The Small Gtpase Rif Is an Alternative Trigger for the Formation of Actin Stress Fibers in Epithelial Cells

Research Article 1247 The small GTPase Rif is an alternative trigger for the formation of actin stress fibers in epithelial cells

Lifei Fan, Stephanie Pellegrin, Alice Scott and Harry Mellor* Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol, BS8 1TD, UK *Author for correspondence ([email protected])

Summary Actin stress fibers are fundamental components of the actin cytoskeleton that produce contractile force in non-muscle cells. The formation of stress fibers is controlled by the small GTPase RhoA and two highly related proteins, RhoB and RhoC. Together, this subgroup of actin-regulatory proteins represents the canonical pathway of stress-fiber formation. Here, we show that the Rif GTPase is an alternative trigger of stress-fiber formation in epithelial cells. Rif is distantly related to RhoA; however, we show that the two proteins share a common downstream partner in stress-fiber formation – the Diaphanous-related formin mDia1. Rif-induced stress fibers also depend on the activity of the ROCK protein kinase. Unlike RhoA, Rif does not raise ROCK activity in cells, instead Rif appears to regulate the localization of myosin light chain phosphorylation. This study establishes Rif as a general regulator of Diaphanous-related formins and shows how non-classical Rho family members can access classical Rho pathways to create new signaling interfaces in cytoskeletal regulation.

Key words: Actin stress fibers, Stress fibres, Formin, Rho GTPase, Contractile

Introduction and allows nucleation of new F-actin filaments (Alberts, 2001; Actin stress fibers are long contractile bundles of actin filaments Watanabe et al., 1999). Activated RhoA also binds to the and myosin II that span the body of non-muscle cells. Their ROCK/ROK protein kinase (Ishizaki et al., 1996; Leung et al., organization is broadly similar to that of the sarcomeric arrays of 1995). ROCK can directly phosphorylate MLC2 at Ser19 (Amano muscle cells, but generally looser and less uniform. Actin stress et al., 1996). It also phosphorylates MYPT/MBS, the regulatory fibers are linked mechanically to the extracellular matrix through subunit of myosin phosphatase. Phosphorylation of MYPT inhibits integrin-rich contacts at the plasma membrane, allowing the cell to phosphatase activity, leading to an increase in phosphorylated MLC

Journal of Cell Science apply contractile force to the substratum. This contractile force is (Kawano et al., 1999; Kimura et al., 1996). Phosphorylation of MLC used to power retraction of the cell body during migration; however, increases stress-fiber contractility by increasing myosin ATPase it also allows cells to remodel the extracellular matrix – a function activity (Katoh et al., 2001) and leads to increased bundling of actin that is important during development and in wound healing filaments in the stress fiber. Activation of ROCK produces thick (Pellegrin and Mellor, 2007). stress fibers, but these are poorly organized (Leung et al., 1996). Unlike the sarcomeric actomyosin arrays of muscle cells, actin Activation of mDia1 produces parallel actin filaments, which are stress fibers are dynamic and their formation is controlled by a wide weakly bundled (Watanabe et al., 1997). A combination of both range of cellular stimuli, most notably the rigidity of the substratum signals is needed for proper stress-fiber formation (Watanabe et al., (Couchman and Rees, 1979; Mochitate et al., 1991). Early work 1999). identified RhoA as a crucial regulator of actin-stress-fiber formation. The Rif GTPase is a relatively recent addition to the Rho family; RhoA is a member of the Rho family of small GTPases, which are it is found only in chordates and displays a relatively low homology signaling proteins with a central role in regulation of the actin to other family members (Ellis and Mellor, 2000). Previously, we cytoskeleton. Expression of active RhoA in cells provokes strong showed that Rif controls the formation of filopodia through the stress-fiber formation (Paterson et al., 1990) and RhoA controls the Diaphanous-related formin mDia2 (DIAPH3) (Pellegrin and Mellor, formation of stress fibers in response to a wide-range of extracellular 2005). Filopodia are thin, actin-rich projections that are used by stimuli (Pellegrin and Mellor, 2007; Ridley and Hall, 1992). In cells to sense their external environment (Mellor, 2009). The vertebrates, the gene encoding RhoA has undergone duplication to canonical pathway for their formation is through the Rho family give two additional isoforms: RhoB and RhoC. These two proteins member Cdc42 (Kozma et al., 1995; Nobes and Hall, 1995). Rif is are highly related to RhoA (approximately 85% identity) and also an alternative trigger for filopodia formation, producing filopodia trigger stress-fiber formation (Giry et al., 1995). For simplicity, we with different characteristics to those triggered by Cdc42 (Pellegrin will refer to this group as the RhoA-like proteins. and Mellor, 2005). Intriguingly, recent work has shown that Cdc42 RhoA-like proteins control two activities required for the and Rif work together in the formation of dendritic spines, which formation of actin stress fibers – nucleation of actin filaments and develop from dendritic filopodia (Hotulainen et al., 2009). phosphorylation of myosin light chain 2 (MLC2). Activated RhoA Here, we show that Rif also controls the formation of epithelial binds to the Diaphanous-related formin mDia1 (DIAPH1) through stress fibers. Rif-induced stress fiber formation depends directly on its G-protein binding domain (GBD) (Watanabe et al., 1997). mDia1 and indirectly on the activity of ROCK. This dual role for Binding to the GBD releases an autoinhibitory interaction in mDia1 Rif demonstrates how non-classical Rho GTPases can interface with 1248 Journal of Cell Science 123 (8)

the ancient pathways of cytoskeletal regulation to create new of contractile actin stress fibers is the presence of non-muscle combinations and new morphological outcomes. myosin II in a loose sarcomeric array (Pellegrin and Mellor, 2007). Rif-induced stress fibers showed clear incorporation of myosin in Results a characteristic striated pattern (Fig. 1C). At present, there are no Rif stimulates formation of actin stress fibers known activators of Rif, meaning that we were not able to drive Activated Rif triggers the formation of numerous long filopodia activation of endogenous Rif protein and measure the effect on the that project from the apical surface of the cell (Ellis and Mellor, actin cytoskeleton; however, silencing of the expression of 2000). These structures collapse upon fixation, making it difficult endogenous Rif using RNA interference led to a significant decrease to see underlying F-actin structures in the cell body. Confocal in actin stress fibers under basal conditions (supplementary material sectioning of cells expressing an activated Rif mutant (Rif-QL) Fig. S2), suggesting that Rif contributes to the formation of actin revealed strong stimulation of actin stress fiber formation at the stress fibers, even in resting epithelial cells. base of the cell (Fig. 1A). These stress fibers were indistinguishable Rif-induced stress fibers differed from RhoA-induced stress fibers from those induced by activated RhoA in these cells (supplementary in two ways. Unlike RhoA, the effect of Rif on stress-fiber material Fig. S1); however, they were not dependent on RhoA for formation was restricted to cell lines of an epithelial origin, and Rif formation, because dominant-negative RhoA had no effect on the did not induce stress fibers in fibroblasts (data not shown). This is ability of Rif to induce stress fibers (Fig. 1B). The defining feature similar to Rif-induced filopodia, which also are not observed in fibroblasts (Ellis and Mellor, 2000). RhoA stimulates stress fiber formation in parallel with an increase the size and maturity of focal adhesions (Ridley and Hall, 1992). Rif activation had a variable effect on focal adhesion formation, dependent on epithelial cell type, with an increase in the number of focal adhesions in MDCK cells, but no obvious effect in HeLa cells (Fig. 1A).

Rif-induced stress fibers are dependent on ROCK activity ROCK is an important regulator of RhoA-induced stress fibers (Leung et al., 1996). We examined the effect of inhibiting ROCK activity on Rif-induced stress fibers by silencing endogenous ROCK1 and ROCK2 using RNA interference. Reduction of either isoform led to a significant decrease in Rif-induced stress fibers (supplementary material Fig. S2). As we were unable to achieve >50% silencing of the ROCK isoforms, we examined the effects of using the specific ROCK inhibitor Y-27632 (Uehata et al., 1997) to achieve a complete block of ROCK activity. Inhibition of ROCK returned stress-fiber levels to control values in cells expressing either

Journal of Cell Science wild-type Rif or the activated Rif-QL mutant (Fig. 2A,B). RhoA activates ROCK by direct interaction with a binding site in the C- terminal region of the protein (Dvorsky et al., 2004; Fujisawa et al., 1996). We therefore examined whether ROCK was also a binding partner for Rif. ROCK1 showed a clear interaction with both wild-type Rif and the activated Rif-QL mutant by yeast two- hybrid analysis (Fig. 2C); however, despite many attempts, we were unable to demonstrate an interaction between Rif and ROCK1 by immunoprecipitation (Fig. 2D), or with a truncated mutant of ROCK that lacks the C-terminal autoinhibitory motif (data not shown). ROCK was not localized to Rif-induced stress fibers, but neither was it localized to RhoA-induced stress fibers (data not shown). Some protein-protein interactions in signaling pathways are transient and/or of low affinity and are not stable to extraction. We examined the effect of Rif activation on the phosphorylation of ROCK targets Fig. 1. Rif triggers formation of stress fibers. (A)HeLa and MDCK cells to determine whether Rif increased the cellular activity of ROCK. were transfected with the activated Rif-QL mutant and stained for Rif (top Expression of the activated Rif-QL mutant had no effect on the panels), F-actin (green) and vinculin (red). The top panels show full projections phosphorylation of either MYPT or of MLC (Fig. 2E). Treatment through the cells, the bottom panels show projected images of 4ϫ0.5m of cells with the protein phosphatase inhibitor calyculin A led to a sections at the base of the cells. Activated Rif stimulated the formation of basal dramatic increase in phosphorylation of both MLC and MYPT, stress fibers in both cell lines. An increase in focal adhesion size was seen in demonstrating that the basal level of phosphorylation of these MDCK cells, but not in HeLa cells. (B)Cells were transfected with the proteins was not saturated under these conditions. Taken together, activated Rif-QL mutant (green) and dominant-negative RhoA (RhoA-T19N; red). Cells were stained for F-actin using Alexa Fluor 660 Phalloidin (bottom we conclude that the induction of stress fibers by Rif is dependent panel). Inhibition of RhoA had no effect on the induction of stress fibers by Rif. on the basal level of ROCK activity. It is possible that Rif makes (C)Cells were transfected with the activated Rif-QL mutant (blue) and GFP- a weak or transient direct interaction with ROCK; however, Rif myosin II (green) and stained for F-actin (red). Myosin II shows a periodic does not increase the cellular level of ROCK activity under staining pattern along Rif-induced stress fibers. Scale bars: 10m. conditions where formation of stress fibers is provoked. Rif triggers stress-fiber formation 1249

Fig. 3. mDia1 is a Rif binding partner. (A)Interaction of Rif with the GBDs of the three Diaphanous-related formins was measured in a yeast two-hybrid assay by growth on Trp/Leu/His-deficient medium in the presence 2 mM 3- Fig. 2. Rif-induced stress fibers depend on ROCK activity. (A)HeLa cells amino-1,2,4-triazole. Plasmids encoding inactive (TN), wild-type (WT) or were transfected with the activated Rif-QL mutant and treated with or without activated (QL) Rif or RhoA fused to the Gal4-DNA binding domain were 10M Y-27632 for 2 hours. Representative cells are shown stained for F-actin transfected into AH109 yeast cells together with the respective GBD domains and the positions of the Rif-transfected cells are indicated (arrows). Scale bar: fused to the Gal4 activation domain. The interaction between RhoA and the 10m. (B)Control cells or cells transfected with wild-type Rif or the activated GBD of rhophilin1 was used as a control. Duplicate colonies are shown for Rif-QL mutant were treated with or without 10M Y-27632 for 2 hours. Cells each combination. Rif interacted with all three Diaphanous-related formins in were fixed and stained for F-actin and scored (100 cells for each condition) for this assay, but not with rhophilin1. (B)Cells were transfected with the the presence of actin stress fibers. Data are means ± s.e.m. (n3). activated FLAG-epitope tagged mDia1DAD mutant and either empty vector, (C)Interaction of RhoA and Rif with ROCK1 was measured in a yeast two- wild-type Rif, the inactive Rif-TN mutant or the activated Rif-QL mutant. hybrid assay by growth on Trp/Leu/His/Ade-deficient medium. Plasmids mDia1 was isolated from cell lysates by immunoprecipitation and these encoding inactive (TN), wild-type (WT) or activated (QL) Rif or RhoA fused samples were probed for co-precipitation of Rif by western blotting. Samples to the Gal4-DNA binding domain were transfected into AH109 yeast cells of cell lysates were taken to confirm expression of the various proteins. mDia1 together with ROCK1 fused to the Gal4 activation domain. Duplicate colonies interacted with wild-type Rif and Rif-QL in this assay. are shown for each combination. (D)Cells were co-transfected with Myc- epitope-tagged ROCK1, Rif-QL, or both. ROCK1 was isolated from cell lysates by immunoprecipitation and these samples were probed for co- precipitation of Rif by western blotting. Samples of cell lysates were taken to confirm expression of the various proteins. No interaction between ROCK1 no interaction with the RhoA-binding domain of rhophilin1 in this and Rif was detected. (E)Cells were transfected with or without activated Rif Journal of Cell Science assay (Fig. 3A). We confirmed the interaction between Rif and under the same conditions used for stress fiber assays. The phosphorylation of mDia1 by immunoprecipitation. Rif showed a strong interaction the ROCK1 substrates MYPT and MLC was probed by western blotting of with mDia1 in this assay (Fig. 3B). Unlike the yeast two-hybrid whole-cell extracts. As a control, cells were pre-treated with 25 nM calyculin- A (cal-A) for 30 minutes, which significantly increased phosphorylation of assay with the isolated GBD, this interaction depended on Rif both proteins. activity, and no interaction was seen with the Rif-TN mutant (Fig. 3B). We conclude that, although the isolated GBD is not able to discriminate between active and inactive Rif, the whole protein gains this ability. mDia1 is a Rif binding partner The ROCK data suggested that an additional component was Rif triggers stress-fiber formation through mDia1 required for the induction of stress fibers by Rif. We have shown We used RNA interference to test the role of mDia1 in Rif-induced previously that Rif interacts with the Diaphanous-related formin stress fiber formation. We used two independent siRNA mDia2 to control filopodia formation (Pellegrin and Mellor, 2005). oligonucleotides against DIAPH1, the gene encoding mDia1, each As RhoA-induced stress fibers require the activity of the related of which gave significant downregulation of mDia1 (Fig. 5C). formin, mDia1 (Watanabe et al., 1997), we tested the interaction Silencing of DIAPH1 had a drastic effect on the ability of Rif to between Rif and the GBDs of all three Diaphanous-related formins induce stress fiber formation (Fig. 4A), reducing stress fibers to almost by yeast two-hybrid assay. mDia3 (DIAPH2) undergoes alternative control levels (Fig. 4B). mDia1 has been reported to be a component splicing in its GBD to yield two isoforms called hDia2B and of focal adhesions, which are the sites of stress fiber initiation and hDia2C. Previous work has shown that this alternative splicing alters attachment (Hotulainen and Lappalainen, 2006). This has led to the the specificity for Rho GTPase interaction, with hDia2B binding proposal that mDia1 seeds actin stress fibers through nucleation of RhoA and hDia2C binding RhoD (Gasman et al., 2003). We actin filaments at focal adhesion sites. Activated Rif triggers the included both splice forms in the assay. As we found previously translocation of mDia2 to the tips of Rif-induced filopodia (Pellegrin (Pellegrin and Mellor, 2005), Rif interacts strongly with the GBD and Mellor, 2005), which is consistent with a model for filopodial of mDia2 (Fig. 3A). Surprisingly, Rif also interacted with mDia1 extension where mDia2 seeds actin filaments formation from the and both splice forms of mDia3. In this assay, interaction was filopodial tip. We were interested to determine whether activated Rif independent of Rif activation – in fact, the interaction was strongest altered the cellular localization of mDia1, and whether we could see with the inactive Rif-TN mutant (Fig. 3A). By contrast, Rif showed any evidence of co-localization of Rif, mDia1 and stress-fiber 1250 Journal of Cell Science 123 (8)

attachment sites. Co-transfection of mDia1 with activated Rif led to distended to form bleb-like structures at the apical surface (Fig. a redistribution of mDia1 (Fig. 4C). We did not observe any 5A). Membrane blebbing is driven by actomyosin contractility in concentration of Rif or mDia1 at the stress-fiber attachment site, either the cell cortex (Charras, 2008). ROCK triggers bleb formation by when transfected together (Fig. 4D), or separately (data not shown). increasing MLC phosphorylation at the bleb site; it is also a Instead, we were surprised to see re-localization of mDia1 to Rif- physiological regulator of membrane blebbing during apoptosis induced filopodia. As with mDia2, we saw localization of mDia1 to (Coleman et al., 2001; Sebbagh et al., 2001) and is involved in cell filopodia tips. We also occasionally saw punctuate staining of mDia1 migration through 3D matrices (Sahai and Marshall, 2003). along the filopodial shaft (Fig. 4C,D). Interestingly, the Rif-induced blebs showed focussed staining of phosphorylated MLC at their bases (Fig. 5A). Previous studies have Rif-induced blebbing in the absence of mDia1 shown that there is an antagonistic relationship between mDia1 and We observed that silencing of DIAPH1 led not only to a loss of ROCK (Sahai and Marshall, 2002). We wondered whether loss of Rif-induced stress fibers, but also to profound changes in Rif- mDia1 was freeing Rif for interaction with ROCK and hence driving induced filopodia. Filopodia were still present; however, they were bleb formation. In apparent agreement with this, bleb formation by Rif in the absence of mDia1 was dependent on ROCK activity (Fig. 5B). We examined the total cellular activity of ROCK by measuring the phosphorylation of MYPT and MLC2 – the two relevant substrates for ROCK in actomyosin contractility. Rif activation did not increase the total level of phosphorylation of either protein (Fig. 5C), either in the presence of endogenous mDia1 (stress fiber Journal of Cell Science

Fig. 4. Rif triggers stress fiber formation through mDia1. (A) HeLa cells Fig. 5. Rif-induced blebbing in the absence of mDia1. (A)HeLa cells were treated with siRNA against DIAPH1 or against a control gene (LMNA, which transfected with DIAPH1 siRNA and then with an expression plasmid encodes lamin). The cells were then transfected with the activated Rif-QL encoding the Rif-QL mutant. In the absence of mDia1, Rif activation led to the mutant. Representative cells are shown stained for F-actin and the positions of formation of bleb-like projections from the apical surface. These showed the Rif-transfected cells are indicated (arrows). Silencing of DIAPH1 leads to concentrated staining for Rif (green) and phosphorylated MLC (red). X-Z a loss of Rif-induced stress fibers. Scale bar: 10m. (B)Cells were treated projections through the body of the cells show that phosphorylated MLC is without siRNA, with control siRNA or with two independent DIAPH1 siRNAs concentrated at the base of these apical blebs (top panels). Scale bar: 10m. (mDia1A and mDia1B). The cells were then transfected with activated Rif-QL, (B)HeLa cells were treated with a control siRNA or with DIAPH1 siRNA. or with empty vector. Cells were fixed, stained for F-actin and scored (100 The cells were then transfected with activated Rif-QL, or with empty vector. cells for each condition) for the presence of actin stress fibers. Data are mean ± Cells were then treated with or without 10M Y-27632 for 2 hours. Cells were s.e.m. (n3). (C)Localization of mDia1 in cells expressing active Rif. HeLa fixed, stained for F-actin and scored (100 cells for each condition) for the cells were transfected with mDia1 (green) and the activated Rif-QL mutant presence of apical blebs. Cells were counted as positive for blebs if they (blue) and co-stained for F-actin (red). The panel on the right shows the contained more than ten blebs and negative if they contained fewer than two separate channels at a region of the cell where the stress fibers meet the plasma blebs. There were no cells that showed intermediate numbers of blebs. membrane. There was no concentration of Rif or mDia1 on stress fibers or at Essentially identical results were obtained with a second DIAPH1 siRNA. their attachment points. Scale bar: 10m. (D)Rif causes translocation of Data are means ± s.e.m. (n3). (C)HeLa cells were treated with control mDia1 to filopodia. HeLa cells were transfected with mDia1 (green) either siRNA or with two independent DIAPH1 siRNAs. The cells were then alone (left panel) or together with the activated Rif-QL mutant (red, right transfected with activated Rif-QL, or with empty vector. The phosphorylation panels). The right-hand panels show a magnified region of filopodia showing of the ROCK1 substrates MYPT and MLC was probed by western blotting of mDia1 at the tips. In the presence of activated Rif, mDia1 undergoes whole-cell extracts. Rif activation did not change the total cellular levels of translocation to the tips of filopodia and to puncta along the filopodial shaft. phosphorylated MYPT or phosphorylated MLC, in the either the presence of Scale bar: 10m. absence of mDia1. Rif triggers stress-fiber formation 1251

formation) or in its absence (bleb formation). We conclude that RhoA, Rac and Cdc42 are relatively ancient signaling proteins, and focussed staining of phosphorylated MLC at the base of Rif-induced all three are present in fungi, worms and flies. One can imagine blebs occurs without a net increase in cellular ROCK activity and two ways in which the newer Rho family members might add is, therefore, consistent with a relocalization of ROCK activity, or functionality to cytoskeletal regulation – either through controlling a relocalization of phosphorylated MLC. new signaling pathways, or by making novel interactions with the existing pathways. Rif illustrates the second possibility: the Discussion pathways that Rif controls are old, but the interface with those We show here that Rif is a novel regulator of actin stress fiber pathways is new and different. Many of the complex mechanical formation in epithelial cells. Some cytoskeletal structures, such as movements of cells are created through co-ordinated regulation of filopodia, can be triggered by several Rho family members actin-regulatory pathways by Rho GTPases. The emergence of the (Aspenstrom et al., 2004); however, previously only the RhoA-like non-classical human Rho GTPases parallels the development of the proteins have been shown to control stress fiber formation. adaptive immune system, increasing complexity of the nervous Actomyosin contractility has a number of important roles in system and a host of other processes requiring complex regulation epithelial biology. Contraction at the apical surface is important of cell shape and cell migration. It seems highly likely that it is in during organogenesis and in the closing of epithelial sheets during these processes that we will find non-classical Rho GTPases, such development and in wound healing (Martin and Parkhurst, 2004). as Rif. Contraction at the basal surface is important for remodelling of the extracellular matrix during development and for the secretory Materials and Methods function of exocrine glands in the adult organism (Pellegrin and Reagents A polyclonal antibody against Rif was raised in sheep using the synthetic peptide Mellor, 2007). Epithelial cells can differentiate to form myoepithelial MDAPGALAQTAAPGPGRKELC-NH2, corresponding to the N-terminus of the cells – specialized contractile cells that have prominent stress fibers human sequence. A polyclonal antibody against the myc epitope tag was raised to at the basal surface (Gudjonsson et al., 2005). These cells form a the synthetic peptide MEQKLISEEDLGC-NH2. In each case, the antibodies were affinity purified using the immunising peptide coupled to SulfoLink resin (Pierce). basket-like network around the luminal epithelium of the mammary Monoclonal antibodies against vinculin (VIN-11-5), FLAG epitope tag (M2) and gland and their contraction powers expulsion from the gland. Similar tubulin (B-5-1-2) were from Sigma. Polyclonal rabbit anti-phospho-MLC (Ser19) systems operate in salivary, sweat and lacrimal glands (Pellegrin was from Cell Signaling Technologies. Polyclonal rabbit anti-phospho-MYPT and Mellor, 2007). The regulation of stress fiber contraction in (Thr696) was from Upstate. Rabbit polyclonal anti-mDia1 was from Abcam. Rabbit polyclonal anti-HA epitope tag and monoclonal anti-myc epitope antibodies (9E10) myoepithelial cells is poorly characterized; however, it is important were from Santa Cruz. Y-27632 was from Calbiochem. siRNA oligonucleotides to note that Rif is relatively highly expressed in epithelial tissue duplexes were from Eurofins. Fluorescent secondary antibodies were raised in donkey (Ellis and Mellor, 2000). and purchased from Invitrogen (Alexa Fluor conjugated) or Stratech Scientific (Cy conjugated). Alexa Fluor 488-phalloidin was from Invitrogen. A full list of cDNA We show that Diaphanous-related formins dictate the specificity constructs is included in supplementary material Table S1. of Rif action: activation of mDia2 regulates filopodia formation downstream of Rif, whereas activation of mDia1 mediates Rif- Cell culture and transfection HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) induced stress fibers. The role of ROCK in Rif-induced stress fibers containing 10% heat-inactivated bovine fetal calf serum, 100 g/l streptomycin sulfate is less clear. ROCK activity is required for Rif to stimulate stress and 100 U/ml benzylpenicillin. Cells were transfected with expression vectors using

Journal of Cell Science fibers; however, we saw no increase in the total cellular activity of Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. siRNA ROCK on Rif activation. We favour a model where Rif makes a oligonucleotides were introduced into HeLa cells using calcium phosphate. Briefly, cells were cultured in six-well dishes until 70% confluent (approximately 106 cells). transient interaction with ROCK that does not lead to significant Transfection solutions were prepared by mixing 4 l of 20 M siRNA in 100 l activation of the kinase, but rather serves to increase the local 0.25 M CaCl2 solution with 100 l 50 mM BES, pH 6.95, 280 mM NaCl, 1.5 mM concentration of ROCK activity. This model would explain the Na2HPO4. After a 15 minute incubation, this suspension was added drop-wise to the dish. Samples were incubated at 37°C and 3% CO overnight. The medium was then concentration of phosphorylated MLC at the base of Rif-induced 2 replaced twice and samples returned to 37°C and 5% CO2 for a further 48 hours. The blebs in cells lacking mDia1. Alternatively, in the absence of an sequences of the siRNA oligonucleotides were as follows: Lamin A/C, ability to activate ROCK, Rif might simply require a significant GGUGGUGACGAUCUGGGCU; mDia1A GAAGUUGUCUGUUGAAGAA; mDia1B, AGAACCUAGAUCAGAUGAA; Rif, GCUACGACAACGUCCUCAU; basal ROCK activity to stimulate the formation of stress fibers. ROCK1, GCCAAUGACUUACUUAGGATT; ROCK2, GCAAAUCUGUUAAU - The potential role of mDia1 in Rif-induced filopodia is intriguing. ACUCGTT. Similarly to mDia2, mDia1 showed clear localization to the tips of Rif-induced filopodia; however, we also saw punctate staining along Immunofluorescence microscopy Cells were prepared for confocal immunofluorescence microscopy by fixation with the filopodial shaft. This was exaggerated with an activated mutant paraformaldeyde and permeabilization with Triton X-100, as described previously of mDia1 (data not shown). Importantly, Rif did not require mDia1 (Pellegrin and Mellor, 2005). Confocal microscopy was performed using a Leica to form apical projections; however, in the absence of mDia1 these AOBS SP confocal laser-scanning microscope with an attached Leica DM IRE2 inverted epifluorescence microscope under a Plan Apo BL ϫ63/1.4 NA oil-immersion were often distended into bleb-like structures. mDia2 controls the objective. Fluorophores were excited using the 405 nm line of a diode laser, the 488 nm extension of filopodia by mediating the nucleation of actin filaments line of a Kr/Ar laser and the 543, 594 and 605 nm lines of a He/Ne laser, as appropriate. at the filopodial tip (Faix et al., 2009). The effects of silencing A series of images was taken at 0.5 m intervals through the Z-plane of the cell and DIAPH1 on Rif-induced filopodia suggest a role for mDia1 in the processed to form a projected image. For imaging of actin stress fibers, projections were from 4ϫ0.5 m sections taken at the base of the cell. maintaining the integrity of these structures. Recently, it has been shown that filopodia can contribute F-actin bundles to the formation Immunoprecipitation assays 7 of stress fibers (Anderson et al., 2008; Nemethova et al., 2008). It Cells were transfected as described. After growth overnight, cells (approximately 10 cells per condition) were broken on ice with 1 ml lysis buffer [20 mM HEPES, pH is intriguing to speculate that Rif-induced filopodia might have a 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM DTT, 1 mM EDTA, 0.1 role in the generation of Rif-induced stress fibers, potentially linking mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche)]. Lysates the two structures. were rotated for 20 minutes at 4°C and then clarified by centrifugation at 15,000 g for 15 minutes. Proteins were immunoprecipitated at 4°C by incubation for 1 hour Humans have 21 Rho family members, most of which date back with 1 g anti-FLAG antibody or anti-myc antibody, as appropriate, followed by to the division of the chordate phylum (Wherlock and Mellor, 2002). incubation with 30 l Protein-G-Sepharose beads (packed bead volume) for 1 hour. 1252 Journal of Cell Science 123 (8)

The beads were washed three times in ice-cold lysis buffer and the bound protein Gudjonsson, T., Adriance, M. C., Sternlicht, M. D., Petersen, O. W. and Bissell, M. eluted by heating at 95°C with SDS-PAGE sample buffer. Immunoprecipitated proteins J. (2005). Myoepithelial cells: their origin and function in breast morphogenesis and were analyzed by western blotting. neoplasia. J. Mammary Gland Biol. Neoplasia 10, 261-272. Hotulainen, P. and Lappalainen, P. (2006). Stress fibers are generated by two distinct Yeast two-hybrid assays actin assembly mechanisms in motile cells. J. Cell Biol. 173, 383-394. cDNAs against mRNA encoding RhoA and Rif were expressed as fusions with the Hotulainen, P., Llano, O., Smirnov, S., Tanhuanpaa, K., Faix, J., Rivera, C. and GAL4 DNA-binding domain in the yeast expression plasmid pGBKT7 (Clontech). Lappalainen, P. (2009). Defining mechanisms of actin polymerization and ROCK1 and the GBD domains of the Diaphanous-related formins were expressed depolymerization during dendritic spine morphogenesis. J. Cell Biol. 185, 323-339. as fusion proteins with the GAL4 activation domain in pGADT7 (Clontech). Potential Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatsu, A., Fujita, A., Watanabe, N., Saito, Y., Kakizuka, A., Morii, N. et al. (1996). The small GTP-binding interactions were tested by co-transformation of plasmids into AH109 yeast cells. protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to Double transformants were selected on Leu/Trp-deficient agar plates. After 4 days, myotonic dystrophy kinase. EMBO J. 15, 1885-1893. two independent cultures were grown overnight in Leu/Trp-deficient medium and Katoh, K., Kano, Y., Amano, M., Onishi, H., Kaibuchi, K. and Fujiwara, K. 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