© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 4168-4179 doi:10.1242/jcs.202739

RESEARCH ARTICLE RhoC regulates the actin remodeling required for phagosome formation during FcγR-mediated phagocytosis Youhei Egami*, Katsuhisa Kawai and Nobukazu Araki*

ABSTRACT 2003; Groves et al., 2008; Swanson, 2008). Pseudopod extension is Phagosome formation is a complicated process that requires accompanied by the formation of branched F-actin networks spatiotemporally regulated actin reorganization. We found that generated by the Arp2/3 complex (Campellone and Welch, 2010). RhoC GTPase is a critical regulator of FcγR-mediated phagocytosis During phagocytic cup formation, the actin-nucleating activity of in macrophages. Our live-cell imaging revealed that RhoC, but not Arp2/3 is stimulated via binding to the Wiskott-Aldrich syndrome RhoA, is recruited to phagocytic cups engulfing IgG-opsonized protein (WASP) and WASP-family verprolin-homologous protein erythrocytes (IgG-Es). RhoC silencing through RNAi, CRISPR/Cas- (WAVE) family members (Lorenzi et al., 2000; May et al., 2000; Park mediated RhoC knockout, and the expression of dominant-negative and Cox, 2009; Tsuboi and Meerloo, 2007). Arp2/3-mediated actin- or constitutively active RhoC mutants suppressed the phagocytosis of filament assembly has been proposed as the driving force for the IgG-Es. Moreover, RhoC-GTP pulldown experiments showed that formation of phagocytic cups (May et al., 2000). Importantly, to endogenous RhoC is transiently activated during phagosome complete phagosome formation, actin disassembly at the base of formation. Notably, actin-driven pseudopod extension, which is phagocytic cups needs to occur concurrently with the maximal required for the formation of phagocytic cups, was severely extension of pseudopods around particles (Egami et al., 2011; impaired in cells expressing the constitutively active mutant RhoC- Greenberg et al., 1993). After the closure of the phagocytic cups, the G14V, which induced abnormal F-actin accumulation underneath the newly formed phagosomes mature via a series of interactions with plasma membrane. mDia1 (encoded by DIAPH1), a Rho-dependent endocytic compartments and finally fuse with lysosomes for particle actin nucleation factor, and RhoC were colocalized at the phagocytic degradation (Downey et al., 1999; Fairn and Grinstein, 2012). cups. Similar to what was seen for RhoC, mDia1 silencing through Rho family GTPases are small GTP-binding proteins that primarily RNAi inhibited phagosome formation. Additionally, the coexpression function as molecular switches by cycling between their active GTP- of mDia1 with constitutively active mutant RhoC-G14V or expression and inactive GDP-bound forms. The GTP-bound forms interact with of active mutant mDia1-ΔN3 drastically inhibited the uptake of IgG-Es. their downstream effectors and regulate cytoskeletal dynamics, thereby These data suggest that RhoC modulates phagosome formation be affecting cell polarity and motility. To date, 20 genes encoding Rho modifying actin cytoskeletal remodeling via mDia1. family members have been identified in the human genome (Vega and Ridley, 2007). During FcγR-mediated phagocytosis, several Rho KEY WORDS: RhoC, mDia1, F-actin, FcγR, Phagocytosis, family GTPases (e.g. Rac1, Rac2, Cdc42 and RhoG) are recruited to Macrophages phagocytic cups and modulate phagosome formation (Beemiller et al., 2010; Caron and Hall, 1998; Cox et al., 1997; Hoppe and Swanson, INTRODUCTION 2004; Ikeda et al., 2017; Massol et al., 1998; Tzircotis et al., 2011). Phagocytosis – a specialized form of endocytosis that permits the Among these GTPases, the GTP-bound forms of Cdc42 and Rac1 uptake of large particles (>0.5 μm in diameter) – plays an essential have been shown to stimulate the Arp2/3 complex via WASP and role in the host defense mechanism and in tissue remodeling. WAVE family proteins (Chen et al., 2010; Eden et al., 2002; Lorenzi Professional phagocytes, such as macrophages and neutrophils, et al., 2000; Park and Cox, 2009; Tsuboi and Meerloo, 2007), recognize, internalize and dispose of foreign particles, invading primarily resulting in the formation of a branched F-actin network at microorganisms and apoptotic bodies, thus contributing to the the phagocytic cup. However, the roles of other Rho family members – resolution of infections and the clearance of senescent or damaged such as RhoA, RhoB and RhoC, which promote the polymerization of cells. Phagocytosis of a particle is initiated by the binding of that unbranched F-actin – in phagosome formation have not been fully particle to a specific cell surface receptor, including Fcγ receptors examined. These GTPases share 88% amino acid sequence identity (FcγRs), complement receptors (CRs) and scavenger receptors (Wheeler and Ridley, 2004) but appear to have different functions (Freeman and Grinstein, 2014). Phagocytosis via FcγRs, the best- (Vega et al., 2011). RhoA and RhoC are localized to the plasma characterized pathway of phagocytosis, entails actin polymerization membrane and cytoplasm, while RhoB is observed in the endosomal and reorganization, which drive the extension of pseudopods around membranes and controls endosomal trafficking (Adamson et al., IgG-opsonized particles to form phagocytic cups (Araki et al., 1996, 1992; Heasman and Ridley, 2008). Several lines of evidence indicate that RhoA, but not RhoC, is essential for CR3 (integrin αMβ1)-mediated phagocytosis in macrophages (Caron and Hall, Department of Histology and Cell Biology, School of Medicine, Kagawa University, 1998; Kim et al., 2012; Tzircotis et al., 2011; Wiedemann et al., Miki, Kagawa 761-0793, Japan. 2006). RhoB has been reported to be expressed in macrophages and *Authors for correspondence ([email protected]; [email protected] be involved in mannose receptor-mediated phagocytosis (Zhang u.ac.jp). et al., 2005). Importantly, the involvement of RhoA and RhoC in Y.E., 0000-0003-0457-8195; N.A., 0000-0001-6160-210X FcγR-mediated phagocytosis remains ill defined. Mammalian diaphanous-related formins act as Rho GTPase

Received 16 February 2017; Accepted 1 November 2017 effectors and induce de novo formation of unbranched actin Journal of Cell Science

4168 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 4168-4179 doi:10.1242/jcs.202739 filaments. The formin proteins mDia1/2 (encoded by DIAPH1 and antibody in an immunofluorescence study (Fig. S2). RAW264 cells DIAPH2, respectively) and FMNL1 are involved in several types of were first incubated with human IgG (hIgG)-coated beads for 10 min, phagocytosis (Brandt et al., 2007; Colucci-Guyon et al., 2005; Naj and then fixed and immunostained with the anti-RhoC antibody. et al., 2013; Seth et al., 2006). During FcγR-mediated phagocytosis, Immunofluorescence staining demonstrated that endogenous RhoC FMNL1 is recruited to phagocytic cups in a Cdc42-dependent localized to the phagocytic cups extending along the surfaces of the manner and regulates phagosome formation (Seth et al., 2006). In hIgG-coated particles (Fig. 1D). In contrast, endogenous RhoA was contrast, mDia1/2 – major downstream effectors of RhoA, RhoB scarcely observed at the forming phagosomes. and RhoC – are primarily implicated in CR3-mediated phagocytosis in macrophages (Colucci-Guyon et al., 2005). Intriguingly, in RhoC is transiently activated during FcγR-mediated neutrophils, mDia1 has been shown to be essential for both CR3- phagocytosis and FcγR-mediated phagocytosis (Shi et al., 2009). Therefore, the To quantitatively monitor the activation levels of endogenous RhoC participation of mDia1 in FcγR-mediated phagocytosis in during FcγR-mediated phagocytosis, we took advantage of the macrophages should be investigated. GTP-dependent interaction of RhoC with the Rhotekin Rho- In the present study, by using live-cell imaging, we found that binding domain (RBD) (Ren et al., 1999). We performed a GST RhoC, but not RhoA, is transiently recruited to actin-enriched pulldown assay based on GST-fused RBD to measure the amount of phagocytic cups during FcγR-mediated phagocytosis. Furthermore, the GTP-bound form of RhoC present during phagocytosis in the expression of RhoC or mDia1 mutants, RNAi-based knockdown RAW264 macrophages. As shown in Fig. 2, the activation of RhoC and CRISPR/Cas-mediated knockout (KO) analyses revealed that was readily detected and reached a maximum at ∼2 min after RhoC regulates phagosome formation via mDia1 in macrophages. incubation with IgG-Es. The amount of the GTP-bound form of Our study provides novel insight into the importance of unbranched RhoC then returned to its basal level within 30 min. Although RhoA F-actin remodeling via RhoC/mDia1 signaling in the uptake of was activated during phagocytosis of IgG-Es, the activation of particles during FcγR-mediated phagocytosis. RhoA occurred slightly later than did that of RhoC (Fig. S3). These findings indicate that endogenous RhoC is transiently activated RESULTS during phagosome formation. RhoC, but not RhoA, is recruited to phagocytic cups during FcγR-mediated phagocytosis Expression of RhoC mutants or depletion of endogenous Previous reports have shown that several Rho GTPases (Cdc42, RhoC suppresses FcγR-mediated phagocytosis Rac1, Rac2 and RhoG) are involved in FcγR-mediated The localization of RhoC and the transient activation of this GTPase phagocytosis (Cox et al., 1997; Hoppe and Swanson, 2004; Ikeda at the early stage of phagocytosis strongly imply that RhoC is et al., 2017; Massol et al., 1998; Tzircotis et al., 2011). However, the involved in phagosome formation. To test the functional contribution involvement of two other Rho family members, RhoA and RhoC, of RhoC to the uptake of IgG-Es, we examined the effects of RhoC during FcγR-mediated phagocytosis has not been fully investigated. mutant expression on the binding of IgG-Es to the cell surface and on RhoA is expressed in RAW264 macrophages and bone marrow- their internalization into phagosomes. We transiently transfected derived macrophages (BMMs) (Kim et al., 2012; Nakaya et al., RAW264 cells with wild-type (wt) RhoC-wt, the constitutively active 2006), while RhoC is highly expressed in RAW264 cells and is mutant RhoC-G14V or the dominant-negative mutant RhoC-T19N. detectable at low levels in BMMs by western blotting (data not Quantitative assays of the binding and phagocytosis of IgG-Es were shown). To examine the spatiotemporal dynamics of Rho GTPases then performed in cells expressing each RhoC allele. As shown in during FcγR-mediated phagocytosis, RAW264 macrophages Fig. 3, the expression of GFP–RhoC-G14V slightly decreased the coexpressing GFP–RhoA and TagRFP–RhoC were allowed to binding of IgG-Es to the cells (P<0.05), whereas the binding of IgG- phagocytose IgG-opsonized erythrocytes (IgG-Es) and analyzed by Es was not affected by the expression of RhoC-wt or RhoC-T19N. live-cell imaging with a confocal laser microscope. Prior to Importantly, the expression of either GFP–RhoC-G14V or GFP– phagocytosis, RhoA and RhoC were mainly found in the cytosol, RhoC-T19N inhibited the phagocytosis of IgG-Es, in contrast to as previously reported in other cell types (Adamson et al., 1992). expression of the GFP (mock) control. After the binding of IgG-Es to the cells, RhoC, but not RhoA, was To further substantiate the significance of RhoC GTPase in recruited to the membranes of the phagocytic cups extending along phagosome formation, we adopted the RNAi approach to the surface of the IgG-Es (Fig. 1A, t=2 min; Movie 1). RhoC knockdown endogenous RhoC in RAW264 cells. The cells were subsequently dissociated from the membranes of the nascent transiently transfected with plasmids coding for several short phagosomes (Fig. 1A, t=8 min). Time-lapse imaging showed that hairpin RNAs (shRNAs). As shown in Fig. 4A, the expression of RhoC was localized in the membranes of linear and circular ruffles each RhoC shRNA (GI557339 or GI557340) decreased RhoC (precursor forms of macropinosomes), regardless of the addition of protein levels, as assessed by immunostaining with the anti-RhoC IgG-Es to the cells, as previously reported in other cell species antibody. We then performed a quantitative phagocytosis assay on (Zawistowski et al., 2013). We confirmed that the expression of these transfectants. The efficiency of phagocytosis in cells expressing RhoA does not affect the localization of RhoC (Fig. 1A; Fig. S1). To RhoC shRNAs was compared with that in non-transfected control ascertain the specific recruitment of RhoC to the phagocytic cups, cells. The depletion of endogenous RhoC by shRNA inhibited we performed line-scan analysis of the fluorescence intensities of phagocytosis to a degree comparable with that observed in cells GFP–RhoA and TagRFP–RhoC and quantified their fluorescence expressing dominant-negative mutant RhoC-T19N (Fig. 4B and intensities. As shown in Fig. 1B,C, RhoC clearly accumulated in the Fig. 3, respectively). Similar results were obtained upon siRNA- membranes of phagocytic cups, in contrast with RhoA. mediated RhoC silencing in RAW264 cells (Fig. S4). In contrast, To further examine the localization of endogenous RhoC in siRNA-mediated knockdown of RhoA expression had no observable RAW264 macrophages during FcγR-mediated phagocytosis, effect on phagosome formation (Fig. S5). immunocytochemical analysis using a monoclonal antibody against We have shown the key role of RhoC in phagosome formation

RhoC was performed. First, we validated the specificity of the RhoC through experiments where the dominant-negative mutant RhoC- Journal of Cell Science

4169 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 4168-4179 doi:10.1242/jcs.202739

Fig. 1. RhoC, but not RhoA, accumulates in the phagocytic cups during FcγR-mediated phagocytosis. (A) Live RAW264 cells coexpressing GFP–RhoA (green) and TagRFP–RhoC (red) were put into contact with IgG-Es and observed by confocal laser microscopy. Phase-contrast images are shown in the top panels. The elapsed time is indicated at the top left. The binding of IgG-Es to the cell surface is set as time 0. The insets show higher magnification images of the indicated areas of the cells. TagRFP–RhoC was recruited to the membranes of the phagocytic cups, whereas GFP–RhoA did not accumulate. Representative images from three independent experiments are shown. The corresponding movie is Movie 1. Scale bar: 5 μm. (B) Line-scan analysis performed using MetaMorph software shows the fluorescence intensities of GFP–RhoA (green) and TagRFP–RhoC (red) at the position of the line in the enlarged image of the boxed region in Fig. 1A, 2 min. Strong fluorescent signals of TagRFP–RhoC were detected at the phagocytic cups (arrowheads). (C) Quantitation of the accumulation levels of RhoC and RhoA at the phagocytic cup. Maximum fluorescence intensity values were measured at the phagocytic cup in each cell coexpressing TagRFP–RhoC and GFP–RhoA or expressing TagRFP (Mock). The fluorescence intensity of TagRFP–RhoC, GFP–RhoA or TagRFP was normalized to that of a region in the cytoplasm. Values represent the means±s.e.m. of three independent replicates (n=3; 30 phagocytic cups in more than five cells in each condition were assessed per replicate). *P<0.05 (one-way ANOVA followed by Tukey’s test). (D) RAW264 macrophages were incubated with 2-μm hIgG-coated beads for 10 min before fixation and immunostained with an anti-RhoC antibody, an anti-RhoA antibody or an isotype-matched control antibody. Phase-contrast images are shown. The insets are magnifications of the boxed areas. Note the recruitment of endogenous RhoC to the phagocytic cups. Scale bars: 5 μm.

T19N was overexpressed or endogenous RhoC was knocked down. generated RhoC-deficient RAW264 cells by using the CRISPR/Cas To further confirm our findings and to exclude the possibility that system. Two guiding RNA (gRNA) sequences were tested and shown phagocytosis was inhibited by undesired off-target effects, we to successfully deplete RhoC protein. As shown in Fig. 4C, two KO Journal of Cell Science

4170 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 4168-4179 doi:10.1242/jcs.202739

Fig. 3. Effect of constitutively active and dominant-negative RhoC mutant expression on FcγR-mediated phagocytosis and the binding of IgG-Es. To quantify FcγR-mediated phagocytosis, RAW264 macrophages transfected with GFP (mock), GFP-RhoC-wt, GFP-RhoC-G14V or GFP-RhoC-T19N were incubated with IgG-Es for 20 min at 37°C. The cells were fixed after disruption of the extracellularly exposed IgG-Es. The efficiency of phagosome formation (gray bars) was calculated based on 50 transfected cells and 50 non- transfected cells. The results are expressed as a percentage of control (non- transfected) cells. The means±s.e.m. of three independent experiments are plotted. For the binding assay, RAW264 cells transfected with each indicated Fig. 2. RhoC is transiently activated during phagosome formation. construct were incubated with IgG-Es for 30 min at 4°C. After a brief washing, RAW264 macrophages were incubated with or without (0 min) IgG-Es for the cells were fixed. The efficiency of IgG-Es binding to the cells (open bars) various times at 37°C. Cell lysates were prepared and incubated with GST or was calculated based on 50 transfected cells and 50 non-transfected cells. The GST–RBD. The proteins associated with GST (top row, right-most lane) or data are expressed as the means±s.e.m. of three independent experiments. GST–RBD (top row, other lanes) were pulled down by using glutathione– *P<0.05; **P<0.01 compared to GFP-transfected cells (mock) (one-way Sepharose beads and analyzed by western blotting for RhoC. The lane for GST ANOVA followed by Dunnett’s test). is a negative control for the pulldown assay. The lane labeled GST-RBD next to GST is a positive control. The middle rows show aliquots of the total cell lysates. – Ponceau S staining was used to visualize GST RBD and GST (bottom rows). phagosomes to the cytosol at almost the same time (Fig. 5A). Similar Note the increase in RhoC-GTP levels during FcγR-mediated phagocytosis. The results were obtained when we used cells expressing GFP–RhoC and intensity of the protein bands was measured by densitometry quantitative – analysis of RhoC-GTP and is shown as mean±s.e.m. (n=3) in the corresponding TagRFP LifeAct, which labels F-actin (data not shown). To further bar graph. The protein band intensity of RhoC-GTP was normalized to that of dissect the functional localization of RhoC and actin at the phagocytic total RhoC (GTP-bound plus GDP-bound forms). **P<0.01 compared to time 0 cups, RAW264 cells expressing GFP–RhoC were incubated with (one-way ANOVA followed by Dunnett’s test). IgG-Es and stained with Rhodamine–phalloidin to visualize F-actin structures. Consistent with our live-cell imaging findings, clones did not show any detectable RhoC protein. The expression of GFP–RhoC was localized to F-actin-rich phagocytic cups during RhoA was not affected in these RhoC-KO cells. We also performed FcγR-mediated phagocytosis (Fig. 5C, arrows). immunocytochemical analysis with the monoclonal antibody against Fig. 3 shows that the expression of constitutively active mutant RhoC. No reactivity was observed in RhoC-deficient cells (Fig. S6). RhoC-G14V or dominant-negative mutant RhoC-T19N inhibits RhoC-KO cells showed an increase in cell spreading area compared FcγR-mediated phagocytosis. We next addressed the role of RhoC to that in (control) wild-type cells. Predictably, quantitative analysis in the regulation of actin cytoskeletal remodeling. RAW264 cells of phagocytosis showed that the uptake of IgG-Es was inhibited in the were transfected with RhoC-G14V, RhoC-wt or RhoC-T19N and RhoC-deficient cells (Fig. 4D). These results suggest that RhoC is an then stained with rhodamine phalloidin. Confocal microscopy important component of FcγR-mediated phagocytosis. imaging revealed that the expression of constitutively active mutant RhoC-G14V induces an abnormally thickened layer of cortical F- RhoC regulates the remodeling of the actin cytoskeleton actin underneath the plasma membrane (Fig. 6A, arrows), whereas during phagosome formation the expression of RhoC-wt or dominant-negative mutant RhoC- Rho GTPases such as Rac1 and Cdc42 are known to spatiotemporally T19N does not (Fig. 6A, arrowheads). Intriguingly, the actin-driven remodel the actin cytoskeleton during FcγR-mediated phagocytosis pseudopod extension required to form phagocytic cups was severely (Ikeda et al., 2017; Swanson, 2008). Previous reports have shown that impaired in cells expressing the constitutively active RhoC mutant RhoC regulates actin-rich lamellipodial protrusion during tumor cell (Fig. 6B, arrows). In contrast, phagocytic cup formation was invasion, which requires a dynamic reorganization of the actin frequently observed in cells expressing RhoC-wt or the dominant- cytoskeleton (Donnelly et al., 2014). These facts prompted us to negative RhoC mutant (Fig. 6B, arrowheads). These data suggest examine the involvement of RhoC in the remodeling of the actin that RhoC modulates the actin cytoskeletal remodeling required to cytoskeleton during phagocytosis. We first analyzed the form phagosomes during FcγR-mediated phagocytosis. spatiotemporal relationship between RhoC and actin dynamics in live RAW264 cells coexpressing GFP–RhoC and TagRFP–actin mDia1 functions as a downstream effector of RhoC and during FcγR-mediated phagocytosis. Time-lapse imaging showed controls phagosome formation during FcγR-mediated that both proteins were colocalized and accumulated at the phagocytic phagocytosis cups at the same time (Fig. 5A,B; Movie 2). Subsequently, the mDia1, mDia2 and mDia3 are major effectors of Rho GTPases proteins were relocated from the membranes of the newly formed (e.g. RhoA, RhoB and RhoC) and are involved in unbranched Journal of Cell Science

4171 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 4168-4179 doi:10.1242/jcs.202739

Fig. 4. Effect of RhoC depletion on FcγR-mediated phagocytosis. (A) RAW264 macrophages expressing each indicated shRNA were fixed and immunostained with the antibody against RhoC (red). Cells transfected with shRNA constructs were identified by GFP fluorescence (green). Note the marked decrease of RhoC immunoreactivity in cells expressing RhoC shRNAs. Phase-contrast images are also shown. Scale bars: 5 μm. (B) The efficiency of IgG-E uptake was calculated based on 50 cells transfected with RhoC shRNA or control shRNA construct (mock) and 50 non-transfected cells. The values expressed as a percentage of control (non-transfected) cells represent the means±s.e.m. of three independent experiments. *P<0.05 compared to mock- transfected cells (one-way ANOVA followed by Dunnett’s test). (C) RAW264 cells transfected with pSpCas9(BB)-2A-puro plasmid (mock) or pSpCas9(BB)-2A- puro-RhoC encoding Cas9 protein and gRNA for RhoC were diluted and selected for single clones with puromycin. Each clone was then expanded without puromycin. The knockout of RhoC protein was verified by western blotting using the antibody against RhoC. RhoA and GAPDH were used as internal controls. (D) The efficiency of IgG-E phagocytosis was calculated based on 50 cells knocked out for RhoC and 50 cells transfected with pSpCas9(BB)-2A-puro plasmid (mock) or 50 non-transfected (control) cells. The results are expressed as the mean±s.e.m. percentage compared with control cells for four independent experiments. *P<0.05 compared to mock transfected cells (one-way ANOVA followed by Dunnett’s test).

F-actin nucleation and elongation (Kühn and Geyer, 2014). Based on the above data, we validated the effects of mDia1 and Previous reports have shown that mDia1 specifically interacts RhoC expression on FcγR-mediated phagocytosis and the binding with and is activated by RhoA, RhoB and RhoC. In contrast, of IgG-Es. In a quantitative assay of phagocytosis, the expression of mDia2andmDia3areactivatedbyotherRhofamilymembers, GFP–mDia1-wt alone had no effect on the uptake of IgG-Es such as Cdc42 (Young and Copeland, 2010). In macrophages, (Fig. 8A). Importantly, both the coexpression of mDia1-wt with the mDia1 primarily participates in CR3-dependent phagocytosis constitutively active mutant RhoC-G14V and the expression of (Colucci-Guyon et al., 2005). Interestingly, mDia1 has been mDia1-ΔN3 (a constitutively active mutant of mDia1) drastically reported to be required for both FcγR- and CR3-mediated inhibited phagosome formation. In addition, the efficiency of phagocytosis in neutrophils (Shi et al., 2009). Therefore, we particle binding was slightly reduced in cells coexpressing mDia1- examined the possible role of mDia1 as a downstream effector of wt with RhoC-G14V or expressing mDia1-ΔN3; in contrast, the RhoC in regulating FcγR-mediated phagocytosis in macrophages. expression of mDia1-wt alone did not affect the binding of IgG-Es Time-lapse observations of live cells coexpressing GFP–mDia1 to the cell. Confocal microscopic imaging showed that the and TagRFP–RhoC showed that mDia1 and RhoC are distributed expression of the constitutively active mutant of mDia1 promotes throughout the cytosol before the onset of phagocytosis. After the a thickened layer of cortical F-actin (Fig. S8, arrows), whereas the binding of IgG-Es to the cell, both proteins accumulated at the expression of mDia1-wt does not (Fig. S8, arrowheads). We next same time and were colocalized at the phagocytic cups (Fig. 7A; addressed the role of endogenous mDia1 in FcγR-mediated Movie 3). Line-scan analysis found that the RhoC and mDia1 phagocytosis by using an siRNA approach. As shown in Fig. 8B, fluorescence intensities were correlated (Fig. 7B). Importantly, the the transfection of mDia1 siRNA decreased in its expression in recruitment of mDia1 to phagocytic cup formation sites was RAW264 macrophages. Notably, the efficiency of IgG-E uptake enhanced by the coexpression of mDia1 with RhoC (Fig. 7A,C,D; was reduced in mDia1-knockdown cells, whereas that of IgG-Es Fig. S7). In contrast, the coexpression of mDia1 with RhoA did not binding was not affected (Fig. 8C). Moreover, treatment with promote the relocation of mDia1 to the phagocytic cups. After SMIFH2, an inhibitor of formin-mediated actin assembly, also closure of the phagocytic cups, mDia1 and RhoC dissociated inhibited phagosome formation (Fig. 8D). Taken together, these simultaneously from the nascent phagosomes. findings indicate that mDia1 – a downstream effector of RhoC – is Journal of Cell Science

4172 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 4168-4179 doi:10.1242/jcs.202739

Fig. 5. Confocal imaging showing the spatiotemporal relationship between RhoC and actin during phagosome formation. (A) Live RAW264 cells coexpressing GFP–RhoC (green) and TagRFP–actin (red) were allowed to interact with IgG-Es and observed by confocal laser microscopy. Phase- contrast images are also shown (top panels). The elapsed time is indicated at the top left. Note the colocalization of RhoC and actin at the phagocytic cups. Representative images from three independent experiments are shown. The corresponding movie is Movie 2. Scale bar: 5 μm. (B) Quantification of the levels of RhoC and actin accumulation at the phagocytic cup. Maximum fluorescence intensity values were measured at the phagocytic cup in each cell coexpressing GFP–RhoC and TagRFP–Actin or expressing GFP (mock). The fluorescence intensity of GFP–RhoC, TagRFP–Actin or GFP was normalized to that of a region in the cytoplasm. Values represent the means±s.e.m. of three independent replicates (n=3; 30 phagocytic cups in more than 5 cells in each condition were assessed per replicate). *P<0.05; **P<0.01 (one-way ANOVA followed by Tukey’s test). (C) RAW264 macrophages transfected with GFP–RhoC (green) were incubated with IgG-Es for 10 min at 37°C, fixed and stained with Rhodamine–phalloidin (red). Consistent with the above finding, GFP– RhoC was localized to F-actin-rich phagocytic cups (arrows). Scale bar: 5 μm.

an important component of FcγR-mediated phagocytosis and In contrast to RhoC, RhoA did not accumulate at the phagocytic cups. regulates phagosome formation. Moreover, the silencing of RhoA had no effect on phagosome formation. These data suggest that RhoA is not involved in FcγR- DISCUSSION dependent phagosome formation. Intriguingly, we found that both The present study provides the first evidence that RhoC is involved RhoA and RhoC are activated during phagocytosis. It is noteworthy in FcγR-mediated phagocytosis. Our live-cell imaging and that the activation of RhoA occurs slightly later than that of RhoC. immunocytochemical analysis showed that RhoC is recruited to the Previous reports have shown that RhoA is activated during FcγR- membranes of phagocytic cups and then dissociates from the mediated phagocytosis and regulates superoxide formation (Kim membranes of nascent phagosomes. Importantly, our RhoC-GTP et al., 2004; Li et al., 2012). Therefore, RhoA may play a major role in pulldown assay demonstrated that RhoC is transiently activated the regulation of superoxide formation during FcγR-mediated during phagosome formation. Furthermore, we found that the phagocytosis. RhoB activation has been reported as being required expression of constitutively active mutant RhoC-G14V or for mannose receptor-mediated phagosome formation (Zhang et al., dominant-negative mutant RhoC-T19N suppresses FcγR-mediated 2005). However, the precise localization of RhoB during FcγR- phagocytosis. We therefore postulate that the activation–inactivation mediated phagocytosis remains unclear. Investigating the role of cycling of RhoC is required for the uptake of IgG-opsonized particles. RhoB in phagocytosis should be the subject of further research. Journal of Cell Science

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The functional molecule regulating the spatiotemporal translocation of RhoC to the phagocytic cups remains to be identified. Our confocal microscopy observations revealed that RhoC, but not its close homolog RhoA, accumulates in the membranes of phagocytic cups and readily dissociates from the internalized phagosomes. Furthermore, the pulldown assay for RhoC-GTP demonstrated that RhoC is predominantly activated during phagosome formation. These results imply that specific guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for RhoC are involved in FcγR-mediated phagocytosis. However, these upstream regulators of RhoC in the process of phagosome formation are currently unknown. Further studies should identify the specific GEFs and GAPs for RhoC to better understand the molecular details of the signal transduction pathway during phagosome formation. Recently, Patel et al. have reported that RhoC shows a higher degree of membrane association than does RhoA (Patel et al., 2016). In their study, these authors demonstrated that arginine 188 of RhoC promotes membrane binding. In contrast with RhoC, RhoA has a serine residue at position 188 in place of an arginine residue. During phagosome formation, protein kinase A (PKA) is targeted to nascent phagosomes (Pryzwansky et al., 1998). Importantly, RhoA phosphorylation on serine 188 by PKA has been shown to induce RhoA translocation from membranes to the cytosol (Forget et al., 2002; Lang et al., 1996). PKA is thus thought to phosphorylate serine 188 of RhoA in the process of FcγR-mediated phagocytosis, thereby inhibiting RhoA recruitment to the phagocytic cups. The local remodeling of the actin cytoskeleton during phagosome formation is regulated by the recruitment and activation of the actin- nucleating activity of Arp2/3. The activation of Cdc42 and Rac1 has been shown to stimulate the Arp2/3 complex via WASP and WAVE family proteins to form a branched F-actin network at the phagocytic cup (Cox et al., 1997; Lorenzi et al., 2000; Massol et al., 1998; May et al., 2000; Park and Cox, 2009; Tsuboi and Meerloo, 2007). Interestingly, the GTP-bound form of Cdc42 also functions as an upstream regulator of the formin protein FMNL1, which promotes the formation of unbranched actin filaments during FcγR-mediated phagocytosis (Otomo et al., 2005; Romero et al., 2004; Seth et al., 2006). Until now, the modulation of unbranched actin polymerization has been comparatively less studied than the regulation of the branched F-actin network at the phagocytic cup in FcγR-mediated phagocytosis. We found that the actin-driven pseudopod extension required to form phagocytic cups is severely inhibited in cells expressing the constitutively active mutant RhoC- G14V, which induces an abnormal cortical F-actin layer and potentially promotes the polymerization of unbranched actin filaments. Moreover, the expression of RhoC-G14V slightly reduces the binding of IgG-Es to the cell. These data suggest that the remodeling of unbranched cortical F-actin underneath the Fig. 6. Effect of RhoC mutant expression on actin polymerization and plasma membrane is required for the process of particle binding and phagocytic cup formation. (A) RAW264 cells expressing GFP–RhoC-G14V, subsequent phagocytic cup formation. At present, the significance GFP–RhoC-wt, GFP–RhoC-T19N or GFP (green) were fixed and stained with of cortical F-actin remodeling during the initial stage of Rhodamine–phalloidin (red) to visualize F-actin. The expression of phagocytosis remains unknown. This remodeling may release constitutively active mutant RhoC-G14V increased cortical F-actin levels monomeric G-actin from cortical F-actin for incorporation into new (arrows), whereas the expression of RhoC-wt, the dominant-negative mutant RhoC-T19N or the GFP control had no effect on the cellular content of filaments to form phagocytic cups. Alternatively, in the process of polymerized F-actin (arrowheads). Scale bars: 5 μm. (B) RAW264 phagocytic target capture, the remodeling of cortical F-actin may macrophages transfected with each indicated construct were incubated with promote the mobility of FcγRs for particle binding (Jaumouille and IgG-Es for 10 min at 37°C, fixed and stained with rhodamine-phalloidin. Grinstein, 2011; Mao et al., 2009). Although phagocytic cup formation was severely impaired in cells expressing It is important to determine both the downstream effector of GFP-RhoC-G14V (arrows), pseudopod extension along the surfaces of IgG- RhoC during FcγR-mediated phagocytosis and how RhoC Es was occasionally observed in cells expressing GFP-RhoC-wt, GFP-RhoC- T19N or GFP control (arrowheads). Scale bars: 5 μm. mechanistically regulates phagosome formation. Our time-lapse imaging demonstrated that RhoC and mDia1, one of the major Journal of Cell Science

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Fig. 7. Time-lapse images showing localization of mDia1 and RhoC during FcγR-mediated phagocytosis. (A) Live RAW264 cells coexpressing GFP–mDia1 (green) and TagRFP–RhoC (red) were allowed to contact IgG-Es and observed by confocal laser microscopy. Note the colocalization of mDia1 and RhoC at the phagocytic cups. The corresponding movie is Movie 3. Scale bar: 5 μm. (B) A line-scan analysis performed with MetaMorph software shows the fluorescence intensities of GFP–mDia1 (green) and TagRFP–RhoC (red) at the position of the line in the enlarged image of the boxed region (Fig. 7A, 3 min). (C) RAW264 macrophages coexpressing TagRFP–mDia1 (red) and GFP–RhoA (green) were incubated with IgG-Es and observed by confocal laser microscopy. Scale bar: 5 μm. (D) Quantification of the recruitment levels of mDia1 to the phagocytic cup. Maximum TagRFP–mDia1 fluorescence intensity values were measured at the phagocytic cup in cells coexpressing GFP–RhoC, GFP–RhoA or GFP. The fluorescence intensity of TagRFP–mDia1 was normalized to that of a region in the cytoplasm. Values represent the means±s.e.m. of three independent replicates (n=3; 30 phagocytic cups in more than five cells in each condition were assessed per replicate). *P<0.05 (one-way ANOVA followed by Tukey’s test). effectors of Rho GTPases, are colocalized at the phagocytic cups. layer underneath the plasma membrane. Similar to what was seen Moreover, a quantitative assay of phagocytosis found that both the with RhoC-G14V, mDia1-ΔN3 expression induced an abnormal F- coexpression of mDia1-wt with the GTP-locked mutant RhoC- actin layer in RAW264 macrophages, whereas the expression of G14V and the expression of activated mutant mDia1-ΔN3 result in a mDia1-wt did not. Collectively, these findings indicate that mDia1 remarkable decrease in the rate of IgG-Es uptake. A previous report is activated at the phagocytic cups in a RhoC-GTP-dependent has indicated that the expression of activated mutant mDia1-ΔN3 manner and that it functions as a downstream effector of RhoC induces a dramatic increase in the basal level of polymerized F-actin during FcγR-mediated phagocytosis. We observed that RhoC (Vicente-Manzanares et al., 2003). In this study, we revealed that the depletion increases the cell spreading area in RAW264 expression of constitutively active mutant RhoC-G14V, which macrophages. A previous report has shown that RhoC knockdown activates mDia1, facilitates the formation of an abnormal F-actin facilitates cell spreading and induces Rac1 activation around the Journal of Cell Science

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Fig. 8. Effect of mDia1 expression and downregulation on FcγR-mediated phagocytosis and the binding of IgG-Es. (A) RAW264 macrophages expressing the GFP–mDia1 construct and/or TagRFP–RhoC-G14V were incubated with IgG-Es for 20 min at 37°C. The efficiency of phagosome formation (gray bars) and of IgG-E binding (open bars) was calculated based on 50 transfected cells and 50 non-transfected cells. Cdc42-G12V (constitutively active mutant) was used as a positive control for the assay. The results are expressed as a percentage of that in control (non-transfected) cells. The data represent the means±s.e.m. of three independent experiments. *P<0.05; ***P<0.001 versus corresponding mock-transfected cells. ##P<0.01; ###P<0.001 versus corresponding cells transfected with mDia1-wt. †P<0.05 versus corresponding cells transfected with Cdc42-G12V. ‡P<0.05 versus corresponding cells transfected with mDia1-wt and Cdc42-G12V (one-way ANOVA followed by Tukey’s test). (B) RAW264 cells were transfected with mDia1 siRNA or mock siRNA. After 48 h, the cells were collected. The knockdown of mDia1 protein was verified by western blotting using the antibody specific for mDia1. mDia2 levels were used as an inter2nal control. (C) The efficiency of IgG-Es phagocytosis (gray bars) and of IgG-E binding to the cells (open bars) was calculated based on 50 cells transfected with mDia1 siRNA and 50 cells transfected with mock siRNA or 50 non-transfected (control) cells. The results are expressed as a percentage of that in control (non-transfected) cells. The means±s.e.m. of four independent experiments are plotted. **P<0.01 compared to cells transfected with mock siRNA (Student’s t-test). (D) The efficiency of IgG-E phagocytosis (gray bars) and of IgG-E binding to the cells (open bars) was calculated based on 50 cells treated with 10 μM SMIFH2 and 50 cells treated with DMSO (mock) or 50 untreated (control) cells. The results are expressed as a percentage of that in control (untreated) cells. The means±s.e.m. of four independent experiments are plotted. **P<0.01 compared to cells treated with DMSO (Student’s t-test). periphery in the lamellipodia of PC3 cells (Vega et al., 2011). antibody (ARH04, Cytoskeleton, Denver, CO), mouse monoclonal anti- Recently, we have demonstrated that the expression of a mDia1 antibody (clone 51) (610849, BD Biosciences, San Jose, CA), rabbit constitutively active Rac1 mutant suppresses FcγR-mediated polyclonal anti-mDia2 antibody (C-terminal region) (DP3491, ECM phagocytosis (Ikeda et al., 2017). RhoC may also modulate Rac1 Biosciences, Versailles, KY), mouse monoclonal anti-glyceraldehyde-3- activity, thereby regulating phagosome formation. phosphate dehydrogenase (GAPDH) antibody (AM4300, Ambion, Huntingdon, UK), rabbit anti-sheep erythrocyte IgG (ICN55806, Organo In conclusion, although RhoC may have multiple downstream γ Teknika-Cappel, Durham, NC), goat anti-rabbit-IgG conjugated to Alexa effectors involved in Fc R-mediated phagocytosis, our study Fluor 594 (A11037, Molecular Probes, Eugene, OR), goat anti-mouse IgG emphasizes the importance of RhoC in the modulation of conjugated to Alexa Fluor 488 (A11029, Molecular Probes, Eugene, OR), phagosome formation by activating mDia1, which can regulate anti-mouse- and anti-rabbit-IgG conjugated to horseradish peroxidase the remodeling of cortical F-actin. (HRP) (W4021 and W4011, Promega, Madison, WI), purified human IgG (I4506, Sigma, St. Louis, MO), monoclonal rabbit IgG isotype control MATERIALS AND METHODS (DA1E) (3900, Cell Signaling Technology, Danvers, MA), monoclonal Reagents mouse IgG isotype control (G3A1) (5415, Cell Signaling Technology, Bovine serum albumin (BSA) and Dulbecco’s modified Eagle’s medium Danvers, MA), Rhodamine-conjugated phalloidin (Molecular Probes, (DMEM) were purchased from Sigma (St. Louis, MO). Fetal bovine serum Eugene, OR), SMIFH2 (Merck Millipore, Nottingham, UK) and 2-μm- (FBS) was obtained from BioSolutions International (Melbourne, diameter polystyrene microspheres (Polysciences, PA) were commercially Australia). Rabbit monoclonal anti-RhoC antibody (D40E4) (3430, Cell obtained. Other reagents were purchased from Wako Pure Chemicals (Osaka,

Signaling Technology, Danvers, MA), mouse monoclonal anti-RhoA Japan) or Nakalai Tesque (Kyoto, Japan) unless otherwise indicated. Journal of Cell Science

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Cell culture were tested: 5′-GGCTGCGATCCGAAAGAAGC-3′ and 5′-CTATATA Mouse macrophage RAW264 cells (RCB0535, Tsukuba, Japan) were GCCGACATCGAAG-3′. After ligation of the synthesized sequences into cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ pSpCas9(BB)-2A-puro, the pSpCas9(BB)-2A-puro-RhoC constructs were ml penicillin and 100 μg/ml streptomycin, as described in the manuals verified by sequencing. Plasmid transfection was performed by using the of the cell line bank (ATCC , TIB-71) (growth medium). Before the Neon Transfection System. At 12 h after transfection, the cells were selected experiments, the culture medium was replaced with Ringer’s buffer (RB) with 5 μg/ml puromycin for 24 h. Subsequently, a limiting dilution of the consisting of 155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,2mM surviving cells was made. The resultant single colonies were then expanded Na2HPO4, 10 mM D-glucose, 10 mM HEPES-NaOH (pH 7.2) and without puromycin. RhoC-knockout clones were confirmed by both DNA 0.5 mg/ml BSA. sequencing and western blotting.

DNA constructs and transfection Protein expression and purification The full-length cDNA coding region of mouse RhoC was amplified by pGEX-2T-RBD encoding the GST-Rho-binding domain (RBD) (amino PCR. The fragment was cloned into the EcoRI and BamHI restriction sites acids 7–89) of Rhotekin was Addgene plasmid #15247 (deposited by of the pEGFP-C1 vector (Clontech, Palo Alto, CA). pEGFP-RhoC-T19N Martin Schwartz; Ren et al., 1999). We expressed GST or GST–RBD (dominant-negative mutant) and pEGFP-RhoC-G14V (constitutively active protein in Escherichia coli [BL21(DE3)] using methods similar to those mutant) were generated by using the QuikChange II site-directed described previously (Egami et al., 2015). Cells grown in LB medium mutagenesis kit (Stratagene, La Jolla, CA). pTagRFP-RhoC, pTagRFP- were incubated in the presence of 0.1 mM isopropyl-1-thio-β-D- RhoC-T19N and pTagRFP-RhoC-G14V were generated by the replacement galactopyranoside (15 h at 25°C). All subsequent purification steps were of EGFP with TagRFP. pTagRFP-Actin was purchased from Evrogen performed at 4°C. After centrifugation, the resulting cell pellets were (Moscow, Russia). pcDNA3-EGFP-RhoA-wt was Addgene plasmid resuspended in a buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM #12965 (deposited by Gary Bokoch; Subauste et al., 2000). YFP-Cdc42 dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 50 units/ml (V12) was kindly provided by Dr. Joel A. Swanson (University of aprotinin, 2 μg/ml leupeptin and 2 μg/ml pepstatin A) and subjected to Michigan, Ann Arbor, MI). pEGFP-Cdc42-G12V (constitutively active sonication. The cell debris was removed by centrifugation, and the resultant mutant) was generated by replacing EYFP with EGFP. The full-length supernatant was used as an E. coli lysate. Purification of the GST-fusion cDNA coding region of mouse Dia1 (mDia1) and the region encoding the proteins to near homogeneity was achieved by using glutathione-Sepharose constitutively activate mutant (amino acids 543–1182) of mouse Dia1 4B affinity chromatography (GE Healthcare, Piscataway, NJ). The purity of (mDia1-ΔN3) were amplified by PCR. The fragments were cloned into the the samples was at least 80%, as confirmed by Coomassie Brilliant Blue BglII and SalI restriction sites of the pEGFP-C1 and pTagRFP-C (Evrogen) staining of SDS-PAGE gels. vectors. All constructs were verified by sequencing prior to use. Transfection of the plasmids into RAW264 cells was performed by using GST pulldown assay and western blotting the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the RAW264 cells were washed with ice-cold phosphate-buffer saline (PBS) manufacturer’s instructions. The transfected cells were seeded onto 25-mm and suspended in lysis buffer containing 25 mM Tris-HCl pH 7.2, 150 mM coverslips and maintained in growth medium. Experiments were performed NaCl, 5 mM MgCl2, 1% NP-40, 5% glycerol and protease inhibitor cocktail 12–24 h after transfection. (Nacalai Tesque, Kyoto, Japan). The cell lysates were briefly sonicated at 4°C and separated from the pellets after centrifugation at 12,100 g for Silencing of endogenous RhoC through shRNA 15 min. Protein concentrations were estimated with the BCA protein assay Short hairpin RNAs (shRNAs) were used to knockdown RhoC expression in reagent. Glutathione–Sepharose beads coupled to GST or GST–RBD were RAW264 cells. RhoC shRNAs cloned into the pGFP-V-RS vector were incubated for 2 h at 4°C with 500 μg of the cell lysates. After the beads were purchased from Origene (Rockville, MD). Two different RhoC shRNAs washed four times with lysis buffer, the proteins bound to the beads were (GI557339 and GI557340) and a control shRNA (TR30008, negative analyzed on 12.5% SDS-PAGE gels followed by western blotting. The control shRNA pGFP-V-RS non-effective tGFP plasmid) were used for samples were subjected to SDS-PAGE and transferred to a polyvinylidene transfection. The transfectants were cultured for 6 days, tested for the difluoride (PVDF) membrane (Bio-Rad, Richmond, CA). Western blotting expression of RhoC through immunocytochemistry and used for the was conducted using the ECL Prime Western Blotting detection system (GE phagocytosis assay. Healthcare, Piscataway, NJ). The membrane was blocked with 5% nonfat dried milk in PBS containing 0.1% Tween 20 for 30 min at room siRNA transfection temperature and probed with anti-RhoC antibody (1:2000), anti-RhoA RAW264 cells were transfected with siRNA duplexes specific for RhoC, antibody (1:1000) or anti-GAPDH antibody (1:10,000) at 4°C overnight. RhoA, mDia1 or bock siRNA (MISSION siRNA Universal Negative After washing, the membrane was incubated with HRP-conjugated anti- Control, Sigma) by using Viromer Blue (Lipocalyx Halle, Germany) rabbit-IgG or anti-mouse-IgG secondary antibody (dilution 1:10,000) for according to the manufacturer’s instructions. Transfection of RhoC siRNA 2 h at room temperature, developed using an ECL Prime regent and exposed was performed twice. At 48 h after the initial transfection with RhoC siRNA to Hyperfilm (GE Healthcare, Piscataway, NJ). GST and GST–RBD was or mock siRNA, the cells were transfected again with the same siRNA and stained with Ponceau S. The intensity of the protein bands was measured maintained in growth medium for 72 h. The cells were then analyzed by using Photoshop CS software. western blotting and used for the phagocytosis assay. The cells transfected with RhoA, mDia1 or mock siRNA were cultured for 48 h and used for Phagocytosis assay subsequent experiments. The siRNA target sequences were: RhoC siRNA 1, Sheep erythrocytes were opsonized with rabbit anti-sheep erythrocyte IgG 5′-CAUCCUCAUGUGUUUCUCCAUUGAC-3′; RhoC siRNA 2, 5′- (1:200, Organo Teknika-Cappel) and resuspended in PBS as described AGGAUCAGUGCCUUUGGCUACCUCG-3′; RhoA siRNA 1, 5′- previously (Araki et al., 1996). For the quantitative assay of phagocytosis, GACAUGCUUGCUCAUAGUCUUC-3′ (Guilluy et al., 2011); RhoA IgG-opsonized erythrocytes (IgG-Es) were added to adherent RAW264 siRNA 2, 5′-GAAGUCAAGCAUUUCUGUC-3′ (Yoon et al., 2016); and macrophages. After 20 min of incubation with IgG-Es at 37°C, the cells on mDia1 siRNA, 5′-GCGACGGCGGCAAACAUAAGAAAUU-3′ (Yamana the coverslips were dipped into distilled water for 20 s to disrupt the et al., 2006). extracellularly exposed IgG-Es, then fixed with 4% paraformaldehyde and 0.1% glutaraldehyde for 15 min. The number of internalized IgG-Es was CRISPR/Cas-mediated knockout of RhoC counted in 50 cells randomly chosen under phase-contrast and fluorescence The pSpCas9(BB)-2A-puro (PX459) plasmid was Addgene plasmid microscopy. The phagocytic index (i.e. the mean number of IgG-Es taken up #48139 (deposited by Feng Zhang; Ran et al., 2013). For knockout per cell) was then calculated. The index obtained for the transfected cells of RhoC in RAW264 macrophages, the following gRNA sequences was divided by the index obtained for the non-transfected (control) cells and Journal of Cell Science

4177 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 4168-4179 doi:10.1242/jcs.202739 expressed as a percentage of that found for the control cells. For the binding Supplementary information assay, RAW264 cells were incubated with IgG-Es for 30 min at 4°C, briefly Supplementary information available online at washed in ice-cold PBS to remove the unbound IgG-Es and fixed. The http://jcs.biologists.org/lookup/doi/10.1242/jcs.202739.supplemental number of cell-bound IgG-Es was then counted, and the binding index (i.e. the mean number of bound IgG-Es per cell) was calculated. The binding index was expressed as a percentage of that in non-transfected (control) References cells. For immunocytochemistry, 106 polystyrene beads were washed in Adamson, P., Paterson, H. F. and Hall, A. (1992). Intracellular localization of the PBS and incubated in ∼10 mg/ml human IgG (hIgG) for 1 h at 37°C. After P21rho proteins. J. Cell Biol. 119, 617-627. Araki, N., Johnson, M. T. and Swanson, J. A. (1996). A role for phosphoinositide 3- washing with PBS, hIgG-opsonized beads were added to the adherent kinase in the completion of macropinocytosis and phagocytosis by macrophages. RAW264 cells. The cells were then incubated for 10 min at 37°C and fixed J. Cell Biol. 135, 1249-1260. with 4% paraformaldehyde. Araki, N., Hatae, T., Furukawa, A. and Swanson, J. A. (2003). Phosphoinositide-3- kinase-independent contractile activities associated with Fcgamma-receptor- Live-cell imaging and data analysis mediated phagocytosis and macropinocytosis in macrophages. J. Cell Sci. 116, 247-257. RAW264 macrophages were cultured onto 25-mm circular coverslips. Beemiller, P., Zhang, Y., Mohan, S., Levinsohn, E., Gaeta, I., Hoppe, A. D. and Each coverslip was assembled into an RB-filled chamber on the Swanson, J. A. (2010). A Cdc42 activation cycle coordinated by PI 3-kinase thermocontrolled stage (Tokai Hit, Shizuoka, Japan). Phase-contrast and during Fc receptor-mediated phagocytosis. Mol. Biol. Cell 21, 470-480. fluorescence images of live cells were sequentially acquired with an Axio Brandt, D. T., Marion, S., Griffiths, G., Watanabe, T., Kaibuchi, K. and Grosse, R. Observer Z1 inverted microscope equipped with a laser scanning unit (2007). Dia1 and IQGAP1 interact in cell migration and phagocytic cup formation. (LSM700, Zeiss) and Plan-Apochromat 63× NA 1.4 lens under the control J. Cell Biol. 178, 193-200. of ZEN2009 software (Zeiss), as previously described (Egami et al., Campellone, K. G. and Welch, M. D. (2010). A nucleator arms race: cellular control of actin assembly. Nat. Rev. Mol. Cell Biol. 11, 237-251. 2015). Time-lapse images of phase-contrast and fluorescence microscopy Caron, E. and Hall, A. (1998). Identification of two distinct mechanisms of were taken at 15 s intervals and assembled into QuickTime movies. At phagocytosis controlled by different Rho GTPases. Science 282, 1717-1721. least three examples were observed in each experiment. MetaMorph 7.8 Chen, Z., Borek, D., Padrick, S. B., Gomez, T. S., Metlagel, Z., Ismail, A. M., and Photoshop CS5 software were used to process images subsequent to Umetani, J., Billadeau, D. D., Otwinowski, Z. and Rosen, M. K. (2010). data acquisition. To quantify the accumulation levels of the proteins, the Structure and control of the actin regulatory WAVE complex. Nature 468, 533-538. maximal values of TagRFP and/or GFP signal intensity at the phagocytic Colucci-Guyon, E., Niedergang, F., Wallar, B. J., Peng, J., Alberts, A. S. and Chavrier, P. (2005). A role for mammalian diaphanous-related formins in cup were measured by using MetaMorph software. The intensity of the complement receptor (CR3)-mediated phagocytosis in macrophages. Curr. Biol. TagRFP and/or GFP signal was normalized to the fluorescence intensity of 15, 2007-2012. a region in the cytoplasm. The mean±s.e.m. values for three independent Cox, D., Chang, P., Zhang, Q., Reddy, P. G., Bokoch, G. M. and Greenberg, S. experiments were plotted. (1997). Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes. J. Exp. Med. 186, 1487-1494. Donnelly, S. K., Bravo-Cordero, J. J. and Hodgson, L. (2014). Rho GTPase Immunostaining isoforms in cell motility: Don’t fret, we have FRET. Cell Adh Migr 8, 526-534. For immunostaining with anti-RhoC antibody, RAW264 cells grown on Downey, G. P., Botelho, R. J., Butler, J. R., Moltyaner, Y., Chien, P., Schreiber, coverslips were fixed in 4% paraformaldehyde for 15 min, rinsed three times A. D. and Grinstein, S. (1999). Phagosomal maturation, acidification, and with PBS and permeabilized with 0.1% Triton X-100 in PBS for 2 min. inhibition of bacterial growth in nonphagocytic cells transfected with Fixed samples were blocked with 1% BSA in PBS, then incubated twice for FcgammaRIIA receptors. J. Biol. Chem. 274, 28436-28444. 1 h, first with anti-RhoC antibody (diluted 1:500 in 1% BSA in PBS) and Eden, S., Rohatgi, R., Podtelejnikov, A. V., Mann, M. and Kirschner, M. W. then with goat anti-rabbit-IgG conjugated to Alexa Fluor 594 (1:500). To (2002). Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418, 790-793. visualize F-actin, cells were fixed in 4% paraformaldehyde and 0.1% Egami, Y., Fukuda, M. and Araki, N. (2011). Rab35 regulates phagosome glutaraldehyde for 15 min, briefly rinsed with PBS and permeabilized with formation through recruitment of ACAP2 in macrophages during FcgammaR- 0.1% Triton X-100 in PBS for 2 min. Specimens were then stained with mediated phagocytosis. J. Cell Sci. 124, 3557-3567. Rhodamine-conjugated phalloidin (1.25 U/ml final concentration) for Egami, Y., Fujii, M., Kawai, K., Ishikawa, Y., Fukuda, M. and Araki, N. (2015). 30 min. Activation-inactivation cycling of Rab35 and ARF6 is required for phagocytosis of Zymosan in RAW264 macrophages. J. Immunol. Res. 2015, 429439. Fairn, G. D. and Grinstein, S. (2012). How nascent phagosomes mature to become Statistical analysis phagolysosomes. Trends Immunol. 33, 397-405. Two-tailed Student’s t-tests or one-way ANOVA followed by a Tukey’s test Forget, M.-A., Desrosiers, R. R., Gingras, D. and Béliveau, R. (2002). or Dunnett’s test were performed. All P-values were considered significant Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho at P<0.05. GDP dissociation inhibitor and their extraction from biological membranes. Biochem. J. 361, 243-254. Freeman, S. A. and Grinstein, S. (2014). Phagocytosis: receptors, signal Acknowledgements integration, and the cytoskeleton. Immunol. Rev. 262, 193-215. The authors would like to thank Dr. Katsuya Miyake for his helpful discussion, as well Greenberg, S., Chang, P. and Silverstein, S. C. (1993). Tyrosine phosphorylation as Mr. Kazuhiro Yokoi and Ms. Yukiko Iwabu for their skillful assistance. is required for Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med. 177, 529-534. Competing interests Groves, E., Dart, A. E., Covarelli, V. and Caron, E. (2008). Molecular mechanisms The authors declare no competing or financial interests. of phagocytic uptake in mammalian cells. Cell. Mol. Life Sci. 65, 1957-1976. Guilluy, C., Swaminathan, V., Garcia-Mata, R., O’Brien, E. T., Superfine, R. and Author contributions Burridge, K. (2011). The Rho GEFs LARG and GEF-H1 regulate the mechanical Conceptualization: Y.E., N.A.; Methodology: Y.E., K.K.; Software: Y.E.; Validation: response to force on integrins. Nat. Cell Biol. 13, 722-727. Y.E., N.A.; Formal analysis: Y.E.; Investigation: Y.E.; Resources: Y.E., K.K., N.A.; Heasman, S. J. and Ridley, A. J. (2008). Mammalian Rho GTPases: new insights Data curation: Y.E., N.A.; Writing - original draft: Y.E., N.A.; Writing - review & editing: into their functions from in vivo studies. Nat. Rev. Mol. Cell Biol. 9, 690-701. Hoppe, A. D. and Swanson, J. A. (2004). Cdc42, Rac1, and Rac2 display distinct Y.E., N.A.; Visualization: Y.E.; Supervision: Y.E., N.A.; Project administration: patterns of activation during phagocytosis. Mol. Biol. Cell 15, 3509-3519. Y.E., N.A.; Funding acquisition: Y.E., K.K., N.A. Ikeda, Y., Kawai, K., Ikawa, A., Kawamoto, K., Egami, Y. and Araki, N. (2017). Rac1 switching at the right time and location is essential for Fcgamma receptor- Funding mediated phagosome formation. J. Cell Sci. 130, 2530-2540. This study was supported by the Japan Society for the Promotion of Science (JSPS) Jaumouillé, V. and Grinstein, S. (2011). Receptor mobility, the cytoskeleton, and (KAKENHI grant number 16K08468 to Y.E., and was also supported in part by JSPS particle binding during phagocytosis. Curr. Opin. Cell Biol. 23, 22-29. KAKENHI grant number 26670094 to N.A. and grant number 26860136 to K.K.). The Kim, J.-S., Diebold, B. A., Kim, J.-I., Kim, J., Lee, J.-Y. and Park, J.-B. (2004). work was partially funded by Kagawa University Scientific Research Encourages Rho is involved in superoxide formation during phagocytosis of opsonized

Research Funding 2015. zymosans. J. Biol. Chem. 279, 21589-21597. Journal of Cell Science

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