The Rockefeller University Press $30.00
J. Cell Biol. Vol. 200 No. 1 81–93
Correspondence to Ralph R. Isberg: Ralph.Isberg@tufts.edu
Abbreviations used in this paper: CBD, Cdc42-binding domain; FcR, Fc re-
ceptor; FRET, fluorescence resonance energy transfer; KD, knockdown; MBP,
maltose-binding protein; MEF, mouse embryonic fibroblast; NWASP, neural
WASP; PBD, p21-binding domain; RE, recycling endosome; ROI, region of interest;
shCdc42, Cdc42 shRNA; shScr, scrambled shRNA; TfR, transferrin receptor;
WASP, Wiskott–Aldrich syndrome protein.
Internalization of particles >0.5 µm is commonly referred to as
phagocytosis, a process that is essential for homeostasis and
immune defense. The requirement for phagocytosis in maintaining
homeostatic balance is first manifested during embryonic de-
velopment (Kerr et al., 1972) in which dead cells are cleared
through phagocytosis to ensure proper organ sculpture (Vaux
and Korsmeyer, 1999) and continues unabated after birth dur-
ing which large numbers of cells (108–109) undergo apoptosis
every day and need to be cleared (Ren and Savill, 1998). Phago-
cytosis is also crucially important in combating disease. In ad-
dition to destroying invading pathogens, phagocytes create a
bridge between innate and acquired immunity by presenting an-
tigens to T cells, thereby enabling the development of long-term
immunity (Savina and Amigorena, 2007). The crucial roles
phagocytosis plays in homeostasis and immunity make it one of
the most fundamental processes in multicellular organisms.
Integrin- and Fc receptor (FcR)–mediated uptake are ex-
amples of receptor-mediated phagocytosis, through which par-
ticles are internalized into membrane-bound vacuoles called
phagosomes. FcRs mediate uptake of antibody-opsonized parti-
cles (e.g., invading pathogens; Aderem and Underhill, 1999),
and integrins mediate internalization and adhesion by binding
to a diverse cadre of ligands (Hynes, 2002; Dupuy and Caron,
2008). For example, uptake of apoptotic cells involves integrin-
mediated phagocytosis (Savill et al., 2002). Additionally, the
bacterial pathogen Yersinia pseudotuberculosis enters both phago-
cytic and nonphagocytic cells by integrin-mediated uptake through
the action of the surface protein invasin. Invasin binds tightly to
1 integrins at the same site as the cell adhesion ligand fibro-
nectin (Tran Van Nhieu and Isberg, 1993) and is sufficient for
mediating uptake (Rankin et al., 1992). Both integrin- and FcR-
mediated uptake require the generation of contractile force by
actin polymerization for membrane to surround the phagocytic
particle (Dupuy and Caron, 2008; Swanson, 2008).
A complex array of signaling molecules is involved in or-
chestrating the actin polymerization during engulfment. Several
Rho GTPases have been implicated in phagocytosis, though the
set of Rho GTPases involved in this process depends on the
phagocytic event studied. For instance, bacterial uptake promoted
by invasin or uptake of Salmonella enterica serovar Typhimurium
immunity, yet the molecular determinants of uptake are
not well characterized. Cdc42, a Rho guanosine triphos-
phatase, is thought to orchestrate critical actin remodel-
ing events needed for internalization. In this paper, we
show that Cdc42 controls exocytic events during phago-
some formation. Cdc42 inactivation led to a selective de-
fect in large particle phagocytosis as well as a general
decrease in the rate of membrane flow to the cell surface.
he process of phagocytosis in multicellular organ-
isms is required for homeostasis, clearance of for-
eign particles, and establishment of long-term
Supporting the connection between Cdc42 and exocytic
function, we found that the overproduction of a regulator
of exocytosis, Rab11, rescued the large particle uptake
defect in the absence of Cdc42. Additionally, we demon-
strated a temporal interaction between Cdc42 and the
exocyst complex during large particle uptake. Further-
more, disruption of exocyst function through Exo70 de-
pletion led to a defect in large particle internalization,
thereby establishing a functional role for the exocyst com-
plex during phagocytosis.
Cdc42 interacts with the exocyst complex to
Sina Mohammadi2 and Ralph R. Isberg1,2
1Howard Hughes Medical Institute and 2Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111
© 2013 Mohammadi and Isberg 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 200 • NUMBER 1 • 2013 82
shown to require Cdc42 (Price et al., 1998). As cell spreading
could equate to the phagocytosis of an infinitely large particle,
one explanation for these results is that Cdc42 is only required
for integrin-dependent processes that involve surface areas
larger than Y. pseudotuberculosis. Alternatively, these results
could be explained by invasin and fibronectin engaging integ-
rins in different ways.
To analyze the role of Cdc42 in integrin-mediated pro-
cesses, cell spreading on invasin-coated surfaces was character-
ized after RNAi inactivation. Cells were transfected with
plasmids encoding shRNAs, which efficiently reduced Cdc42
protein levels as compared with scrambled shRNA (shScr) con-
trols (Fig. S1 A). The depleted cells were replated on invasin-
coated coverslips, and the extent of cell spreading was followed
by live phase-contrast microscopy. Cdc42-depleted cells dis-
played a severe kinetic defect in spreading when compared with
control cells, with almost no spreading taking place over 30 min
(Fig. 1, A and B; and Video 1). This is consistent with data on
spreading on fibronectin and argues that the absence of a re-
quirement for Cdc42 in invasin-promoted processes is not be-
cause invasin engagement of integrins has special features not
found in other integrin–ligand interactions.
To test the model that particle size determines Cdc42 re-
quirement, control and Cdc42-depleted cells were challenged
with particles of varying sizes coated with two different ligands.
Y. pseudotuberculosis (1–2 µm) were used to represent small
phagocytic particles, whereas 2.9- and 4.5-µm beads coated
with purified invasin were used as medium and large particles,
respectively. Consistent with previous results using interfering
forms of Cdc42 (Alrutz et al., 2001), depletion of Cdc42 had no
effect on the uptake of Y. pseudotuberculosis and caused only a
small depression in the uptake of 2.9-µm beads (Fig. 1 C). In
contrast, depletion of Cdc42 resulted in a strong uptake defect
for large particles (4.5-µm beads; Fig. 1 C). The size-dependent
reduction was specific to Cdc42 depletion, as Rac1 depletion
reduced the uptake efficiency of all tested particle sizes (Fig. 1 C).
Strikingly, the size-dependent uptake defect in Cdc42-depleted
cells was not limited to integrin-mediated uptake. Cdc42-depleted
cells, expressing FcRIIA and challenged with IgG-coated beads,
also displayed a size-dependent uptake defect (Fig. 1 D), indi-
cating that this phenomenon is widespread among receptor–
Previous results indicated that integrin-promoted up-
take was independent of NWASP (Alrutz et al., 2001), one of
the key downstream effectors of Cdc42 that stimulates Arp2/3
complex function and actin rearrangement (Symons et al.,
1996). To determine whether NWASP deficiency also leads
to a size-dependent uptake defect, NWASP/ mouse embry-
onic fibroblasts (MEFs) were challenged with invasin-coated
small and large particles. Control experiments demonstrated
that 1.5-µm invasin-coated beads behaved similarly to Y. pseu-
dotuberculosis during uptake (unpublished data), thus 1.5-µm
beads were used to represent small particles. Uptake of 4.5-µm
beads by NWASP/ cells was defective, whereas no such
defect was observed with 1.5-µm beads (Fig. 1 E). Rac1 medi-
ates actin remodeling through the activation of WASP family
Verprolin-homologous family proteins in a manner similar
appears to occur in a Rac1- and RhoG-dependent and Cdc42-
independent fashion (Patel and Galán, 2006; Mohammadi and
Isberg, 2009). In contrast, uptake of antibody-coated erythro-
cytes requires both Cdc42 (Caron and Hall, 1998) and its down-
stream signaling molecules Wiskott–Aldrich syndrome protein
(WASP) and neural WASP (NWASP; Park and Cox, 2009),
with spatiotemporal activation of Cdc42 occurring during the
uptake of erythrocytes (Beemiller et al., 2010). Integrin-mediated
cell adhesion and spreading similarly requires several Rho
GTPases, including Cdc42 (Clark et al., 1998; Price et al., 1998;
Partridge and Marcantonio, 2006).
The processes of phagocytosis and cell adhesion and spread-
ing involve many of the same molecular determinants and share sim-
ilar signaling patterns during encounter with ligands (Cougoule
et al., 2004). In fact, cell spreading could be viewed as the frus-
trated phagocytosis of an infinitely large particle. Furthermore,
both processes require membrane delivery from internal sources
to the cell surface (Cox et al., 1999; Gauthier et al., 2009). It was
previously assumed that cell size decreases during phagocytosis.
However, electrophysiological measurements and cell spread-
ing assays have shown that cell surface area in fact increases
(Holevinsky and Nelson, 1998; Cox et al., 1999). This increase is
believed to be a result of membrane delivery from internal sources
to the site of particle uptake, which has been termed focal exocyto-
sis (Huynh et al., 2007). The recycling endosome (RE) is a major
source of membrane delivery to the forming phagosome. The
RE has a tubular structure and delivers membranes to regions of
the cell surface that are undergoing dramatic reorganization
(van Ijzendoorn, 2006), such as nascent phagosomes.
The initial impetus behind this study was to resolve a
seeming discrepancy in the requirement for Cdc42 during inte-
grin-mediated phagocytosis. Cdc42 inactivation appears not to
affect integrin-mediated uptake of bacteria (Alrutz et al., 2001),
whereas others found that Cdc42 inactivation negatively affects
the integrin-mediated uptake of large (4 µm—larger than aver-
age bacteria) beads (Wiedemann et al., 2001). This discrepancy
led us to hypothesize that Cdc42 dependence during uptake is
governed by the size of the phagocytic particle. Accordingly,
we found that although Cdc42-depleted cells internalized small
particles efficiently, large particle uptake was defective. An in-
triguing and unique link between Cdc42 and the secretory
machinery was then uncovered, leading to the discovery of the
interaction between Cdc42 and the exocyst complex during
Impaired Cdc42 signaling leads to a
selective defect in large particle uptake
Previous results indicated that integrin-promoted uptake of
Y. pseudotuberculosis (1–2 µm in size) required active Rac1 but
was unaffected by interfering forms of Cdc42 (Alrutz et al.,
2001). Conflicting results indicate that Cdc42 is indeed required
for uptake of 4-µm invasin-coated beads (Wiedemann et al.,
2001). Also, in conflict with the bacterial uptake data is the ob-
servation that cell spreading on fibronectin, a ligand that en-
gages many of the same integrin receptors as invasin, has been
83Cdc42 modulates exocytic function during uptake • Mohammadi and Isberg
measuring the interaction between activated endogenous Cdc42
and the Cdc42-binding domain (CBD) of WASP (Beemiller
et al., 2010) was used. Both small and large particles were found
to activate Cdc42 (Fig. S2 and Videos 2 and 3), similar to pre-
vious observations with FcR-mediated uptake (Beemiller et al.,
2010), indicating that size dependence is not caused by varia-
tions in Cdc42 activation.
The size-dependent uptake defect in Cdc42-depleted
cells could be caused by differences in the surface area be-
tween small and large particles. If so, increasing the total sur-
face area of small phagocytic particles encountered by a cell
should recapitulate the uptake defect. To address this possibil-
ity, uptake of small and large particles by the same cell was
assessed in control and Cdc42-depleted cells (Fig. 3, A and E).
Differing quantities of small particles were added so that the
surface area ratio of small to large particles was low (Fig. 3,
A–D) or similar (Fig. 3, E–H). Interestingly, even when the
total small particle surface area was similar to the total large
particle surface area, identical results were obtained regarding
size dependence and Cdc42 requirement (Fig. 3 F). Small par-
ticles were internalized efficiently regardless of total surface
area and Cdc42 depletion status. Notably, the same Cdc42-
depleted cell that failed to internalize large particles efficiently
internalized large numbers of small particles, indicating that
to NWASP (Eden et al., 2002). In contrast to NWASP/
cells, WAVE2/ MEFs were defective for the uptake of
small particles (Fig. 1 F), indicating that only the downstream
effector of Cdc42 displays a size-dependent phenotype in this pro-
cess, similar to the contrast between the roles of Rac1 and
Cdc42 in uptake.
Rac1 activity can be influenced by the level and activation
state of Cdc42 (Nobes and Hall, 1995; Price et al., 1998), so the
size-dependent uptake defect observed in Cdc42-depleted cells
may be caused by a selective reduction in Rac1 activity. By this
model, the Cdc42 requirement would reflect a need for increased
amounts of active Rac1 during large particle uptake relative to
small particles. Therefore, fluorescence resonance energy trans-
fer (FRET) microscopy was used to quantify Rac1 activation
levels at nascent phagosomes in control and Cdc42-depleted
cells (Fig. 2, A and B). Rac1 was found to be activated to the
same level regardless of particle size or Cdc42 level (Fig. 2,
C and D), indicating that Rac1 activity is not altered by a reduction
in Cdc42 levels in this phagocytic system and that the cause of
Cdc42 dependence lies elsewhere.
Another possibility for the observed differential require-
ment for Cdc42 is that receptor engagement by small particles
leads to less Cdc42 activation than large particle engagement.
To investigate this possibility, a ratiometric imaging method,
Figure 1. Defects in Cdc42 signaling result in a size-dependent uptake defect. (A) Cdc42 depletion leads to a kinetic defect in cell spreading on invasin.
Control (shScr)- and Cdc42 (shCdc42)-depleted COS1 cells were plated onto invasin-coated glass coverslips, and spreading was visualized by phase-
contrast time-lapse imaging. Numbers indicate time (in minutes). Bar, 10 µm. (B) Quantification of A. The area occupied by the cell was measured at each time
point. Data are presented as fold increase in area, normalized to time = 0. (C) Cdc42-depleted cells internalize small particles but fail to internalize large
particles efficiently. Control, Cdc42-depleted, and Rac1 (Rac1 shRNA [shRac1])-depleted COS1 cells were incubated with small (Y. pseudotuberculosis),
medium (2.9-µm beads), or large (4.5-µm beads) invasin-coated particles. Uptake was then quantified microscopically. (D) FcR-mediated uptake in Cdc42-
depleted cells is size dependent. Small (1.5 µm) or large (4.5 µm) IgG-coated beads were incubated with control or Cdc42-depleted COS1 cells express-
ing FcRIIA, and uptake was quantified microscopically. (E) NWASP is required for the internalization of large particles but not for small particle uptake.
NWASP+/+ and NWASP/ MEFs were incubated with 4.5- and 1.5-µm particles, and uptake was quantified microscopically. (F) WAVE2 is required for
internalization of small particles. WAVE2+/+ and WAVE2/ MEFs were incubated with Y. pseudotuberculosis, and uptake was quantified microscopically.
Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
JCB • VOLUME 200 • NUMBER 1 • 2013 84
(referred to as Lyn-GFP) was used (Teruel et al., 1999). Control
and Cdc42-depleted cells expressing Lyn-GFP were challenged
with small and large particles, and phagosome formation was
visualized by time-lapse microscopy. Membrane morphology
during small particle (Y. pseudotuberculosis) uptake was unaf-
fected by Cdc42 levels. Control and Cdc42-depleted cells inter-
nalized Y. pseudotuberculosis with relatively little membrane
perturbation (Fig. 4, A and B; and Videos 4 and 5). Uptake of
4.5-µm beads by control depleted cells, however, stimulated a
great deal of membrane perturbation (Fig. 4 C, 2 and 3 min im-
ages; and Video 6). A large degree of flux in GFP signal was
each phagosome forms independently and that the molecular
requirements for internalization are distinct for large and
Defects in Cdc42 signaling interfere with
A series of experiments were performed to determine reasons
for the size-dependent requirement for Cdc42. First, phagosome
morphology in the presence and absence of Cdc42 was ana-
lyzed. To visualize nascent phagosome structure, a membrane-
targeted GFP carrying the myristoylation site of Lyn kinase
Figure 2. Cdc42 depletion does not affect Rac1
activation levels at nascent phagosomes. (A and B)
Rac1 activity at the site of particle binding is not
affected by Cdc42 depletion. Control (shScr) and
Cdc42 (shCdc42)-depleted COS1 cells, expressing
mCFP-Rac1 and PBD-mYFP were incubated with Y.
pseudotuberculosis or 4.5-µm invasin-coated beads.
Each condition was imaged microscopically for FRET
analysis. Arrowheads denote regions of particle
binding. Bar, 20 µm. (C and D) Quantification of A
and B. The FRET signal at the site of particle attach-
ment was quantified for each scenario. Error bars
Figure 3. Variations in phagocytic particle
surface area do not affect uptake efficiency.
(A and E) The effects of varying small particle
surface area upon uptake efficiency were
quantified after coincubating cells with small
and large particles. (A–H) Control (shScr)
and Cdc42 (shCdc42)-depleted COS1 cells
were challenged with a fixed MOI and 4.5-µm
beads simultaneously, with 1.5-µm beads at
low (A–D) or high (E–H) multiplicity, and up-
take by the same cell of the two differently
sized particles was quantified microscopically
on a per-cell basis. The total number of small
(C and G) and large (D and H) beads asso-
ciated with each cell was used to determine
multiplicity empirically. In the low multiplicity
scenario (A–D), the large/small surface area
ratio was 5, and in the high multiplicity sce-
nario (E–H), the large/small surface area
ratio was 1.3. Error bars represent SEM.
***, P < 0.001.
85 Cdc42 modulates exocytic function during uptake • Mohammadi and Isberg
of these proteins to small phagosomes (Fig. 4, E–G). The extent
of localization was further characterized by quantifying the TfR
pixel intensity at individual nascent phagosomes. This analysis
showed that, per unit surface area in contact with the particles,
more TfR associated with large phagosomes than small phago-
somes (Fig. 4 H). Additionally, confocal microscopy showed a
punctate pattern of TfR localization around large nascent phago-
somes (unpublished data), indicating potential sites for exocytic
vesicle fusion on the nascent phagosome. Collectively, these
data are consistent with large particles selectively using the
cell’s secretory functions during uptake.
To determine whether a connection exists between the re-
quirement for Cdc42 and the presence of secretory markers, the
influence of Cdc42 inactivation upon bulk membrane flow to
the cell surface was examined using fluorescently tagged trans-
ferrin in the absence of phagocytic particles. Cells, pulsed with
Alexa Fluor 488–conjugated transferrin were chased in media
observed shortly after encounter with the large particle, which
subsided as the particle was internalized. This was in dramatic
contrast to Cdc42-depleted cells, which displayed very little
membrane perturbation in response to large particle binding
(Fig. 4 D and Video 7). It is unlikely that differences between
control and Cdc42-depleted cells are caused by variations in the
rate of actin polymerization because F-actin levels remain
unchanged after Cdc42 depletion (unpublished data). These
results are consistent with Cdc42 playing a role in modulating
membrane structure at the site of large particle uptake.
The localization of endocytic markers to nascent phago-
somes was examined to determine whether the size of the
phagocytic particle affects recruitment patterns. Two endocytic
markers, transferrin receptor (TfR) and Rab11, have been found
on nascent phagosomes during erythrocyte engulfment (Cox
et al., 2000). Interestingly, although we found TfR and Rab11
on phagosomes of large particles, there was very little recruitment
Figure 4. Reduced membrane perturbation at the nascent phagosome in Cdc42-depleted cells. (A–D) Characterizing phagosome morphology using time-
lapse microscopy. Y. pseudotuberculosis or 4.5-µm beads were added to control (shScr) or Cdc42 (shCdc42)-depleted COS1 cells expressing Lyn-GFP and
imaged every 15–20 s. At each time point, a bright-field and a GFP image were collected. Numbers indicate time (in minutes). Bars, 5 µm. (E) Transferrin
receptor (TfR) localizes to large nascent phagosomes. COS1 cells were challenged with 1.5 (top)- and 4.5 (bottom)-µm beads and fixed, and endogenous
TfR was detected using an anti-TfR antibody. In merged images, extracellular particles and TfR are pseudocolored red and green, respectively. Arrowheads
point to phagosomes magnified in insets to emphasize the localization pattern. (F) Rab11 localizes to large nascent phagosomes. COS1 cells expressing
Myc-Rab11 were challenged with 1.5 (top)- and 4.5 (bottom)-µm beads, and Myc-Rab11 was detected using an anti-Myc antibody. Bar, 10 µm; applies to
E also. (G) Quantification of E and F. At least 50 phagosomes per condition were scored by eye for TfR or Rab11 recruitment. (H) The extent of TfR recruit-
ment to phagosomes was determined by immunostaining for TfR and quantifying mean pixel intensity for ≥20 small and large phagosomes (Iphagosome). TfR
staining at a nearby cytosolic region, devoid of attached particles, was determined also (Icytoplasm). Extent of recruitment is presented as the quantity Iphagosome/Icytoplasm.
(I and J) Inactivation of Cdc42 or NWASP leads to reduced transferrin efflux. COS1 cells expressing dominant-negative Cdc42 and control cells (I) and
NWASP+/+ and NWASP/ MEFs (J) were pulsed with fluorescent transferrin and chased in its absence. Remaining transferrin amounts were quantified
microscopically. Error bars represent SEM. **, P < 0.01; ***, P < 0.001.
JCB • VOLUME 200 • NUMBER 1 • 2013 86
sufficient to overcome uptake defects observed with both defec-
tive Cdc42 signaling and the loss of a downstream effector. Nota-
bly, this rescue was not a general feature of Rab protein expression.
Rab7 is a GTPase required for late endosome maturation (Wang
et al., 2011) and is not involved in membrane flow to the surface.
We found that constitutively active Rab7 (Rab7Q67L) failed to
rescue the large particle uptake defect in Cdc42-depleted cells
(Fig. 5 C). Additionally, the efficiency of large particle internaliza-
tion was unaffected by dominant-negative Rab7 (Rab7T22N)
expression (Fig. 5 D).
The effect on phagocytosis of decreasing membrane flow
to the cell surface was examined next. Expression of dominant-
negative Rab11 in cells has been shown to slow trafficking to
the cell surface (Zeng et al., 1999) and reduce the uptake effi-
ciency of IgG-coated erythrocytes (Cox et al., 2000), so cells
expressing dominant-negative Rab11 (Rab11S25N) were chal-
lenged with small and large particles. Expression of Rab11S25N
led to a size-dependent uptake defect, similar to that observed in
Cdc42-depleted cells (Fig. 5, D and E), consistent with a size-
dependent requirement for membrane delivery to the cell surface.
This defect in uptake was not caused by changes in steady-state
levels of 1 integrin receptors on the plasma membrane. Flow
cytometry showed that neither Cdc42 depletion nor the absence
of NWASP reduced 1 integrin cell surface staining (unpub-
lished data). Similarly, neither constitutively active nor domi-
nant-negative Rab11 expression altered 1 integrin cell surface
staining (unpublished data).
Because Rab11 expression could rescue the large particle up-
take defect of Cdc42-depleted cells, the notion of a physical inter-
action between Cdc42 and Rab11 was examined. Cells expressing
wild-type and two constitutively active forms of Cdc42 (Cdc42G12V
and Cdc42F28L) along with wild-type Rab11 were lysed, and in-
teraction between Cdc42 and Rab11 was assayed by immunopre-
cipitation and Western blotting. No interaction was observed,
regardless of coimmunoprecipitation combination (Fig. S3 A).
containing excess unlabeled transferrin and fixed at various
time points, and the cell-associated fluorescence was quantified
microscopically. The presence of an interfering form of Cdc42
caused a kinetic defect in transferrin efflux relative to the vector
alone (Fig. 4 I), similar to previous work connecting Cdc42
with the secretory pathway (Kroschewski et al., 1999). Similar
results were obtained using NWASP/ MEFs when compared
with NWASP+/+ MEFs (Fig. 4 J), indicating that at least one
well-characterized Cdc42 effector is involved in this process.
These data indicate that Cdc42 signaling influences the rate of
bulk membrane delivery to the cell surface.
Rab11 expression rescues the
large particle uptake defect
in Cdc42-depleted cells
Focal exocytosis of endomembranes has been implicated during
phagosome formation (Bajno et al., 2000), so we postulated that
decreased exocytic activity associated with Cdc42 inactivation
(Fig. 4) could be responsible for defects in large particle uptake.
This is supported by previous observations that Cdc42 acts as
a regulator of endomembrane trafficking (Kroschewski et al.,
1999; Garrett et al., 2000). Rab11 expression has been shown to
affect the rate of membrane flow to the cell surface from the RE,
as constitutively active Rab11 increases and dominant-negative
Rab11 decreases this rate (Ren et al., 1998; Cox et al., 2000). To
determine whether increased Rab11 activity could overcome
the loss of Cdc42 function, control and Cdc42-depleted cells,
expressing constitutively active Rab11 (Rab11Q70L), were chal-
lenged with 4.5-µm beads, and uptake efficiency was quantified.
Expression of Rab11Q70L rescued the uptake defect in Cdc42-
depleted cells but had no significant effect on the internalization
efficiency of control depleted cells (Fig. 5 A). Rab11Q70L
expression was also found to rescue the large particle uptake
defect observed in NWASP/ cells (Fig. 5 B). These data
indicate that increasing membrane flow to the cell surface is
Figure 5. Rab11 expression compensates for defective Cdc42 signaling during large particle internalization. (A) Rab11 expression rescues large par-
ticle uptake defect in Cdc42-depleted cells. Control (shScr) and Cdc42 (shCdc42)-depleted COS1 cells were transfected with constitutively active Rab11
(Rab11Q70L) or a control plasmid and challenged with 4.5-µm beads. Uptake was quantified microscopically. (B) Rab11 expression rescues large particle
uptake defect in NWASP/ MEFs. NWASP/ and NWASP/ MEFs, expressing Rab11Q70L, were challenged with 4.5-µm beads, and uptake was
quantified. (C) Rab7 expression does not rescue large particle uptake defect in Cdc42-depleted cells. Experiment performed as in A, using constitutively
active Rab7 (Rab7Q67L). (D) Rab7 inactivation does not affect large particle uptake efficiency. COS1 cells were transfected with a control plasmid or a
dominant-negative Rab7 (Rab7T22N) and challenged with 4.5-µm beads, and uptake was quantified. (E) Rab11 inactivation leads to a size-dependent
uptake defect. COS1 cells were transfected with a control plasmid, dominant-negative Rab11 (rab11S25N), or dominant-negative Rac1 (Rac1T17N).
Rac1T17N was included as a negative control. These cells were then challenged with 1.5- and 4.5-µm beads, and uptake was quantified. Error bars
represent SEM. **, P < 0.01; ***, P < 0.001.
87 Cdc42 modulates exocytic function during uptake • Mohammadi and Isberg
evidence links Cdc42 and the exocyst complex: expression of
“active” mutants of the exocyst subunit, Exo70, has been shown
to rescue the temperature sensitivity of a cdc42 mutant in Sac-
charomyces cerevisiae (Wu et al., 2010). Exo70 localizes to the
plasma membrane through interaction with inner leaflet lipids
and is thought to create docking sites for the rest of the exocyst
complex, thereby demarcating the site of assembly (Liu et al.,
2007). Because the expression of an active form of Exo70 can
bypass the requirement for Cdc42 in yeast, we examined
whether the expression of human Exo70 could rescue large par-
ticle uptake defects in the absence of Cdc42 function. Control
and Cdc42-depleted cells, expressing Exo70, were challenged
with 4.5-µm beads. Exo70 expression had no effect on uptake in
control depleted cells (Fig. 7 A). In contrast, Exo70 expression
in Cdc42-depleted cells increased uptake efficiency to levels in-
distinguishable from control samples (Fig. 7 A), suggesting that
an increase in the number of tethered exocyst complexes at the
plasma membrane could rescue the large particle uptake defect
in the absence of Cdc42 signaling. Exo70 expression did not
rescue defective uptake of large particles by Rac1-depleted
cells (Fig. 7 B), indicating that the rescue was specific to the ab-
sence of Cdc42 and that Exo70 could not bypass a requirement
for cytoskeletal rearrangements associated with Rac1 activity.
Interestingly, Exo70 expression did not rescue defective uptake
of large particles in NWASP/ MEFs (Fig. 7 C).
To determine whether exocyst function is specifically re-
quired for uptake of large particles, the effect of reduced Exo70
activity on uptake of differently sized beads was examined. En-
dogenous Exo70 was depleted using synthetic RNAi-mediated
knockdown (KD). Quantitative RT-PCR and Western analysis
were used to monitor the extent of Exo70 depletion, and both
mRNA and protein levels were found to be greatly reduced
compared with the control (Fig. S1, B and C). Exo70-depleted
cells did not internalize 4.5-µm beads efficiently (Fig. 7 D),
whereas uptake of 1.5-µm beads was unaffected (Fig. 7 F), indi-
cating that robust exocyst function is selectively required during
large particle uptake. Intriguingly, Exo70 was also required for
the FcR-mediated internalization of large particles (Fig. 7 E),
whereas its depletion did not affect small particle uptake (Fig. 7 G).
These data indicate that the requirement for exocyst function is
not limited to 1 integrin–mediated uptake and is likely a gen-
eral requirement for efficient phagocytosis.
Similarly, dominant-negative Rab11 did not interact with Cdc42
either (Fig. S3 B), whereas a known binding partner (CBD do-
main from WASP) coimmunoprecipitated with Cdc42 efficiently
(Fig. S3 C). These results indicate that Cdc42 influences exocytic
functions using binding partners other than Rab11.
Cdc42 interacts with the exocyst complex
during large particle phagocytosis
An alternate site of interaction with the exocytic network could
be proteins that act downstream of Rab11/RE. One potential tar-
get is the exocyst, a multicomponent complex involved in tether-
ing post-Golgi vesicles to the plasma membrane (Munson and
Novick, 2006), whose components were found in a proteomic
analysis of Drosophila melanogaster phagosomes (Stuart et al.,
2007). In mammalian cells, constitutively active Cdc42 interacts
with the exocyst complex when two exocyst components associ-
ated with secretory vesicles, Sec8 and Sec3 (Sakurai-Yageta
et al., 2008), are both expressed. However, there are no studies of
wild-type Cdc42 associating with any exocyst components in
response to an activation signal such as phagocytosis. Thus, to
examine the possibility of Cdc42–exocyst interaction during
phagocytosis, coimmunoprecipitation experiments were con-
ducted using cells expressing wild-type Myc-Cdc42, Sec8-HA,
and Sec3-FLAG. These cells were challenged with 4.5- and 1.5-µm
beads, lysed at various time points, and Myc-Cdc42 immuno-
precipitated, and the amount of coprecipitated Sec8-HA was de-
termined using Western blotting. Remarkably, 4.5-µm beads
stimulated a transient Cdc42/Sec8 association (Fig. 6, A and C).
An increase in association was observed within 2 min after the
addition of beads to cells, peaking at 5 min (Fig. 6 C). Associa-
tion levels then diminished, reaching original levels at 15 min.
This is in contrast to 1.5-µm beads, which showed no increase
in Cdc42/Sec8 association throughout the experiment (Fig. 6,
B and C). These data indicate that large particles selectively
stimulate Cdc42 association with exocyst components.
The exocyst complex plays a functional role
during large particle uptake
Because we found that Cdc42–exocyst interaction is stimulated
by large particle uptake, the interplay between Cdc42 and the
exocyst complex during phagocytosis was examined further.
In addition to the interaction data discussed in Fig. 6, genetic
Figure 6. Kinetic association of Cdc42 with the
exocyst complex during large particle uptake.
(A and B) Sec8 associates with Cdc42 during large
particle uptake. 293T cells were cotransfected with
Myc-Cdc42wt, Sec8-HA, and Sec3-FLAG and were
incubated with 4.5 (A)- or 1.5 (B)-µm beads. At the
indicated times, cells were lysed, and Myc-Cdc42
was precipitated from the cleared lysates using anti-
Myc antibody resin. Input and immunoprecipitate (IP)
samples were analyzed by SDS-PAGE and Western
blotting. (C) Quantification of A and B. Densitometry
was performed on Western blots, and the quantity of
Sec8-HA that precipitated with Myc-Cdc42 was nor-
malized to input quantities and divided by starting
amounts to derive the fold Sec8 immunoprecipitate.
This assay was repeated multiple times, yielding simi-
lar results, and data are shown from one representa-
JCB • VOLUME 200 • NUMBER 1 • 2013 88
interfering form of Rab11 was examined. We observed that
Exo70 expression almost completely rescued the defect in up-
take resulting from the expression of dominant-negative Rab11
(Fig. 8 B). Next, we quantified uptake in Exo70-depleted cells,
expressing constitutively active Rab11, to determine whether
Rab11 activity could bypass a requirement for Exo70. In con-
trast to the observation that Exo70 expression could rescue the
uptake defect in the absence of Rab11 activity, depletion of Exo70
could not be bypassed by constitutively active Rab11 expression
(Fig. 8 C). These data are consistent with the model that Exo70
acts downstream of Rab11, demonstrating a critical role for the
exocyst complex during phagosome formation.
Rho family GTPases have long been known to be important regu-
lators of particle uptake, and their central role in the process has
been presumed to involve coordination of critical cytoskeletal re-
arrangements involved in phagosome closure (Niedergang and
Chavrier, 2005). In this work, we demonstrate that Cdc42 is im-
portant for membrane delivery to the phagosome, thereby estab-
lishing a connection between the cytoskeletal and secretory
networks during phagocytosis. Data presented here point to a
model in which size governs the Cdc42-mediated requirement for
exocytosis during phagosome formation. In contrast to small par-
ticles, large particles were associated with the recruitment of the
exocytic machinery (Fig. 4) and stimulated the temporal associa-
tion of the exocyst complex with Cdc42 (Fig. 6). Additionally,
only large particles required the exocytic proteins Rab11 and
Exo70 for uptake (Figs. 5 and 7), demonstrating a functional role
for the exocyst complex during receptor-mediated uptake that
was previously uncharacterized.
In this work, we show several functional similarities be-
tween integrin- and FcR-mediated uptake. We show that up-
take in the absence of Cdc42 is size dependent in both cases
(Fig. 1), that Cdc42 activation kinetics during uptake are similar
As there was a transient Cdc42–exocyst interaction during
large particle uptake (Fig. 6) and because Exo70 depletion in-
terfered with uptake of large particles (Fig. 7), we decided to
test whether Exo70 stabilizes the interaction between Cdc42
and the exocyst complex. Coimmunoprecipitation could not
demonstrate stable Cdc42–Exo70 interaction in cells express-
ing constitutively active Cdc42 and wild-type Exo70 (unpub-
lished data). It is possible that the Cdc42 interaction with an
Exo70-containing complex is transient or that Exo70 plays a
role in stabilizing a complex that allows Cdc42 to recognize
other exocyst components. Therefore, we investigated whether
the interaction of Cdc42 with the exocyst complex required the
presence of Exo70. Using the strategy used in Fig. 6, we tested
whether Exo70 was required for Cdc42 interaction with a Sec8/
Sec3-containing complex. Coimmunoprecipitation of constitu-
tively active Myc-Cdc42 (Cdc42V12), Sec8-HA, and Sec3-
FLAG was tested in control and Exo70-depleted cells and
analyzed by Western blotting. Interestingly, Exo70 depletion
clearly reduced the efficiency of Sec8 and Sec3 coassociation
with Cdc42 (Fig. 8 A, quantified on the right). These results in-
dicate that Exo70 is required for high efficiency interaction
between Cdc42 and Sec3/Sec8 and is indeed necessary for
maintaining the integrity of the interaction complex.
The interplay between Exo70 and Rab11 during large par-
ticle uptake was examined next to examine the likely order that
these two proteins function during uptake. A recent study has
shown that the depletion of either Rab11 or Exo70 stalls cargo
delivery at the plasma membrane, which indicates that both pro-
teins are intimately involved in the tethering of vesicles to the
plasma membrane (Takahashi et al., 2012). We show that the
inactivation of either Rab11 or Exo70 inhibited large particle
uptake (Figs. 5 and 7, respectively), but the molecular sequence
of action between these two proteins during uptake is unknown.
Two experiments were performed to examine the epistatic rela-
tionship between Rab11 and Exo70 during phagocytosis. First,
the effect of exogenous Exo70 expression in the presence of an
Figure 7. Exocyst function is required for large particle uptake. (A) Expression of Exo70 rescues large particle uptake defect in Cdc42-depleted cells.
Control (shScr) and Cdc42 (shCdc42)-depleted COS1 cells were transfected with FLAG-Exo70 or a control plasmid and challenged with 4.5-µm beads.
Uptake was quantified microscopically. (B) Exo70 expression does not rescue large particle uptake defect in Rac1-depleted cells. Control and Rac1 (Rac1
shRNA [shRac1])-depleted COS1 cells, expressing FLAG-Exo70, were challenged with 4.5-µm beads, and uptake was quantified. (C) Large particle uptake
defect in NWASP/ cells is not rescued by Exo70 expression. NWASP+/+ and NWASP/ cells were transfected with FLAG-Exo70 or a control plasmid
and challenged with 4.5-µm beads. Uptake was quantified microscopically. (D and E) Exo70 depletion leads to defective large particle uptake. Control
(siGLO) and Exo70 (siExo70)-depleted HeLa cells (D) or FcRIIA-expressing HeLa cells (E) were challenged with invasin-coated (D) or IgG-coated (E) 4.5-µm
beads, and uptake was quantified microscopically. (F and G) Exo70 depletion does not interfere with internalization of small particles. Control (siGLO)
and Exo70 (siExo70)-depleted HeLa cells (F) or FcRIIA-expressing HeLa cells (G) were challenged with invasin-coated (D) or IgG-coated (E) 1.5-µm beads,
and uptake was quantified microscopically. Error bars represent SEM. ***, P < 0.001.
Cdc42 modulates exocytic function during uptake • Mohammadi and Isberg
process in a fashion that is independent of the exocyst complex.
Alternatively, NWASP signaling could be occurring in a Cdc42-
independent manner, which has been observed previously
(Rohatgi et al., 2001). Although no direct role for NWASP has
been described during exocytosis, WASP-like proteins have been
implicated in vesicle delivery to the plasma membrane (Kim et al.,
2007), and the interaction of exocyst components and actin nu-
cleation–promoting factors, such as NWASP, has been the sub-
ject of recent investigations (Liu et al., 2012).
Although we have shown that Exo70 is required for Cdc42–
exocyst interaction (Fig. 8), the precise nature of the complex is
unclear. In yeast, Cdc42 interacts with the N terminus of the
Sec3 component of the exocyst complex (Zhang et al., 2001),
but the N-terminal sequence and subcellular localization pat-
terns for yeast and mammalian Sec3 differ (Matern et al., 2001).
Thus, Cdc42 and Sec3 may not interact in mammalian systems.
Several small GTPases have been shown to interact with exocyst
components in mammalian systems, namely Sec5, Sec10, Sec15,
and Exo70 (Sugihara et al., 2002; Inoue et al., 2003; Prigent
et al., 2003; Zhang et al., 2004; Zuo et al., 2011), and Cdc42
could potentially interact with these subunits during uptake.
To examine the nature of the exocyst localization during
phagosome formation more closely, we constructed fluorescently
tagged versions of each exocyst complex member and studied
recruitment patterns to the nascent phagosome (Fig. S5). Exo70
was efficiently recruited to large phagosomes, whereas Sec5
and Sec15 showed weaker levels of localization. This finding is
in contrast to previous work showing that most exocyst compo-
nents (all except Sec6 and Exo84) are found on nascent phago-
somes (Stuart et al., 2007). As most of the exocyst components,
when expressed as fluorescently tagged fusions, showed diffuse
cytoplasmic localization (Fig. S5 B), concentration at the nascent
phagosome was not readily detected. Previous work using purified
(Fig. S2; Beemiller et al., 2010), and that both processes require
exocyst function (Fig. 7). Collectively, these data indicate that
the molecular requirements for particle internalization are largely
independent of cell type and receptor–ligand combinations and
that size dependence and a requirement for efficient exocytic
activity are general features of phagocytic systems.
We showed that large particle uptake defect in NWASP/
cells could be rescued by Rab11 expression (Fig. 5) but not with
Exo70 (Fig. 7). This dichotomy implies that the mode of rescue
is different for the two molecules. Exo70 is thought to act as a
docking and assembly site for the exocyst complex (Liu et al.,
2007), and its exogenous expression likely increases the num-
ber of functional exocyst complexes in the cell. Rescue in
Cdc42-depleted cells could come from the combination of re-
sidual Cdc42 levels that stimulate low level NWASP activity,
which in combination with Exo70 lead to increased uptake. Ex-
ogenous expression of Rab11 has been shown to increase the
rate of trafficking to the plasma membrane (Ren et al., 1998;
Cox et al., 2000), so rescue by Rab11 expression is most likely
caused by a general increase in membrane trafficking. Increas-
ing exocyst assembly sites through Exo70 expression without a
large-scale increase in membrane flow could be sufficient for
rescuing the RNAi-mediated depletion of Cdc42 but is not suf-
ficient for rescuing the genetic absence of NWASP. However,
the increase in membrane flow caused by Rab11 expression
may be sufficiently robust for rescuing uptake in both Cdc42-
depleted and NWASP/ cells.
The precise role of NWASP in the context of membrane
exocytosis is unknown. We show that although both Cdc42 and
NWASP are required for large particle uptake (Fig. 1), the ab-
sence of NWASP could not be rescued by the exogenous ex-
pression of Exo70 (Fig. 7). This phenomenon could be described
by several possibilities. NWASP may be involved in the uptake
Figure 8. Exo70 interaction with the exocyst complex and Rab11 during phagosome formation. (A) Cdc42–exocyst association requires Exo70. Control
(siGLO) and Exo70 (siExo70)-depleted 293T cells were transfected with constitutively active Cdc42 (Myc-Cdc42V12), Sec8-HA, and Sec3-FLAG. Cells
were lysed, and Myc-Cdc42 was precipitated from the cleared lysates using anti-Myc antibody resin. Input and immunoprecipitate (IP) samples were ana-
lyzed by SDS-PAGE and Western blotting. Densitometry was used to quantify the amount of Sec8 or Sec3 that precipitated with Myc-Cdc42, which was
then normalized to input quantities. (B) Exo70 expression rescues large particle uptake defect in the absence of Rab11 activity. HeLa cells expressing either
Exo70 or dominant-negative Rab11 (Rab11S25N) or both were challenged with 4.5-µm beads, and uptake was quantified microscopically. (C) Control
(siGLO) or Exo70 (siExo70)-depleted HeLa cells were transfected with an empty plasmid or constitutively active Rab11 (HA-Rab11Q70L) and challenged
with 4.5-µm beads. Uptake was quantified microscopically. Error bars represent SEM. **, P < 0.01.
JCB • VOLUME 200 • NUMBER 1 • 2013 90
Materials and methods
Cell culture and transfection
COS1, HeLa, and 293T cells (obtained from American Type Culture Col-
lection) and NWASP/ and WAVE2/ MEFs (gifts from S. Snapper,
Massachusetts General Hospital, Boston, MA) were maintained in DMEM
supplemented with 10% heat-inactivated fetal bovine serum. Cells were
transfected using Lipofectamine 2000 (Invitrogen) or FuGENE HD (Roche)
per the manufacturers’ recommendations. Typically, cells were analyzed
16–20 h after transfection, except for RNAi transfections, which were per-
formed for 40–48 h. All plasmids used in this study are listed in Table S1.
Cdc42 and Rac1 were depleted using plasmids encoding shRNAs. For
these experiments, a new plasmid based on pRNAT-U6.1/neomycin (Gen-
Script) was constructed that encodes mCherry (Shaner et al., 2004), whose
expression could be used a proxy for protein depletion. shRNA sequences
used are as follows: scrambled KD, 5-AATTCTCCGAACGTGTCACGT-3;
Cdc42 KD, 5-AAGTGGGTGCCTGAGATAACT-3 (Wilkinson et al., 2005);
and Rac1 KD, 5-AAGGAGATTGGTGCTGTAAAA-3 (Chan et al., 2005). The
extent of KD was examined by Western blotting using monoclonal anti-
Cdc42, anti-Rac1 (both obtained from BD), and antitubulin (Sigma-Aldrich)
to control for loading (Fig. S1). Exo70 was depleted using a synthetic
duplex RNA (Thermo Fisher Scientific) with the following sequence:
5-GGUUAAAGGUGACUGAUUAUU-3 (Zuo et al., 2006). Efficiency of
Exo70 depletion was examined using quantitative real-time PCR and West-
ern blotting with an anti-Exo70 antibody (Abcam).
All live-cell imaging was performed on a wide-field microscope (Axiovert
200M; Carl Zeiss) using a 10×, NA 0.3 Plan Neofluar or a 63×, NA 1.4
Plan Apochromat lens. The microscope was equipped with an environmen-
tal control chamber, standard FITC, TRITC, and DAPI filter cube sets (Chroma
Technology Corp.), and a charge-coupled device camera (C4742-95-
12ERG; Hamamatsu Photonics), all controlled by the Openlab imaging
software package (PerkinElmer).
Fixed samples (FRET experiments and all quantitative uptake assays)
were analyzed on a wide-field microscope (TE300; Nikon) equipped with
60×, NA 1.4 Plan Apochromat and 100×, NA 1.3 Plan Fluor lenses, stan-
dard YFP, CFP, and YFP/CFP FRET filter cube sets (Chroma Technology Corp.),
and a charge-coupled device camera (C4742-98-24NR; Hamamatsu Pho-
tonics), controlled by the IPLab/iVision software package (Scanalytics/BD).
All image analysis was performed using either Openlab or IPLab/
iVision. Figures were assembled using Photoshop (Adobe).
Cell spreading on invasin
8-well chambered coverslips (LabTek) were coated with 5 µg/ml maltose-
binding protein (MBP)-Inv497 (C-terminal 497 aa of invasin fused to MBP)
in PBS overnight at 4°C. Control and Cdc42-depleted cells were lifted using
PBS + 10 mM EDTA, plated onto the coated dishes by centrifugation, and
moved immediately to the microscope with a heated stage and imaged
using a 10× objective lens for 30 min at 15-s intervals. For quantification,
the area occupied by each cell was manually delineated and measured using
the Openlab software package. Number of image sequences used for
quantification is as follows: shScr, n = 5; and shCdc42, n = 9.
Quantifying uptake efficiency
Bacterial uptake was quantified on a per-cell basis as previously described
(Mohammadi and Isberg, 2009) using a virulence plasmid-deficient
Y. pseudotuberculosis strain. In brief, overnight cultures of bacteria were
diluted 1:40 into fresh media and grown to mid–exponential phase (optical
density of 0.7). Bacteria were added to cells grown on coverslips at a
multiplicity (MOI) of 10, and uptake was allowed to proceed for 20 min.
Cells were then fixed, and uptake was quantified microscopically on a per-
For experiments involving bead uptake, 1.5-, 2.9-, or 4.5-µm latex
beads (Polysciences, Inc.) were incubated overnight at 4°C with 1.5, 0.75,
or 0.5 mg/ml purified MBP-Inv497, respectively. Beads were washed and
stored at 4°C until use. Coated beads were added to cells grown on cover-
slips (MOI of 5–10) and briefly centrifuged (2 min at 300 g). Uptake was
allowed to proceed for 30 min. Cells were then fixed, and uptake efficiency
was quantified microscopically on a per-cell basis. Anti-MBP antibodies
(New England Biolabs, Inc.) were used to detect MBP-Inv497.
For antibody coating, 1.5- or 4.5-µm beads were coated with 1 or
3 mg/ml rabbit IgG (Sigma-Aldrich), respectively. Beads were then washed
phagosomes and mass spectrometry (Stuart et al., 2007) was
able to overcome this problem by isolation from cytoplasmic
contamination. Given the lack of clear concentration of exocyst
components about the membrane and the fact that we could not
detect interaction between Cdc42 and Exo70 by coimmunopre-
cipitation (unpublished data), we believe that exocyst complex
formation during phagocytosis is transient and highly suscepti-
ble to perturbations, such as component depletion or exogenous
expression of individual components.
The small GTPase RalA has been implicated in exocyst
complex assembly and stability (van Dam and Robinson, 2006)
and has been shown to mediate the formation of Cdc42-dependent
structures such as filopodia (Sugihara et al., 2002) and S. en-
terica invasion foci (Nichols and Casanova, 2010). However, we
found that RalA inactivation does not affect integrin-mediated
uptake efficiency and that RalA is not activated during large
particle phagocytosis (Fig. S4), indicating that RalA is not play-
ing a role in the processes described in this work. Processes such
as ciliogenesis (Das and Guo, 2011) that require constitutive
exocytosis do not appear to need Ral function, and although
no Ral homologue is found in the budding yeast S. cerevisiae
(Lipschutz and Mostov, 2002), the exocyst assembles and func-
tions stably (TerBush et al., 1996). It is possible that the regula-
tory proteins described in this work, namely Rab11 and Cdc42,
modulate critical exocytic events without contribution from RalA.
Our data suggest that small particles are internalized qui-
etly from a signaling standpoint: few changes in membrane
morphology at the site of attachment (Fig. 4) are accompanied
by a brief peak of Cdc42 activity that subsides quickly (Fig. S2)
with no requirement for membrane delivery from internal sources
(Figs. 5–7). The quiet nature of small particle entry could be ad-
vantageous to invading particles, such as intracellular bacterial
pathogens. The bacterial pathogen could be stimulating few
enough pathways to be phagocytosed but escape immune detec-
tion during the entry process, which may be deleterious to the
Phagocytic uptake of apoptotic bodies shares several key
characteristics with the large particle phagocytic system tested
here in our study: apoptotic cells are internalized by zippering
phagocytosis (Krysko et al., 2006), they are internalized by
binding to integrin receptors (Savill et al., 2002), among oth-
ers, and internalization of apoptotic cells requires both Cdc42
and WASP (Leverrier and Ridley, 2001; Leverrier et al., 2001).
Thus, it is tempting to compare the implications of our ob-
served defects in large particle uptake to defects in apoptotic
cell clearance. Several autoimmune diseases are associated with
impaired clearance of dead cells (Nagata, 2007). Thus, impair-
ments in signaling that lead to defects in apoptotic cell phago-
cytosis, such as defects in Cdc42, Rab11, or Exo70 function
described in our work, may potentially contribute to the devel-
opment of autoimmunity.
In this work, we have demonstrated that the size of the
phagocytic particle is a critical determinant of signaling potential
during uptake. We have established a molecular link between the
cytoskeletal and the exocytic machineries that was previously
unknown. Additionally, we have established a functional role for
the exocyst complex during receptor-mediated uptake.
91 Cdc42 modulates exocytic function during uptake • Mohammadi and Isberg
(Invitrogen) and 100 µM desferrioxamine (Sigma-Aldrich). At the indicated
times, cells were fixed and imaged to quantify remaining transferrin
amounts. The mean fluorescence at two ROIs per cell was determined, and
≥20 cells were analyzed per time point. Values were normalized to the
mean fluorescence values at time = 0.
Cdc42–exocyst coimmunoprecipitation assay
293T cells, expressing Myc-Cdc42wt, Sec8-HA, and Sec3-FLAG, were se-
rum starved for 3–4 h. They were then incubated with 1.5- or 4.5-µm
beads (MOI of 1) for 0, 2, 5, 10, 20, and 30 min. At each time point,
cells were lysed using 500 µl lysis buffer and collected by scraping with a
rubber policeman. Lysates were cleared by centrifugation, and 20 µl was
saved as input. 400 µl cleared lysate was incubated with 25 µl anti-Myc
resin (Santa Cruz Biotechnology, Inc.) for 1 h at 4°C with agitation. Beads
were then washed 3× in wash buffer (lysis buffer with 0.1% Triton X-100),
resuspended in 50 µl sample buffer, and eluted by boiling for 5 min. 10 µl
of these samples was loaded on SDS-PAGE gels; input samples were di-
luted 20-fold, and 10 µl was loaded. For experiments with Myc-Cdc42V12,
no beads were added. Cells were treated exactly the same otherwise.
Rab11/Cdc42 coimmunoprecipitation assay
This experiment was performed essentially like the Cdc42–exocyst immuno-
precipitation experiment described in the previous section. In brief, 293T
cells, expressing Myc-Cdc42wt, G12V, or F28L (Lin et al., 1997) with
HA-mYFP, HA-mYFP-Rab11wt, or HA-mYFP-Rab11S25N were lysed, and
Cdc42 was precipitated using the anti-Myc antibody resin. Beads were then
washed and eluted by boiling in sample buffer. Eluate (immunoprecipitation)
and input samples were analyzed by Western blotting using anti-Myc and
anti-HA antibodies (both obtained from Santa Cruz Biotechnology, Inc.).
RalA activation assay
293T cells were serum starved for 3–4 h and then incubated with 4.5-µm
beads (MOI of 1) for 0, 2, 5, 10, 20, and 30 min. At each time point,
300 µl lysis buffer was added, and cells were collected by scraping with
a rubber policeman. Lysates were cleared by centrifugation, and 20 µl
was saved as input. 50 µl cleared lysate was incubated with 25 µl GST-
Sec5RBD (Ral-binding domain of Sec5 fused to GST) beads (gift from
R. Wong and L. Feig, Tufts University School of Medicine, Boston, MA) for
30 min at 4°C with agitation. Sec5RBD binds to endogenous RalA-GTP,
allowing for the isolation of active RalA from the inactive. Beads were then
washed 3× in wash buffer (same as lysis buffer except with 0.1% Triton
X-100), resuspended in 50 µl sample buffer, and eluted by boiling for
5 min. 10 µl of these samples were loaded on SDS-PAGE gels; input sam-
ples were diluted 20-fold, and 10 µl was loaded. A monoclonal anti-RalA
antibody (BD) was used to detect endogenous RalA.
Each assay presented here was performed at least three times. Unless oth-
erwise indicated, graphs depict mean values ± SEM. Microscopic assays
on fixed samples were performed in triplicate (three coverslips) and quanti-
fied by scoring 30–50 cells per coverslip. Images presented here are rep-
resentative of each sample analyzed. Data were analyzed using Prism
(GraphPad Software), and p-values were generated using a two-tailed un-
paired t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Online supplemental material
Fig. S1 shows RNAi-mediated depletion of Cdc42, Rac1, and Exo70.
Fig. S2 shows that Cdc42 is activated in response to small and large
particles. Fig. S3 shows that coimmunoprecipitation experiments show
no Cdc42 association with Rab11. Fig. S4 shows that RalA inactivation
does not affect uptake efficiency and RalA is not activated in response
to large particles. Fig. S5 shows the localization pattern of exocyst com-
plex components. Table S1 shows expression plasmids used in this study.
Video 1 shows cell spreading on invasin-coated glass surface in con-
trol and Cdc42-depleted cells. Video 2 shows ratiometric imaging to
quantify Cdc42 activity at small nascent phagosomes. Video 3 shows
ratiometric imaging to quantify Cdc42 activity at large nascent phago-
somes. Video 4 shows small phagosome morphology in cells expressing
Cdc42. Video 5 shows small phagosome morphology in Cdc42-depleted
cells. Video 6 shows large phagosome morphology in cells express-
ing Cdc42. Video 7 shows large phagosome morphology in Cdc42-
depleted cells. Online supplemental material is available at http://www
We would like to thank Larry Feig, Ka-Wing Wong, and Elizabeth Creasey
for helpful discussions and members of the Isberg laboratory for critical review
and stored at 4°C until use. Cells were transfected with FcRIIA overnight
(gift from E. Caron, Imperial College, London, England, UK), and beads
were added and centrifuged briefly as in the previous paragraph. Cells
were fixed after 30 min, and uptake was quantified exactly as described
for MBP-Inv497 beads except that particles were visualized using anti–
rabbit fluorescent antibodies.
Rac1 FRET imaging
Cells expressing mCFP-Rac1wt and p21-binding domain (PBD)-mYFP were
incubated with small or large particles for 20 min. Cells were then washed,
fixed, and processed for imaging. Imaging and analysis were performed
exactly as previously described (Wong and Isberg, 2005). In brief, CFP,
YFP, and FRET images were captured for each sample, and regions of in-
terest (ROIs) at each nascent phagosome (i.e., with partially internalized
particles) and at cytosolic regions with no bound particles were selected.
For each ROI, sensitized FRET values (sFRET), which are corrected for cross-
excitation and bleed through, were calculated. Subsequently, FRET values
that were normalized to the expression levels of the donor and acceptor
(nFRET) were calculated using the following equation:
I represents the mean intensity for the indicated fluorescence channel. The
fold increase in Rac1 activation was calculated by dividing the phagosome
nFRET value by the cytosolic nFRET value.
The kinetics of Cdc42 activation in response to small and large particles
was quantified using ratiometric imaging, essentially as previously described
(Beemiller et al., 2010). In brief, cells were transfected with mCitrine-CBD
(gift from J. Swanson, University of Michigan Medical School, Ann Arbor,
MI) and mCherry in 35-mm glass-bottom dishes (MatTek). Cells were serum
starved for ≥3 h before the addition of phagocytic particles. Imaging com-
menced immediately after the addition of small or large particles. Cells
were imaged every 20 s for 20–30 min. Phase-contrast, YFP, and RFP
images were captured at each time point. Image analysis was performed
essentially as described previously (Henry et al., 2004). Image sequences
were synchronized to the frame at which a bead came in contact with the
cell, and a circular ROI, covering the phagocytic particle, was drawn on
the phase-contrast image. The mean intensity of this ROI was determined
for the YFP and RFP images, and a ratio of YFP to RFP was calculated for
each time point (termed Rp). Next, mean intensities for YFP and RFP were
determined for the whole cell, and a ratio of YFP to RFP was calculated
(Rc). Finally, a recruitment index was calculated for each phagosome at
each time point by dividing Rp by Rc. The number of image sequences
used for quantification is as follows: 1.5 µm mCitrine/mCherry, n = 8; 1.5 µm
mCitrine-CBD/mCherry, n = 13; 4.5 µm mCitrine/mCherry, n = 5; and
4.5 µm mCitrine-CBD/mCherry, n = 5.
Time-lapse microscopy with Lyn-GFP
Cells were grown and transfected with Lyn-GFP (gift from T. Meyer, Stan-
ford University School of Medicine, Stanford, CA) in 35-mm glass-bottom
dishes. Cells were serum starved 3 h before imaging. Imaging was initi-
ated right after phagocytic particles were added to dishes and proceeded
for 20–30 min.
TfR and Rab11 staining
COS1 cells were incubated with coated 1.5- or 4.5-µm beads and fixed
after 30 min. The extracellular portions of the phagocytic particles were
stained using anti-MBP antibodies (New England Biolabs, Inc.). Cells were
then permeabilized and incubated with the anti-TfR antibody (Invitrogen).
Coverslips were washed, and images were captured microscopically. To
visualize Rab11 localization to nascent phagosomes, COS1 cells express-
ing Myc-Rab11 were incubated with 1.5- and 4.5-µm beads and fixed.
Extracellular particles were visualized using anti-MBP antibodies, and anti-
Myc antibodies (Santa Cruz Biotechnology, Inc.) were used to detect Myc-
Rab11. The mean percentage of phagosomes that displayed positive
staining was graphed.
Quantifying transferrin efflux
COS1 cells or MEFs were serum starved in serum-free media (DMEM) and
loaded with 20 µg/ml iron-loaded Alexa Fluor 488–conjugated transferrin
(Invitrogen) for 1 h. Cells were washed (time = 0) and allowed to efflux trans-
ferrin in serum-free media supplemented with 100 µg/ml apotransferrin
JCB • VOLUME 200 • NUMBER 1 • 2013 92
Hynes, R.O. 2002. Integrins: bidirectional, allosteric signaling machines. Cell.
Inoue, M., L. Chang, J. Hwang, S.H. Chiang, and A.R. Saltiel. 2003. The exocyst
complex is required for targeting of Glut4 to the plasma membrane by
insulin. Nature. 422:629–633. http://dx.doi.org/10.1038/nature01533
Kerr, J.F., A.H. Wyllie, and A.R. Currie. 1972. Apoptosis: a basic biological
phenomenon with wide-ranging implications in tissue kinetics. Br. J.
Cancer. 26:239–257. http://dx.doi.org/10.1038/bjc.1972.33
Kim, S., K. Shilagardi, S. Zhang, S.N. Hong, K.L. Sens, J. Bo, G.A. Gonzalez,
and E.H. Chen. 2007. A critical function for the actin cytoskeleton in tar-
geted exocytosis of prefusion vesicles during myoblast fusion. Dev. Cell.
Kroschewski, R., A. Hall, and I. Mellman. 1999. Cdc42 controls secretory and
endocytic transport to the basolateral plasma membrane of MDCK cells.
Nat. Cell Biol. 1:8–13. http://dx.doi.org/10.1038/8977
Krysko, D.V., G. Denecker, N. Festjens, S. Gabriels, E. Parthoens, K. D’Herde,
and P. Vandenabeele. 2006. Macrophages use different internalization
mechanisms to clear apoptotic and necrotic cells. Cell Death Differ.
Leverrier, Y., and A.J. Ridley. 2001. Requirement for Rho GTPases and PI 3-
kinases during apoptotic cell phagocytosis by macrophages. Curr. Biol.
Leverrier, Y., R. Lorenzi, M.P. Blundell, P. Brickell, C. Kinnon, A.J. Ridley, and
A.J. Thrasher. 2001. Cutting edge: the Wiskott-Aldrich syndrome pro-
tein is required for efficient phagocytosis of apoptotic cells. J. Immunol.
Lin, R., S. Bagrodia, R. Cerione, and D. Manor. 1997. A novel Cdc42Hs mu-
tant induces cellular transformation. Curr. Biol. 7:794–797. http://dx.doi
Lipschutz, J.H., and K.E. Mostov. 2002. Exocytosis: the many masters of the
exocyst. Curr. Biol. 12:R212–R214. http://dx.doi.org/10.1016/S0960-
Liu, J., X. Zuo, P. Yue, and W. Guo. 2007. Phosphatidylinositol 4,5-bisphos-
phate mediates the targeting of the exocyst to the plasma membrane for
exocytosis in mammalian cells. Mol. Biol. Cell. 18:4483–4492. http://dx
Liu, J., Y. Zhao, Y. Sun, B. He, C. Yang, T. Svitkina, Y.E. Goldman, and W.
Guo. 2012. Exo70 stimulates the Arp2/3 complex for lamellipodia for-
mation and directional cell migration. Curr. Biol. 22:1510–1515. http://
Matern, H.T., C. Yeaman, W.J. Nelson, and R.H. Scheller. 2001. The Sec6/8 com-
plex in mammalian cells: characterization of mammalian Sec3, subunit in-
teractions, and expression of subunits in polarized cells. Proc. Natl. Acad.
Sci. USA. 98:9648–9653. http://dx.doi.org/10.1073/pnas.171317898
Mohammadi, S., and R.R. Isberg. 2009. Yersinia pseudotuberculosis virulence
determinants invasin, YopE, and YopT modulate RhoG activity and localization.
Infect. Immun. 77:4771–4782. http://dx.doi.org/10.1128/IAI.00850-09
Munson, M., and P. Novick. 2006. The exocyst defrocked, a framework of
rods revealed. Nat. Struct. Mol. Biol. 13:577–581. http://dx.doi.org/10
Nagata, S. 2007. Autoimmune diseases caused by defects in clearing dead cells
and nuclei expelled from erythroid precursors. Immunol. Rev. 220:237–
Nichols, C.D., and J.E. Casanova. 2010. Salmonella-directed recruitment of
new membrane to invasion foci via the host exocyst complex. Curr. Biol.
Niedergang, F., and P. Chavrier. 2005. Regulation of phagocytosis by Rho
GTPases. Curr. Top. Microbiol. Immunol. 291:43–60. http://dx.doi.org/10
Nobes, C.D., and A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assem-
bly of multimolecular focal complexes associated with actin stress fibers,
lamellipodia, and filopodia. Cell. 81:53–62. http://dx.doi.org/10.1016/
Park, H., and D. Cox. 2009. Cdc42 regulates Fc gamma receptor-mediated
phagocytosis through the activation and phosphorylation of Wiskott-
Aldrich syndrome protein (WASP) and neural-WASP. Mol. Biol. Cell.
Partridge, M.A., and E.E. Marcantonio. 2006. Initiation of attachment and gen-
eration of mature focal adhesions by integrin-containing filopodia in cell
spreading. Mol. Biol. Cell. 17:4237–4248. http://dx.doi.org/10.1091/mbc
Patel, J.C., and J.E. Galán. 2006. Differential activation and function of Rho
GTPases during Salmonella–host cell interactions. J. Cell Biol. 175:453–
Price, L.S., J. Leng, M.A. Schwartz, and G.M. Bokoch. 1998. Activation of
Rac and Cdc42 by integrins mediates cell spreading. Mol. Biol. Cell.
of the text. We thank Emmanuelle Caron, Scott Snapper, Joel Swanson,
Tobias Meyer, and Larry Feig for providing reagents.
This work was supported by Howard Hughes Medical Institute, by
award R37AI23538 and training grant 5T32AI007422 from the National
Institute of Allergy and Infectious Diseases, and by Program Project Award
grant P30DK34928 from the National Institute of Diabetes and Digestive
and Kidney Diseases.
Submitted: 17 April 2012
Accepted: 5 December 2012
Aderem, A., and D.M. Underhill. 1999. Mechanisms of phagocytosis in mac-
rophages. Annu. Rev. Immunol. 17:593–623. http://dx.doi.org/10.1146/
Alrutz, M.A., A. Srivastava, K.W. Wong, C. D’Souza-Schorey, M. Tang, L.E.
Ch’Ng, S.B. Snapper, and R.R. Isberg. 2001. Efficient uptake of Yersinia
pseudotuberculosis via integrin receptors involves a Rac1-Arp 2/3 path-
way that bypasses N-WASP function. Mol. Microbiol. 42:689–703.
Bajno, L., X.R. Peng, A.D. Schreiber, H.P. Moore, W.S. Trimble, and S.
Grinstein. 2000. Focal exocytosis of VAMP3-containing vesicles at sites
of phagosome formation. J. Cell Biol. 149:697–706. http://dx.doi.org/
Beemiller, P., Y. Zhang, S. Mohan, E. Levinsohn, I. Gaeta, A.D. Hoppe, and
J.A. Swanson. 2010. A Cdc42 activation cycle coordinated by PI 3-kinase
during Fc receptor-mediated phagocytosis. Mol. Biol. Cell. 21:470–480.
Caron, E., and A. Hall. 1998. Identification of two distinct mechanisms of phago-
cytosis controlled by different Rho GTPases. Science. 282:1717–1721.
Chan, A.Y., S.J. Coniglio, Y.Y. Chuang, D. Michaelson, U.G. Knaus, M.R.
Philips, and M. Symons. 2005. Roles of the Rac1 and Rac3 GTPases in
human tumor cell invasion. Oncogene. 24:7821–7829. http://dx.doi.org/
Clark, E.A., W.G. King, J.S. Brugge, M. Symons, and R.O. Hynes. 1998. Integrin-
mediated signals regulated by members of the rho family of GTPases.
J. Cell Biol. 142:573–586. http://dx.doi.org/10.1083/jcb.142.2.573
Cougoule, C., A. Wiedemann, J. Lim, and E. Caron. 2004. Phagocytosis, an
alternative model system for the study of cell adhesion. Semin. Cell Dev.
Cox, D., C.C. Tseng, G. Bjekic, and S. Greenberg. 1999. A requirement for
phosphatidylinositol 3-kinase in pseudopod extension. J. Biol. Chem.
Cox, D., D.J. Lee, B.M. Dale, J. Calafat, and S. Greenberg. 2000. A Rab11-
containing rapidly recycling compartment in macrophages that promotes
phagocytosis. Proc. Natl. Acad. Sci. USA. 97:680–685. http://dx.doi.org/
Das, A., and W. Guo. 2011. Rabs and the exocyst in ciliogenesis, tubulogenesis
and beyond. Trends Cell Biol. 21:383–386. http://dx.doi.org/10.1016/
Dupuy, A.G., and E. Caron. 2008. Integrin-dependent phagocytosis: spreading
from microadhesion to new concepts. J. Cell Sci. 121:1773–1783. http://
Eden, S., R. Rohatgi, A.V. Podtelejnikov, M. Mann, and M.W. Kirschner. 2002.
Mechanism of regulation of WAVE1-induced actin nucleation by Rac1
and Nck. Nature. 418:790–793. http://dx.doi.org/10.1038/nature00859
Garrett, W.S., L.M. Chen, R. Kroschewski, M. Ebersold, S. Turley, S.
Trombetta, J.E. Galán, and I. Mellman. 2000. Developmental control of
endocytosis in dendritic cells by Cdc42. Cell. 102:325–334. http://dx.doi
Gauthier, N.C., O.M. Rossier, A. Mathur, J.C. Hone, and M.P. Sheetz. 2009.
Plasma membrane area increases with spread area by exocytosis of a
GPI-anchored protein compartment. Mol. Biol. Cell. 20:3261–3272.
Henry, R.M., A.D. Hoppe, N. Joshi, and J.A. Swanson. 2004. The uniformity of
phagosome maturation in macrophages. J. Cell Biol. 164:185–194. http://
Holevinsky, K.O., and D.J. Nelson. 1998. Membrane capacitance changes asso-
ciated with particle uptake during phagocytosis in macrophages. Biophys.
J. 75:2577–2586. http://dx.doi.org/10.1016/S0006-3495(98)77703-3
Huynh, K.K., J.G. Kay, J.L. Stow, and S. Grinstein. 2007. Fusion, fission, and
secretion during phagocytosis. Physiology (Bethesda). 22:366–372.
93 Cdc42 modulates exocytic function during uptake • Mohammadi and Isberg
Wong, K.W., and R.R. Isberg. 2005. Yersinia pseudotuberculosis spatially con-
trols activation and misregulation of host cell Rac1. PLoS Pathog. 1:e16.
Wu, H., C. Turner, J. Gardner, B. Temple, and P. Brennwald. 2010. The Exo70
subunit of the exocyst is an effector for both Cdc42 and Rho3 func-
tion in polarized exocytosis. Mol. Biol. Cell. 21:430–442. http://dx.doi
Zeng, J., M. Ren, D. Gravotta, C. De Lemos-Chiarandini, M. Lui, H. Erdjument-
Bromage, P. Tempst, G. Xu, T.H. Shen, T. Morimoto, et al. 1999.
Identification of a putative effector protein for rab11 that participates in
transferrin recycling. Proc. Natl. Acad. Sci. USA. 96:2840–2845. http://
Zhang, X., E. Bi, P. Novick, L. Du, K.G. Kozminski, J.H. Lipschutz, and W.
Guo. 2001. Cdc42 interacts with the exocyst and regulates polarized se-
cretion. J. Biol. Chem. 276:46745–46750. http://dx.doi.org/10.1074/jbc
Zhang, X.M., S. Ellis, A. Sriratana, C.A. Mitchell, and T. Rowe. 2004. Sec15
is an effector for the Rab11 GTPase in mammalian cells. J. Biol. Chem.
Zuo, X., J. Zhang, Y. Zhang, S.C. Hsu, D. Zhou, and W. Guo. 2006. Exo70
interacts with the Arp2/3 complex and regulates cell migration. Nat. Cell
Biol. 8:1383–1388. http://dx.doi.org/10.1038/ncb1505
Zuo, X., B. Fogelgren, and J.H. Lipschutz. 2011. The small GTPase Cdc42 is
necessary for primary ciliogenesis in renal tubular epithelial cells. J. Biol.
Chem. 286:22469–22477. http://dx.doi.org/10.1074/jbc.M111.238469
Prigent, M., T. Dubois, G. Raposo, V. Derrien, D. Tenza, C. Rossé, J. Camonis,
and P. Chavrier. 2003. ARF6 controls post-endocytic recycling through
its downstream exocyst complex effector. J. Cell Biol. 163:1111–1121.
Rankin, S., R.R. Isberg, and J.M. Leong. 1992. The integrin-binding domain of
invasin is sufficient to allow bacterial entry into mammalian cells. Infect.
Ren, M., G. Xu, J. Zeng, C. De Lemos-Chiarandini, M. Adesnik, and D.D.
Sabatini. 1998. Hydrolysis of GTP on rab11 is required for the direct de-
livery of transferrin from the pericentriolar recycling compartment to the
cell surface but not from sorting endosomes. Proc. Natl. Acad. Sci. USA.
Ren, Y., and J. Savill. 1998. Apoptosis: the importance of being eaten. Cell
Death Differ. 5:563–568. http://dx.doi.org/10.1038/sj.cdd.4400407
Rohatgi, R., P. Nollau, H.Y. Ho, M.W. Kirschner, and B.J. Mayer. 2001. Nck
and phosphatidylinositol 4,5-bisphosphate synergistically activate actin
polymerization through the N-WASP-Arp2/3 pathway. J. Biol. Chem.
Sakurai-Yageta, M., C. Recchi, G. Le Dez, J.B. Sibarita, L. Daviet, J. Camonis,
C. D’Souza-Schorey, and P. Chavrier. 2008. The interaction of IQGAP1
with the exocyst complex is required for tumor cell invasion down-
stream of Cdc42 and RhoA. J. Cell Biol. 181:985–998. http://dx.doi.org/
Savill, J., I. Dransfield, C. Gregory, and C. Haslett. 2002. A blast from the
past: clearance of apoptotic cells regulates immune responses. Nat. Rev.
Immunol. 2:965–975. http://dx.doi.org/10.1038/nri957
Savina, A., and S. Amigorena. 2007. Phagocytosis and antigen presentation in
dendritic cells. Immunol. Rev. 219:143–156. http://dx.doi.org/10.1111/
Shaner, N.C., R.E. Campbell, P.A. Steinbach, B.N. Giepmans, A.E. Palmer, and
R.Y. Tsien. 2004. Improved monomeric red, orange and yellow fluores-
cent proteins derived from Discosoma sp. red fluorescent protein. Nat.
Biotechnol. 22:1567–1572. http://dx.doi.org/10.1038/nbt1037
Stuart, L.M., J. Boulais, G.M. Charriere, E.J. Hennessy, S. Brunet, I. Jutras, G.
Goyette, C. Rondeau, S. Letarte, H. Huang, et al. 2007. A systems biol-
ogy analysis of the Drosophila phagosome. Nature. 445:95–101. http://
Sugihara, K., S. Asano, K. Tanaka, A. Iwamatsu, K. Okawa, and Y. Ohta. 2002.
The exocyst complex binds the small GTPase RalA to mediate filopodia
formation. Nat. Cell Biol. 4:73–78. http://dx.doi.org/10.1038/ncb720
Swanson, J.A. 2008. Shaping cups into phagosomes and macropinosomes. Nat.
Rev. Mol. Cell Biol. 9:639–649. http://dx.doi.org/10.1038/nrm2447
Symons, M., J.M. Derry, B. Karlak, S. Jiang, V. Lemahieu, F. Mccormick, U.
Francke, and A. Abo. 1996. Wiskott-Aldrich syndrome protein, a novel
effector for the GTPase CDC42Hs, is implicated in actin polymerization.
Cell. 84:723–734. http://dx.doi.org/10.1016/S0092-8674(00)81050-8
Takahashi, S., K. Kubo, S. Waguri, A. Yabashi, H.W. Shin, Y. Katoh, and K.
Nakayama. 2012. Rab11 regulates exocytosis of recycling vesicles at the
plasma membrane. J. Cell Sci. 125:4049–4057. http://dx.doi.org/10.1242/jcs
TerBush, D.R., T. Maurice, D. Roth, and P. Novick. 1996. The Exocyst is a mul-
tiprotein complex required for exocytosis in Saccharomyces cerevisiae.
EMBO J. 15:6483–6494.
Teruel, M.N., T.A. Blanpied, K. Shen, G.J. Augustine, and T. Meyer. 1999. A
versatile microporation technique for the transfection of cultured CNS
neurons. J. Neurosci. Methods. 93:37–48. http://dx.doi.org/10.1016/S0165-
Tran Van Nhieu, G., and R.R. Isberg. 1993. Bacterial internalization mediated
by beta 1 chain integrins is determined by ligand affinity and receptor
density. EMBO J. 12:1887–1895.
van Dam, E.M., and P.J. Robinson. 2006. Ral: mediator of membrane traffick-
ing. Int. J. Biochem. Cell Biol. 38:1841–1847. http://dx.doi.org/10.1016/
van Ijzendoorn, S.C. 2006. Recycling endosomes. J. Cell Sci. 119:1679–1681.
Vaux, D.L., and S.J. Korsmeyer. 1999. Cell death in development. Cell. 96:245–
Wang, T., Z. Ming, W. Xiaochun, and W. Hong. 2011. Rab7: role of its protein
interaction cascades in endo-lysosomal traffic. Cell. Signal. 23:516–521.
Wiedemann, A., S. Linder, G. Grassl, M. Albert, I. Autenrieth, and M.
Aepfelbacher. 2001. Yersinia enterocolitica invasin triggers phagocytosis
via beta1 integrins, CDC42Hs and WASp in macrophages. Cell. Microbiol.
Wilkinson, S., H.F. Paterson, and C.J. Marshall. 2005. Cdc42-MRCK and Rho-
ROCK signalling cooperate in myosin phosphorylation and cell invasion.
Nat. Cell Biol. 7:255–261. http://dx.doi.org/10.1038/ncb1230