Rab35 Controls Actin Bundling by Recruiting Fascin as an Effector Protein

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Science  04 Sep 2009:
Vol. 325, Issue 5945, pp. 1250-1254
DOI: 10.1126/science.1174921


Actin filaments are key components of the eukaryotic cytoskeleton that provide mechanical structure and generate forces during cell shape changes, growth, and migration. Actin filaments are dynamically assembled into higher-order structures at specified locations to regulate diverse functions. The Rab family of small guanosine triphosphatases is evolutionarily conserved and mediates intracellular vesicle trafficking. We found that Rab35 regulates the assembly of actin filaments during bristle development in Drosophila and filopodia formation in cultured cells. These effects were mediated by the actin-bundling protein fascin, which directly associated with active Rab35. Targeting Rab35 to the outer mitochondrial membrane triggered actin recruitment, demonstrating a role for an intracellular trafficking protein in localized actin assembly.

A dynamic actin network is required for normal cell morphology, cell locomotion, and cytokinesis (1, 2). These processes require polymerization of globular actin monomers into filaments and bundling of the filaments under the control of actin-binding proteins (ABPs). Certain ABPs cross-link filamentous actin (F-actin) into ordered parallel bundles that maintain the structural integrity of the cell and are structural components of filopodia, stereocilia, and microvilli (24). It is unclear how cells initiate the dynamic assembly of actin at the right times and places during development, physiological stresses, injury, and disease.

The importance of F-actin bundling during development is readily apparent during bristle formation in Drosophila. Bristles are mechanosensory organs found in genetically controlled locations on the cuticle. Their shapes and growth are dependent on actin bundles (57). The largest bristles, macrochaetae, are formed by a “shaft” cell that extrudes a cytoplasmic extension. This extension contains evenly distributed microtubules and F-actin bundles located just beneath the plasma membrane (Fig. 1, F and G). Bristle morphologies reflect the organization of actin bundles and can be used to study the regulation of actin in vivo.

Fig. 1

Reducing Rab35 function causes morphological defects in bristle development. (A to E) Rab35DN expressed in the peripheral nervous system induced sharp bends, kinks, and forked ends [arrows in (B), (D), and (E)] in bristles compared to control flies expressing Rab35WT [arrows in (A) and (C)]. Scale bars, 0.2 mm (A), 10 μm [(C) and (E)]. (F) Developing bristle, transverse section. Shaft cells (red) assemble and extend 7 to 11 plasma membrane–associated actin bundles. Microtubules are evenly distributed across the center of the developing shaft. (G and H) Phalloidin staining of packed actin bundles in developing bristles of Rab35WT-producing (G) and Rab35DN-producing (H) flies. Rab35DN caused wavy, loose, thin actin structures (H) relative to controls (G) at 45 to 47 hours after puparium formation. Texas Red–phalloidin staining detected the packed actin bundles. Scale bar, 10 μm. (I to K) Relative to YFP-alone controls (I), YFP-Rab35WT (J) but not YFP-Rab35DN (K) induced filopodia-like membrane protrusions when expressed in S2 cells. Green, YFP proteins; red, phalloidin (filamentous actin). Scale bar, 5 μm. (L to N) Latrunculin A administration completely suppressed the morphological changes caused by Rab35 (M) relative to vehicle control-treated cells (L). Treatment with nocodazole did not block the morphological changes (N). Scale bar, 5 μm. (O to Q) Flies expressing Rab35RNAi in the peripheral nervous system had abnormal bristle morphology (P) relative to controls (O). A mouse Rab35WT transgene reversed this phenotype (Q). Scale bar, 0.2 mm.

Rab proteins constitute the largest subset of Ras-family small guanosine triphosphatases (GTPases). Rab proteins control formation, motility, and docking of vesicles in specific trafficking pathways (8, 9) by recruiting specific effector proteins to different membrane compartments. Rab proteins are evolutionarily conserved: Each of the >70 types of mammalian Rab proteins is related to a particular Drosophila Rab protein. We tested all 31 Drosophila Rab GTPases systematically for their abilities to influence fly development, with the use of dominant negative (DN) mutant proteins produced in specific cell types (10). Rab activities are controlled by a cycle of associations with GTP or guanosine diphosphate (GDP). The DN mutants contained a Thr/Ser → Asn mutation that causes the proteins to bind preferentially to GDP and remain inactive. Overproduced DN proteins presumably tie up interacting proteins such as Rab exchange factors in nonproductive associations.

Only one of the 31 Drosophila Rab proteins had a strong effect on the actin cytoskeleton. Flies producing DN Rab35 (Rab35DN) in the peripheral nervous system (driven by prospero-gal4) exhibited unique and specific bristle morphology defects not seen with any other Rab DN gene. Rab35DN caused the development of adult bristles that had sharp bends, kinks, and forked ends in the thorax (Fig. 1, A to E) and other body regions including the head (fig. S6B). Bristles from Rab35DN-expressing flies, stained with the actin-binding dye phalloidin at 45 to 47 hours after puparium formation, had a wavy, loose, and disconnected actin organization (Fig. 1H and fig. S2B) relative to the wild-type control (Fig. 1G and fig. S2A). Similar phenotypes are observed in mutants deficient for certain ABPs (57), which suggests that Rab35 may function as an ABP or through ABP(s). In the thoracic cuticle, production of Rab11DN or Rab5DN, which block Rab proteins that regulate endocytic trafficking, caused extensive defects in membrane growth and bristle distribution; these phenotypes are distinct from the Rab35DN effect (10). Rab35 was ubiquitously expressed, with transcripts and proteins especially abundant in the developing nervous system (fig. S1, C to H).

To test whether the defective-bristle actin phenotype was due to reduced Rab35 function, we expressed UAS-Rab35 hairpin RNA interference (RNAi) in flies to reduce Rab35 mRNA (fig. S1A). The RNAi caused the same phenotypes as did Rab35DN (Fig. 1P), which confirmed that the DN protein was inhibiting the intended target. The bristle phenotype caused by Rab35RNAi was completely rescued upon expression of a mouse wild-type Rab35 gene (Fig. 1Q). In an otherwise wild-type genetic background, mouse Rab35DN caused the same phenotype as did fly Rab35DN (fig. S4C) in the peripheral nervous system. Thus, at least some functions of Rab35 protein are conserved from flies to mammals, an evolutionary span of ~500 million years.

Expressing wild-type Rab35 (Rab35WT) in cultured cells induced multiple filopodia-like cellular extensions (Fig. 1J and figs. S3, A, B, C, D, and I, and S4E). No such effects were seen upon expression of Rab35DN (Fig. 1K and figs. S3, E to I, and S4F). Similarly, Rab35 induces peripheral processes in Jurkat T cells (11) and neurite outgrowth in PC12 and N1E-115 cells (12). The effect of extra Rab35 on cultured cells might reflect its role in vivo, allowing shaft cells to sprout protrusions during bristle development. Rab35DN, in contrast, stopped filopodia growth in vitro (Fig. 1K and figs. S3, E to H, and S4F) and caused defects in growing bristles in vivo (Fig. 1, B, D, and E). Ubiquitous Rab35DN expression driven by tubulin-gal4 caused lethality in embryos and, over a period of days, the death of cultured cells.

Treating Drosophila S2 cells with the actin polymerization inhibitor latrunculin A, but not with the microtubule-disrupting agent nocodazole, efficiently blocked the Rab35-driven morphology change (Fig. 1, L to N). Thus, Rab35 appeared to regulate actin assembly.

To explore how Rab35 influences the actin cytoskeleton, we set out to identify effector proteins that bind Rab35 directly. Rab effectors bind specific Rab proteins; binding is dependent on the Rab protein being in its GTP-bound, active state. The effectors have diverse functions in vesicle sorting, motor protein binding, vesicle trafficking, membrane fusion (13), and other roles yet to be defined. We used affinity chromatography to purify proteins that preferentially bind Rab35-GTP, which has been used to identify other Rab effectors (14). Purified glutathione S-transferase (GST)–tagged Rab35WT protein was loaded with guanosine 5′-O-(3′-thiotriphosphate) (GTP-γ-S) or with GDP and incubated with bovine brain cytosolic extracts. Several proteins were found to bind Rab35–GTP-γ-S specifically (fig. S5A and table S1). Mass spectrometry revealed a prominent 55-kD polypeptide that bound Rab35–GTP-γ-S to be fascin. Myc-tagged Rab35 and FLAG-tagged fascin coimmunoprecipitated from cell extracts. Fascin bound more strongly to Rab35WT than to Rab35DN (Fig. 2A). Purified GST-Rab35 fusion protein bound fascin in vitro (Fig. 2B). Thus, Rab35 binds fascin directly. Fascin bound more strongly to Rab35WT preloaded with GTP-γ-S than to Rab35WT preloaded with GDP in immunoprecipitations of endogenous Rab35 from fly cells and in GST pull-downs (fig. S5, B and C), consistent with the identification of fascin as a Rab35 effector.

Fig. 2

Rab35 directly interacts with fascin. (A) HeLa cells were cotransfected with myc-Rab35 and FLAG-tagged fascin. Cell lysates were subjected to immunoprecipitation with antibody to myc, followed by immunoblotting with monoclonal antibody to FLAG to detect fascin (top panel). Lower panel: Rab35 was detected with antibodies to myc. At least 6 times as much fascin (6.59 ± 0.63) associated with Rab35WT as with Rab35DN (n = 3, P < 0.05). (B) Immunoblot of GST pull-down of Rab35 proteins with purified fascin. At least 3 times as much fascin (3.27 ± 0.19) bound to Rab35WT as to Rab35DN (n = 3, P < 0.05). No binding was observed between fascin and Rab5 or Rab2. Coomassie Blue–stained GST proteins were loading controls. (C) Transgenic flies expressing UAS-Rab35RNAi alone, and flies expressing both UAS-fascin and UAS-Rab35RNAi, were crossed to prospero-gal4, which triggers expression in developing bristles. Overproducing fascin significantly reduced bristle defects (arrows in right panel) relative to flies expressing UAS-Rab35RNAi (arrows in left panel) only. Scale bar, 0.2 mm.

Fascin is an actin cross-linking protein that organizes F-actin into tightly packed parallel bundles in protruding (filopodia) and nonprotruding (microspike) structures at the leading edges of cells (15, 16). Higher than normal fascin levels have been associated with cancer cell migration, so the protein has been proposed as a marker for cancer diagnosis and a therapeutic target (1719). Fascin is produced in many tissues and is especially abundant in the nervous system (20). In Drosophila, fascin mutants (called singed) are female sterile and have aberrant mechanosensory bristles (21, 22) due to dysfunctional actin structures. The Drosophila egg chamber is composed of a germline cyst surrounded by a somatic follicular epithelium. Each cyst consists of 15 nurse cells and one oocyte. Cortical cytoskeletal structures are required during late oogenesis when nurse cell cytoplasm is rapidly transferred to the oocyte. The sterility phenotype of singed led us to examine the influence of Rab35DN in nurse cells (driven by tubulin-gal4 at 22°C) or follicle cells (driven by CY2-gal4). Both caused female sterility (94%). The interfering Rab35DN caused reduced ovarian actin levels relative to control flies, and ovary structure was abnormal (fig. S6, D, F, H, and J).

We tested whether the physical interaction between Rab35 and fascin was reflected in a genetic interaction. Rab35RNAi, produced in peripheral neurons, was used to damage bristle development. Altered bristle morphology was suppressed when extra fascin was supplied to the same cells (Fig. 2C). Increased fascin compensated for reduced Rab35 function, which suggests that fascin is at least one of the major proteins regulated by Rab35.

Purified GST-fascin and GST-Rab35 were mixed together or separately with purified nonmuscle F-actin in vitro. Actin-bundling activity increased with fascin concentration (fig. S7, A and B). No effect of Rab35 on actin bundling was observed, alone or in combination with fascin (fig. S7, A and C). Thus, Rab35 has no discernible effect on actin bundling in vitro, but its association with fascin may be a means to control when or where actin is bundled in vivo.

Perhaps activated Rab35 recruits fascin to a subcellular location where fascin stimulates actin bundling. To explore this idea, we first examined the relative locations of Rab35 in different cell types and its association with fascin in mammalian cells. Rab35WT was enriched near the plasma membrane (Fig. 3, D to F, and figs. S6G and S8A) and colocalized with fascin at the leading edge of filopodia and within microspikes in lamellipodia (Fig. 3, D to F). Rab35DN caused severe alteration of cellular structure and accumulated to a greater extent in the cytosol than along the membrane (Fig. 3, G to I, and figs. S6H and S8B). In the presence of Rab35DN, less membrane-associated fascin was observed (Fig. 3, G to I). The membrane association of Rab35 and fascin was confirmed by a cell fractionation analysis; 33.4% of Rab35 fused to yellow fluorescent protein (YFP-Rab35) and 17.7% of fascin fractionated with the membrane fraction (Fig. 3J). The association of fascin with the membrane fraction was at least partially dependent on Rab35, because less Rab35 and less fascin associated with the membrane in cells where Rab35DN was expressed. Thus, Rab35 may bring fascin to the plasma membrane to influence subcortical actin structure and initiate filament bundling.

Fig. 3

Rab35 associates with fascin near the plasma membrane. (A to C) NIH 3T3 cells expressing YFP-Rab35 [(C) and green in (A)] were costained with actin [(B) and red in (A)] to show the colocalization of the two proteins at plasma membranes, membrane ruffles, and leading edges of cells. Scale bar, 5 μm. (D to I) NIH 3T3 cells producing either YFP-Rab35 WT [(F) and green in (D)] or YFP-Rab35DN [(I) and green in (G)] were costained with antibodies to fascin [(E) and (H) and red in (D) and (G)] to show the colocalization of the Rab35 and fascin proteins. Scale bars, 5 μm. Insets in (A), (D), and (G) show higher-magnification views of the edges of cells. (J) Rab35WT enrichment in a membrane fraction of cells that had undergone the same treatment as in (D) to (I) (lane 7, upper panel), compared to the fractionation properties of Rab35DN (lane 8, upper panel; 33.4% increase in membrane-associated Rab35 normalized to the total Rab35 level). More fascin was observed in the membrane fraction with Rab35WT (lane 7, lower panel) than with Rab35DN (lane 8, lower panel; 17.7% enrichment of membrane-associated fascin with Rab35WT normalized to the total fascin level); 10% of total protein lysates were loaded as controls (lanes 1 to 4). (K) Subcellular fractionation of NIH 3T3 cells. (a) Three markers were used to show the separation of different membrane compartments. (b) Cells treated as in (D) to (J) were fractionated. Rab35WT and fascin fractionated mainly with the plasma membrane marker Na+- and K+-dependent ATPase (enriched in fractions 3 to 5), but not with the endoplasmic reticulum marker calnexin or the mitochondria marker prohibitin. (c) Cells expressing YFP-Rab35-mito were also fractionated. An enrichment of fascin in the mitochondrial fraction was observed (fractions 6 to 8). From left to right, 2.5 to 25% iodixanol gradients.

If fascin mediates the effects of Rab35, then Rab35-induced filopodia formation should be reduced when fascin function is blocked. Fascin was depleted in Rab35-expressing cells by introducing fascin small hairpin RNA (16). The interfering RNA significantly blocked filopodia formation induced by Rab35 (fig. S9, B and C). Phosphorylation of fascin at Ser39 is important for its actin-bundling activity and proper localization to filopodia (15, 16). We produced point mutants that mimic the active dephosphorylated (Ser39 → Ala, S39A) or inactive phosphorylated (Ser39 → Asp, S39D) forms of fascin (15, 16). Expression of tdTomato-tagged S39A or S39D fascin mutants in NIH 3T3 cells along with Rab35 had opposite effects on filopodia formation (figs. S9, D to F, and S10, A to I). The S39A mutant in combination with Rab35WT caused more protein to accumulate at the tip of cell extensions (figs. S9E and S10, D to F) relative to wild-type fascin plus Rab35WT, but no significant increase in the number of filopodia was observed (fig. S10J). In contrast, S39D in combination with Rab35 reduced the number of filopodia (fig. S10J). In these cells most of the Rab35WT protein remained near the plasma membrane (figs. S9F and S10, G to I). These results are consistent with a role for fascin as a downstream effector of Rab35 in filopodia formation.

We constructed a gene encoding a modified form of Rab35 targeted to the outer mitochondrial membrane, a location that normally has modest levels of fascin or assembled actin and no detectable Rab35 (Fig. 4, A to D). When Rab35DN was used in this experiment, the modified protein was on the surface of mitochondria (Fig. 4F). No discernible change in actin structure was observed in the vicinity of mitochondria (Fig. 4H and fig. S11, C and D). In contrast, when Rab35WT was brought to the mitochondrial outer membrane (Fig. 4, I to L), the mitochondria were consistently decorated with increased actin meshworks (Fig. 4L and fig. S11, A and B). As a control, Rab5WT targeted to mitochondria in the same manner did not cause actin assembly in the vicinity of mitochondria (Fig. 4, M to P). Fascin relocation to mitochondria was confirmed by cell fractionations. Mitochondrial enrichment of fascin was observed when Rab35WT-mito was produced (Fig. 3K). Thus, relocation of Rab35 can drive the location of actin assembly.

Fig. 4

Localized stimulation of actin bundling by Rab35 recruitment of fascin. (A to D) NIH 3T3 cells were stained with MitoTracker (Invitrogen; green) to show mitochondria and actin (red). Box in (C) is shown in (D) at higher magnification. Arrowheads indicate areas surrounding mitochondria. Scale bar, 5 μm. (E to P) NIH 3T3 cells producing YFP-Rab35DN-mito, YFP-Rab35WT-mito, and YFP-Rab5-mito (all in green) were stained with phalloidin (red) to detect actin. In 20 to 30% of cells, localization of Rab35WT to mitochondria induced actin assembly in the vicinity of mitochondria (K). In parallel experiments in which Rab35DN and Rab5 were localized to mitochondria, no such enrichment of actin was observed [(G) and (O), respectively]. Boxes in (C), (G), (K), and (O) are shown in (D), (H), (L), and (P) at higher magnification. Arrowheads indicate areas surrounding mitochondria. Detectable accumulation of actin near mitochondria was observed only in (L). Scale bars, 5 μm.

Our results show that a Rab35 effector protein, fascin, is able to stimulate local actin bundling and thus control bristle and filopodia formation (fig. S11E). The exact ways in which such a mechanism may be used probably vary among cell and tissue types. In cultured cells, active Rab35 recruits fascin to drive actin bundling at the leading edge of cell protrusions. During Drosophila bristle development, Rab35 may recruit fascin and induce actin bundling to initiate the cytoplasmic extension required for bristle extension. Inadequate Rab35 function leads to bends and kinks in the bristles.

Conflicting results about Rab35 function have been obtained from different cell types and models. Rab35RNAi revealed a function in cytokinesis in Drosophila S2 cells (23); in HeLa cells, Rab35 also plays a role in cytokinesis (23). No cytokinesis phenotype was observed in mutants of Caenorhabditis elegans Rab35, but Rab35 transports yolk receptors in oocytes (24). In HeLa-CIITA, a cell line in which major histocompatibility complex (MHC) class II is expressed, Rab35 regulates a recycling pathway in a clathrin-, AP2-, and dynamin-independent manner (25). In Jurkat T cells, Rab35-mediated recycling appears to be clathrin-dependent (11). In PC12 and N1E-115 cells, activated Rab35 stimulates neurite outgrowth via a Cdc42-dependent pathway (12). Drosophila Rab35, like mammalian Rab35, is found near the plasma membrane, on intracellular vesicles, and in the cytosol (23) (Fig. 3, A to C). This broad distribution contrasts with more discrete locations of other Rab proteins, including many we studied (10), which suggests that Rab35 may have diverse functions.

Interfering with Rab35 in living flies showed its importance for normal bristle morphology and its function in regulating actin assembly. The powerful influence of Rab35 on the cytoskeleton can now be at least partly explained by localization of fascin and its consequent influence on actin filament bundling.

Supporting Online Material

Materials and Methods

Figs. S1 to S12

Table S1


References and Notes

  1. We thank M. Fish for DNA injections; X. Huang, E. Bustamante, and C. Gauthier for help with initial experiments; Scott lab members for valuable discussion and comments; the Stanford Cell Sciences Imaging Facility for assistance with scanning electron microscopy studies; and S. Pfeffer, A. Ghabrial, and R. Rohatgi for critical reading and comments on the manuscript. Supported by a Jane Coffin Childs Memorial Fund for Medical Research fellowship (J.Z.) and by the NIH National Technology Center for Networks and NIH Pathway grant U54 RR020843 (M.F. and M.B.). The research reported here was supported by the Howard Hughes Medical Institute. M.P.S. is an Investigator of the HHMI.
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