Profilin binding couples chloride intracellular channel protein CLIC4 to RhoA-mDia2 signaling and filopodium formation

Abstract

Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with β1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin-binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA-mDia2-regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.

Keywords: G protein-coupled receptor; G-actin; Rho (Rho GTPase); actin; cell adhesion; cell motility; epidermal growth factor (EGF); filopodia; formin; lysophosphatidic acid; membrane trafficking; profilin.

Figure 1.

LPA and EGF induce translocation of CLIC4 to the plasma membrane. A, live-cell imaging of CLIC4 translocation to the plasma membrane. Cells were seeded on glass coverslips and transfected with YFP–CLIC4. LPA (2 μm, top) and EGF (100 ng/ml) were added 2 min after starting imaging. Frames from time-lapse movies at the indicated time points are shown. Scale bar, 10 μm. B–E, quantification of LPA- and EGF-induced CLIC4 translocation. B and D, translocation was quantified by measuring YFP fluorescence at the plasma membrane (PM, blue line) and in cytoplasm (Cyt., yellow line). Mean ± S.E. of normalized plasma membrane and cytosolic CLIC4 fluorescence are plotted over time (LPA, n = 16 cells; EGF n = 18 cells, from two independent experiments). C and E, net translocation is expressed as mean ± S.E. of the normalized PM/Cyt. fluorescence ratio (LPA, n = 16 cells; EGF n = 18 cells, from two independent experiments).

Figure 2.

LPA- and EGF-induced translocation of CLIC4 depends on RhoA activation. A, kinetics of RhoA activation by LPA and EGF and dependence on CLIC4. shControl and shCLIC4 knockdown cells were transfected with a RhoA biosensor (17). RhoA activity is plotted as normalized YFP/CFP ratio over time (LPA: shControl = 10 cells, shCLIC4 #3 = 15 cells, and shCLIC4 #5 = 15 cells, from at least two independent experiments; EGF: shControl = 15 cells, shCLIC4 #3 = 15 cells, and shCLIC4 #5 = 10 cells, from at least two independent experiments). B and C, RhoA pulldown assays. shControl and shCLIC4 knockdown HeLa cells were serum-starved overnight and either stimulated with LPA (2 μm) or EGF (100 ng/ml) for 3 min or left untreated. GTP-bound RhoA was pulled down as described under “Experimental procedures.” GTP-bound and total RhoA were detected by immunoblot analysis using anti-RhoA antibodies. CLIC4 knockdown was monitored by immunoblot analysis of total cell lysates using anti-CLIC4 antibodies. Actin was used as loading control. Representative blots of one out of six independent experiments are shown. Densitometric analysis (mean ± S.E.) of six experiments is shown in C along with the results of one-way ANOVA with Dunnett's multiple comparisons test (*, p < 0.05; **, p < 0.01). D, HeLa cells were plated on glass coverslips and cotransfected with mCherry–CLIC4 and either dominant-negative (RhoAN19, top) or constitutively active RhoA (RhoAV14, bottom) in a bicistronic IRES vector expressing GFP. Cells were serum-starved and either stimulated with LPA (2 μm) or EGF (100 ng/ml). RhoAN19- and RhoAV14-transfected cells express monomeric GFP. Frames from time-lapse movies at the indicated time points are shown. Scale bars, 10 μm. E, quantification of agonist-induced CLIC4 translocation in RhoAN19-expressing (blue trace) and RhoAV14-expressing (red trace) cells. LPA-induced (top) and EGF-induced (bottom) net translocation are expressed as mean ± S.E. of the PM/Cyt. fluorescence ratio.

Figure 3.

LPA-induced translocation of CLIC4 relies on mDia2 and its activity. A and B, live-cell imaging of CLIC4 translocation in mDia knockdown HeLa cells. Stable mDia1 and mDia2 knockdown HeLa cells were obtained as described under “Experimental procedures.” shControl, shmDia1 (#2 (18)), and shmDia2 (#1 (44)) cells were seeded on glass coverslips and transfected with YFP–CLIC4. shControl cells were pre-treated with SMIFH2 (50 μm, 20 min) or left untreated. LPA (A) (2 μm) or EGF (B) (100 ng/ml) were added 2 min after starting imaging. SMIFH2 was maintained during stimulation. Frames from time-lapse movies at the indicated time points are shown. Scale bar, 10 μm. C, quantification of LPA-induced translocation in shControl (blue trace, n = 8 cells), shmDia1 (red trace, n = 8 cells), shmDia2 (green trace, n = 12 cells), and SMIFH2-treated cells (yellow trace, n = 12 cells) from two independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was performed comparing the highest values of the curves (***, p < 0.001; ****, p < 0.0001, ns, non-significant). D, quantification of EGF-induced translocation in shControl (blue trace, n = 16 cells), shmDia1 (red trace, n = 9 cells), shmDia2 (green trace, n = 12 cells), and SMIFH2-treated cells (yellow trace, n = 12 cells), from two independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was performed comparing the highest values of the curves (*, p < 0.05; ****, p < 0.0001, ns, nonsignificant). Net translocation in C and D is expressed as mean ± S.E. of the normalized PM/Cyt. ratio obtained from the analyzed cells. E, knockdown validation was achieved by immunoblotting (IB) using anti-mDia1 and anti-mDia2 antibodies (see also Fig. S4C). Actin was used as loading control. F, Cdc42 pulldown assays. shControl and shCLIC4 knockdown HeLa cells were serum-starved overnight and either stimulated with LPA (2 μm) or EGF (100 ng/ml) for 3 min or left untreated. GTP-bound Cdc42 was pulled down as described under “Experimental procedures.” GTP-bound and total Cdc42 were detected by immunoblot analysis using anti Cdc42 antibody. Left, representative blots of one out of three independent experiments are shown. Right, bar graph shows normalized Cdc42–GTP levels as mean ± S.E. of three independent experiments (one-way ANOVA with Dunnett's multiple comparisons test, *, p < 0.05).

Figure 4.

CLIC4 binds profilin-1 via distinct residues that make up an open cleft. A, profilin-1 coimmunoprecipitates (IP) with CLIC4 in cells. 3×FLAG-CLIC4 WT (wt) and its mutant in Cys-35 (C35A) were cotransfected with GFP-profilin-1 in HEK293 cells. CLIC4 was immunoprecipitated from cell lysate (1 mg) using anti FLAG antibodies. Coimmunoprecipitated profilin-1 and CLIC4 were detected by immunoblotting (IB) using anti-GFP and anti-FLAG antibodies, respectively. Representative blots of one out of three independent experiments are shown. B, densitometric analysis shows coimmunoprecipitated GFP–profilin-1 (mean ± S.D.) from three independent experiments (one-way ANOVA with Tukey's multiple comparisons test, **, p < 0.01; ****, p < 0.0001). C and D, CLIC4 directly binds profilin-1 in vitro. C, purified immobilized GST–CLIC4 WT and GST–CLIC4 C35A mutant were incubated with profilin-1 for 2 h on ice and pulled down with GST–agarose beads. The amount of profilin-1 pulled down by GST–CLIC4 was detected by immunoblotting using anti-profilin-1 antibodies. The reciprocal experiment was performed using GST–profilin-1 and CLIC4 WT and the C35A mutant (D). GST alone was used as a control. Ponceau staining showed equal loading of the GST-fusion proteins. Representative blots of one out of three independent experiments are shown. E, YFP–CLIC4 (WT) and the indicated mutants were transfected into HEK293 cells. Total-cell lysate (1 mg) was incubated with GST or GST–profilin-1. The amount of YFP–CLIC4 WT and mutants pulled down by profilin-1 was detected by immunoblot analysis using anti-GFP antibodies. Ponceau staining showed equal loading of the GST-fusion proteins. Representative blots of one out of two independent experiments are shown. F and G, molecular modeling of profilin-1 interactions. F, HADDOCK computational model showing CLIC4 (orange) and profilin-1 (gold). The CLIC4 residues discussed under “Results,” and the N and C termini of both proteins are indicated. G, crystal structure of profilin-1 (gold) binding to G-actin (blue) (PDB code 2PAV). See text for further details. H, equilibrium dissociation constant of the CLIC4–profilin-1 is 33.5 ± 7.2 μm. Graph shows steady-state response measured by SPR (RU = resonance units) at the indicated profilin-1 concentrations. The equilibrium dissociation constant (K_D) is expressed as mean ± S.D., and the coefficient of determination of the fitting (R²) is 0.95.

Figure 5.

Role of profilin-1 in LPA-induced CLIC4 translocation. A, live-cell imaging of CLIC4 translocation in profilin-1 knockdown HeLa cells. Stable profilin-1 knockdown cells were obtained as described under “Experimental procedures” using two distinct PFN1-targeting shRNAs. Cells were seeded on glass coverslips and transfected with YFP–CLIC4. LPA (2 μm) and EGF (100 ng/ml) were added 2 min after starting imaging. Frames from time-lapse movies at the indicated time points are shown. Scale bar, 10 μm. B, characterization of profilin-1 knockdown HeLa cells. Total-cell lysates obtained from shControl (C), shprofilin-1 #1 and shprofilin-1 #3 (#1 and #3) knockdown cells were immunoblotted (IB) with profilin-1 antibodies; actin was used as loading control. Hairpin #1 and #3 reduced profilin-1 protein levels by 95 ± 2 and 92 ± 4% (mean ± S.E., n = 3). RT-qPCR was used to independently validate the level of PFN1 knockdown (relative mRNA expression of PFN1 (mean ± S.D., n = 3): shControl = 1 ± 0.092; shprofilin-1 #1 = 0.038 ± 0.149; shprofilin-1 #3 = 0.075 ± 0.084). C, quantification of LPA-induced CLIC4 translocation (n_shControl = 7 cells; n_{shprofilin-1 #1} = 6 cells; n_{shprofilin-1 #3} = 12 cells, from two independent experiments). One-way ANOVA with Dunnett's multiple comparisons test was performed comparing the highest values of the curves (*, p < 0.05). D, quantification of EGF-induced CLIC4 translocation (n_shControl = 11; n_{shprofilin-1 #1} = 14; n_{shprofilin-1 #3} = 11 cells, from two independent experiments). One-way ANOVA with Dunnett's multiple comparisons test was performed comparing the highest values of the curves, **, p < 0.01; ***, p < 0.001. Net translocation in C and D is expressed as mean ± S.E. of the normalized PM/Cyt. ratio obtained from the analyzed cells.

Figure 6.

CLIC4 does not bind F-actin and has no effect on actin polymerization. A, CLIC4 does not bind F-actin in vitro. Cosedimentation assays were performed mixing recombinant purified full-length CLIC4 (2.5 and 5 μm) with BSA (0.3 μm) and either F-actin (2.5 μm) or F-actin buffer as described under “Experimental procedures.” The same percentages of soluble (S) and pelleted (P) fractions were subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining. Data are representative of three independent experiments performed using three different actin and CLIC4 preparations. B and C, CLIC4 does not affect spontaneous actin polymerization or mDia2-mediated actin nucleation in vitro. Bulk actin polymerization was assayed as described under “Experimental procedures” using purified recombinant CLIC4 (10 and 30 μm in B) 10 μm in C, actin (2 μm in B, 1 μm in C), mDia2-FH1–FH2 (0.1 μm), in the presence (+) or absence (−) of profilin-1 (5 μm) as indicated.

Figure 7.

CLIC4 depletion increases filopodium formation. A, effect of CLIC4 depletion on filopodia. Control and CLIC4 knockdown HeLa cells were seeded on collagen-I–coated glass coverslips, serum-starved overnight, and fixed. Maximal projections of confocal Z-stacks show actin cytoskeleton and nuclear stained with phalloidin (gray) and DAPI (blue), respectively. Scale bar, 10 μm. B, quantification of filopodium length and density. Data represent filopodium length and density measured in two independent experiments as described under “Experimental procedures” (n_shControl = 28 cells (9 images), n_{shCLIC4 #3} = 34 cells (10 images), and n_{shCLIC4 #5} = 31 (10 images)). Filopodial length: box represents the 25th to 75th percentiles with line and whiskers indicating the median and the 5th and the 95th percentiles, respectively. Bar graph depicts filopodial density as mean ± S.E. One-way ANOVA with Tukey's multiple comparisons test (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001, ns, nonsignificant). Note that all cells formed filopodia. C, fascin staining. Cells were treated as in A. Confocal images of cells stained with actin (green), fascin (red), and DAPI (blue) are shown. Insets show 1.31-fold magnifications of boxed areas in the main image. Scale bar, 10 μm. D, super-resolution imaging of mDia2 in filopodia. Cells were seeded on collagen-I–coated glass coverslips, transfected with FLAG-mDia2 WT, and serum-starved overnight. Super-resolution images of mDia2 WT (green) and phalloidin (actin, red). Scale bar, 500 nm. E, SMIFH2 treatment reduces filopodium length. Cells were seeded on collagen-I–coated glass coverslips, serum-starved overnight, and incubated with SMIFH2 (50 μm, 20 min) or mock-treated (DMSO) before fixation. Maximal projections of confocal Z-stacks show actin cytoskeleton and nuclei stained with phalloidin (gray) and DAPI (blue), respectively. Scale bar, 10 μm. F, rescue of filopodium length in CLIC4 knockdown cells using CLIC4 WT and CLIC4 C35A. CLIC4 knockdown HeLa cells were seeded on collagen-I–coated glass coverslips and transfected with either WT 3×FLAG-mCLIC4 (mCLIC4 WT) or 3×FLAG-mCLIC4 C35A (mCLIC4(C35A)) as Mus musculus CLIC4 is insensitive to the employed shRNAs. Cells were serum-starved overnight and fixed. Top, maximal projections of confocal Z-stacks show CLIC4, actin cytoskeleton, and nuclei stained with anti-FLAG antibody (green), phalloidin (gray), and DAPI (blue), respectively. Note rescue of both low and high CLIC4 expressors. Scale bar, 10 μm. Bottom left, representative blot showing the expression levels of WT (wt) and C35A 3×FLAG–mCLIC4. Bottom right, data representing filopodium length measured in two independent experiments were plotted and analyzed as in B (n_{nontransfected (−)} = 25 cells, n_{3×FLAG-mCLIC4 WT} = 26 cells, n_{3×FLAG-mCLIC4(C35A)} = 25 cells; ****, p < 0.0001, ns, nonsignificant). G, effect of LPA stimulation on filopodium length. shControl and shCLIC4 cells were seeded on collagen-I–coated coverslips and serum-starved overnight. Cells were stimulated with LPA (2 μm, 2 min) or left untreated before fixation. Maximal projections of confocal Z-stacks stained with phalloidin (gray) are shown. Scale bar, 10 μm. Data representing filopodium length measured in two independent experiments were plotted and analyzed as in B (n_{shControl starvation} = 25 cells, n_{shControl LPA} = 24 cells, n_{shCLIC4 #3 starvation} = 30 cells, n_{shCLIC4 #3 LPA} = 22 cells; *, p < 0.05; ****, p < 0.0001).

Figure 8.

CLIC4 translocation controls the filopodium retraction rate. A, CLIC4 depletion reduces the filopodium retraction rate in LPA-stimulated cells. Control (shControl) and CLIC4 knockdown (shCLIC4 #3) HeLa cells were seeded on collagen-I–coated glass-bottom Petri dishes and serum starved overnight. Subsequently, cells were labeled with Sir-Actin, and filopodia were tracked as described under “Experimental procedures.” Left, frames taken from Movies 3 and 4 show selected filopodia in control and CLIC4 knockdown cells, respectively, at the indicated times before and after LPA stimulation. Arrowheads mark the tips of filopodia that were tracked and quantified. Scale bar, 2 μm. Right, length of 10 different filopodia obtained from control (red trace) and CLIC4 knockdown (green trace) cells (8 and 7 cells, respectively, from two independent experiments) was normalized and plotted against time (sec. = seconds) as mean ± S.E. Segmental linear regression was used to fit the retractions traces (dashed black lines) and calculate the slopes. B and C, proposed signaling scheme of how agonist-induced CLIC4 translocation regulates filopodium formation. B, EGF and LPA activate RhoA and Cdc42, leading to their translocation to the plasma membrane (PM). C, activated RhoA signals through mDia2 to promote actin polymerization in a profilin-1 (PFN-1)-regulated manner. This triggers rapid CLIC4 translocation to the plasma membrane, a process that requires CLIC4 binding to profilin-1 (PFN-1). At the plasma membrane, CLIC4 counteracts mDia2 downstream of both RhoA and Cdc42 and thereby modulates CLIC4 translocation and filopodium formation in a negative feedback loop.