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Three cell-permeant compounds, cytochalasin D, latrunculin A and jasplakinolide, which perturb intracellular actin dynamics by distinct mechanisms, were used to probe the role of filamentous actin and actin assembly in clathrin-mediated endocytosis in mammalian cells. These compounds had variable effects on receptor-mediated endocytosis of transferrin that depended on both the cell line and the experimental protocol employed. Endocytosis in A431 cells assayed in suspension was inhibited by latrunculin A and jasplakinolide, but resistant to cytochalasin D, whereas neither compound inhibited endocytosis in adherent A431 cells. In contrast, endocytosis in adherent CHO cells was more sensitive to disruption of the actin cytoskeleton than endocytosis in CHO cells grown or assayed in suspension. Endocytosis in other cell types, including nonadherent K562 human erythroleukemic cells or adherent Cos-7 cells was unaffected by disruption of the actin cytoskeleton. While it remains possible that actin filaments can play an accessory role in receptor-mediated endocytosis, these discordant results indicate that actin assembly does not play an obligatory role in endocytic coated vesicle formation in cultured mammalian cells.
Receptor-mediated endocytosis in mammalian cells requires clathrin, adaptors and dynamin[1–3]. In contrast, endocytic vesicle formation in the yeast, Saccharomyces cerevisae, is only partially inhibited by mutations in clathrin[4,5] and occurs independently of the yeast adaptor- or dynamin-related proteins. Instead, both receptor-mediated and fluid-phase endocytosis in yeast appear to be functionally coupled to organization and assembly of the actin cytoskeleton[8–10]. Mutations in yeast actin and in several actin binding proteins inhibit endocytosis[11–13]. Correspondingly, many of the so-called ‘end’ mutations or ‘dim’ mutations[15,16], isolated based on their effects on endocytosis, also disrupt the organization of the yeast cortical actin cytoskeleton. Thus, it remains possible that distinct mechanisms mediate endocytosis in yeast as compared to mammalian cells.
Nonetheless, a functional link between clathrin-mediated endocytosis and the actin cytoskeleton in mammalian cells is being forged by recent findings that several components of the mammalian endocytic machinery interact, either directly or indirectly, with the actin cytoskeleton. Overexpression of a dominant-negative mutant of dynamin-1 in HeLa cells leads to redistribution of actin stress fibers to the cell cortex. These effects may be mediated by dynamin binding partners, because the neuronal isoform of dynamin interacts both in vivo and in vitro with profilin and with syndapin, an SH3 domain containing protein that binds to the actin regulatory protein, N-WASp (neuronal Wiskott-Aldrich syndrome protein). Two other proteins involved in clathrin-mediated endocytosis in mammalian cells, eps15 and amphiphysin, have been reported to also interact with the actin cytoskeleton[21,22]. Endocytosis is inhibited by constitutively active mutants of Rho and Rac, although these effects appeared to be independent of actin assembly.
Whether either filamentous actin or actin assembly play a direct role in clathrin-mediated endocytosis in mammalian cells has yet to be established. In contrast to the well-defined and rapid onset temperature-sensitive genetic lesions used to study actin and endocytosis in yeast, studies in mammalian cells have relied on three cell-permeant toxins which perturb actin assembly and disassembly by distinct mechanisms, namely: 1) cytochalasin D (cytoD) which caps actin filaments preventing assembly and, owing to the dynamic nature of actin filaments, ultimately leads to actin filament disassembly; 2) latrunculin A (latA) which also causes actin filament disassembly, in this case by sequestering actin monomers; and 3) jasplakinolide (jas) which, like phalloidin, binds to and stabilizes actin filaments promoting their assembly. These toxins appear to have variable effects on clathrin-mediated endocytosis in mammalian cells. Several studies have reported that early endocytic events are at least partially inhibited by cytochalasins[27–29]; however, others have reported no effect on receptor-mediated endocytosis[23,30–32]. CytoD treatment selectively inhibited fluid-phase uptake at the apical surface of polarized MDCK and Caco-2 cells without affecting endocytosis at the basolateral surface. In contrast, treatment with jas stimulated the uptake and accumulation of fluid-phase endocytic tracers at the basolateral surface of MDCK cells without affecting endocytosis at the apical surface. Receptor-mediated endocytosis of transferrin (Tfn) was also unaffected. Finally, although both cytoD and latA cause actin filament disassembly, only latA inhibited endocytosis in human adenocarcinoma, A431 cells.
A role for actin in endocytosis was also demonstrated in vitro using a perforated cell assay. Several proteins that sequester actin monomers, including DNAse I, gelsolin fragments and, in particular, β-thymosins, were shown to inhibit endocytic vesicle formation as measured by the receptor-mediated endocytosis of Tfn. In contrast, neither cytoD nor phalloidin (which stabilizes actin filaments) had any effect. While these studies suggested that active actin assembly might be required for endocytosis in mammalian cells, the possibility remained that actin plays a more passive role, for example, in simply maintaining the structural integrity of the plasma membrane.
Given these discrepancies, we have reinvestigated the role of actin in endocytic coated vesicle formation in mammalian cells using several methods to perturb actin dynamics. The results reported here suggest that actin assembly is not directly required for endocytic coated vesicle formation. Instead, they suggest that actin assembly and the organization of a cortical actin network can play, at best, a modulatory role that is more pronounced under certain conditions of growth and membrane differentiation.
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We used a well-established perforated cell assay for receptor-mediated endocytosis of Tfn[2,36] to test the hypothesis that de novo assembly of actin filaments might play an active role in endocytic vesicle formation. To this end, we assessed the ability of actin-depleted cytosol to support endocytic coated vesicle formation. As assessed by western blotting,> 95% of actin was depleted from bovine brain cytosol by passage over DNase I-Sepharose. Unexpectedly, the actin-depleted cytosol supported the sequestration of biotinylated-Tfn from exogenously added avidin indistinguishably from untreated cytosol (data not shown). This was true even when assays were performed in the presence of 1 μM phalloidin to stabilize any actin filaments associated with the perforated cell preparations, as these might have provided an alternate source of monomeric actin.
This inability to detect a direct requirement for actin assembly in endocytic coated vesicle formation in perforated cells, coupled to inconsistencies in the reported effects of actin disrupting drugs on endocytosis in intact cells led us to re-examine the requirements for actin filament assembly in endocytosis in vivo using cytochalasin D and latrunculin A. The effects of preincubating A431 cells for 30 min at 37°C with either buffer alone (dashed lines), 5 μM cytoD (open circles) or 10 μM latA (open squares) on internalization of Tfn are shown inFig. 1. Preincubation at these concentrations of cytoD and latA resulted in complete disruption of the actin cytoskeleton, as assessed by Alexa488-phalloidin staining or indirect immunofluorescence using anti-actin antibodies (Fig. 2). Endocytosis assays were performed in three ways: 1) cells were gently removed from tissue culture dishes with PBS/EDTA and both preincubation and internalization assays were performed in suspension (panel a); 2) adherent cells were preincubated with toxins and then released by PBS/EDTA treatment on ice for internalization assays in suspension in the continuous presence of the drugs (panel b); and 3) both preincubation and internalization assays were performed on adherent cells grown on 35 mm tissue culture dishes (panel c). Importantly, the rates (∼20%/min) and extents (∼150% at steady-state) of Tfn internalization in untreated cells were indistinguishable, regardless of the protocol used (compare dashed lines in panels a,b and c). (The fact that the extent of internalization of Tfn exceeded the amount bound on the surface at 4°C reflected the recycling and reinternalization of the substantial intracellular pool of Tfn receptors.) In contrast, the effects of actin filament disruption on endocytosis varied depending on the experimental conditions. Consistent with our previous findings obtained with A431 cells pretreated and assayed in suspension, latA inhibited the rate of endocytosis by ∼40%, whereas cytoD had little or no effect (Fig. 1a). The opposite result was obtained when A431 cells were preincubated on plates and then assayed in suspension (Fig. 1b). Under these conditions cytoD inhibited endocytosis by 60–70%, while latA had little effect. Finally, neither actin filament disrupting drug inhibited endocytosis when A431 cells were pretreated and assayed while remaining adherent (Fig. 1c), even though actin filaments were dramatically disrupted by both toxins under these conditions (Fig. 2, panels b and c).
Figure 1. Effects of cytochalasin D and latrunculin A on receptor-mediated endocytosis of transferrin in A431 cells. A431 cells were preincubated in SFM alone (●), containing 5 μM cytoD (○) or 10 μM latA (□) either in suspension (panel a) or on plates (panels b and c) for 30 min at 37°C. Cells were returned to ice for addition of B-XX-Tfn and endocytosis assays were performed as described in Materials and Methods in cells either in suspension (panels a and b) or on plates (panel c). Results presented are the averages (±SD shown for control cells only) of four independent experiments.
Figure 2. Immunofluorescence labeling of actin filaments in A431 cells and CHO cells treated with actin disrupting toxins. A431 cells were preincubated without (panel a) or with 5 μM cytoD (Panel b), 10 μM latA (Panel c) or 1 μM jas (panel d) for 30–45 min prior to fixation and labeling with Alexa488-phalloidin (panels a–c) or with anti-actin mAb (panel d) as described in Materials and Methods. The cell boundaries in panel c were determined by phase contrast microscopy and have been traced.
Similar inconsistencies in the effects of actin disrupting drugs on endocytosis were observed in TRVb-1 CHO cells which express the human transferrin receptor (Fig. 3), although with these cells cytoD and latA had similar effects. When CHO cells were preincubated either in suspension or on plates with cytoD and latA and then assayed in suspension (panels a and b), both the rate and extent of Tfn endocytosis were partially inhibited. In contrast to results obtained with A431 cells, adherent CHO cells treated with latA or cytoD and assayed on plates showed significant (>70%) inhibition in both the rate and extent of Tfn endocytosis. The structural changes in the actin cytoskeleton induced in CHO cells treated with these toxins (Fig. 6, panels b and c) were indistinguishable from those seen in similarly treated A431 cells (Fig. 2b,c). As for A431 cells, the efficiency of endocytosis in untreated CHO cells was not significantly different when assayed in suspension or on plates (compare dashed lines inFig. 3b,c), although a reduction in endocytosis rates was observed during more prolonged incubations in suspension (compareFig. 3a with b and c, see alsoFig. 4a). For both A431 cells and CHO cells, decreases in the extent of Tfn accumulation at steady-state reflect alterations in the relative cell surface versus intracellular pool sizes of TfnR after treatment with the toxins. Inhibition of endocytosis invariably led to increases in surface bound TfnR (not shown), presumably at the expense of intracellular receptors. A decrease in the extent of Tfn accumulation at steady-state might also reflect effects of the actin cytoskeleton on later stages of trafficking through endosomal compartments, as previously reported.
Figure 3. Effects of cytochalasin D and latrunculin A on receptor-mediated endocytosis of transferrin in TRVb-1 CHO cells. TRVb-1 CHO cells were preincubated in SFM alone (●), containing 5 μM cytoD (○) or 10 μM latA (□) either in suspension (panel A) or on plates (panels b and c) for 30 min at 37°C. Cells were returned to ice for addition of B-XX-Tfn and endocytosis assays were performed as described in Materials and Methods in cells either in suspension (panels a and b) or on plates (panel c). Results presented are the averages (±SD shown for control cells only) of three independent experiments.
Figure 6. AP2 distribution in TRVb-1 CHO cells treated with actin disrupting toxins. TRVb-1 CHO cells cultured on coverslips were incubated for 30–45 min at 37°C in SFM alone (panel a) or containing 5 μM cytoD (panel b), 10 μM latA (panel c) or 1 μM jas (panel d). They were fixed and processed for immunofluorescence as described in Materials and Methods. Panels a–d show staining with ALEXA488-phalloidin, panels a′–d′ show indirect immunofluorescence using AP.6 anti-α-adaptin monoclonal antibody and Texas Red-conjugated secondary antibody.
Figure 4. Effect of cytochalasin D, latrunculin A and jasplakinolide on receptor-mediated endocytosis in cells grown in suspension. TRVb-1 CHO cells (panel a) or K562 human erythroleukemic cells (panel b), cultured in suspension, were preincubated at 37°C in SFM alone (●), containing 5 μM cytoD (○), 10 μM latA (□) or 1 μm jas (▵) for 30–45 min. B-XX-Tfn was added on ice and the cells incubated for the indicated times at 37°C to allow for endocytosis. B-XX-Tfn internalization was measured as described in Materials and Methods.
These data suggest that the sensitivity of endocytosis to actin disruption is affected by the adherence properties of each cell type. Because CHO cells can be cultured both on plates and in suspension, we could examine the effects of actin disruption on cells cultured in suspension as compared to those grown in adherent cultures. Although TRVb-1 CHO cells cultured in suspension internalized Tfn less efficiently than when grown on plates, receptor-mediated endocytosis in suspension-grown CHO cells was completely unaffected by either latA or cytoD (Fig. 4a). Similarly, the efficient endocytosis of Tfn in human K562 erythroleukemic cells, which grow only in suspension, was unaffected by preincubation with either latA or cytoD (Fig. 4b).
Jasplakinolide (jas) is a recently discovered membrane permeant compound that binds to and stabilizes actin filaments much like phalloidin[26,38]. Interestingly, cytoD and jas have opposing effects on fluid-phase endocytosis from distinct plasma membrane domains in polarized MDCK cells: cytoD inhibits endocytosis selectively at the apical surface, whereas jas enhances endocytosis selectively at the basolateral surface. We therefore tested the effects of jas on endocytosis, assayed both in suspension and on plates, in several cell lines. Incubation of cells with 1 μM jas for 45 min at 37°C resulted in dramatic changes in the morphology of actin filaments and thick, short actin bundles were detected by indirect immunofluorescence using anti-actin antibodies (Fig. 2, panel d, not shown for CHO cells). No detectable labeling was observed using Alexa-phalloidin, indicating that the phalloidin binding sites were now occupied with jas (Fig. 6d, not shown for A431 cells).Fig. 5 shows that as for latA and cytoD, treatment with jas gave variable effects on endocytosis depending on the cell line and assay conditions. Endocytosis in A431 cells was sensitive to jas only when assayed in suspension (compareFig. 5 a with b). A complementary pattern of inhibition and resistance was seen in 3T3-L1 fibroblasts (Fig. 5c,d). Consistent with results obtained using actin disrupting drugs (Fig. 3) endocytosis in CHO cells cultured on plates was sensitive to jas under either assay condition (Fig. 5e,f). In contrast, endocytosis in the Cos-7 fibroblast cell lines was not significantly affected by jas under either condition (Fig. 5g,h), even when treated with 4 μM jas (not shown). Endocytosis in K562 and CHO cells grown in suspension was also unaffected by 1 μM jas (Fig. 4, triangles).
Figure 5. Cell type dependent effects of jasplakinolide on receptor-mediated endocytosis. The indicated cells were preincubated for 45 min with (○) or without (●) 1 μM jas and assayed either in suspension (panels a,c,e and g) on or on plates (panels b,d,f and h) as described in Materials and Methods.
Together these data suggest that neither filamentous actin nor actin assembly are directly required for clathrin-mediated endocytosis in mammalian cells. In yeast, cortical actin patches were found associated with invaginations of the plasma membrane and it has been suggested that these might correspond to sites of endocytosis. In mammalian cells, disruption of the actin cytoskeleton by latA leads to partial dispersal of highly localized endocytic ‘hot-spots’ . We therefore examined the effect of actin disruption on the distribution of the plasma membrane-specific adaptor protein, AP2, in adherent TRVb-1 CHO cells in which receptor-mediated endocytosis was most strongly affected (Fig. 6). In control cells, AP2 is distributed in small puncta at the plasma membrane and a diffuse cytosolic pool is also detectable (Fig. 6a). Cells treated with either cytoD (Fig. 6b), latA (Fig. 6c) or jas (Fig. 6d) appear to retract towards their nucleii leaving thin membrane sheets adherent to the glass surface. The AP2-staining puncta on these flattened membranes appeared larger than in control cells. Whether these slight changes in AP2 staining patterns relate to the decreased rates of endocytosis is unknown.
Using quick freeze deep-etch methodology, we examined the ultra-structural relationship between cortical actin filaments and clathrin-coated pits. We focused our attention on adherent A431 cells (Fig. 7), Cos cells and CHO cells (Fig. 8a and b, respectively) as receptor-mediated endocytosis in these cell types was differentially sensitive to disruption of the actin cytoskeleton. Endocytosis in adherent CHO cells was more sensitive to disruption of actin cytoskeleton than that in A431 cells or Cos cells (compareFig. 1c withFig. 3c orFig. 5 panels a, f and h). The images inFigs 7 and 8 are displayed as three dimensional anaglyphs that should be viewed with red/green or red/blue glasses. Several coated pits at various stages of invagination can be seen in A431 cells (Fig. 7). While actin filaments are sometimes seen around the base of coated pits (top panel), several others appeared to bud from areas devoid of actin filaments (bottom panel). In contrast, caveolae, which appear as smaller diameter vesicles covered by a ‘whorling’ coat structure and whose uptake has been shown to be actin dependent[41,42], appear to line up along actin filaments. Cos-7 cells have an elaborate cortical actin cytoskeleton (Fig. 8, top) and coated pits can be seen to bud from areas devoid of actin. At higher magnification in CHO cells (Fig. 8, bottom), actin filaments can be seen to run adjacent to the coated pit on the left and above the coated pit on the right.
Figure 7. Three-dimensional anaglyph images of the inner plasma membrane surface of A431 cells viewed by quick-freeze deep-etch microscopy. The images show coated pits at different stages of invagination, without any consistent structural interaction with actin filaments. This is especially visible in the lower panel, where clathrin pits appear to be forming at areas devoid of actin while caveolae, which are recognizable by their small uniform diameters and the ‘whorl’ appearance of their coats, can be seen to line up along a feather-like array of short F-actin filaments. ×200 000
Figure 8. Three-dimensional anaglyph images of endocytic coated pits in Cos cells and CHO cells. Top panel: The highly developed cortical actin network on the under surface of the Cos cell plasma membrane is visible. Coated pits appear to bud from areas devoid of actin. ×120 000. Bottom panel: Higher magnification image of coated pits in CHO cells shows that actin filaments are often seen close to coated pits. The actin filament appears to pass over the clathrin lattice on the right, but adjacent to the lattice on the left. ×250 000
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Much remains to be understood regarding the mechanisms of clathrin-mediated endocytosis. While the roles played by adaptors, clathrin and dynamin are being elucidated, the function of other proteins implicated in endocytosis, including amphiphysin, eps15, endophilin, etc. remain largely obscure. Several workers have shown that clathrin-mediated endocytosis is inhibited upon disruption of the actin cytoskeleton[27,28,31,33,43], suggesting a role for actin in endocytosis. However, given previous conflicting results[23,29,30,33,34], we sought to determine whether filamentous actin or actin assembly play direct roles in endocytic vesicle formation. Assuming that the fundamental machinery driving coat assembly, receptor recruitment and vesicle detachment at the plasma membrane is the same in all mammalian cells, then our results suggest that neither filamentous actin nor actin assembly/disassembly are obligatorily required for clathrin coated vesicle formation. Consistent with this, electron microscopic analysis failed to detect a specific structural relationship between actin filaments and endocytic coated pits at any stage of vesicle formation. Instead, these data would suggest that actin filaments have, at most, an accessory function in endocytic vesicle formation required only under select conditions.
A more direct functional link between endocytosis and actin assembly and organization has been suggested to occur in S. cerevisiae . However, even in yeast, the correlation between actin disruption and inhibition of endocytosis is not absolute. For example, there exist mutant alleles of myo5 (affecting a type I unconventional myosin)[44,45], pfy1 (affecting profilin) and tpm1 (affecting tropomyosin I) that disrupt actin organization in yeast but do not inhibit endocytosis (AL Munn, E Kubler and H Reizman, unpublished results, but see[8,14]). Recent genetic analysis of the end5-1 allele has yielded similar results (SN Naqvi and AL Munn, personal communication). The end5-1 mutation in yeast disrupts both actin organization and endocytosis. Cloning and further characterization revealed end5p to be a proline-rich protein weakly homologous to WIP, a human WASp-interacting protein and two hybrid analysis confirmed interactions between end5p and las17p, the yeast homologue of WASp, which is also necessary for endocytosis. Importantly, some mutations that suppress the temperature-sensitive growth defect associated with end5-1 have been identified which selectively suppress either the actin organization defect or the endocytic defect of end5-1 (SN Naqvi and AL Munn, personal communication). These results provide further evidence that actin assembly and cortical actin organization might not be obligatorily coupled to all forms of endocytosis, even in yeast.
If not directly required for endocytic clathrin coated vesicle formation, what accessory role(s) might actin be playing? Actin filaments might be required when endocytosis must be highly localized at specific sites; for example at the base of microvilli in polarized epithelial cells[33,49,50], at endocytic hot-spots adjacent of active sites in the synapse[51,52], or after patching and capping of receptors on lymphocytes. Alternatively, actin might be differentially required when endocytosis occurs from the adherent versus nonadherent surfaces of cells, depending on the complexity of the cortical actin cytoskeleton underlying the plasma membrane. Actin might have a more dominant role in endocytosis from membranes that are made more rigid by contacts with the extracellular matrix or from membranes that have more elaborate cortical networks than those found in the cultured cells studied here. Actin and perhaps actin based motors might also be required to facilitate movement of detached vesicles through the cell cortex, especially where there is a well-developed structure such as at the terminal web or within microvilli in polarized epithelial cells. Indeed, actin tails were seen to assemble onto newly formed pinocytic vesicles in cultured mast cells. This role might be analogous to the facilatory role of microtubules and microtubule-based motors, whose requirements for vesicular trafficking are most apparent over longer distances. Importantly, our biochemical measurements assess the formation of sealed endocytic vesicles: subsequent events such as the movement of newly formed endocytic vesicles through the cell cortex or trafficking through the endosomal compartment are not assessed. It is likely, based on results of others, that actin plays a role in these later events associated with endocytosis and endosomal trafficking[28,55].
Our results provide insight into the discrepancies in the published literature regarding the effects of actin disruption on clathrin-mediated endocytosis in that endocytosis in different cell types or the same cell type assayed under different conditions, was found to be differentially sensitive to actin disrupting toxins. Moreover, they suggest that perturbations of the actin cytoskeleton can impact the rates and extents of clathrin-mediated endocytosis under conditions related to the adherent properties of a given cell line. Based on these variable effects of actin disruption on endocytosis, we conclude that neither actin assembly nor actin filament organization play an obligatory role in endocytic coated vesicle formation in mammalian cells.