The microtubule-associated Rho activating factor GEF-H1 interacts with exocyst complex to regulate vesicle traffic

Abstract

The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.

Figure 1. Perturbation of GEF-H1 function leads to accumulation of vesicular structures

(A)HeL a cells were transfected with either control siRNAor two different GEF -H1 specific oligonucleotide siRNA (#8 and #9). After 72 hrs, the cells were visualized using DIC. Scale bar, 10 μm. See also movies S1–3. (B) Percentage of cells showing accumulation of vesicles of varying sizes is shown for the control and GEF-H1-knockdown cells. Data represent means ± SD for approximately 80 cells/sample and results from three separate experiments are shown. Significance values were evaluated using two-tailed Student’s t test (^***p <0.0005). (C) Cells were transfected with GEF-H1 specific siRNA (oligo #9) to deplete the endogenous protein. After 48 hrs, depleted cells were transfected with siRNA resistant GEF-H1^wtor catalytically inactive mutant (GEF -H1^Y393A). Proteins were allowed to express for 20 hrs, and then cells were visualized under DIC (vesicles indicated by ▶). Scale bar, 10 μm. (D) The percentage of cells showing the accumulation of vesicles was calculated. More than 50 cells were analyzed for each sample. Data represent means ± SD from three independent experiments. Significance values were evaluated using two-tailed Student’s t tests (^**p< 0.005; ^***p <0.0005). (E) HeLa cells plated on plastic bottom dishes were transfected with control or GEF-H1 siRNA. 72hrs post -transfection, cells were processed using the Gilula et al. method and visualized using electron microscope(arrows represent vesicles). See also Figure S1 and Movies S1–3

Figure 2. Depletion of GEF-H1 leads to accumulation of Rab11 positive vesicles and affects localization and recycling of Transferrin

(A) Control and GEF-H1-depleted cells were immunostained for Rab11. Cells were then visualized by fluorescence microscopy. Scale bar, 10 μm. (B) The fluorescence intensity associated with Rab11 outside the perinuclear endocytic recycling compartment was quantified and expressed relative to the control. > 60 cells were used to quantify the fluorescence intensity and results for three independent experiments are shown. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (*p<0.05).(C ) Control and GEF-H1-depleted cells were incubated with Alexa 568 labeled transferrin for 10 min. Cells were then subjected to a brief acid wash to remove surface bound Tfn, fixed and visualized under fluorescence microscope. (D) Kinetics of Tfn uptake was measured for control and GEF-H1-depleted cells as described in methods. A representative plot, of four independent experiments is shown. (E) To study the effect of GEF-H1 depletion on Tfn recycling, the cells were incubated with labeled Tfn for 1 hr, this is the first time point (0 min). Most of the fluorescence in the control cells is associated with ERC and small endocytic vesicles. In GEF-H1-depleted cells besides localization to the ERC a significant fraction was contained within large vesicles (marked by an ▶). (F) After washing out the labeled Tfn, cells were incubated with excess of unlabeled Tfn to enable recycling. Samples were taken out at different time points, subjected to acid wash, fixed and visualized by fluorescence microscopy. The fluorescence intensity associated with each cells was quantified and expressed as percentage of the intensity at time point 0 min. Representative graph from three different experiments is shown.

Figure 3. GEF-H1 is involved in exocytic trafficking of VSVG

(A) Cells were transfected with control or GEF-H1 specific siRNA. After 48 hrs, the cells were transfected again with GFP-VSV-G and immediately transferred to 40°C for 20 hrs. After removing the first sample (0 min), cells were shifted to 32°C. Samples were then taken at 30, 60 and 90 min post temperature shift, fixed, and immunostained with 8G5 antibody without permeabilization and observed using confocal microscope. Scale bar, 10 μm. (B)Fluorescence intensity associated with 8G5 staining of GEF-H1-depleted cells at time points 60 and 90 min was quantified and expressed relative to control cells. >80 cells were quantified for each sample and results of three independent experiments are plotted. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (^***p <0.0005) (C) VSV-G trafficking assay was performed with cells co-transfected with GFP-VSV-G and empty vector, HA-GEF-H1^wt, GEF-H1^Y393A and GEF-H1^C53R as described earlier. Scale bar, 10 μm (D) Fluorescence intensity associated with 8G5 staining of the samples at time points 60 and 80 min was quantified and expressed relative to cells transfected with empty vector. > 60 cells were quantified for each sample and results of three independent experiments are plotted. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (*p<0.05).

Figure 4. GEF-H1 depletion perturbs localization of exocyst component Exo70 and Sec8 as well as assembly/stability of exocyst complex

(A) Control and GEF-H1-depleted cells were immunostained for endogenous Exo70. The white boxes are enlarged in the bottom panel to show, in detail, features of Exo70 localization at/near the PM. Scale bar, 10 μm. (B) Number of Exo70-positive vesicles in control and GEF-H1-depleted cells were counted and the percentage of cells carrying indicated number of vesicles/cell were plotted. >50 cells were analyzed from three separate experiments. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (^**p< 0.005). (C) Immuno-staining of cells transfected with either control or GEF-H1 specific siRNA with α-Sec8 antibody. Scale bar, 10 μm. (D) Percentage of cells exhibiting leading edge localization of endogenous Sec8 in control and GEF-H1-depleted cells was plotted. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (** p<0.005). (E) Lysates from cells transfected with empty vector, GEF-H1^wt, GEF-H1^Y393A or GEF-H1^C53R were subjected to immunoprecipitation (IP) with α-Sec8 antibody and the blots were then probed with α-Exo70 antibodies. The western blot against actin was used to show equal loading. (F)The band intensities were quantified using Image J software and the amount of Exo70 pulled-down from each sample was normalized to the levels of precipitated Sec8 and expressed relative to the sample transfected with empty vector. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (*p<0.05). See also Figure S2.

Figure 5. GEF-H1 interacts with exocyst protein Sec5

(A) EGFP-GEF-H1 and HA-Sec5 were co-expressed in HeLa cells. HA-Sec5 was then IPed using αHA antibody and probed for co-precipitation for EGFP-GEF-H1 by α-GFP antibody. (B) GST tagged truncation mutants of GEF-H1 (described in the bottom panel) were expressed in E. coli and purified using glutathione beads. The purified proteins were then used to pull-down Sec5 from lysates of cells expressing HA-Sec5. The pull-downs were analyzed with western blotting. (C) HA-Sec5 was in vitro translated using rabbit reticulocyte lysate system and added to recombinant GST or GST-GEF-H1 fragments described in the lower panel. Association between GST-GEF-H1 and HA-Sec5 was determined by pulling down GEF-H1 using glutathione beads and then probing for Sec5 using α-HA antibody. (D) HA-Sec5 was IPed from cell lysates co-expressing Sec5 and GFP-GEF-H1^wt or constitutively active, microtubule binding-deficient mutant (GEF-H1^C53R), using α-HA antibody. Co-precipitation of GEF-H1 was analyzed using anti GFP antibody. (E) HeLa cells were co-transfected with GFP-VSV-G and GST-GEF-H1^(aa119-236) or GST vector and the VSV-G assay was done as described earlier. Scale bar, 10 μm. (F) Fluorescence intensity associated with 8G5 staining of the samples at time points 60 and 90 min was quantified and expressed relative to that of the cells transfected with empty vector. >40 cells were quantified for each sample and results from three independent experiments are plotted. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (*p<0.05, **p<0.005). See also Figure S3.

Figure 6. Ral GTPases regulate interaction between GEF-H1 and Sec5 and RhoA activation

(A) Endogenous GEF-H1 was IPed from HeLa cell lysates expressing empty vector, Flag-RalA^wt, constitutively active mutant RalA^G23V or fast-cycling active mutant RalA^F39L. (B) The band intensities were quantified and the amount of Sec5 pulled-down from each sample was normalized to the levels of immunoprecipitated GEF-H1 and expressed relative to the sample with empty vector. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (*p<0.05). (C) GTP-bound active Rho was pulled down from extracts of cells expressing empty vector (EV), RalA^wt, RaLA^G23V, RalA^F39L, Sec5 binding deficient mutant (E38R), and Exo84 binding deficient mutant (A48W), using Rhotekin-RBD beads (GST tagged Rhotekin-Rho binding domain). The blots were then probed for RhoA. (D) The band intensities of GTP-RhoA and total RhoA were quantified. GTP-RhoA was normalized to the total RhoA levels in the lysates and expressed relative to the control cells with EV. Since enhanced RhoA activation has been reported in response to microtubule depolymerization, nocodazole treatment was used as a positive control (Chang et al., 2008). Results shown are average of four independent experiments. Data represent means ± SD. p values calculated by Student’s t-test, ^**p< 0.005, ***p<0.0005. (E) EV and active RalA mutants (G23V and F39L) were expressed in control and GEF-H1-depleted cells. Active RhoA was then pulled down from cell extracts using Rhotekin-RBD beads. The blots were probed with α-RhoA antibody. (F) RhoA activation induced by RalA in presence or absence of GEF-H1 was expressed as percentage of total RhoA in the lysate, and relative to the levels observed for control or GEF-H1depleted cells with EV, respectively (number of experiments=3; data represent means ± SD; *p<0.05, **p<0.005). (G) Lysates from cells transfected with indicated plasmids were used for RBD pull-down. (H) The band intensities of GTP-RhoA and total RhoA were quantified. Rho activation for cells expressing indicated proteins relative to the sample transfected with EV was calculated as described above (number of experiments=3; data represent means ± SD; p values; *p<0.05, **p<0.005).

Figure 7. Rho GTPase regulates exocyst assembly/stability and localization

(A) Sec5 was IPed from cells expressing HA-Sec5 and either GFP-RhoA^wt, GFP-RhoA^T19N, or GFP-RhoA^Q63L. Co-IPof Rho proteins was detected by immunoblotting with α-GFP antibody. (B) The band intensities were quantified and the amount of RhoA associated with Sec5 was normalized to the levels of IPed Sec5. The fold change of interaction was expressed relative to the RhoA^wt sample. n=3; data represent means ± SD; ***p< 0.0005. (C) To analyze the effect of Rho mutants on interaction between Sec8 and Exo70 cells were transfected with EV the wild type, constitutively active (Q63L) or dominant negative (T19N) mutants. Sec8 was then IPed using α-Sec8 antibody from the lysates and co-IP with Exo70 was detected using α-Exo70 antibody. (D) The band intensities from the western blots were quantified and the amounts of Exo70 co-IPed were normalized to the levels of Sec8 pulled down in respective samples. The fold change in interaction was expressed relative to the sample transfected with EV. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (*p<0.05). (E) Cells were transfected with control or RhoA and B specific siRNA oligonucleotides for 48 hrs. Cells were then fixed, immunostained with α-Exo70 antibody and visualized using fluorescence microscopy. Scale bar, 10 μm. (F) Number of Exo70-positive vesicles in control, RhoA-depleted, and RhoA and B co-depleted cells were counted and percentage of cells carrying the indicated number of vesicles/cell were plotted. >50 cells were analyzed from three separate experiments. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (*p<0.05, **p<0.005). (G) GFP-VSV-G-transfected HeLa cells were treated with cell permeable C3 transferase for 4 hrs before performing the trafficking assay. Quantification of the fluorescence intensity associated with 8G5 staining of the samples for time points 60, 80 and 90min was expressed relative to that of the untreated cells (no. cells > 40; data represent means ± SD; *p<0.05, **p<0.005). Scale bar, 10 μm. (H,I) HeLa cells were transfected with 20 nM of RhoA or RhoA+B specific siRNA oligonucleotides for 48 hrs. Then cells were transfected again with 20 nM of siRNA and GFP-VSV-G for 24 hrs. VSV-G trafficking assay was performed as described in methods. VSV-G insertion into the PM was assessed by staining the cells with 8G5 antibody and measuring the intensities at time points; 60, 80 and 90 min and expressing the values relative to that of the control siRNA transfected cells. > 50 cells were analyzed for each sample from three separate experiments. Data represent means ± SD. Significance values were calculated using two-tailed Student’s t test (*p<0.05, **p<0.005, ***p< 0.0005). Scale bar =10μm. See also Figure S4 and Figure S5.