Journal: Journal of Cell Science

Article Title: RHO binding to FAM65A regulates Golgi reorientation during cell migration

doi: 10.1242/jcs.198614

Figure Lengend Snippet: FAM65A is an adaptor protein that links RHO to CCM3, MST3 and MST4. (A) FAM65A interacts with all RHO proteins, as well as CCM3, MST3, MST4 and several YWHA isoforms. Averaged Log2 of SILAC ratios from replicates of two reciprocally labelled mixtures of GFP–FAM65A versus GFP-only anti-GFP immunoprecipitations (IP) ( Table S2 ) were plotted. Proteins that significantly ( P <0.05) interacted with GFP–FAM65A are marked in red. FAM65A, RHOA, RHOB, RHOC, CCM3, MST3, MST4 and various YWHA isoforms are depicted on the graph. FAM65A (bait) is marked in blue. H/L, heavy:light ratios. (B) Analysis of the relative stoichiometry of FAM65A-interacting proteins by iBAQ ( Table S3 ). iBAQ values of FAM65A-interacting proteins in GFP–FAM65A immunoprecipitations were subtracted by their corresponding iBAQ values in GFP-only immunoprecipitations and normalised to FAM65A levels before being averaged between two reciprocally labelled experiments. Values from two duplicate experiments were plotted. (C) Interaction of endogenous CCM3, MST3 and MST4 with FAM65A is independent of RHO binding. HeLa cells were transfected with expression vectors for GFP–FAM65A, or GFP-only as control, and subjected to TAT-C3 or mock treatment for 4 h before lysis and immunoprecipitation with anti-GFP antibody. Input lysates as well as anti-GFP immunoprecipitation eluates were subsequently analysed by immunoblotting using the indicated antibodies. Although the RHOA and YWHA interaction with FAM65A was abrogated upon TAT-C3 treatment, interactions of CCM3, MST3 and MST4 were unaffected. Quantification of protein levels in each immunoprecipitation condition relative to the input are displayed below the blots (arbitrary units). Quantifications were performed in three independent experiments. Error bars=s.d. Significance P -value was calculated using two-tailed heteroscedastic t -test analysis. n.s., not significant ( P >0.05). (D) Schematic representation of FAM65A regions. The N-terminal of FAM65A contains an HR1 domain (amino acids 138–205) and the C-terminal comprises an ARM domain (amino acids 1050–1202). (E) The N-terminus of FAM65A interacts with RHOA and YWHA proteins, whereas the C-terminal interacts with CCM3, MST3 and MST4. HeLa cells were transfected with expression vectors for GFP-tagged full-length, N-terminal-deleted or C-terminal-deleted FAM65A mutants, or GFP-only as control, and subjected to immunoprecipitation with anti-GFP antibody. Input lysates as well as anti-GFP immunoprecipitation eluates were subsequently analysed by immunoblotting using the indicated antibodies. Quantification of protein levels in each immunoprecipitation condition relative to the input are displayed below the blots (arbitrary units). Quantifications were performed on three independent experiments. Error bars=s.d. Significance P -value was calculated using two-tailed heteroscedastic t -test analysis. n.s., not significant ( P >0.05). (F) FAM65A acts as an adaptor protein, linking active RHOA to MST3 and MST4. HeLa cells were co-transfected with empty vector or an expression vector for Myc–RHOA-Q63L (constitutively active), along with GFP-tagged full-length FAM65A, or the C-terminal-deleted GFP–FAM65A mutant, or GFP-only as control, and subjected to immunoprecipitation with anti-Myc antibody. Input lysates as well as anti-Myc immunoprecipitation eluates were subsequently analysed by immunoblotting using the indicated antibodies. Ectopic expression of full-length but not the C-terminal-deleted FAM65A mutant resulted in co-immunoprecipitation of endogenous MST3 and MST4 with constitutively active RHOA. Quantification of MST3 and MST4 levels in each immunoprecipitation condition relative to the input is displayed on the right-hand side of the blots (arbitrary units). Quantification was performed on three independent experiments. Error bars=s.d. Significance P -value was calculated using two-tailed heteroscedastic t -test analysis. n.s., not significant ( P >0.05).

Article Snippet: Mouse monoclonal antibodies against RHOA (sc-418), RHOB (sc-8048), MST3 (sc-135993), MST4 (sc-376649), CCM3 (sc-365586), Ezrin (sc-58758) and myosin light chain 2 (MYL9, MYL12A and MYL12B) (sc-28329) were purchased from Santa Cruz Biotechnology.

Techniques: Multiplex sample analysis, Binding Assay, Transfection, Expressing, Control, Lysis, Immunoprecipitation, Western Blot, Two Tailed Test, Plasmid Preparation, Mutagenesis

Journal: Journal of Cell Science

Article Title: RHO binding to FAM65A regulates Golgi reorientation during cell migration

doi: 10.1242/jcs.198614

Figure Lengend Snippet: RHO and FAM65A do not regulate MST kinase activity. (A) The majority of GCKIII kinase activity in HeLa cells comes from MST4, with MST3 contributing to the remainder, both independently of RHO activity. HeLa cells were transfected with the indicated siRNA pools or non-targeting siRNA pool as control. 72 h post transfection, the cells were treated as indicated with TAT-C3 (C3) for 4 h, before lysis and analysis by immunoblotting with the indicated antibodies. P prefix indicates phosphorylated forms of the indicated proteins. Active phosphorylated GCKIII (pGCKIII) is resolved as a doublet, with the lower more-intense band corresponding to MST4, and the higher weaker band corresponding to MST3. TAT-C3 inactivated RHO, as manifested by a reduction in pMLC and pEzrin (pEZR) levels, but neither TAT-C3 nor YSK1 depletion affected pGCKIII levels. Bar graphs on the right-hand side of the blots display the quantifications of the indicated phosphorylated proteins. pGCKIII and pEzrin levels were normalised to total Ezrin levels as loading control, whereas pMLC was normalised to total MLC levels (arbitrary units). Quantification was performed on three independent experiments. Error bars=s.d. (B) GCKIII kinase activity comes from MST3 and MST4 and is not regulated by RHO. Wild-type (WT), MST3, MST4 or MST3 MST4 (MST3/4) double CRISPR knockout (KO) HeLa cells were starved for 24 h and treated as indicated with TAT-C3 for 4 h, before being stimulated by 10% FBS (15 min). The cells were then lysed and analysed by immunoblotting with the indicated antibodies. Stimulation activated RHO, as revealed by an increase in pMLC levels, whereas TAT-C3 inhibited RHO. Neither treatment affected pGCKIII levels, whereas double MST-knockout abrogated pGCKIII. Bar graphs below the blots display the quantifications of the indicated phosphorylated proteins. pGCKIII and pEzrin were normalised to total Ezrin levels as loading control, whereas pMLC was normalised to total MLC levels (arbitrary units). Quantification was performed on three independent experiments. Error bars=s.d. (C) GCKIII kinase activity is not regulated by FAM65A or RHO. WT or FAM65A CRISPR KO HeLa cell lines were starved for 24 h and treated as indicated with TAT-C3 for 4 h, before being stimulated by 10% FBS (15 min). The cells were then lysed and analysed by immunoblotting with the indicated antibodies. Neither FAM65A loss nor RHO activation–inactivation by FBS or TAT-C3 affected pGCKIII levels. Bar graphs on the right-hand side of the blots display the quantifications of the indicated phosphorylated proteins. pGCKIII was normalised to either total Ezrin (light grey bars) or total MST3 and MST4 (dark grey bars). pEzrin was normalised to total Ezrin, whereas pMLC was normalised to total MLC (arbitrary units). Quantification was performed on three independent experiments. Error bars=s.d.

Article Snippet: Mouse monoclonal antibodies against RHOA (sc-418), RHOB (sc-8048), MST3 (sc-135993), MST4 (sc-376649), CCM3 (sc-365586), Ezrin (sc-58758) and myosin light chain 2 (MYL9, MYL12A and MYL12B) (sc-28329) were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Transfection, Control, Lysis, Western Blot, CRISPR, Knock-Out, Activation Assay

Journal: Journal of Cell Science

Article Title: RHO binding to FAM65A regulates Golgi reorientation during cell migration

doi: 10.1242/jcs.198614

Figure Lengend Snippet: Active RHO and FAM65A mediate Golgi reorientation by inhibiting MST3 and MST4 function. (A) RHO inhibition impairs Golgi reorientation towards the direction of cell migration. Mock or TAT-C3 (C3)-treated migrating HeLa cells in a wound healing assay were fixed and immunostained with an anti-GM130 antibody (red) and DAPI (blue) before confocal analysis. Transmission light microscopy was used to visualise the boundaries of the cells. Scale bars: 10 µm. Arrows indicate the direction of the Golgi. (B) Quantification of Golgi orientations from A. The percentage of the edge cells with their Golgi reoriented towards the wound were calculated from five wound healing assays ( n =5). 111–136 cells were quantified per condition. Error bars=s.d. Significance P -values were calculated using two-tailed heteroscedastic t -test analysis. (C) FAM65A loss mimics the RHO inhibition effect on Golgi reorientation, whereas double loss of MST3 and MST4 (MST3/4 KO) rescues this effect. Golgi reorientation in wild-type (WT), FAM65A, MST3, MST4 and double MST3 and MST4 CRISPR knockout (KO) HeLa cells was analysed as described in A. Scale bars: 10 µm. Arrows indicate the direction of the Golgi. (D) Quantification of Golgi orientation from C. The percentage of the edge cells with their Golgi reoriented towards the wound were calculated from five wound healing assays per condition ( n =5). 85–153 cells were quantified per condition. Error bars=s.d. Significance P -values were calculated using two-tailed heteroscedastic t -test analysis; n.s., not significant ( P >0.05).

Article Snippet: Mouse monoclonal antibodies against RHOA (sc-418), RHOB (sc-8048), MST3 (sc-135993), MST4 (sc-376649), CCM3 (sc-365586), Ezrin (sc-58758) and myosin light chain 2 (MYL9, MYL12A and MYL12B) (sc-28329) were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Migration, Wound Healing Assay, Transmission Assay, Light Microscopy, Two Tailed Test, CRISPR, Knock-Out

Journal: Journal of Cell Science

Article Title: RHO binding to FAM65A regulates Golgi reorientation during cell migration

doi: 10.1242/jcs.198614

Figure Lengend Snippet: FAM65A mediates directional migration by inhibiting MST3 and MST4 function. (A) Loss of FAM65A impairs directional migration, whereas double loss of MST3 and MST4 does not affect directional migration. Migrating wild-type (WT), FAM65A and double MST3 and MST4 (MST3/4) CRISPR knockout (KO) HeLa cells in a wound healing assay were then analysed for 24 h by using time-lapse microscopy. Images show the wound area at indicated timepoints. (B) Quantification of the wound closure from experiments shown in A. The average distances migrated by the wound edges were calculated from three wound healing assays per condition ( n =6). Error bars=s.d. Significance P -values were calculated using two-tailed heteroscedastic t -test analysis; n.s., not significant ( P >0.05). (C) Impairment of directional migration upon FAM65A depletion is rescued by double loss of MST3 and MST4. Migration of WT or MST3/4 CRISPR knockout (KO) HeLa cells that had been transfected with FAM65A-specific or non-targeting (NT) siRNA pools was analysed as described in A. Images show the wound at indicated timepoints. (D) Quantification of the wound closure from the experiments shown in C. The average distances migrated by the wound edges were calculated from three wound healing assays per condition ( n =6). Error bars=s.d. Significance P -values were calculated using two-tailed heteroscedastic t -test analysis; n.s., not significant ( P >0.05). NTi, non targeting siRNA; FAM65Ai, siRNA targeting FAM65A.

Article Snippet: Mouse monoclonal antibodies against RHOA (sc-418), RHOB (sc-8048), MST3 (sc-135993), MST4 (sc-376649), CCM3 (sc-365586), Ezrin (sc-58758) and myosin light chain 2 (MYL9, MYL12A and MYL12B) (sc-28329) were purchased from Santa Cruz Biotechnology.

Techniques: Migration, CRISPR, Knock-Out, Wound Healing Assay, Time-lapse Microscopy, Two Tailed Test, Transfection

Journal: Journal of Cell Science

Article Title: RHO binding to FAM65A regulates Golgi reorientation during cell migration

doi: 10.1242/jcs.198614

Figure Lengend Snippet: CCM3 acts as an adaptor linking MST kinases to FAM65A. (A) Depletion of CCM3 does not fully abrogate MST activity. HeLa cells were transfected with non-targeting control (NT) or the indicated siRNA pools. At 72 h post transfection, cells were lysed and analysed by immunoblotting with indicated antibodies. Despite a reduction in the total levels of MST3 and MST4, active phosphorylated MST kinases (pGCKIII) were still present in CCM3-depleted cells. (B) RHO-induced MST4 relocation from the Golgi is dependent on CCM3. HeLa cells that had been transfected with CCM3-specific or non-targeting control siRNA pools were seeded at low density, before being serum-starved for 24 h. Cells were treated with TAT-C3 (C3) for 4 h and stimulated with 10% FBS (15 min) as indicated, before being fixed and immunostained with the indicated antibodies for confocal analysis. Scale bars: 10 µm. (C) Quantification of colocalisation from experiments shown in B. The Pearson correlation coefficients between green (MST4) and red (GM130) channels were calculated and averaged from a minimum of three independent fields of view ( n =3) from three experiments, each comprised 2–11 cells per field. Error bars=s.d. Significance P -values were calculated using two-tailed heteroscedastic t -test analysis; n.s., not significant ( P >0.05). (D) Interaction of MST3 and MST4 with FAM65A is CCM3 dependent. HeLa cells were serially transfected with CCM3-specific or non-targeting control siRNA pools followed by expression vectors for GFP–FAM65A or GFP-only as control and subjected to lysis and immunoprecipitation (IP) with anti-GFP antibody. Input lysates (WCL) as well as anti-GFP immunoprecipitation eluates were analysed by immunoblotting (IB) using the indicated antibodies. Depletion of CCM3 abrogates the MST3 and MST4 (MST3/4) interaction with FAM65A. Quantification of MST3 and MST4 (MST3/4) levels in each immunoprecipitation condition relative to the input is displayed on the right-hand side of the blots (arbitrary units). Quantification was performed on three independent experiments. Error bars=s.d. Significance P -value was calculated using two-tailed heteroscedastic t -test analysis. (E) The proposed mechanism for regulation of Golgi reorientation by RHO. When RHO proteins are inactive, the FAM65A–CCM3–MST complex is localised to the Golgi, where MST proteins act to inhibit reorientation. Upon RHO activation, the FAM65A–CCM3–MST complex is relocated away from the Golgi owing to its interaction with RHO, thus relieving the inhibitory effect of MST on Golgi reorientation.

Article Snippet: Mouse monoclonal antibodies against RHOA (sc-418), RHOB (sc-8048), MST3 (sc-135993), MST4 (sc-376649), CCM3 (sc-365586), Ezrin (sc-58758) and myosin light chain 2 (MYL9, MYL12A and MYL12B) (sc-28329) were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Transfection, Control, Western Blot, Two Tailed Test, Expressing, Lysis, Immunoprecipitation, Activation Assay