynamics involves cortactin, which activates the actin
nucleation factor Arp2/3 and leads to stimulation of branched
actin filament assembly [48–50]. It is believed that dynamin
recruits cortactin through interaction of the proline-rich C-
terminus of dynamin with the SH3-domain of cortactin [51].
To disrupt the interaction between clathrin and Hip1R, we used
the dominant-negative clathrin LCb-EED/QQN fused to EGFP
[47], a mutant protein that binds normally to clathrin heavy chain
but is deficient in its interaction with Hip1R [44,46] We found
that transient overexpression of EGFP-LCb-EED/QQN in Swiss
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3T3 cells stably expressing LCb-DsRed or in U373 astrocytes
resulted in complete elimination of plaques but that it had no
discernable effects on the formation and properties of normal pits
(Figures 3B and 4A). In agreement with earlier observations
obtained with HeLa cells [47], overexpression of EGFP-LCb-
EED/QQN had no discernable effects on the clathrin-dependent
receptor-mediated uptake of fluorescent transferrin in astrocytes
(Figure S5), a result that is consistent with the small relative
contribution of plaques to endocytic traffic (see below). As
expected, the mutant light chain colocalized with and replaced
wild-type LCa-DsRed (Figure 3A), with an efficiency of approx-
imately 85% as determined by the extent in the decrease of the
fluorescence signal of LCa-DsRed at any clathrin spot. Control
experiments performed by expression of wild-type EGFP-LCb
showed no effects on the formation of clathrin plaques or pits
(Figures 3B and 4A) even though the replacement level was similar
to that achieved with EGFP-LCb EED/QQN (Figure 3A).
The role of Hip1R in plaque formation was confirmed by the
loss of plaques, but not of pits in cells depleted of Hip1R by RNAi
(Figure 3E). Under these conditions, the average lifetime and
maximum fluorescence intensity of the remaining pits at the
bottom surface do not change.
Depletion of clathrin LCa and light chain b (LCb) is another
way to disrupt the linkage between clathrin and Hip1R because it
also leads to accumulation of CI-MPR in the TGN without
affecting uptake of transferrin or EGF [47,52]. To test whether loss
of light chains also results in absence of plaques, we depleted LCa
and LCb in U373 cells stably expressing s2-EGFP (Figure 3C
antibody staining) by using specific small RNA interference
(sRNAi) probes to both light chains [47,52]. We found a complete
absence of coated plaques, whereas formation of coated pits
remained normal (Figure 3D).
In the course of the light chain replacement experiments
performed by expression of EGFP-LCb EED/QQN, we saw a
clear association between absence of plaques and a marked
increase in cell motility (Figure 4A and 4B). This increase was not
due merely to expression of exogenous light chains, as expression
of wild-type EGFP-LCb had no detectable effect (Figure 4B).
Cortactin appears at endocytic clathrin structures during late
stages of coat formation in mammalian cells [8,10]. We compared
the recruitment of cortactin to clathrin plaques and pits, using
Swiss 3T3 cells stably expressing LCa-DsRed and transiently
expressing cortactin-EGFP. Cortactin recruitment to coated
plaques was low, but it increased steadily during growth, with a
peak just prior to the onset of uncoating (Figure 5). In contrast,
coated pits had very low levels of cortactin fluorescence
throughout the growth phase (Figure 5). The overall cortactin
level in coated pits was approximately 4-fold lower than in
plaques. Our results with coated plaques are similar to those
reported by Merrifield and colleagues when analyzing relatively
long-lived clathrin structures at the adherent surface of the cells
[10]. Because cortactin activates Arp2/3, we also followed
recruitment of Arp2/3 to endocytic clathrin structures in Swiss
3T3 cells stably expressing LCa-DsRed and Arp2/3-GFP. Arp2/3
was barely detectable at coated pits. In contrast, following low
levels of recruitment during most of the lifetime of a coated plaque,
Arp2/3 peaked at the onset of uncoating (Figure 5B). This
observation is consistent with the acute recruitment of Arp2/3
seen at the time of invagination and uncoating of the long-lived
clathrin structures analyzed by Merrifield and coworkers [37].
Based on these observations, we suggest that assembly of actin
and its associated proteins is necessary for both early and late
phases in the formation of a clathrin-coated plaque, but that it is
not critical for the formation of clathrin-coated pits.
Comparative Dynamics of Clathrin-Coated Pits and
Plaques
We analyzed the incorporation of different coat constituents by
complementary optical imaging methods. The fluorescent spots
representing coated pits, imaged by spinning disk confocal
microscopy, WF, or TIR fluorescence illumination, are of
diffraction-limited dimensions, whereas the plaques give brighter
spots that are often larger than the diffraction limit and that
change shape during their extended lifetime (compare the time
series of the representative pit and plaque in Figure 1C; see also
Video S4). We analyzed temporal differences between the two
classes of coated structure, by applying a power-spectrum analysis
to detect principal frequency components in the time course of
fluorescence variation. The data shown in Figure S2 were
obtained by spinning disk confocal imaging recorded from the
adherent surface of LCa-DsRed–expressing Swiss 3T3 cells. The
primary difference between the two structures is that the
fluctuations of fluorescence intensity in mature plaques are
short-lived in comparison with the lifetime of coated pits.
Dissociation of coats of pits [5,14] or of plaques (this study; [10])
represents an endocytic event. Detailed analysis of the time-lapse
series used to generate the data in Figure 2 shows 11 dissolutions of
plaques and 62 dissolutions of pits occurring in 308 mm2of bottom
membrane during a period of 250 s (50 frames), indicating that the
contribution of plaques to endocytosis is relatively small.
We used a combination of WF and TIR illumination to
examine the displacement of coated structures along the optical z-
axis as a function of time (Figure 6). The signal acquired by WF
fluorescence microscopy is proportional to the total number of
fluorophores, whereas the TIR signal is also inversely related to
the distance from the coverslip of the same fluorescing object. The
TIR-to-WF ratio obtained by this approach (first used with
clathrin structures by Merrifield and coworkers [9]) is proportional
to the average distance from the coverslip of the clathrin assembly
as it moves inward during endocytosis. We carried out the relevant
measurements on images recorded from the bottom surface of
Swiss 3T3 cells stably expressing LCa-DsRed (Figure 6). The
clathrin centroid of a conventional coated pit moves steadily
inwards into the cell and away from the coverslip (Figure 6A and
6D), as expected for a coat that grows continuously from a shallow
dome into a larger, nearly spherical coated vesicle. Plaques also
move inward (i.e., away from the coverslip), but only during the
last 10–20 s of their lifetime (Figure 6B, 6C, 6E, and 6F). The
plaque properties are precisely those outlined by Merrifield and
colleagues in their description of endocytic clathrin structures in
the adherent surface of the same Swiss 3T3 cell line [9,10].
We have shown that in BSC1 cells, the AP-2 adaptor complex is
recruited together with clathrin during the early stages of
endocytic coated-pit formation (up to two-thirds of the total
assembly time), but that adaptor incorporation then levels off
during the final third of the process [14]. We devised an approach
we have called differential evanescence nanometry (DiNa)—an extension
of the TIR-to-WF ratio method described in the preceding
paragraph—to measure the relative z-axis positions of two
fluorophores with a precision of approximately 10 nm [14]. We
determined the relative displacements of clathrin (tagged with
Tomato-LCa) and AP-2 (tagged with s2-EGFP) and showed that
AP-2 is enriched in the ‘‘upper’’ hemispheres of the coated pits
that form on the adherent surface of a BSC1 cell [14] (Figure 7C).
Thus, the oldest part of the pit contains most of the adaptor,
consistent with the decline in its incorporation rate, relative to that
of clathrin, as the pit nears completion. These observations help
explain the asymmetric distribution of density, in cryoelectron
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tomograms of brain-derived coated vesicles, which we attribute to
adaptor molecules [19].
We used U373 astrocytes to extend these observations to a cell
type in which coated plaques could be studied. We confirmed that
in these cells, as in BSC1 cells, AP-2 incorporates selectively during
the earlier stages of conventional coated-pit formation (Figure 8A).
DiNa measurements in U373 cells further showed that for late-
stage coated pits, the z-centroids of the clathrin and AP-2
distributions are displaced (Figure 7B, pit), just as in BSC1 cells
(Figure 7C, pit). Coated plaques do not show the same differential
pattern of incorporation and displacement. Instead, clathrin and
AP-2 co-incorporate at all stages (Figure 8C), and the z-centroids
of clathrin and AP-2 remain coincident throughout the coated-
plaque lifetime, even just prior to plaque dissolution, after the coat
of the plaque has moved toward the cell interior (Figure 7B,
plaque). Thus, clathrin and AP-2 move together into the cell
interior as the plaque assembles and buds, carrying endocytic
traffic toward endosomal compartments.
Recruitment of Epsin and Dynamin
Epsin has a modular structure, including an N-terminal
homology (ENTH) domain that binds phosphoinositides, ubiquitin
interaction motifs that bind ubiquitin-containing proteins, and
bindingsitesfor clathrin, AP-2,and Eps15[53,54]. LikeEps15 [55],
epsin does not incorporate into a budded coated vesicle [53], even
though its accumulation parallels that of clathrin during coat
formation [13,16]. Epsin accumulates with clathrin during the
formation of pits or plaques in Swiss 3T3 (Figure 8B and 8D) or
BSC1 (unpublished data) cells transiently expressing Epsin1-EGFP
and stably expressing either LCa-DsRed or Tomato-LCa. DiNa of
conventional coated pits in Swiss 3T3 cells (Figure 7B, pit) or in
BSC1 cells (Figure 7C) shows that as the pit matures, most epsin
molecules remain at the growing edge of the coat, in association
with the cell membrane, which in turn adheres to the coverslip.
Localization of epsin at the edge of a coat is in accord with the
enrichment of epsin and Eps15 at the growing edge of a coated pit,
seen by immunoelectron microscopy.[54–56]. In a coated plaque,
however, the average axial positions of clathrin and epsin always
overlap, not only during the full lifetime of the plaque, but also
during the final stages prior to dissolution, when the clathrin coat
movesinward(Figures6Eand7Aand7B, plaque).Thisobservation
agrees with the absence of a preferred location for epsin in extended
arrays of clathrin observed by electron microscopy [54,57].
Dynamin associates with coated pits during the growing phase
of the coat and then in a stronger burst after completion and just
prior to uncoating (Figure 5C) [22,39]. Dynamin associates
similarly with plaques, most prominently just before uncoating
(Figure 5C), as previously described [9].
Discussion
Pits and Plaques
Our analysis of endocytic clathrin coats in living cells in culture
shows two distinct kinds of coated structure (see model, Figure 9).
One type, seen at both the free and adherent surfaces of the cell,
corresponds to the canonical coated pits. Its coat assembles
progressively into a curved lattice, which deforms the membrane
as it grows. Pinching off of a coated vesicle (dynamin dependent
but actin independent) and coat disassembly complete the clathrin
cycle. The other type, seen only at the adherent surface,
corresponds to clathrin-coated plaques. The coat is roughly planar
(or moderately domed, as suggested by some of Heuser’s
micrographs [27,29,57]); after some minutes, it moves inward
toward the cell interior, bringing with it a portion of the
underlying membrane, which buds off in an actin- and
dynamin-dependent process. In the presence of an actin
depolymerizer, latrunculin A, new coated plaques do not form.
Those already formed continue to accumulate, but they freeze in
place and do not bud. Ablation of the clathrin-Hip1R interaction,
by mutation or depletion of the light chains, or by depletion of
Figure 3. Disruption of plaque formation by interference with the function of clathrin light chains. Loss of plaques upon expression of
EFGP-LCb-EED/QQN (A and B) or due to depletion of clathrin light chains or Hip1R by RNAi (C, D, and E). (A) Effective replacement of LCa-DsRed with
EGFP-LCb or EFGP-LCb-EED/QQN. Swiss 3T3 cells stably expressing LCa-DsRed were transfected with a modified form of clathrin EGFP-LCb, in which
the conserved three critical residues EED required for the interaction of the light chains with Hip1R were mutated to QQN. As control, cells were
transfected with no plasmid or with wild-type EGFP-LCb. The fluorescence images show the characteristic punctate pattern of clathrin light chains
elicited by the LCa constructs and, due to replacement, the corresponding loss of LCb fluorescence. The estimated replacement level is 85% (n=170).
(B) Expression of EGFP-LCb-EED/QQN prevents formation of plaques, but not of pits. The panels are representative semi-logarithmic plots of
maximum fluorescence versus time for pits and plaques from two Swiss 3T3 cells expressing similar amounts of EGFP-LCb or EGFP-LCb-EED/QQN. The
average lifetime of canonical pits on the bottom of the Swiss 3T3 cells expressing EGFP-LCb is 106631 s (n=45) and 107631 s (n=71) in cells
expressing EFGP-LCb-EED/QQN. The maximum fluorescence intensities of these pits are 2,1316968 and 2,52661,392, respectively. The average
lifetime and maximum fluorescence intensity of plaques on the bottom of the Swiss 3T3 cells expressing EGFP-LCb are 291676 s and 3,70161,451
(n=27). The differences in average lifetime or in maximum fluorescence intensity between pits and plaques in cells expressing EGFP-LCb is
statistically significant (p,0.0001). There is no statistically significant difference in average lifetime or in maximum fluorescence intensity between pits
of cells expressing EGFP-LCb or EFGP-LCb-EED/QQN. (C and D) Loss of plaques upon depletion of clathrin light chains. Both clathrin light chains of
U373 astrocytes were depleted by transfection with Oligofectamin using a pool of sRNAi specific for the light chains. As control, cells were transfected
with Oligofectamin using a scrambled sRNAi sequence (Materials and Methods). (C) The extent of light-chain depletion (,85%) was estimated using
mAb CON.1 immunofluorescence labeling; the mAb recognizes both light chains. (D) The panels are representative semi-logarithm plots of maximum
fluorescence versus time for pits and plaques from two different cells containing normal or depleted amounts of clathrin light chains. The average
lifetime and maximum fluorescence intensity of the pits in the control cell were 63623 and 1,5626888 (n=304), respectively. The average lifetime
and maximum fluorescence intensity of the pits in the cell depleted of clathrin light chains were 68631 and 1,2596589 (n=168), respectively; the
differences are not statistically significant. The average lifetime and maximum fluorescence intensity of the plaques in the control cell were 243691
and 3,39661,388 (n=43), respectively; the differences in average lifetime and in maximum fluorescence intensity between pits and plaques in the
control cells are statistically significant (p,0.0001). The plaques are absent in the cell depleted of clathrin light chains. (E) Loss of plaques upon
depletion of Hip1R. Hip1R of U373 astrocytes transiently expressing EGFP-LCa were depleted by transfection with Oligofectamin using a pool of
sRNAi specific for Hip1R. As control, cells were transfected with Oligofectamin using a scrambled sRNAi sequence (Materials and Methods). The panels
are representative semi-logarithmic plots of maximum fluorescence versus time for pits and plaques from two different cells containing normal or
depleted amounts of Hip1R. The average lifetime and maximum fluorescence intensity of the pits in the control cell were 58626 and 1,3346761
(n=209), respectively. The average lifetime and maximum fluorescence intensity of the pits in the cell depleted of Hip1R were 56627 and 8666570
(n=237), respectively; the differences are not statistically significant. The average lifetime and maximum fluorescence intensity of the plaques in the
control cell were 194647 and 2,84061,133 (n=36), respectively; the differences in average lifetime and in maximum fluorescence intensity between
pits and plaques in the control cells are statistically significant (p,0.0001). Plaques are absent in the cell depleted of Hip1R.
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Hip1R also prevents coated-plaque formation. Coated plaques
tend to be larger and longer-lived than coated pits, and they show
more variable assembly dynamics. As we discuss below, a key
result is the recognition that pits and plaques have distinct
functional properties. Understanding the differences helps recon-
cile the apparently disparate properties of clathrin-dependent
endocytosis in various cell types and at various cellular surfaces.
Our imaging experiments used improved optics and other
instrumentation that allowed longer-term imaging with minimal
photobleaching. We recorded data from both the free and
adherent surfaces of three different types of cells, and we excluded
from the analysis all clathrin-containing clusters for which a high
mobility indicated endosomal localization [5]. We also used AP-2
and epsin to restrict our analysis to coats at the plasma membrane.
Our experiments show that clathrin plaques are present only at the
adherent surfaces of Swiss 3T3, U373 astrocytes, and HeLa cells
and that they are essentially absent from BSC1 cells. Canonical
coated pits form with similar characteristics at both free and
adherent surfaces of all four cell types. A subset of the coated pits
form at preferred sites, or hot spots, but we find such sites
primarily at the adherent surface of Swiss 3T3 cells and astrocytes,
as described previously in COS cells [6].
Assigning previous observations to one or the other class of
objects resolves apparent discrepancies in the published literature.
Figure 4. Increase of cell motility correlates with loss of plaques. (A) Expression in U373 astrocytes of EGFP-LCb-EED/QQN prevents plaque
formation. U373 astrocytes were transfected with EGFP-LCb-EED/QQN or with EGFP-LCb as control, and their clathrin structures imaged 48 h after
transfection. The semilogarithmic plots represent maximum fluorescence versus time of pits and plaques from two cells expressing similar amounts
of EGFP-LCb or EGFP-LCb-EED/QQN. The average lifetimes are 69623 s (n=129) for canonical pits tagged with EGFP-LCb is and 232678 s (n=30) for
plaques; the corresponding fluorescence intensity maxima are 1,4206536 and 2,0876455, respectively. The difference in maximum fluorescence
intensity is statistically significant (p,0.0001). The right panels are snapshots from two different time points of a video acquired from cells expressing
EGFP-LCb-EED/QQN using the WF fluorescence combined with bright-field phase illumination; the images show the elongated shape of the cells and
the change in their position. Control cells (unpublished data) are more symmetric and remain stationary. (B) Increased motility of cells correlates with
absence of plaques. Each panel shows four motility tracks, each from a different cell that either expresses its endogenous clathrin light chains (not
transfected), or where they have been replaced, by overexpression, with EGFP-LCb or EGFP-LCb-EED/QQN.
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Merrifield and coworkers [9,10] combined TIR and WF
fluorescence microscopy to examine formation of clathrin
structures, tagged with LCa-DsRed, at the adherent surface of
Swiss 3T3 cells. Most of their reported structures have a constant
fluorescence signal and are relatively long-lived (.200 s). These
and other published tracings [13,15,38,43] show the final interval
in the lifetime of these objects but do not illustrate the growth
phase. A characteristic of all the structures they examine is an
abrupt inward movement coincident with membrane budding
shortly before dissolution of the coat. These steps represent bona
fide endocytic events, by the criterion that entrapped transferrin-
receptor, tagged with a pH-sensitive fluorophore, does not respond
to pH changes in the medium once the structure has begun to
uncoat [10]. We have now been able to use a similar TIR-WF
protocol, but with substantially longer acquisition periods, to
capture unambiguously the complete lifecycle of all clathrin
structures, whether long or short. We specifically included in our
analysis the Swiss 3T3 cell line studied by Merrifield et al. We
confirmed that all the long-lived structures at the adherent surface
indeed move inward abruptly just before losing their coats, in
contrast to the continuous inward movement of clathrin and AP-2
during the growing phase of a canonical coated pit. We conclude
that the long-lived endocytic clathrin structures analyzed by
Merrifield et al., are the same as the clathrin-coated plaques
described here. They are probably also equivalent to the persistent
clathrin-containing structures observed by TIR in primary
adipocytes [28]. These assemblies, which represent more than
half the total clathrin signal at any time point, have variable size,
are on average 2.5 times larger than pits, and have lifetimes longer
than 3 min. Taking into consideration that plaques are relatively
larger and that they invaginate at a considerably smaller frequency
than pits, and including the contribution of pits forming on the
free surface, we estimate that plaques carry approximately 11% of
the transferrin uptake mediated by clathrin. This estimate is in
agreement with the insignificant change of transferrin uptake in
cells lacking plaques due to perturbations in the interaction
between clathrin light chains and Hip1R (this study; see also [47])
or due to Hip1R ablation by RNAi [34].
Do the long-lived coated structures correspond to the gently
domed, extended arrays of clathrin seen on the adherent surface of
unroofed cells by freeze-etch electron microscopy? Several lines of
evidence argue that they do. First, the extended arrays are seen
only on the cytoplasmic surfaces of adherent plasma membranes
containing plaques as detected by live-cell imaging—e.g., the
HeLa cells imaged in Figure 1—and not on the corresponding
surfaces of cells—e.g., the BSC1 cells in Figure 1—that do not
contain plaques. Second, the sizes of the arrays seen by electron
microscopy and the plaques described here are similar. The largest
of the hexagonal arrays in published micrographs are about 500–
1,000 nm in diameter, and most are smaller (this study and [24–
27]). The plaques have dimensions that range from diffraction
limited to several hundred nanometers. Third, the DiNa
measurements described here show that the coated plaques we
have characterized are essentially flat, as are those seen by electron
microscopy. Fourth, extended fluorescent patches induced during
InlB-dependent uptake of Listeria monocytogenes [58] probably
correspond to hexagonal flat arrays seen by microscopy during
engulfment of latex beads by macrophages [24].
Why do the available electron micrographs of thinly sectioned
samples not show deeply invaginated plaques? A simple explana-
tion is provided in the graphical representation presented in Figure
S6; the data were obtained from a time-lapse series, and show, for
every frame of the video, the number of structures scored as pit or
plaque, and the number of pits scored as deeply invaginated (the
Figure 5. Recruitment of cortactin, Arp2/3, and dynamin into
clathrin-coated pits and plaques. Spinning disk fluorescence
intensity profiles obtained from time-series images of clathrin
assemblies forming at the adherent (bottom) surface of Swiss 3T3
fibroblasts stably expressing Clathrin LCa-DsRed and transiently
expressing (A) cortactin-EGFP, (B) Arp3-EGFP, and (C) dynamin2-EGFP.
Fluorescence images for each set of these proteins were simultaneously
acquired with a beam splitter using 200-ms exposures obtained every
5 s. The points represent averages of complete datasets, normalized to
the maximum intensity and to the lifetime of the object. Each point
represents average plus or minus standard deviation. N is the number
of coated pits and plaques analyzed. The maximum recruitments of
clathrin LCa-DsRed, cortactin-EGFP, Arp3-EGFP, or dynamin2-EGFP by
plaques are statistically higher (p,0.0001) than recruitment by pits.
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last 10–15 s of their lifetime) or plaques scored as displaced from
the substrate (by 100–150 nm during the last 5 s of their lifetime).
This analysis reveals detection of a total of 799 structures scored as
pits and 544 as plaques, with 197 images of deeply invaginated pits
and only 11 substantially displaced plaques. Thus, the likelihood of
visualizing deeply invaginating plaques by electron microscopy of
thin sections is extremely low, and presently it is unrealistic to
expect acquisition of a sufficient number of images to draw
confident conclusions.
Coated Plaque Assembly
Spontaneous clathrin assembly under suitable conditions in vitro
yields closed structures that range in diameter from 60 to 200 nm.
Reconstructions from cryoEM images of D6-barrel and tetrahedral
coats show that curvature is built into the pucker at the apex of a
triskelion and that side-by-side packing of the legs can vary so as to
create much flatter lattices [21]. The hexagonal arrays seen by
electron microscopy on the inward-facing surface of membranes
apposed to plastic substrates or bacterial cell walls are quite
imperfect, with frequent defects, as if their enforced planar assembly
had introduced strain, just as expected if we were to extrapolate to
zero curvature the variation in lateral contacts seen in more sharply
curved coat structures. Thus, the extended flat arrays could arise
because adhesion to the external substrate resists introduction of
curvature, and essentially planar arrays grow until the accumulated
strain is best compensated by a defect in the lattice. But the
requirement of an intact light-chain-Hip1R-actinlinkage for plaques
to appear and the correlation of plaques with cell motility both
suggest that cytoskeletal interactions may help direct coated plaque
formation. AP-2 is also essential, for both coated plaques and
canonical coated pits at the plasma membrane (this paper). It is
therefore possible that pits and plaques initiate similarly, through an
AP-2 dependent mechanism, but that the organization of the actin
cytoskeleton dictated by adhesion to a substrate redirects clathrin
assembly into planar arrays. To outline a specific mechanism for this
redirection wouldrequire amoredetailed picture thannow available
of how Hip1R associates with its partners. We note that initiation of
clathrin coats that is AP-2 independent can also occur, as the Listeria-
directed arrays neither incorporate AP-2 nor require it [59].
Epsin
Epsin accumulates along with clathrin during assembly of a
canonical coated pit. Our measurements of the relative z-
displacement of epsin and clathrin show that the former remains
in the plane of the surrounding membrane as the coated pit
invaginates. That is, most of epsin does not move into the domed
region, nor is it present in the mature coated vesicle. It therefore
must accumulate largely around the rim of the pit, as does its
partner, Eps15 [55,56]. A limited number of published [54,57]
and unpublished electron micrographs (L. Traub, personal
communication) suggest an accumulation of epsin on the rim of
the pits, but the number of available images are not sufficient to
Figure 6. Movement of clathrin-coated pits and coated plaques into the cell interior. (A and B) Fluorescence intensity profile from a single
canonical coated pit(A)orcoatedplaque(B) acquiredfrom a Swiss3T3 cells stablyexpressingclathrin LCa-DsRed byalternating TIRandWFillumination.
The clathrin structures were imaged as they assembled on the bottom surface of the cell in direct contact with the coverslip. The TIR/WF cycles were
acquired at 10-s intervals using exposures of 200 and 1,000 msec taken 200 ms apart. The WF fluorescence plot was normalized to its maximum
fluorescence value to correct for variations in the intensity, whereas the TIR signals were normalized to that of the WF at the early stages of coat
formation but allowed to diverge as the coat was formed. This corrects for differences in illumination and number of fluorophores in the spot [14]. Stars
denote the endpoint used to calculate the lifetime. (C) Summary of TIR and WF data collected from all plaques during the last 40 s before their
dissolution, with both datasets normalized to their corresponding maximum intensity, as in the work of Merrifield et al. [9]; each time point represents
the average plus or minus the standard deviation (n=40 plaques). (D and E) The average axial position (z, nm) for the ensemble of clathrin LCa-DsRed
captured in the coat of pits (D) or plaques (E), plotted as a function of their respective lifetimes as calculated using, z(t)~{90|logTIR(t)
WF(t)
??
with an
evanescentTIRfieldpenetrationof90 nmdeterminedexperimentally[14].Eachpointrepresentstheaverageplusorminusthestandarddeviationforall
pits (n=51) and plaques (n=40). The coats of canonical pits shift continuously inward as they assemble, whereas plaques move abruptly inwards only
just before dissolution. (F) The average axial position (displacement just before dissolution) of the clathrin plaques as calculated from averageintensities
presented in (C) using the same equation as in (E). This method of calculation is identical to the one used in Merrifield et al. [9]; using this method, we
detect a similar movement away from the surface at the end of the plaque lifetime, as shown in (E).
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draw a firm conclusion. At very high concentrations, epsin can
tubulate liposomes, apparently by insertion of an N-terminal
amphipathic helix into the outer leaflet of the bilayer [60]. This
property has led to the suggestion that a function of epsin in
coated-pit formation is to stabilize membrane curvature [60]. The
location of most of the epsin, at the base of the invaginating pit, a
position at which there is little net curvature, is not consistent with
this proposal. Moreover, epsin is present in the essentially
uncurved coated plaques, where its fluorescence intensity
fluctuates in parallel with clathrin, and it moves inwards together
with clathrin when the plaque internalizes. Because there is no
curvature, and hence no differential z-displacement, we cannot tell
from the DiNa measurements whether epsin is present throughout
the plaque, or mostly at its rim as in a canonical coated pit.
Published electron micrographs suggest that epsin may be
distributed throughout the lattice [54,57], but the studies have
not been extensive enough to draw firm conclusions. Epsin
contains multiple ubiquitin-interacting motifs (UIMs), and it is
believed to facilitate incorporation of polyubiquitinated cargo into
clathrin structures [57], whereas association of epsin with
monoubiquitinated substrates is reported to exclude interaction
with clathrin coats [61]. How epsin’s putative function as a
receptor for ubiquitinated cargo relates to its localization remains
to be determined.
Membrane Budding and Uncoating
The clearly defined, inward shift of a coated plaque, which
invariably occurs shortly before dissolution, depends on actin and
dynamin. As this shift is uniform across the plaque, there is no
need to invoke any reorganization of the clathrin lattice into a
curved structure [18,29]; membrane uptake is clearly determined
by other processes. Local reorganization of the actin cytoskeleton
is the best candidate for the driving force of this invagination.
There is considerable evidence that the cortactin-activated, Arp2/
3 branching mechanism may have a role [10,15,34]. For example,
Merrifield et al. [10] report cortactin recruitment at late stages of
coat formation, and they link its arrival to internalization.
Engqvist-Goldstein et al. [34] show that recruitment of Hip1R
into long-lived clathrin coats (which we interpret as coated
plaques) follows clathrin accumulation (as might be expected from
its direct interaction with light chains), whereas cortactin peaks just
before internalization. Our data on coated plaques are in full
Figure 7. Relative positions of clathrin, AP-2, and epsin in coated pits and plaques. (A) Models representing the distribution of clathrin, AP-
2, and epsin during the formation of a canonical clathrin-coated pit or a clathrin-coated plaque. (B) Difference in the axial positions of clathrin
Tomato-LCa or clathrin LCa-DsRed with respect to AP-2 s2-EGFP (Dz, red) or to epsin1-EGFP (Dz, blue) determined for individual pits or plaques
selected for DiNa analysis in U373 astrocytes (52 pits and 40 plaques) or Swiss 3T3 fibroblasts (49 pits and 45 plaques) cells. The difference of axial
position within each pit is plotted as a function of time; to facilitate the comparison between pits of different lifetimes, the time component is
normalized as a fraction of each lifetime. DiNa results were obtained from data acquired three Swiss and three U373 astrocytes. The axial separations
become statistically significant (p,0.0001) when the pits reach a maturity of 50% or more. (C) Difference in the axial position of clathrin Tomato-LCa
with respect to AP-2 s2-EGFP (Dz, red) or to epsin1-EGFP (Dz, blue) determined for 15 and 14 individual pits selected for DiNa analysis in BSC1 cells,
respectively. Similar DiNa results were obtained from data acquired in three additional Swiss and three additional astrocytes (unpublished data). Error
bars in (B) and (C) indicate plus or minus the standard deviation.
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agreement with these observations. As Hip1R is essential for
plaque initiation, we cannot determine whether its linkage to actin
is also critical for internalization.
Our data suggest that uncoating of a plaque proceeds by an
auxilin- and Hsc70-dependent mechanism, as for canonical coated
vesicles. Auxilin is recruited during the last seconds of a coated
plaque lifetime, but it is also incorporated in variable amounts
during the course of assembly. It is possible that auxilin and Hsc70
catalyze exchange of clathrin into and out of the plaque during its
lifetime [43].
Plaques, Actin, and Yeast Cells
Ample genetic evidence points to a pathway that requires both
clathrin and actin for endocytosis in yeast cells (reviewed in [30]).
Moreover, live-cell fluorescence microscopy shows that clathrin,
actin, and some of their interacting proteins are recruited to
distinct sites at the plasma membrane, beginning with clathrin and
Las17 (a regulator of Arp2/3) followed by actin and ending with a
number of kinases and phosphatases, which facilitate efficient
disassembly of different endocytic coat proteins [62]. This
sequence of molecular associations parallels inward movement of
the clathrin fluorescent spot [4,11,62]. Tubular invaginations of up
to 50 nm in diameter and 180 nm in length, decorated with
clathrin at their tips, can be seen emanating from the plasma
membrane [63]. Thus, actin-based activities appear to drive a
partially coated clathrin structure inward. Clathrin may provide
the membrane anchor, and some additional activity may then
drive membrane scission. We suggest that the endocytic pathway
and its mechanisms characterized in yeast are closely related to the
uptake of coated plaques in more complex eukaryotic cells.
Functions of Clathrin-Coated Pits and Coated Plaques
The endocytic functions of canonical coated pits have been well
documented. The canonical structures are the principal carriers
for uptake of transferrin receptor and many other membrane-
inserted proteins and for rapid reuptake of membrane components
at synapses [2,3]. Coated plaques can also take up transferrin (and
presumably other receptor-bound ligands) [9,10,28], but their
Figure 8. Recruitment of AP-2 and epsin into clathrin-coated pits and plaques. WF fluorescence intensity profiles obtained from time-series
images of clathrin assemblies forming at the bottom surface of cells using 1-s simultaneous exposure with 10-s intervals. The data were obtained
from (A and C) U373 astrocytes stably expressing AP-2 s2-EGFP and transiently expressing Tomato-LCa, or (B and D) Swiss 3T3 fibroblasts stably
expressing Clathrin LCa-DsRed and transiently expressing epsin1-EGFP. Left panels represent examples from single pits or plaques as a function of
time. Right panels represent averages from the complete data sets normalized to the length of each lifetime. Each point represents average plus or
minus the standard deviation; the data from the astrocytes presented in (A) and (C) were obtained from the three cells; the data from the fibroblasts
presented in (B) and (D) are also from three cells; n is the number of analyzed pits and plaques.
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principal physiological role remains to be determined. When they
do form, coated plaques appear to require contact with an external
substrate. Conversely, the increased cell motility we see when
coated plaques are ablated suggests that loss of plaques is linked to
a loss of firm attachment. The close interplay between the actin
and clathrin systems responsible for plaque internalization may
also have been hijacked by invaders such as certain bacteria [59]
and by large viruses [36]. Clathrin, dynamin, actin, and Arp2/3
are all required for invasion by L. monocytogenes and for pinocytosis
of beads coated with the invasin InlB [58,59,64]. Extended arrays
of clathrin have been seen on the adherent surface of osteoclasts
[25,26], on the cytoplasmic face of the invaginated cell membrane
during bead uptake [24], and extended clathrin arrays are also
present on endosomes [65]. The latter arrays are not associated
with the usual heterotetrameric adaptor proteins.
In summary, we have established criteria for identifying coated
plaques, and we have described many of their properties as
detected by live-cell fluorescence microscopy. The properties of
coated plaques, as worked out by quantitative analysis of our live-
cell data, are distinct from the properties of canonical coated pits
and are fully consistent with all published observations of clathrin-
coated structures by electron microscopy, including the new
electron microscopy data presented here. The participation of
actin in coated-plaque internalization is the most obvious
difference between these modes of membrane remodeling, but
we have documented others, including the dynamics of coat
assembly and the relationship to processes such as adhesion and
motility. Clathrin is clearly a scaffold for a number of
mechanistically distinct, membrane-associated process.
Materials and Methods
Preparation of Plasmids, Expressor Cells,
Immunofluorescence, and RNAi
Swiss 3T3 cells expressing LCa-DsRed were kindly provided by
Dr. W. Almers, BSC1, and U373 cells expressing s2-EGFP were
previously characterized in [5,22]. LCa-DsRed was made by
fusing rat LCa DNA to the 59 end of the coding sequence for
DsRed (Clonetech). Epsin-EGFP was created by fusing the rat
epsin-1 DNA to the 59 end of the coding sequence of EGFP
(Clonetech). Tomato-LCa was created from rat LCa as described
in [22]. BSC1 cells stably expressing epsin-EGFP were obtained by
transfection using Fugene 6 (Roche Applied Science). A single
clone of low-expressing Epsin-EGFP was maintained by selection
with G418. All cells were maintained at 37uC and 5% CO2in
Dulbecco’s modified Eagle medium (DMEM) with 10% fetal
bovine serum and 0.4 mg/ml G418. As a control, BSC1 cells were
transfected with LCa-DsRed and imaged 48 h after transfection.
The dynamics of the clathrin spots under these conditions (Figure
S3) show no difference to those in EGFP- or Tomato-LCa–
expressing cells. U373 astrocyte cells stably expressing s2-EGFP
were used in conjunction with transient expression of Tomato-
LCa and imaged after 48 h of transfection. Swiss 3T3 cells stably
expressing LCa-DsRed were used in conjunction with transient
expression of epsin-EGFP and imaged after 48 h of transfection.
Cells transiently expressing cortactin-EGFP (gift of Dr. D.
Drubin), EGFP-LCb, or EGFP-LCb-EED/QQN (gift of Dr. P.
McPherson) were also imaged after 48 h of transfection. All
transfections were carried out using Fugene 6 according to
manufacturer instructions. For immunofluorescence, a monoclo-
nal antibody (mAb), CON.1, specific for clathrin LCa and LCb
was generated from hybridoma cells obtained from the American
Type Collection. Prior to staining, the cells were fixed with 3.75%
paraformaldehyde, and then sequentially labeled with CON.1 and
a Cy3-labeled antimouse polyclonal antibody. Depletion of the
clathrin light chains was achieved by sRNAi-mediated gene
silencing experiments by transfecting U373 astrocytes stably
expressing s2-EGFP with a mixture containing 200 nM of LCa
oligo (D-004002-01; Dharmacon) and 200 nM of LCb oligo (D-
004003-03; Dharmacon) with Oligofectamine (Invitrogen); the
treatment was repeated after 48 h, and the cells were replated after
62 h onto glass coverslips for imaging 10 h after. A scrambled
oligo sequence (Dharmacon; gift from Dr. J. Liberman) was used
as control. The efficiency of LCa and LCb depletion was
determined by measuring the loss of immunofluorescence using
CON.1 specific for both light chains. Depletion of Hip1R was
achieved by sRNAi-mediated gene silencing experiments by
transfecting U373 astrocytes stably expressing s2-EGFP with
Figure 9. Models representing the sequential formation of a clathrin-coated pit and a coated plaque. Coated pits are small, rapidly
forming structures that by progressive recruitment of clathrin, adaptors, and other regulatory proteins deform the underlying membrane as they bud
inward to form coated vesicles. Coated plaques are larger, less dynamic, and relatively planar structures that move uniformly inward from the cell
surface shortly before the membrane pinches off. Presence and/or remodeling of the actin cytoskeleton is essential for the formation, inward
movement, and dissolution of clathrin-coated plaques, but it is not required for formation and budding of the rapidly growing clathrin-coated pits.
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200 nM of a mixture containing Hip1R oligos (L-027079-00;
Dharmacon) and with Oligofectamine (Invitrogen) or by first
transfecting U3T3 astrocytes with the mixture of Hip1R oligos,
which where then transfected 2 d later with EGFP-LCb.
Handling of Cells Prior to Imaging
Roughly 20,000–40,000 cells were plated on glass #1.5
coverslips (25 mm in diameter) 10 h before the actual imaging
experiment. Up to this point, the cells were maintained at 37uC
and 5% CO2in DMEM with 10% fetal bovine serum. Cells were
imaged in Gibco’s CO2independent medium and in the absence
of phenol red (Invitrogen). The temperature of the sample holder
(20/20 Technologies) was kept at 37uC using a Peltier-controlled
holding device. The holding device and the stage were surrounded
by a custom-designed air-controlled environmental chamber kept
at 33–35uC.
Imaging
All spinning disk confocal imaging experiments were conducted
by using the microscope setup previously described [5]. We further
modified the microscope by insertion of a computer-controlled
Spherical Aberration Correction unit (SAC; Intelligent Imaging
Innovations), which drastically reduced the spherical aberration
and thus increased the sensitivity. In addition, this setup was
modified with lasers equipped at 473 nm and 561 nm (Cobolt)
and 660 nm (Crystal Lasers). The lasers input to the fiber optic is
controlled through a PCAOM AOTF module (Neos). The
emission is collected after the spinning disk unit and passes
through a dual view unit (Roper Scientific), equipped with a
565DCXR dichroic mirror and the HQ525/40 and HQ620/50
(Chroma) to separate the fluorescence of EGFP and Tomato/
DsRed on two sides of the same CCD chip. The camera has been
updated to a Cascade 512B (Roper Scientific), which is operated
with multiplication gain and no binning. The Dual view unit and
the AOTF allow for simultaneous or for fast switching between
excitation with 473 and 561 nm. The sample is only exposed to
the laser light while the CCD is acquiring, and is immediately
turned off during the frame transfer time of the CCD, which can
take 50–200 ms, depending on mode of readout. This shutoff is
achieved by synchronizing the AOTF with the camera frame
readout TTL signal (Intelligent Imaging Innovations). Using this
setup, we have been able to image an exposure as low as 50–
100 ms, reducing phototoxicity and allowing longer measurement
times. Images were captured with SlideBook 4 software (Intelligent
Imaging Innovations). All TIR/WF measurements were carried
out using the conditions previously described [14].
Image Processing
Clathrin clusters were identified using a sequence of deconvolu-
tion, 2D Gaussian and Laplacian filtering followed by threshold-
ing, which created a mask as previously explained in [5,22]. The
masks were tracked using SlideBook 4 (Intelligent Imaging
Innovations). The coordinates of the center of these masks versus
time were exported along with the images. A MATLAB routine
[14] was used to read the intensity profile for each object from the
corresponding images. DiNa measurements were carried as
previously described [14]. Clathrin-coated structures were selected
according to the following criteria [5,14,22]: (1) the fluorescent
objects appeared and disappeared within the time series; (2) the
objects displayed the limited movement expected for membrane-
bound clathrin structures in the horizontal plane during their
growth phase (500 nm/lifetime); and (3) the objects did not collide
with each other. Cell motility as a function of time was determined
in cells imaged in WF bright illumination, by tracking the
temporal change in the position of the center of the nucleus.
Electron Microscopy
BSC1 and HeLa cells were plated on glass coverslips and grown
for 4 h (BSC1) or overnight (HeLa) at 37uC and 5% CO2in
DMEM with 10% fetal bovine serum. To visualize the adherent
surface of the cells, the samples were first broken open
(‘‘unroofed’’) by standard methods, followed by chemical fixation,
rapid freezing, deep etching, and rotary shadowing. [40]. The
samples were visualized using a JEOL JEM 1200EX microscope
operating at a nominal magnification of 30,000 and 80 kV, and
imaged digitally with an AMTKK CCD camera.
Supporting Information
Figure S1
dynamics of pits and plaques. The kymograph is a schematic
representation as a function of time of the effect of actin
depolymerization on the lifetime of pits (gray) and plaques (black)
in the adhered surface of a Swiss 3T3 cell stably expressing LCa-
DsRed. The data derived from the time series obtained from a cell
imaged using spinning disk confocal microscopy at 10-s intervals
and 100-ms exposure time (Video S3). Acquisition of the time
series was started at t=0, and 5 mM latrunculin A was added at
t=540 sec. As shown before with similarly treated BSC1 cells
[33], actin depolymerization results in the accumulation of clathrin
structures on the plasma membrane but does not affect the
dynamics of coated pits. Formation of new plaques ceases, and
existing plaques completely freeze upon latrunculin A treatment.
Found at: doi:10.1371/journal.pbio.1000191.s001 (2.12 MB TIF)
Effect of latrunculin A on the lifetime and
Figure S2
Concatenated tracings of different coated pits (gray) and plaques
(black). The characteristic times for intensity fluctuations within
the lifetime of a plaque are much shorter than the lifetime of a
coated pit. A formal autocorrelation analysis (unpublished data)
confirms this conclusion. Thus, the properties of a plaque cannot
be attributed to those of a series of coated pits forming one after
another at the same location.
Found at: doi:10.1371/journal.pbio.1000191.s002 (2.43 MB TIF)
Intensity profiles of coated pits and plaques.
Figure S3
expressing clathrin LCa-DsRed. Images from a time series
acquired by spinning disk confocal microscopy from the adherent
surface of a BSC1 cell transiently expressing clathrin LCa-DsRed.
The cell was imaged 48 h after transfection, using 3-s intervals for
a duration of 300-s and 150-ms exposures. The average lifetime of
the clathrin structures (53617 s) is similar to the average lifetime
of canonical pits previously characterized in BSC1 cells stably or
transiently expressing EGFP-LCa [5].
Found at: doi:10.1371/journal.pbio.1000191.s003 (1.68 MB TIF)
Formation of clathrin structures in BSC1 cells
Figure S4
tures forming at the cell surfaces. In these double
logarithmic plots, each dot corresponds to the duration (lifetime)
and the corresponding size (maximum fluorescence intensity prior
to dissolution) of a clathrin or AP-2 fluorescent spot recorded at the
top or bottom plasma membrane of adherent cells. Each panel
contains data obtained from three cells and includes the
observations obtained from the single cells depicted in the
experiments associated with Figures 2, 3, and 4. (A) Average
lifetime of the canonical coated pits on the top of Swiss 3T3 cells
stably expressing Clathrin LCa-DsRed is 54.6629 s (n=193). The
maximum fluorescence intensity is 1,98861,387. (B) Average
lifetime of the canonical coated pits on the top of U373 astrocytes
Sizes and lifetimes of clathrin-coated struc-
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