The Rockefeller University Press $30.00
J. Cell Biol. Vol. 195 No. 3 525–536
Correspondence to Matteo Bonazzi: email@example.com; Frances
M. Brodsky: Frances.Brodsky@ucsf.edu; or Pascale Cossart: firstname.lastname@example.org
Matteo Bonazzi’s present address is UMR 5236, Centre d’études d’agents
Pathogènes et Biotechnologies pour la Santé (CPBS), Centre National de la
Recherche Scientifique, Université Montpellier 1, Université Montpellier 2, 34293
Abbreviations used in this paper: CHC, clathrin heavy chain; CLC, clathrin light
chain; EPEC, enteropathogenic Escherichia coli; MALDI, matrix-assisted laser
desorption/ionization; TOF, time of flight; wt, wild type.
Clathrin coats membranes of vesicles formed during receptor-
mediated endocytosis and organelle biogenesis from the trans-
Golgi network (Brodsky et al., 2001). Clathrin can also form
extended lattices with no curvature at cell–substrate interfaces
(plaques; Saffarian et al., 2009) and patches on endosomes
(Popoff et al., 2009; Raiborg and Stenmark, 2009). The clath-
rin coat itself is formed by self-assembly of triskelion-shaped
molecules composed of three clathrin heavy chains (CHCs) and
associated clathrin light chain (CLC) subunits (Brodsky et al.,
2001). Clathrin coats form at membranes by binding a variety of
adaptor molecules that select the cargo molecules sequestered
into the coat for sorting. During internalization of receptors that
stimulate Src family kinases, including the receptor tyrosine
kinase (RTK) EGF receptor (EGFR) and T and B lymphocyte
receptors, CHC is modified by tyrosine phosphorylation (Wilde
et al., 1999; Stoddart et al., 2002; Crotzer et al., 2004). The
function or molecular details of this modification have not been
fully defined, but Src family kinase phosphorylation of CHC
is specifically required for uptake of these signaling receptors
(Crotzer et al., 2004). Clathrin is also required for the inter-
nalization of large objects such as bacteria (Veiga and Cossart,
2005; Veiga et al., 2007; Eto et al., 2008; Chan et al., 2009), fungi
hyphae (Moreno-Ruiz et al., 2009), and large viruses (Cureton
et al., 2009) in a process that involves cooperation with actin.
In the case of Listeria monocytogenes (Sousa et al., 2007;
Bonazzi et al., 2008), pathogenic adhesion and infection
involve signaling through Src family tyrosine kinases triggered
that host–pathogen interactions induce tyrosine phosphory-
lation of clathrin heavy chain. This modification was critical
for recruitment of actin at bacteria–host adhesion sites dur-
ing bacterial internalization or pedestal formation. At the
bacterial interface, clathrin assembled to form coated pits
of conventional size. Because such structures cannot inter-
nalize large particles such as bacteria, we propose that
acterial pathogens recruit clathrin upon interaction
with host surface receptors during infection. Here,
using three different infection models, we observed
during infection, clathrin-coated pits serve as platforms to
initiate actin rearrangements at bacteria–host adhesion
sites. We then showed that the clathrin–actin interdepen-
dency is initiated by Dab2 and depends on the presence of
clathrin light chain and its actin-binding partner Hip1R, and
that the fully assembled machinery can recruit Myosin VI.
Together, our study highlights a physiological role for
clathrin heavy chain phosphorylation and reinforces the
increasingly recognized function of clathrin in actin cyto-
skeletal organization in mammalian cells.
Clathrin phosphorylation is required for actin
recruitment at sites of bacterial adhesion
Matteo Bonazzi,1,2,3 Lavanya Vasudevan,4,5,6 Adeline Mallet,7 Martin Sachse,7 Anna Sartori,7 Marie-Christine Prevost,7
Allison Roberts,4,5,6 Sabrina B. Taner,4,5,6 Jeremy D. Wilbur,4,5,6 Frances M. Brodsky,4,5,6 and Pascale Cossart1,2,3
1Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris F-75015, France
2Inserm, U604, Paris, F-75015 France
3Institut National de la Recherche Agronomique, USC2020, Paris F-75015, France
4Department of Bioengineering and Therapeutic Sciences, 5Department of Microbiology and Immunology, and 6Department of Pharmaceutical Chemistry,
The G.W. Hooper Foundation, University of California, San Francisco, San Francisco, CA 94143-0552
7Institut Pasteur, Plateforme de Microscopie Ultrastructurale, Imagopole, Paris F-75015, France
© 2011 Bonazzi et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub-
lication date (see http://www.rupress.org/terms). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 195 • NUMBER 3 • 2011 526
Figure 1. Infection induces CHC phosphorylation. (a) Quantification of the intensity of chemiluminescence of three independent Western blots of Jeg3
cells treated as indicated or HeLa cells infected with EPEC for the indicated times. After each treatment, CHC was immunoprecipitated and tyrosine phos-
phorylation was assayed with the anti-phosphotyrosine antibody PY20. Error bars indicate mean ± SD. (b) MALDI-TOF mass spectrometry spectrum of
in vitro phosphorylated CHC fragment (1074–1675). Src kinase was incubated with recombinant CHC followed by digestion with trypsin. Tyr 1487 is
527 Clathrin in actin recruitment during bacterial infection • Bonazzi et al.
found within a peptide with a predicted monoisotopic mass of 1942.9 D and within the phosphopeptide with a predicted mass of 2022.9 D. Tyr 1477
is found within a peptide with a predicted monoisotopic mass of 2355.1 D and within the phosphopeptide with a predicted mass of 2435.1 D. Arrows
indicate the native and phosphorylated peptides. (c) Purified recombinant his-tagged-hub fragment of CHC (residues 1074–1675) was treated with or
without recombinantly purified Src kinase for 3 h at room temperature. Samples were immunoblotted with antibody against the histidine tag on the hub
(His, as loading control), 4G10 (for phospho-tyrosine), and anti-phospho-CHC antibody (pCHC) purified by affinity column and by membrane-based deple-
tion. (d) Anti-pCHC (affinity purified by column) was tested for binding to the 1477&1487-phospho-peptide in the presence of the nonphospho-peptide,
1477-phospho-peptide, 1487-phospho-peptide, or 1477&1487-phospho-peptide at the indicated dilutions by ELISA. (e) Jeg3 cells were infected with
L. innocua(InlA) for 30 min and HeLa cells were infected with L. innocua(InlB) for 5 min or with EPEC for 6 h. After infections, cells were fixed and labeled
for total CHC (CHC, red) and phospho-CHC (pCHC, green), and F-actin was labeled with fluorescent phalloidin (actin, blue). Arrows indicate colocaliza-
tion between bacteria, pCHC, and actin. Bars 10 µm.
by bacterial binding to host-cell receptors. Similarly to L. mono
cytogenes, enteropathogenic Escherichia coli (EPEC) require
clathrin during infection, but unlike L. monocytogenes, EPEC are
not internalized by host cells and form actin-based pedestals at
the sites of interaction with the infected cell (Wong et al., 2011).
In this study, we address whether Src family kinase-mediated
tyrosine phosphorylation of CHC occurs during bacteria–cell
interaction and whether the infectious process might reveal a
function for CHC phosphorylation in the context of clathrin–
Our understanding of clathrin–cytoskeleton interac-
tions during clathrin-mediated processes is evolving. The
participation of clathrin in uptake of pathogens challenges
the concept of a size limit for clathrin cargo and implicates
clathrin–actin interactions in pathogen internalization path-
ways (Pizarro-Cerdá et al., 2010). During infection, clathrin
and actin accumulate at sites of bacteria–host interactions,
and clathrin recruitment precedes actin rearrangements that
are critical both for bacterial internalization and for the for-
mation of bacteria-induced actin pedestals (Veiga et al., 2007).
These observations coincide with an increasingly recognized
connection between clathrin and the actin cytoskeleton in
mammalian cells (Merrifield et al., 2002; Yarar et al., 2005;
Hyman et al., 2006) that goes beyond colocalization (Ferguson
et al., 2009). The two mammalian CLCs (LCa and LCb) both
interact with actin-binding proteins Hip1 and Hip1R via a
shared 22–amino acid sequence in their N terminus (Chen
and Brodsky, 2005; Legendre-Guillemin et al., 2005). Bind-
ing of CLCs to Hip proteins regulates their interaction with
actin and affects actin dynamics (Engqvist-Goldstein et al.,
2001; Engqvist-Goldstein et al., 2004; Wilbur et al., 2008).
The importance of a functional link between clathrin and
actin, via the CLC–Hip interaction, has been well-established
in yeast, where the Hip homologue Sla2p is essential for
clathrin-mediated endocytosis (Newpher and Lemmon, 2006);
its role in various forms of mammalian cell endocytosis is
still being defined (Kaksonen et al., 2006; Saffarian et al.,
2009). Actin is present at clathrin plaques, which requires
CLC–Hip interaction, and is also situated where the neck
of a clathrin-coated pit elongates, apparently directing
coated vesicles away from the plasma membrane (Collins
et al., 2011). This latter activity is needed for coated vesicles
to form at membranes with high surface tension (Boulant
et al., 2011). The molecular details of how clathrin and
actin coordinate during bacterial interaction with the host
cell surface are defined in the investigation reported here.
By analysis of clathrin coat formation, the role of clathrin
phosphorylation, and the interaction of clathrin with actin
during bacteria–host cell attachment and internalization, we
establish the sequence of molecular events during early
stages of infection. We show that bacteria–host adhesion
involves the adaptor Dab2 to form clathrin-coated pits that
support actin recruitment via CLC and Hip1R. The motor
protein Myosin VI is then recruited to provide a pulling force
for bacterial internalization. Moreover, we show that, similar
to the endocytosis of signaling receptors, the phosphoryla-
tion of CHC is triggered by bacterial infection and that this
modification is critical for effective function of the clathrin
platform as an actin organizer during bacterial internaliza-
tion and pedestal formation.
CHC phosphorylation is required
for bacterial internalization or
Signaling through tyrosine kinases promotes the localization
of clathrin at the plasma membrane, dependent on tyrosine
phosphorylation of CHC (Wilde et al., 1999; Crotzer et al.,
2004). This phosphorylation is mediated by Src family kinases
(Src, Lyn, or Lck; Wilde et al., 1999; Stoddart et al., 2002;
Crotzer et al., 2004). The same kinases are also activated dur-
ing the clathrin-dependent internalization of L. monocyto
genes by the InlA pathway (Sousa et al., 2007; Veiga et al.,
2007; Bonazzi et al., 2008). We therefore tested the possibility
that bacteria, which use clathrin to invade host cells (Veiga
et al., 2007), trigger the phosphorylation of CHC. Epithelial
cells were incubated for 1 h with either L. monocytogenes,
or with the noninvasive strain Listeria innocua expressing
either one of the two Listeria invasion proteins InlA or InlB
(L. innocua(InlA) and L. innocua(InlB)) to specifically fol-
low the two internalization pathways used by Listeria during
infection (Jonquières et al., 1999; Sousa et al., 2007; Veiga
et al., 2007; Bonazzi et al., 2008). Alternatively, HeLa cells
were infected with EPEC for 3, 6, and 8 h. In addition, to
directly test the possibility that bacterial signaling is involved
in CHC phosphorylation, cells were also incubated with either
purified InlA or InlB for 1 h. CHC was then immunoprecipi-
tated and tyrosine phosphorylation was analyzed by Western
blotting. In all cases, we could detect a significant degree of
CHC phosphorylation (Fig. 1 a and Fig. S1, a and b), with the
strongest phosphorylation of CHC upon exposure to InlA and
JCB • VOLUME 195 • NUMBER 3 • 2011 528
We next tested the role of the CHC phosphorylation in
Listeria internalization and in the formation of EPEC-induced
actin pedestal. To do so, endogenous CHC was depleted by
siRNA in Jeg3 and HeLa cells, and CHC expression was res-
cued by transfecting with either wild-type (wt) CHC-GFP or
a mutant CHC-GFP with tyrosines 1477 and 1487 changed
to phenylalanines (Y1477, 1487F). Both constructs carried
point mutations to avoid siRNA sensitivity (Fig. 2 a). Jeg3 and
HeLa cells were then infected with L. monocytogenes or with
EPEC, respectively. Bacterial internalization was evaluated
by differential immuno-labeling, and actin-based pedestals
were quantified by labeling F-actin with fluorescent phal-
loidin. As previously reported (Veiga et al., 2007), clathrin
depletion inhibited both Listeria internalization and EPEC
pedestal formation (Fig. 2, b and c). Strikingly, the expression
of wt CHC-GFP restored bacterial entry and pedestal forma-
tion to 70% and 60% of control cells, respectively, whereas
the expression of the Y1477, 1487F mutant CHC-GFP failed
to do so (Fig. 2, b and c), establishing that CHC phosphoryla-
tion is critical for efficient L. monocytogenes internalization
and EPEC pedestal formation.
Ultrastructural analysis of clathrin
recruitment at bacterial adhesion and
Since our first study of a role for clathrin in bacterial inter-
nalization, a key issue has been to explain how structures that
do not normally exceed a diameter >200 nm can mediate the
internalization of much larger particles such as bacteria and
fungus hyphae (Veiga and Cossart, 2005; Veiga et al., 2007;
Moreno-Ruiz et al., 2009). The optical resolution of conven-
tional fluorescence microscopy is not sufficient to determine
whether clathrin accumulation during infection corresponds
to the formation of a homogeneous coat, of multiple coated pits,
or represents the recruitment of clathrin-coated vesicles from
other intracellular compartments. Here we investigated the
ultrastructural organization of clathrin at the Listeria entry sites
and at enteropathogenic E. coli (EPEC)-induced actin pedestals
by electron microscopy. To study Listeria uptake, cells were
infected with either L. innocua (InlA) or L. innocua(InlB) for
15 and 30 min. From previous studies, these correspond to the
time points when clathrin was more frequently observed at Listeria
after 8 h of EPEC infection (Fig. 1 a). Because Src is activated
during the InlA-mediated internalization of L. monocytogenes,
we tested the possibility that, at least in this pathway, Src me-
diates CHC phosphorylation. Indeed, treatment of Jeg3 cells
with the Src family kinase inhibitor PP1, before InlA binding,
totally prevented CHC phosphorylation (Fig. S1 c).
We previously identified CHC tyrosine 1477 as a target
site for Src family kinases by peptide mapping and mutagenesis
(Wilde et al., 1999). Further mass spectrometry analysis of
the purified recombinant hub fragment of clathrin (residues
1,074–1,675) incubated with purified Src kinase revealed
that tyrosine 1487 is also a target phosphorylation site (Fig. 1 b).
To be able to specifically identify phosphorylated clathrin
in vivo, we raised a polyclonal antibody against a phospho-
peptide comprising these two target sites (anti-pCHC). This
antibody was purified by depletion with nonphosphorylated
peptide, followed by affinity purification with the 1477&1487-
phospho-peptide. Western blotting showed a preferential
affinity of this antibody for Src-phosphorylated clathrin hub
fragment compared with unphosphorylated hub (Fig. 1 c). By
inhibition of antibody binding to the 1477&1487-phospho-
peptide with nonphospho-peptide or peptides phosphorylated
only at 1477 or 1487 or the 1477&1487-phospho-peptide, the
affinity-purified antibody was shown to primarily recognize
an epitope dependent on phosphorylation of both target sites
(Fig. 1 d). The anti-pCHC had minimal reactivity with pep-
tides phosphorylated at each single site or the nonphospho-
peptide. Analysis of cells stimulated with EGF showed that
the anti-pCHC recognized the plasma membrane clathrin that
accumulates upon EGF signaling and did not detect the intra-
cellular clathrin recognized by the X22 monoclonal antibody
(Fig. S2; Brodsky, 1985).
The anti-pCHC antibody was then used to label cells dur-
ing infection. In addition, F-actin was labeled by fluorescent
phalloidin. Jeg3 cells were infected with L. innocua(InlA) for
30 min and HeLa cells were infected with L. innocua(InlB) or with
EPEC for 5 min or 6 h, respectively. In all three cases, phospho-
CHC was highly enriched around all bacteria that recruited
F-actin, and the intracellular staining of phospho-CHC was neg-
ligible (Fig. 1 d). In contrast, as previously reported (Veiga and
Cossart, 2005), the X22 antibody against total CHC labeled cyto-
solic structures as well as bacteria cell adhesion sites (Fig. 1 d).
Figure 2. CHC phosphorylation is required
for bacterial infection and pedestal formation.
(a) Representative Western blot of Jeg3 cells
transfected with a control siRNA or with a CHC-
targeted siRNA alone or in combination with the
overexpression of wt CHC-GFP or the Y1477,
1487F mutant of CHC-GFP (non-pCHC-GFP).
Comparable results were obtained in HeLa cells.
Note that the GFP tag on the rescue constructs
interferes with reactivity of the TD.1 antibody
against the CHC N-terminal domain (Näthke
et al., 1992) used for immunoblotting. (b and c)
Jeg3 cells and HeLa cells were then infected with
L. monocytogenes and HeLa cells with EPEC.
Bacterial internalization and EPEC pedestal
formation were quantified by immunofluores-
cence. In all cases, values are means ± SD of
three independent experiments (error bars).
529Clathrin in actin recruitment during bacterial infection • Bonazzi et al.
Jeg3 cells with L. innocua(InlA), and used a candidate-based
approach to investigate the molecular machinery that connects
actin dynamics with clathrin coats. Cells were infected with
L. innocua(InlA) or L. innocua(InlB) for 15, 30, and 45 min,
and EPEC infections were performed for 4, 5, and 6 h. These
time points correspond to the maximal frequency of clathrin
recruitment during infection. As the identification of the clathrin
adaptor involved in L. monocytogenes internalization remains
elusive (Veiga and Cossart, 2005; Pizarro-Cerdá et al., 2007),
entry sites. By electron microscopy, we never observed homo-
geneous clathrin coats around invading bacteria, but rather only
isolated clathrin-coated pits, with a size similar to that previ-
ously reported in literature (Fig. 3, a–c). To test the possibility
that clathrin might form atypical coats at bacteria, clathrin local-
ization was also established by immuno-labeling of thawed
cryosections from HeLa cells infected with L. monocytogenes.
Confirming our morphological analysis, clathrin labeling was
never observed on flat membrane surfaces surrounding the bac-
teria, but always in association with membranous invaginations
(Fig. 3 d). These results confirm previous early observations
from our laboratory showing the presence of isolated clathrin-
coated pits that formed at the Listeria entry site (Mengaud et al.,
1996). The observation of clathrin at membrane invaginations
suggests that bacteria trigger the formation of clathrin-coated
pits rather than the recruitment of clathrin-coated vesicles.
Furthermore, complete clathrin-coated vesicles were rarely ob-
served in the vicinity of the bacterial entry sites, which suggests
the presence of stable clathrin-coated pits, rather than active
zones of endocytosis.
As the size of the observed clathrin-coated pits was in-
compatible with that of bacteria, we hypothesized that during
infection, clathrin may serve a different role rather than that
of a vesicle coat protein for endocytosis. Our hypothesis was
further strengthened by the study of EPEC infection. We pre-
viously found that during EPEC infections, clathrin depletion
impairs actin recruitment to bacterial adhesion sites, there-
fore blocking the formation of pedestals (Veiga et al., 2007).
Interestingly, our electron microscopy analysis showed that
clathrin also appeared as coated pits, but not vesicles, at
EPEC-induced actin pedestals (Fig. 3 e). As clathrin is required
for actin recruitment during bacterial internalization and
EPEC-induced pedestal formation, we suggest that, during
infection, stable clathrin-coated pits may serve as a platform
for cytoskeletal rearrangements.
We next tested the possibility that clathrin recruitment
by bacteria during infection affects conventional clathrin-
mediated endocytosis. Jeg3 cells were preincubated with Cy3-
labeled transferrin for 30 min on ice, and transferrin was chased
for 1 h at 37°C in noninfected as well as in L. innocua(InlA)-,
L. innocua(InlB)-, and EPEC-infected cells. Cells were then
acid washed before fixation to remove cell surface–bound transfer-
rin, and the efficiency of transferrin internalization was assessed
by fluorescence microscopy. Infected cells were able to inter-
nalize transferrin as efficiently as noninfected cells (Fig. S3),
which indicates that bacterial entry has no general effect on the
overall active cycle of clathrin coat assembly and disassembly.
Dab2, Myosin VI, and Hip1R orchestrate
actin association with clathrin coats
We previously found that L. monocytogenes infections by the
InlA–E-cadherin or the InlB–Met internalization pathways as
well as EPEC infections require clathrin coat formation up-
stream of the rearrangements of the actin cytoskeleton that re-
sult from host–pathogen interactions (Veiga and Cossart, 2005;
Veiga et al., 2007). To investigate these observations further,
we infected HeLa cells with L. innocua(InlB) or EPEC and
Figure 3. Ultrastructure of clathrin coats during bacterial infections.
(a–c and e) Jeg3 cells were incubated with L. innocua(InlA) (a and b) and
HeLa cells with L. innocua(InlB) (c) or EPEC (e), fixed, and processed for
electron microscopy. (d) HeLa cells were infected with L. monocytogenes
and processed for immuno-labeling on thawed cryosections where clathrin
was localized by antibody labeling. Enlarged views of the boxed regions
are shown on the right. Bars, 1 µm.
JCB • VOLUME 195 • NUMBER 3 • 2011 530
with Dab2 at the Listeria entry sites, but interestingly, we could
never observe the accumulation of Myosin VI at EPEC-induced
actin-based pedestals (Fig. 4 b). We then investigated the re-
cruitment of Hip1R, a key player in actin dynamics at clathrin
coats (Engqvist-Goldstein et al., 2001; Carreno et al., 2004;
Engqvist-Goldstein et al., 2004; Chen and Brodsky, 2005;
Newpher and Lemmon, 2006; Le Clainche et al., 2007; Wilbur
et al., 2008), as well as a component of clathrin-coated plaques
(Saffarian et al., 2009). Hip1R colocalized with actin at sites
of L. innocua(InlA) and L. innocua(InlB) infection by both
the InlA–E-cadherin and the InlB–Met entry pathways, and
at the interface between EPEC and the actin-based pedestals
(Fig. 4 c). Also in this case, the frequency of Hip1R recruitment
was maximal at 30 min of infection with L. innocua(InlA) and
we first studied Dab2, a nonclassical clathrin adaptor that has
recently been associated with the formation of unusually large
clathrin lattices at the plasma membrane (Mettlen et al., 2010).
Dab2 localized, together with actin, at the entry site of both
L. innocua(InlA) and L. innocua(InlB), with a higher frequency
of protein recruitment at 30 and 15 min, respectively (Fig. 4 a).
Dab2 was also present at the interface between EPEC and actin
pedestals throughout the 4–6-h period observed (Fig. 4 a).
In addition to its role as a clathrin adaptor, Dab2 regulates
clathrin-mediated endocytosis by interacting with the molecular
motor Myosin VI (Inoue et al., 2002; Morris et al., 2002), which
drives the internalization of clathrin-coated vesicles by mov-
ing toward the minus end of actin cables (Buss et al., 2001a,b).
In our infection models, the localization of Myosin VI coincided
Figure 4. Recruitment of actin-organizing
machinery during infection. Jeg3 cells were in-
cubated with L. innocua(InlA, red) for 15, 30,
and 45 min, and HeLa cells were incubated
with L. innocua(InlB, red) for 5, 15, and 30 min
or with EPEC for 4, 5, and 6 h, then fixed
and processed for immunofluorescence with
antibodies recognizing Dab2, myosin VI, or
Hip1R (all green). Actin was labeled with fluo-
rescent phalloidin (blue). At each time point of
infection, the frequency of protein recruitment
at bacteria adhesion sites was evaluated by
immunofluorescence. Arrows point to sites of
colocalization between bacteria, the labeled
protein, and actin. Values are means (± SD)
of three independent experiments where 100
bacteria were counted for each condition.
Error bars indicate mean ± SD. Bars, 10 µm.
531 Clathrin in actin recruitment during bacterial infection • Bonazzi et al.
CHC, Dab2, and Myosin VI recruitment (Fig. 6 c). This par-
tial effect is likely caused by decreased CHC stability in the
absence of CLC, reducing CHC presence at the membrane.
Despite its clear impact on Listeria internalization, Myosin VI
depletion did not impair the recruitment of the other members
of the clathrin–actin interaction machinery. These were even
detected in higher quantities at the Listeria adhesion sites in
the absence of Myosin VI, which reflects a lack of internal-
ization (Fig. 6 d). The depletion of Myosin VI also did not
affect protein recruitment at EPEC pedestals (Fig. 6 d). Hip1R
depletion and cytochalasin D treatment had similar effects and
both prevented the recruitment of actin and Hip1R at clath-
rin coats (Fig. 6, e and f). This latter effect likely reflects the
strong influence that Hip1R and actin have on each other
(Wilbur et al., 2008).
We next used the clathrin rescue assay to investigate the
role of CHC phosphorylation in the organization of the clathrin-
actin machinery. After treatment with siRNA, the ectopic ex-
pression of wt CHC-GFP efficiently rescued endogenous CHC
depletion, and CHC-GFP was efficiently recruited at bacte-
rial adhesion sites in all of the three infection models studied.
Conversely, the Y1477, 1487F CHC-GFP mutant was not de-
tected at sites of bacterial entry and adhesion. As previously
observed, Dab2 localization was not significantly affected by
the depletion of endogenous CHC (Figs. 6 and 7), and conse-
quently, the overexpression of either wt or mutant CHC did
not alter Dab2 recruitment at bacterial adhesion sites (Fig. 7).
Notably, however, the recruitment of CLC, Hip1R, Myosin VI,
and actin was restored to control levels in the case of the
ectopic expression of wt CHC and not by the expression of
the Y1477, 1487F mutant. Confirming our previous observa-
tions, Myosin VI was never recruited at EPEC-induced actin
pedestals, regardless of the experimental condition (Fig. 7 c).
Our data indicate that the tyrosine phosphorylation of CHC is
required for the accumulation of clathrin coats at bacteria–cell
interfaces, followed by the assembly of the clathrin–actin
organizing machinery that mediates bacterial internalization
or the formation of actin pedestals (Fig. 8).
15 min of infection with L. innocua(InlB). Hip1R was found at
the interface between EPEC and the actin pedestals throughout
the infectious process.
We then used an siRNA-based approach to study the re-
spective roles of Dab2, Myosin VI, and Hip1R in our infection
models. The mean inhibition of protein expression was 80%,
and no off-target effects on other components of the machin-
ery were observed (Fig. S4). The depletion of Dab2, Myosin VI,
or Hip1R efficiently inhibited L. monocytogenes internaliza-
tion by the InlB pathway, whereas it had a milder but still
significant effect the InlA-mediated internalization pathway
(Fig. 5, a–c). This is in line with our previous observations
of a compensatory role for caveolin that attenuates the clathrin
knockdown phenotype in the InlA pathway (Bonazzi et al.,
2008). Similarly, the formation of EPEC-induced actin ped-
estals was followed in cells depleted of Dab2, Myosin VI,
and Hip1R, and infected with EPEC for 4, 5, and 6 h. Hip1R
depletion efficiently inhibited the formation of EPEC-induced
actin pedestals, whereas the knockdown of Dab2 had a milder
effect (Fig. 5, a–c). In line with its lack of colocalization, the
knockdown of Myosin VI had no effect on the formation of
actin pedestals (Fig. 5 b).
Hierarchical organization of the
To dissect the respective roles of Dab2, CHC, CLC, Myosin
VI, and Hip1R during infection, we observed the effects of sin-
gle protein depletion on the other members of the machinery.
We also used cytochalasin D to impair actin polymerization
before infection. The knockdown of Dab2 had a clear effect
on the recruitment of all members of the machinery including
actin (Fig. 6 a). The depletion of CHC prevented binding of
the CLC subunit and recruitment of Hip1R, and, as previously
described (Veiga et al., 2007), blocked recruitment of actin. It
also partially affected the recruitment of Myosin VI and Dab2
(Fig. 6 b), possibly affecting the latter by reducing coated pit
stability. CLC depletion completely prevented recruitment of
Hip1R and actin, while apparently having a partial effect on
Figure 5. Dab2, Myosin VI, and Hip1R are required during bacterial infections. (a–c) Jeg3 cells and HeLa cells were transfected with two siRNA sequences
(si1 and si2) targeted to Dab2 (a), Myosin VI (b), or Hip1R (c). L. monocytogenes was then used to infect Jeg3 and HeLa cells to follow the InlA or InlB
internalization pathway, respectively, by a gentamicin survival assay. HeLa cells were infected with EPEC to follow actin-based pedestals formation by
immunofluorescence where 200 pedestals were counted for each experiment. Values are means ± SD of three independent experiments (error bars).
Asterisks represent P-values (**, P ≤ 0.01; ***, P ≤ 0.001; Student’s t test).
JCB • VOLUME 195 • NUMBER 3 • 2011 532
as a prelude for actin cytoskeleton reorganization during bacterial
adhesion and internalization. We also describe key components of
the molecular machinery that orchestrates actin dynamics at clath-
rin coats, reinforcing the increasingly recognized link between
clathrin and actin in mammalian cells (Kaksonen et al., 2006; Veiga
et al., 2007; Cureton et al., 2009; Saffarian et al., 2009).
Using three infection models, we provide here the first func-
tional demonstration of a physiological role for the signaling-
induced tyrosine phosphorylation of CHC. This posttranslational
modification is needed for clathrin accumulation at the membrane
Figure 7. CHC phosphorylation is required to assemble the clathrin-actin machinery. (a–c) Jeg3 cells were transfected with a control siRNA or with a CHC-
targeted siRNA, alone or in combination with the overexpression of wt CHC-GFP or the Y1477, 1487F mutant of CHC-GFP (non-p-CHC). Cells were then
incubated with L. innocua(InlA), L. innocua(InlB), or EPEC, then fixed and processed by immunofluorescence. The frequency of recruitment of the different
component of the clathrin–actin machinery at the bacterial adhesion sites was evaluated by fluorescence microscopy and normalized to that of control cells.
Values are means ± SD of three independent experiments (error bars) where 100 bacteria were counted for each condition.
Figure 6. Ordered recruitment of the clathrin-actin machinery established by siRNA treatment. (a–f) Jeg3 and HeLa cells were transfected with siRNA
targeted to either Dab2 (a), CHC (b), CLC (c), Myosin VI (d), or Hip1R (e); or incubated with cytochalasin D for 30 min (f). Cells were incubated with
L. innocua(InlA), L. innocua(InlB), or EPEC, then fixed and processed by immunofluorescence. The frequency of recruitment of the different component of
the clathrin/actin machinery at the bacterial adhesion sites was evaluated by fluorescence microscopy and normalized to that of control cells. Values are
means (± SD) of three independent experiments where 100 bacteria were counted for each condition. Red horizontal lines mark the control level of
protein recruitment. Error bars indicate mean ± SD.
Clathrin in actin recruitment during bacterial infection • Bonazzi et al.
has been reported during epithelial morphogenesis (Yang et al.,
2007). Interestingly, AP-2 was never observed during L. mono
cytogenes entry, and its knockdown does not affect bacterial
internalization (Veiga and Cossart, 2005). However, a role of
Dab2 independent of AP-2 has been described (Maurer and
Cooper, 2006). Furthermore, the NPXY consensus sequence
recognized by Dab2 is not present in E-cadherin or Met, the
host receptors for L. monocytogenes, which suggests that Dab2
must be associated with additional components that remain
to be identified.
CLC is a Hip protein interactor and a regulator of clathrin
assembly (Chen and Brodsky, 2005; Legendre-Guillemin et al.,
2005; Wilbur et al., 2010). Here we show that the interaction
between CLC and Hip1R plays a key role in recruiting and
organizing actin at clathrin coats. In our infection models, the
knockdown of either CLC or Hip1R impedes the recruitment of
actin at the bacterial adhesion sites, resulting in impaired bacte-
rial internalization. Importantly, CLC depletion prevents Hip1R
recruitment, which locates CLC upstream of Hip1R and dem-
onstrates its essential role in the recruitment of Hip1R, hence
actin, at clathrin coats.
Infection triggers the tyrosine
phosphorylation of CHC
Clathrin-mediated internalization of cell surface receptors that
signal through Src family kinases is accompanied by the tyro-
sine phosphorylation of CHC. Here we provide evidence for
an essential role of this posttranslational modification during
bacterial infections. By developing new tools for the study of
CHC phosphorylation, we were able to show that the initial
step of bacteria–host interactions triggers the phosphorylation
of CHC, which occurs at tyrosines 1477 and 1487. In line with
our previous observations (Sousa et al., 2007; Bonazzi et al.,
2008), during InlA-mediated infections, CHC phosphoryla-
tion is mediated by Src family kinases. Indeed, the pretreat-
ment of cells with the Src family kinase inhibitor PP1 before
purified InlA incubation resulted in decreased phosphorylation
of CHC. Our previous studies led to the hypothesis that the
tyrosine phosphorylation of CHC might favor the accumula-
tion of clathrin-coated pits so that they could be available for
coordinated receptor uptake upon dephosphorylation and syn-
chronized budding at the end of receptor signaling (Stoddart
et al., 2002; Crotzer et al., 2004). In the case of bacterial inter-
action, the phosphorylated coated pits apparently serve as plat-
forms for actin organization. However, completion of vesicle
invagination may eventually serve an endocytic purpose later
during infection, where clathrin-mediated endocytosis could
play a role in vacuole maturation. Such an additional and more
conventional role for clathrin at a later stage of infection would
explain our earlier findings that dynamin and Hrs are also key
proteins needed for successful infection (Veiga and Cossart,
2005). By analogy with the bacterial–host interactions we have
observed, we speculate that phosphorylation-stabilized or “fro-
zen” clathrin coated pits at the edge of immune cell signal-
ing domains might also play a role in actin nucleation at the
immunological synapse, whose organization is both clathrin
and actin dependent (Calabia-Linares et al., 2011). Thus, phos-
phorylation of CHC may have a dual role of stabilizing clathrin
as an actin-nucleating platform and providing a pool of coated
pits that can be coordinately activated for endocytosis upon
subsequent dephosphorylation. How tyrosine phosphorylation
of CHC contributes to coated pit stability is not yet established.
Because of its location on the proximal leg region, this modifi-
cation could potentially block further addition of triskelia and
freeze lattice growth through direct inhibition of regional inter-
action or recruitment of a blocking protein.
Defining components of clathrin–actin
interaction during infection
The surprising observation that clathrin assembles into coated
pits of conventional size at bacteria–host interfaces suggested
that clathrin may serve a role during infection other than that
of an endocytic protein. Here we demonstrate the sequential re-
cruitment of Dab2, CHC, CLC, Hip1R, and Myosin VI to sites
of host–pathogens interaction to orchestrate the recruitment of
actin at sites of bacterial adhesion/internalization. Dab2 has
been identified as the clathrin adaptor for the internalization
of UPEC, where it functions in association with AP-2 (Eto
et al., 2008), and a role for Dab2 in E-cadherin endocytosis
Figure 8. Proposed model for the clathrin-actin machinery triggered by
bacterial infection. Bacteria activate host receptor downstream signaling,
which results in the recruitment of the clathrin adaptor Dab2 and of the
CHC–CLC complex. Clathrin coats are then stabilized by the tyrosine phos-
phorylation of CHC (P), followed by the sequential recruitment of Hip1R
and actin. Myosin VI is also recruited in the case of invading bacteria,
probably providing the pulling force for bacterial internalization, whereas
it is excluded from EPEC-induced actin pedestals. CHC remains phosphory-
lated at the bacterial adhesion or internalization sites where clathrin and
actin are colocalized.
JCB • VOLUME 195 • NUMBER 3 • 2011 534
with the immunizing peptide. The affinity-purified antibody was used di-
rectly for immunofluorescence. This affinity purified antibody was tested for
peptide specificity by ELISA. The antibody at 1:1,000 dilution was incu-
bated with purified peptides with two sites phosphorylated, one site phos-
phorylated, or nonphosphorylated in solution at the specified concentrations,
then tested for binding to the double phospho-peptide using a secondary
antibody coupled to horseradish peroxidase. For analysis by immunoblotting,
the affinity-purified anti-pCHC antibody was further depleted by expo-
sure to nonphosphorylated recombinant hub fragments immobilized on
membrane as follows. 10 sets of 15 µg of purified hub were run on an
SDS-PAGE gel and transferred to nitrocellulose membrane. Hub bands
were cut out of the membrane and blocked with 5% BSA in PBS for 2 h.
100 µl of 9 µg/ml anti-pCHC was put onto the hub-loaded membrane for
1 h and pipetted every 10 min. After 1 h, the anti-pCHC was moved onto
another-hub loaded membrane, and the process repeated. In total, the anti-
pCHC was exposed to hub-loaded membranes 10 times. The depleted
anti-pCHC was then tested by immunoblotting for binding to recombinant
purified Hub protein (Liu et al., 1995) with or without exposure to purified
Src kinase (Sigma-Aldrich).
Plasmids and siRNA sequences
siRNA targeting the CHC sequence 1, 5-GGGAAUUCUUCGUACUC-
CATT-3; and 2, 5-GAUUAUCAAUUACCGUACATT-3. The CLC sequence
1, 5-AAAGACAGTTATGCAGCTATT-3; and 2, 5-AAGGAACCAGCGC-
CAGAGTGA-3. Control siRNA sequences were obtained from Invitrogen.
siRNA targeting the Hip1R sequence 1, 5-CUCCGACAUGCUGUACUU-
CTT-3; and 2, 5-ACCGGAGAATCTCATTGAGATCTT-3. The CHC sequ-
ence used for rescue analysis was 5-GCAAUGAGCUGUUUGAAGA-3.
Control siRNA sequences were obtained from QIAGEN. siRNA targeting
the Myosin VI sequence 1, 5-GCUGGCAGUUCAUAGGAAUTT-3; and 2,
5-AUUCCUAUGAACUGCCAGCTT-3. Control siRNA sequences were
obtained from Eurogentec. siRNA targeting the Dab2 sequence 1,
5-GCAAAGAUAUCCUGUUAGUTT-3; and 2, 5-GAACCAGCCUUCAC-
CCUUUTT-3. Control siRNA sequences were obtained from Thermo
Fisher Scientific. For CHC rescue analysis, human CHC cDNA was made
to be resistant to the CHC siRNA sequences used and inserted into a
pcDNA3.1/Zeocin vector (Invitrogen) using the HindIII and NotI digestion
sites. The siRNA-resistant human CHC cDNA was further mutated by site-
directed mutagenesis using Quikchange II XL (Agilent Technologies) so that
tyrosines 1477 and 1487 were mutated to code for phenylalanines. The
resulting cDNA was then cloned into a pcDNA3.1/Zeocin vector using the
HindIII and NotI digestion sites.
For siRNA transfection, 2 × 105 cells/ml were mixed with siRNA and Lipo-
fectamine RNAiMAX (Invitrogen) before plating according to manufacturer’s
instructions for reverse transfections. Cells were probed for siRNA efficacy
72 h after transfection by Western blotting. For CHC rescue experiments,
48 h after siRNA transfection, cells were transfected with the indicated clath-
rin constructs using Gene Juice according to manufacturer’s instructions.
Cells grown on glass coverslips were fixed in 4% paraformaldehyde at
room temperature for 20 min, rinsed in PBS, and incubated for 20 min in
blocking solution (0.5% BSA and 50 mM NH4Cl in PBS, pH 7.4) supple-
mented with 0.05% saponin. Cells were then incubated for 30 min at room
temperature with the appropriate primary antibodies diluted in blocking
solution, washed five times in PBS, and incubated for 30 min at room
temperature with Alexa Fluor–coupled secondary antibodies (Invitrogen).
Where needed, Alexa Fluor–coupled phalloidin was added to the second-
ary antibodies to label the cell cytoskeleton. Cells were washed five times
in PBS and mounted on microscope slides using Fluoromount mounting
medium (Electron Microscopy Sciences). Preparations were analyzed using
an epifluorescence microscope (Axiovert 135; Carl Zeiss) connected to a
charge-coupled device camera (CoolSnap HQ; Photometrics) and oper-
ated by Metamorph software (Molecular Devices). Images were acquired
using a 63× oil-immersion objective lens. Image analysis was performed
using ImageJ (National Institutes of Health).
Transmission electron microscopy
Cells were grown on glass-bottomed Petri dishes and infected with either
L. innocua(InlA) or L. innocua(InlB) at an MOI of 50. Cells were fixed in
2.5% glutaraldehyde in 0.1 M Hepes buffer, pH 7.2, overnight at 4°C,
washed in 0.1 M Hepes buffer, postfixed for 1 h in 1% osmium + 1.5%
K3[Fe(CN)6] in 0.1M cacodylate buffer, pH 7.2, and washed in 0.1 M
cacodylate buffer. Cells were then incubated for 1 h with 1% tannic acid
Knockdown of the motor protein Myosin VI does not
reduce the recruitment of all proteins of the clathrin–actin
machinery at bacterial adhesion sites but prevents bacterial inter-
nalization. This identifies Myosin VI as the last component of
such machinery that provides the pulling force for bacterial
internalization, followed by the disassembly of clathrin coats.
The absence of Myosin VI at pedestals likely reflects the fact that
these are not internalization structures. Myosin VI recruitment
at EPEC pedestals may be prevented by a bacterial type III
effector, secreted upon adhesion to host cells.
In summary, by ectopic expression of a nonphosphorylat-
able mutant of CHC, we showed herein that the phosphoryla-
tion of CHC triggered by infection is the initiating event for the
accumulation of clathrin coats at bacterial adhesion sites. These
coats, via CLC–Hip1R interactions, in turn coordinate actin re-
cruitment at bacterial–host adhesion sites (Fig. 8), leading either
to internalization in conjunction with recruited Myosin VI or to
pedestal formation. Thus, using bacterial infection models, we
provide evidence that the clathrin–actin interaction mediated
by CLC and Hip1R goes beyond its function in conventional
coated vesicle endocytosis (Newpher and Lemmon, 2006;
Boulant et al., 2011; Collins et al., 2011). We show here that,
via the CLC–Hip1R interaction, the clathrin lattice can serve as
a functional actin-organizing platform for uptake of structures
that exceed the size limits of clathrin-coated vesicles and sup-
ports additional actin-based activities such as pedestal forma-
tion, unrelated to endocytosis. We further demonstrate that this
novel function for clathrin in actin organization is dependent on
CHC phosphorylation, thereby defining the functional conse-
quence of CHC modification by Src family kinases, which are
stimulated when signaling receptors are engaged during infec-
tion and initiation of endocytosis.
Materials and methods
Bacterial strains and cell lines
L. monocytogenes EGD (BUG 600) was grown in brain–heart infusion.
L. innocua transformed with pRB474 harboring the inlA gene (BUG 1489)
was grown in brain–heart infusion with 7 µg/ml chloramphenicol. L. innocua
transformed with pP1-B3 harboring the inlB gene (BUG 1531) was grown
in brain–heart infusion with 5 µg/ml erythromycin. EPEC strain JPN15
(BUG 2361) was grown in Luria-Bertani broth. Jeg3 cells (human epithelial
placental cells ATCC n°: HTB-36) and HeLa cells (human cervix epithelial
cells ATTC n°: CCL-2) were grown in MEM medium containing Glutamax,
nonessential amino acids, sodium pyruvate, and 10% FBS.
Antibodies and reagents
PY20 anti-phosphotyrosine monoclonal antibody and anti-Hip1R poly-
clonal antibody were obtained from Millipore. Anti-Dab2 polyclonal anti-
body and anti-Myosin VI polyclonal antibody were obtained from Santa
Cruz Biotechnology, Inc. Anti-actin monoclonal antibody was obtained
from Sigma-Aldrich. Anti-CLC polyclonal antibody was produced as de-
scribed previously (Acton et al., 1993), monoclonal antibodies against
CHC (X22 for immunofluorescence and TD.1 for blotting were produced in
our laboratory; Brodsky, 1985; Näthke et al., 1992), anti-InlA polyclonal
antibody R302, anti-InlA monoclonal antibody L7.7, anti-InlB B4-6 mono-
clonal antibody, and anti–L. innocua polyclonal antibody R11 were pro-
duced in our laboratory. The 4G10 anti-phospho-tyrosine monoclonal
antibody was obtained from the Bishop laboratory at University of Califor-
nia, San Francisco. Cytochalasin D was obtained from Sigma-Aldrich. To
obtain the anti-pCHC antibody, rabbits were immunized with a phospho-
peptide comprising CHC residues from 1473–1491, with phosphorylated
tyrosines 1477 and 1487. The antibody was affinity purified by depletion with
nonphosphorylated peptide 1473–1491, followed by positive purification
535 Clathrin in actin recruitment during bacterial infection • Bonazzi et al.
supernatants were discarded. Beads were then washed in lysis buffer and
resuspended in Laemmli loading buffer. Samples were then loaded on an 8%
poly-acrylamide gel and blotted for protein analysis.
Src kinase was incubated with recombinant CHC (Hub) followed by diges-
tion with trypsin and collection of peptide masses by matrix-assisted laser de-
sorption/ionization (MALDI) time of flight (TOF). Peptides masses in the mass
spectrum were manually matched to expected peptide masses, and identifi-
cation of phospho-peptides was determined by the presence of two peptides
with an 80-D mass difference. The peptide sequence and the presence of
phospho-tyrosine were confirmed by MALDI TOF/TOF analysis.
Online supplemental material
Fig. S1 shows bacterial infection and incubation with bacterial surface
proteins involved in L. monocytogenes internalization phosphorylate CHC.
When cells are incubated with the L. monocytogenes surface protein InlA,
CHC phosphorylation is dependent on Src family kinases. Fig. S2 shows
that EGF treatment of cells promotes the accumulation of phosphorylated
CHC at the plasma membrane. Fig. S3 shows that bacterial infection does
not affect the clathrin-mediated internalization of transferrin. Fig. S4 shows
the efficiency and specificity of the siRNA interference treatments used in
this study. Online supplemental material is available at http://www.jcb
This work was supported by Institut Pasteur, Institut National de la Santé et de la
Recherche Médicale, Institut National de la Recherche Agronomique, European
Research Council (advanced grant 233348) to P. Cossart, and grant
GM038093 from the National Institutes of Health and grant 15IB-0035 from
the California Breast Cancer Research Program to F.M. Brodsky. M. Bonazzi is
supported by the Pasteur Roux Fellowship. P. Cossart is an international research
scholar of the Howard Hughes Medical Institute.
Submitted: 26 May 2011
Accepted: 3 October 2011
Acton, S.L., D.H. Wong, P. Parham, F.M. Brodsky, and A.P. Jackson. 1993.
Alteration of clathrin light chain expression by transfection and gene dis-
ruption. Mol. Biol. Cell. 4:647–660.
Bonazzi, M., E. Veiga, J. Pizarro-Cerdá, and P. Cossart. 2008. Successive post-
translational modifications of E-cadherin are required for InlA-mediated
internalization of Listeria monocytogenes. Cell. Microbiol. 10:2208–
Boulant, S., C. Kural, J.-C. Zeeh, F. Ubelmann, and T. Kirchhausen. 2011. Actin
dynamics counteract membrane tension during clathrin-mediated endo-
cytosis. Nat. Cell Biol. 13:1124–1131. http://dx.doi.org/10.1038/ncb2307
Brodsky, F.M. 1985. Clathrin structure characterized with monoclonal antibod-
ies. I. Analysis of multiple antigenic sites. J. Cell Biol. 101:2047–2054.
Brodsky, F.M., C.Y. Chen, C. Knuehl, M.C. Towler, and D.E. Wakeham.
2001. Biological basket weaving: formation and function of clathrin-
coated vesicles. Annu. Rev. Cell Dev. Biol. 17:517–568. http://dx.doi
Buss, F., S.D. Arden, M. Lindsay, J.P. Luzio, and J. Kendrick-Jones. 2001a.
Myosin VI isoform localized to clathrin-coated vesicles with a role in
clathrin-mediated endocytosis. EMBO J. 20:3676–3684. http://dx.doi
Buss, F., J.P. Luzio, and J. Kendrick-Jones. 2001b. Myosin VI, a new force in
clathrin mediated endocytosis. FEBS Lett. 508:295–299. http://dx.doi
Calabia-Linares, C., J. Robles-Valero, H. de la Fuente, M. Perez-Martinez,
N. Martín-Cofreces, M. Alfonso-Pérez, C. Gutierrez-Vázquez, M.
Mittelbrunn, S. Ibiza, F.R. Urbano-Olmos, et al. 2011. Endosomal clath-
rin drives actin accumulation at the immunological synapse. J. Cell Sci.
Carreno, S., A.E. Engqvist-Goldstein, C.X. Zhang, K.L. McDonald, and D.G.
Drubin. 2004. Actin dynamics coupled to clathrin-coated vesicle forma-
tion at the trans-Golgi network. J. Cell Biol. 165:781–788. http://dx.doi
Chan, Y.G.Y., M.M. Cardwell, T.M. Hermanas, T. Uchiyama, and J.J. Martinez.
2009. Rickettsial outer-membrane protein B (rOmpB) mediates bacte-
rial invasion through Ku70 in an actin, c-Cbl, clathrin and caveolin 2-
dependent manner. Cell. Microbiol. 11:629–644. http://dx.doi.org/10.1111/
in 0.1 M cacodylate buffer and an additional 30 min with 1% osmium in
0.1 M cacodylate buffer. Cells were then dehydrated through a graded
ethanol series and incubated overnight in a mixture of 50% pure ethanol
and 50% EPON resin. Cells were then incubated in pure EPON and the
resin was polymerized for 72 h at 65°C. For immunolabeling on thawed
cryosections, cells were grown onto 100-mm Petri dishes and incubated
with wt L. monocytogenes at an MOI of 50 for 20 min, then samples were
fixed with a mixture of 2% paraformaldehyde and 0.1% glutaraldehyde
in 0.1 M Sorensen’s phosphate buffer. After quenching of free aldehyde
groups, cells were scraped and pellets were embedded in 12% gelatin
(TAAB). After hardening on ice, blocks of 1 mm3 were cut and infiltrated
overnight at 4°C with 2.1 M sucrose in 0.1 M phosphate buffer. Blocks
were mounted on pins and frozen by plunging in liquid nitrogen. 60-nm
sections were cut using a cryo-microtome (UC6/FC6; Leica) and picked
up using a 1:1 mixture of 2% methylcellulose and 2.1 M sucrose. Sections
were labeled at room temperature with the rabbit anti-CLC followed by
protein A gold (Cell Microscopy Center) and observed with a transmission
electron microscope (1010; Jeol) operated at 80 kV, equipped with a
keenview camera (Olympus Soft imaging).
For the gentamicin survival assays, L. monocytogenes were grown at 37°C to
an OD600 of 0.8–1.0. Before infection, bacteria were washed twice in PBS,
diluted in DME, and incubated with either Jeg3 or HeLa cells (E-cadherin–
expressing Jeg3 cells are mainly infected by the InlA pathway, and HeLa
cells, which do not express E-cadherin, are mainly infected by the InlB
pathway) at an MOI of 50. After 1 h of incubation at 37°C, cells were
washed with complete culture medium and incubated for 2 h at 37°C in
complete culture medium containing 10 µg/ml gentamicin. Cells were
washed and lysed in 0.2% Triton X-100. The number of viable bacteria
was assessed by titering on agar plates. For CHC rescue experiments,
bacterial internalization was analyzed by double labeling bacteria before
and after permeabilization (Veiga and Cossart, 2005), and the particle
analysis plug-in of Image J was used to quantify internalized bacteria in
CHC-GFP–expressing cells. For other assays, Jeg3 or HeLa cells were
washed in serum-free culture medium and incubated with L. innocua(InlA)
or L. innocua(InlB), respectively, at an MOI of 50. Cells were centrifuged
for 2 min at 1,000 rpm and incubated at 37°C for the indicated times.
Alternatively, HeLa cells were infected with EPEC at 37°C for the indicated
times. At each time point, cells were fixed and processed for immunofluores-
cence. Protein recruitment and EPEC-induced actin pedestals formation
were quantified by fluorescence microscopy where 100–200 events were
observed for each condition. To hierarchically organize the components
of the actin–clathrin machinery, values were then normalized versus the
frequency of recruitment of a given protein in control conditions, and
the fluctuations of such value when other members of the machinery are
knocked down were plotted.
Transferrin uptake assay
Jeg3 cells were briefly washed in DME without serum and incubated in the
same medium supplemented with 25 µg/ml Cy3-labeled transferrin and
20 mM Hepes for 30 min on ice. One sample was fixed immediately after
that to measure the amount of surface-bound transferrin. A second sample
was washed in PBS and subjected to a 4-min acid wash (500 mM NaCl,
0.5% acetic acid) to verify that all transferrin was extracellular. All remain-
ing samples were incubated with either L. innocua(InlA), L. innocua(InlB),
or EPEC as described above for 1 h at 37°C. Cells were then washed
in PBS, subjected to the acid wash, and fixed and labeled with DAPI and
with the anti–L. innocua antibody R11 where applicable. Cell-bound and/
or intracellular transferrin was measured by fluorescence microscopy
Immunoprecipitations were performed as described previously (Bonazzi
et al., 2008). In brief, cells were treated as indicated, rinsed in pre-chilled
PBS at 4°C, and incubated in 500 ml of lysis buffer (1% NP-40, 20 mM Tris,
pH 8.0, 150 mM NaCl, 10% glycerol, 20 mM NaF, 5 mM Na3VO4, and
complete protease inhibitors cocktail; Roche). Cells were then scraped and
collected in 1.5 ml Eppendorf tubes and further incubated in the lysis buffer
for 15 min at 4°C on a spinning wheel. Lysates were centrifuged for 15 min
at 4°C at maximum speed and the supernatants were incubated for 1 h
at 4°C with protein A–Sepharose beads to eliminate unspecific binding
of proteins to beads. Lysates were then centrifuged to eliminate beads and
incubated overnight at 4°C on a spinning wheel with the anti-CHC antibody.
Protein A beads were added to samples and incubated at 4°C for 3 h.
Samples were then centrifuged for 2 min at 4°C at maximum speed, and the
JCB • VOLUME 195 • NUMBER 3 • 2011 536 Download full-text
host cells is mediated by a clathrin-dependent mechanism. Cell. Microbiol.
Morris, S.M., S.D. Arden, R.C. Roberts, J. Kendrick-Jones, J.A. Cooper,
J.P. Luzio, and F. Buss. 2002. Myosin VI binds to and localises with
Dab2, potentially linking receptor-mediated endocytosis and the actin
cytoskeleton. Traffic. 3:331–341. http://dx.doi.org/10.1034/j.1600-0854
Näthke, I.S., J. Heuser, A. Lupas, J. Stock, C.W. Turck, and F.M. Brodsky. 1992.
Folding and trimerization of clathrin subunits at the triskelion hub. Cell.
Newpher, T.M., and S.K. Lemmon. 2006. Clathrin is important for normal
actin dynamics and progression of Sla2p-containing patches during en-
docytosis in yeast. Traffic. 7:574–588. http://dx.doi.org/10.1111/j.1600-
Pizarro-Cerdá, J., B. Payrastre, Y.J. Wang, E. Veiga, H.L. Yin, and P. Cossart.
2007. Type II phosphatidylinositol 4-kinases promote Listeria monocyto
genes entry into target cells. Cell. Microbiol. 9:2381–2390. http://dx.doi
Pizarro-Cerdá, J., M. Bonazzi, and P. Cossart. 2010. Clathrin-mediated endo-
cytosis: what works for small, also works for big. Bioessays. 32:496–504.
Popoff, V., G.A. Mardones, S.K. Bai, V.R. Chambon, D.L. Tenza, P.V. Burgos, A.
Shi, P. Benaroch, S. Urbé, C. Lamaze, et al. 2009. Analysis of articu-
lation between clathrin and retromer in retrograde sorting on early endo-
somes. Traffic. 10:1868–1880. http://dx.doi.org/10.1111/j.1600-0854.2009
Raiborg, C., and H. Stenmark. 2009. The ESCRT machinery in endosomal sort-
ing of ubiquitylated membrane proteins. Nature. 458:445–452. http://
Saffarian, S., E. Cocucci, and T. Kirchhausen. 2009. Distinct dynamics of endo-
cytic clathrin-coated pits and coated plaques. PLoS Biol. 7:e1000191.
Sousa, S., D. Cabanes, L. Bougnères, M. Lecuit, P. Sansonetti, G. Tran-
Van-Nhieu, and P. Cossart. 2007. Src, cortactin and Arp2/3 complex
are required for E-cadherin-mediated internalization of Listeria into
cells. Cell. Microbiol. 9:2629–2643. http://dx.doi.org/10.1111/j.1462-
Stoddart, A., M.L. Dykstra, B.K. Brown, W. Song, S.K. Pierce, and F.M.
Brodsky. 2002. Lipid rafts unite signaling cascades with clathrin to
regulate BCR internalization. Immunity. 17:451–462. http://dx.doi.org/
Veiga, E., and P. Cossart. 2005. Listeria hijacks the clathrin-dependent endocytic
machinery to invade mammalian cells. Nat. Cell Biol. 7:894–900. http://
Veiga, E., J.A. Guttman, M. Bonazzi, E. Boucrot, A. Toledo-Arana, A.E.
Lin, J. Enninga, J. Pizarro-Cerdá, B.B. Finlay, T. Kirchhausen, and P.
Cossart. 2007. Invasive and adherent bacterial pathogens co-opt host
clathrin for infection. Cell Host Microbe. 2:340–351. http://dx.doi
Wilbur, J.D., C.Y. Chen, V. Manalo, P.K. Hwang, R.J. Fletterick, and F.M.
Brodsky. 2008. Actin binding by Hip1 (huntingtin-interacting pro-
tein 1) and Hip1R (Hip1-related protein) is regulated by clathrin light
chain. J. Biol. Chem. 283:32870–32879. http://dx.doi.org/10.1074/jbc
Wilbur, J.D., P.K. Hwang, J.A. Ybe, M. Lane, B.D. Sellers, M.P. Jacobson, R.J.
Fletterick, and F.M. Brodsky. 2010. Conformation switching of clathrin
light chain regulates clathrin lattice assembly. Dev. Cell. 18:854–861.
Wilde, A., E.C. Beattie, L. Lem, D.A. Riethof, S.H. Liu, W.C. Mobley, P.
Soriano, and F.M. Brodsky. 1999. EGF receptor signaling stimulates
SRC kinase phosphorylation of clathrin, influencing clathrin redistri-
bution and EGF uptake. Cell. 96:677–687. http://dx.doi.org/10.1016/
Wong, A.R.C., J.S. Pearson, M.D. Bright, D. Munera, K.S. Robinson, S.F. Lee,
G. Frankel, and E.L. Hartland. 2011. Enteropathogenic and enterohaemor-
rhagic Escherichia coli: even more subversive elements. Mol. Microbiol.
Yang, D.H., K.Q. Cai, I.H. Roland, E.R. Smith, and X.X. Xu. 2007. Disabled-2 is
an epithelial surface positioning gene. J. Biol. Chem. 282:13114–13122.
Yarar, D., C.M. Waterman-Storer, and S.L. Schmid. 2005. A dynamic actin
cytoskeleton functions at multiple stages of clathrin-mediated endo-
cytosis. Mol. Biol. Cell. 16:964–975. http://dx.doi.org/10.1091/mbc.E04-
Chen, C.Y., and F.M. Brodsky. 2005. Huntingtin-interacting protein 1 (Hip1)
and Hip1-related protein (Hip1R) bind the conserved sequence of clath-
rin light chains and thereby influence clathrin assembly in vitro and
actin distribution in vivo. J. Biol. Chem. 280:6109–6117. http://dx.doi
Collins, A., A. Warrington, K.A. Taylor, and T. Svitkina. 2011. Structural organi-
zation of the actin cytoskeleton at sites of clathrin-mediated endocytosis.
Curr. Biol. 21:1167–1175. http://dx.doi.org/10.1016/j.cub.2011.05.048
Crotzer, V.L., A.S. Mabardy, A. Weiss, and F.M. Brodsky. 2004. T cell recep-
tor engagement leads to phosphorylation of clathrin heavy chain dur-
ing receptor internalization. J. Exp. Med. 199:981–991. http://dx.doi
Cureton, D.K., R.H. Massol, S. Saffarian, T.L. Kirchhausen, and S.P.J. Whelan.
2009. Vesicular stomatitis virus enters cells through vesicles incompletely
coated with clathrin that depend upon actin for internalization. PLoS
Pathog. 5:e1000394. http://dx.doi.org/10.1371/journal.ppat.1000394
Engqvist-Goldstein, A.E., R.A. Warren, M.M. Kessels, J.H. Keen, J. Heuser,
and D.G. Drubin. 2001. The actin-binding protein Hip1R associates with
clathrin during early stages of endocytosis and promotes clathrin as-
sembly in vitro. J. Cell Biol. 154:1209–1223. http://dx.doi.org/10.1083/
Engqvist-Goldstein, A.E.Y., C.X. Zhang, S. Carreno, C. Barroso, J.E. Heuser,
and D.G. Drubin. 2004. RNAi-mediated Hip1R silencing results in stable
association between the endocytic machinery and the actin assembly ma-
chinery. Mol. Biol. Cell. 15:1666–1679. http://dx.doi.org/10.1091/mbc
Eto, D.S., H.B. Gordon, B.K. Dhakal, T.A. Jones, and M.A. Mulvey. 2008.
Clathrin, AP-2, and the NPXY-binding subset of alternate endocytic
adaptors facilitate FimH-mediated bacterial invasion of host cells.
Cell. Microbiol. 10:2553–2567. http://dx.doi.org/10.1111/j.1462-5822
Ferguson, S.M., A. Raimondi, S. Paradise, H. Shen, K. Mesaki, A. Ferguson, O.
Destaing, G. Ko, J. Takasaki, O. Cremona, et al. 2009. Coordinated ac-
tions of actin and BAR proteins upstream of dynamin at endocytic clathrin-
coated pits. Dev. Cell. 17:811–822. (published erratum appears in Dev.
Cell. 2010. 18:332) http://dx.doi.org/10.1016/j.devcel.2009.11.005
Hyman, T., M. Shmuel, and Y. Altschuler. 2006. Actin is required for endocyto-
sis at the apical surface of Madin-Darby canine kidney cells where ARF6
and clathrin regulate the actin cytoskeleton. Mol. Biol. Cell. 17:427–437.
Inoue, A., O. Sato, K. Homma, and M. Ikebe. 2002. DOC-2/DAB2 is the binding
partner of myosin VI. Biochem. Biophys. Res. Commun. 292:300–307.
Jonquières, R., H. Bierne, F. Fiedler, P. Gounon, and P. Cossart. 1999. Interaction
between the protein InlB of Listeria monocytogenes and lipoteichoic acid:
a novel mechanism of protein association at the surface of gram-positive
bacteria. Mol. Microbiol. 34:902–914. http://dx.doi.org/10.1046/j.1365-
Kaksonen, M., C.P. Toret, and D.G. Drubin. 2006. Harnessing actin dynamics
for clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 7:404–414.
Le Clainche, C., B.S. Pauly, C.X. Zhang, A.E.Y. Engqvist-Goldstein, K.
Cunningham, and D.G. Drubin. 2007. A Hip1R-cortactin complex nega-
tively regulates actin assembly associated with endocytosis. EMBO J.
Legendre-Guillemin, V., M. Metzler, J.F. Lemaire, J. Philie, L. Gan, M.R.
Hayden, and P.S. McPherson. 2005. Huntingtin interacting protein 1
(HIP1) regulates clathrin assembly through direct binding to the regula-
tory region of the clathrin light chain. J. Biol. Chem. 280:6101–6108.
Liu, S.H., M.L. Wong, C.S. Craik, and F.M. Brodsky. 1995. Regulation of clath-
rin assembly and trimerization defined using recombinant triskelion hubs.
Cell. 83:257–267. http://dx.doi.org/10.1016/0092-8674(95)90167-1
Maurer, M.E., and J.A. Cooper. 2006. The adaptor protein Dab2 sorts LDL re-
ceptors into coated pits independently of AP-2 and ARH. J. Cell Sci.
Mengaud, J., H. Ohayon, P. Gounon, R.-M. Mege, and P. Cossart. 1996. E-cadherin
is the receptor for internalin, a surface protein required for entry of L. monocy
togenes into epithelial cells. Cell. 84:923–932. http://dx.doi.org/10.1016/
Merrifield, C.J., M.E. Feldman, L. Wan, and W. Almers. 2002. Imaging actin
and dynamin recruitment during invagination of single clathrin-coated
pits. Nat. Cell Biol. 4:691–698. http://dx.doi.org/10.1038/ncb837
Mettlen, M., D. Loerke, D. Yarar, G. Danuser, and S.L. Schmid. 2010. Cargo-
and adaptor-specific mechanisms regulate clathrin-mediated endocytosis.
J. Cell Biol. 188:919–933. http://dx.doi.org/10.1083/jcb.200908078
Moreno-Ruiz, E., M. Galán-Díez, W. Zhu, E. Fernández-Ruiz, C. d’Enfert, S.G.
Filler, P. Cossart, and E. Veiga. 2009. Candida albicans internalization by