(Received for publication, June 13, 1997)
From the Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 and the ¶ Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Actin filament organization is essential for
endocytosis in yeast. In contrast, the actin-depolymerizing agent
cytochalasin D has yielded ambiguous results as to a role for actin in
receptor-mediated endocytosis in mammalian cells. We have therefore
re-examined this issue using highly specific reagents known to
sequester actin monomers. Two of these reagents, thymosin 4 and
DNase I, potently inhibited the sequestration of transferrin receptors
into coated pits as measured in a cell-free system using perforated
A431 cells. At low concentrations, thymosin
4 but not DNase I was
stimulatory. Importantly, the effects of both reagents were
specifically neutralized by the addition of actin monomers. A role for
the actin cytoskeleton was also detected in intact cells where
latrunculin A, a drug that sequesters actin monomers, inhibited
receptor-mediated endocytosis. Biochemical and morphological analyses
suggest that these reagents inhibit later events in coated vesicle
budding. These results provide new evidence that the actin cytoskeleton
is required for receptor-mediated endocytosis in mammalian cells.
The plasma membrane is directly linked to and functionally integrated with the underlying actin-based cytoskeleton which forms the cell "cortex." Thus, it might be anticipated that vesicular trafficking at the plasma membrane would require the active rearrangement of cortical actin filaments to remove a barrier to vesicular fusion or budding events. Alternatively, actin and actin-based motor proteins might be required to direct vesicle budding and fusion events through the cell cortex at the plasma membrane. Actin filaments can play both inhibitory and facilatory roles in exocytosis. For example, agonist-stimulated secretion appears to require localized disassembly of F-actin at the cell periphery (1, 2). Furthermore, low concentrations of proteins which sequester actin monomers can trigger regulated secretion in permeabilized pancreatic acinar cells suggesting that actin filaments might act as a clamp preventing fusion of docked vesicles (3). In contrast, higher concentrations of these actin-sequestering proteins inhibit regulated secretion, suggesting that actin filament integrity might also play an as yet undefined, facilatory role in regulated exocytosis (3).
Receptor-mediated endocytosis of the mating pheromone -factor is
potently inhibited in the yeast, S. cerevisiae, expressing mutations in either actin or the actin-binding protein, fimbrin (4).
Yeast carrying mutations in three other genes, END3,
END5/VRP1, and END7/RVS167,
which disrupt actin organization, were also shown to be defective in
endocytosis (5, 6). More recently a role for the type I myosin, myo5,
in receptor-mediated endocytosis in yeast was revealed (7). Together
these studies establish that actin filaments and actin-based motor
proteins play an essential role in endocytosis in yeast.
In contrast, the role of actin in endocytosis in mammalian cells remains poorly understood. Cytochalasin D, a drug that destabilizes actin filaments, inhibits receptor-mediated and fluid-phase endocytosis at the apical surface of polarized Madin-Darby canine kidney cells (8) and Caco 2 cells (9), but has no effect on endocytosis at the basolateral surface. There are conflicting results on the effects of cytochalasin D on receptor-mediated endocytosis of transferrin in nonpolarized cells (10-13). Cytochalasin D caps the growing ends of actin filaments and thus causes the depolymerization of actin filaments that are actively turning over, i.e. predominantly the stress fibers. In contrast, cortical actin filaments are more resistant to disruption by cytochalasin D (15), providing a possible explanation for negative results using this reagent. Other lines of evidence have recently implicated actin filament organization in endocytosis. For example, activation of the Rho family GTPases, which trigger actin filament assembly at the cortex, has been shown to stimulate fluid phase pinocytosis (16, 17) but inhibit clathrin-mediated endocytosis (12). These findings prompted us to re-examine the role of actin microfilaments in receptor-mediated endocytosis using reagents which selectively sequester actin monomers, thereby disrupting actin filaments by shifting the equilibrium to the depolymerized state. Here we report that these actin-binding proteins or drugs inhibit the formation of clathrin-coated vesicles at the plasma membrane. These results provide new evidence that the actin cytoskeleton plays an essential role in receptor-mediated endocytosis in mammalian cells.
A431 cells were cultured in Dulbecco's
modified Eagle's medium containing 10% defined fetal calf serum
(Hyclone) as described previously (28, 29). Following trypsinization,
4 × 106 cells were seeded onto 15-cm culture dishes
20-24 h prior to preparing perforated cells, also as described
previously (29). Biotinylated transferrin
(B-Tfn)1 was prepared (18,
29) using either sulfo-NHS-XX-biotin
(6-((6-(biotinoyl)amino)hexanoyl)amino)hexanoic acid, sulfosuccinimidyl
ester, sodium salt) obtained from Molecular Probes (Eugene, OR) or
sulfo-NHS-SS-biotin (sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate) from Pierce. The former biotinylating reagent was used, in general, for avidin sequestration assays as the longer spacer arm gave lower background signals. The
latter cleavable biotinylating reagent was required for the MesNa
(-mercaptoethanesulfonic acid) assay. Thymosin
4 was expressed in
Escherichia coli and purified to >99% homogeneity (based
on Coomassie Blue-stained gels) as described (19). Monomeric actin was
prepared from rabbit skeletal muscle (3). Latrunculin B was obtained
from Alexis Corp. (San Diego, CA), latrunculin A was from Molecular
Probes, DNase I was from Boehringer Mannheim, and cytochalasin D was
from Sigma. All other materials were reagent grade.
Perforated A431 cells were prepared and assays were
performed exactly as described previously (18, 29). For assays
containing T4 or DNase I, perforated cells were incubated with the
drugs at 4 °C for 10 min before the addition of the cytosol. This
treatment appeared to enhance the effects of DNase I and T
4. To
block the effects of DNase I, actin monomers were added to the
perforated cells at 4 °C for 10 min before the addition of DNase I. T
4 was inactivated in vitro by formation of T
4-actin
complexes as described (3). Briefly, 7 µl of 160 µM
T
4 was incubated with 42 µl of 180 µM G-actin
(T
4:actin molar ratio of 1:5) for >1 h on ice and added to
perforated cells.
Adherent A431 cells were dissociated for 5 min at 37 °C in phosphate-buffered saline containing 5 mM EDTA, washed in serum-free culture medium containing 0.2% bovine serum albumin and 20 mM Hepes, pH 7.2 (SFM), and resuspended at 2 × 107 cells/ml in SFM at 4 °C. Cells were then diluted 10-fold into SFM containing the indicated concentrations of latrunculin A or B (prepared as a 2 mg/ml stock solution and stored at 4 °C) and incubated at 37 °C for 1 h. Upon return to ice, B-Tfn was added (to 2 µg/ml) from a 20× stock. Aliquots (50 µl) were removed and kept on ice to determine total surface bound ligand and 4 °C controls, and the remaining suspension was returned to 37 °C. Aliquots were removed after increasing times to determine the kinetics of endocytosis. B-Tfn internalization was measured using the avidin protocol as described (20). Similar results were obtained if adherent cells were pretreated with latrunculin B before their release from the plate by phosphate-buffered saline/EDTA for endocytosis assays in suspension. Control experiments established that at the concentrations used, the solvent alone had no effect on endocytosis.
Electron MicroscopyAnti-human Tfn-R antibody HTR-D65 (obtained from I. Trowbridge, Salk Institute, La Jolla, CA) was conjugated with 10-nm gold particles (BBI International) as described previously (28). Incubations for morphological studies with intact cells were performed exactly as described for biochemical analysis except that they were scaled up 6-fold. After pelleting, cells were resuspended in 0.2 M cacodylate buffer (pH 7.2) containing 2% glutaraldehyde and processed for conventional epon sectioning as described (28). Samples were viewed on a Jeol 1200 at 60 kV. Quantitation of gold particles was performed at the microscope by random examination of cell profiles at a magnification of 20,000.
Thymosins are abundant and highly specific actin
monomer-binding proteins ubiquitously expressed in vertebrate cells
(21). T4 forms a 1:1 complex with
-actin and sequesters actin
monomers (22). As a result, actin monomers are not available for
polymerization, and actin filaments are depolymerized. Although T
4
can directly interact with and depolymerize F-actin at high
concentrations, at lower concentrations (<20 µM) it can
neither sever nor cap actin filaments (23). Recent studies on the
effects of T
4 on secretion in permeabilized acinar cells established
the specificity of this reagent for disruption of actin filaments (3).
Since T
4 disrupts actin filaments by a mechanism completely distinct from that of cytochalasin D, we tested its effects on receptor-mediated endocytosis in perforated A431 cells. Receptor-mediated endocytosis in
this cell-free assay is dependent on cytosol and an ATP-regenerating system and is detected by the sequestration (either in constricted coated pits or sealed coated vesicles) of receptor-bound biotinylated ligands from exogenously added avidin (24, 29).
Titration of T4 into perforated A431 cells showed that it had a
biphasic effect on receptor-mediated endocytosis. T
4 concentrations up to 0.5 µM stimulated ligand sequestration by ~50%
(Fig. 1, A and
inset), whereas higher concentrations led to a complete inhibition of endocytosis (half-maximum inhibition at ~10
µM). Similar biphasic effects of T
4 were observed on
secretion in permeabilized acinar cells (3). Importantly, the
specificity of these effects could be verified by first inactivating
T
4 in vitro with excess actin monomers before its
addition to perforated cells. Under these conditions, both the
activation of sequestration seen at low concentrations of T
4 and the
inhibition seen at higher concentrations were abrograted (Fig.
1B). Exogenous actin on its own had no effect on
receptor-mediated endocytosis. Together these results suggested
involvement of the actin cytoskeleton in receptor-mediated endocytosis.
Effect of DNase I on Receptor-mediated Endocytosis
DNase I is
a structurally distinct actin-sequestering protein with an
exceptionally high affinity (Kd ~ 1 nM) for monomeric actin. DNase I is characterized by its
unique ability to increase the depolymerization rate constant of actin
at the pointed filament end without severing the actin filaments (25). As a result, the depolymerization of filaments capped at their barbed
ends is accelerated, and actin monomers are sequestered. We therefore
examined the effects of DNase I on receptor-mediated endocytosis in
perforated cells and found that it also potently inhibited the
sequestration of transferrin into coated pits (Fig. 1C,
open circles). Inhibition by DNase I was
concentration-dependent with half-maximal inhibition
occurring at <5 µM. In contrast to T4, no stimulation
of endocytosis could be observed at low concentrations of DNase I. However, this could be explained by the difference in affinity for
actin seen between DNase I and T
4 (~1 nM
versus ~1 µM, respectively) and the
differences in their effects on actin filament depolymerization. Again,
the specificity of DNase I action on the actin network was confirmed
since its inhibitory effects on endocytosis were neutralized in the
presence of actin monomers (Fig. 1C, closed
circles). Together with the effects seen with T
4, these results
suggest that actin filaments are required for receptor-mediated
endocytosis in perforated mammalian cells.
The perforated
A431 cell system has been extensively characterized both biochemically
and morphologically and appears to faithfully reconstitute many of the
biochemically distinct events required for the formation of endocytic
clathrin-coated vesicles (12, 18, 20, 24, 28). Nonetheless, it remained
possible that the requirement for actin assembly seen in perforated
cells might reflect an artificial effect due to disorganization of the
actin cytoskeleton as a result of the mechanical disruption of the
plasma membrane. Therefore, we examined the effect of latrunculin A on receptor-mediated endocytosis in intact A431 cells. Latrunculins are a
new class of membrane-permeable, actin-disrupting agents which show
powerful and specific effects on the actin-based cytoskeleton of
nonmuscle cells (26). In vitro, latrunculin affects the
kinetics of polymerization of actin by forming a nonpolymerizable 1:1
molar complex with G-actin (27). Thus, like T4 and DNase I but
unlike the cytochalasins, latrunculins destabilize actin filaments by sequestering actin monomers and shifting the equilibrium to the disassembled state.
To determine whether actin filaments are required for receptor-mediated
endocytosis in intact cells, A431 cells were exposed to increasing
concentrations of latrunculin A for 1 h. This treatment induced
dramatic changes in the morphology of adherent A431 cells which became
round and contracted. As described previously (28) these morphological
effects were similar to those seen when cells were incubated with 10 µg/ml cytochalasin D (data not shown). The morphology of A431 cells
in suspension was not dramatically altered by latrunculin A treatment.
The data in Fig. 2A show that latrunculin A significantly inhibited the rate of receptor-mediated endocytosis in intact cells in a concentration-dependent
manner. The extent of inhibition (~50%) was consistent with those
reported for the effects of cytochalasin D treatment in Hep2 cells
(14). Half-maximal inhibition was obtained at ~4 µg/ml latrunculin
A. While these concentrations are somewhat higher than those needed to
destabilize stress fibers, they are not inconsistent with
destabilization of more resistant elements of the cortical actin
network. Latrunculin B had similar effects although at slightly higher
concentrations (not shown; but see below), consistent with this
analogue being less potent. As previously shown (11, 12), cytochalasin
D (10-50 µg/ml) had no effect on endocytosis in A431 cells.
TfnR undergo constitutive endocytosis and recycling. As a result, the number of surface TfnR reflects the relative rates of internalization and recycling. As can be seen in Fig. 2B, cells incubated with latrunculin A show a concentration-dependent increase in surface TfnR. Thus, the effects of latrunculin A on intact cells are consistent with an inhibition in endocytosis without a concomitant effect on TfnR recycling. A similar finding was reported following cytochalasin D treatment in Hep2 cells (14). Together, these results confirm a requirement for actin in receptor-mediated endocytosis both in intact and perforated mammalian cells.
Actin Filaments Are Not Required for Clustering of TfnR into Coated PitsEfficient receptor-mediated endocytosis requires both the
concentration of receptor-bound ligands into coated pits and the subsequent budding of coated vesicles. Thus, it remained possible that
coated vesicle formation continued in the presence of these actin-monomer sequestering agents, but TfnR clustering in coated pits
was impaired. To test this we used the gold-conjugated anti-TfnR monoclonal antibody D65 and conventional thin section electron microscopy to examine the distribution of TfnR relative to coated pits
in A431 cells treated with or without 25 µM latrunculin
B. This treatment resulted in a 40% inhibition of TfnR endocytosis measured in parallel using biochemical assays. The micrographs in Fig.
3 show that neither the morphology of
coated pits nor their ability to cluster TfnR was affected.
Quantitation of these results showed that 37% of surface D65-gold was
found associated with coated pits in both control (100 of 270 gold
particles counted) and latrunculin B-treated (114 of 308 gold particles
counted) samples.
Actin Filaments Are Required for Clathrin-coated Vesicle Budding
Confirmation that actin filaments were required for
coated vesicle formation was obtained using the perforated cell assay system to selectively measure the budding of preformed coated pits.
Previous characterization of this system has established that
detachment of preformed coated pits can be measured selectively using
the small membrane-impermeant reducing agent MesNa as a probe for the
internalization of biotinylated ligands into sealed coated vesicles
(24, 28). Using this assay the formation of constricted coated pits and
coated vesicle budding are detected when receptor-bound biotinylated
ligands are internalized into sealed vesicles that are inaccessible to
MesNa. Using the MesNa assay, we again found a biphasic response to
thymosin 4. The data in Fig. 4 show
that ligand internalization, like sequestration, was stimulated at low
concentrations and inhibited at higher concentrations of T
4. The
inhibition seen at high concentrations of T
4 is consistent with the
results of ultrastructural analysis and suggest that actin filaments
are required for late events (either for coated pit constriction,
coated vesicle detachment or both) in endocytic coated vesicle
formation. The stimulation of coated vesicle budding seen at low
concentrations of T
4 could reflect destabilization of actin
filaments that otherwise act as a barrier to vesicle budding and
detachment as observed for exocytosis (3).
In summary, the use of highly specific actin modulatory proteins that sequester actin monomers has revealed a requirement for actin filaments in receptor-mediated endocytosis in mammalian cells. These results resolve apparent discrepancies and suggest a similarity in the mechanisms of receptor-mediated endocytosis in yeast and mammalian cells. It will be important to determine whether the actin requirement in mammalian cells reflects a direct involvement of actin filaments in coated vesicle budding or instead reflects a more general requirement for the structural integrity of the cell cortex in plasma membrane function. Additional evidence for the involvement of other actin binding proteins or actin-based type I myosins, as appears to be the case for endocytosis in yeast (4, 7), may help to distinguish these two possibilities.
We thank Sergei Bannykh for preparing the Epon sections and Mike McCaffery for help with the electron microscopy, which was performed in the EM Core Facility supported by NCI, National Institutes of Health Grant CA 58689.