TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility

Artículo de publicación ISICellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility.Fondecyt 11121239 11140064 1120240 1121078 1120126 NIH R01 NS042225 Conicyt Millennium Institute on Immunology and Immunotherapy P09-016-

TRPM4 controls cellular migration <i>via</i> Rac1 GTPase activity.

<p>A) MEFs were incubated with DMSO (0.1% v/v final) vehicle or 10 μM 9-phenanthrol for 16 h. F-actin was labeled with the F-actin stain Alexa 555 phalloidin (red) and Hoechst (blue). B) Quantified results from A. The percentage of wound closure is expressed as mean ± standard deviation; s.d. (n = 3, p<0.05). *, significant difference (p<0.05) versus DMSO controls. Statistical analysis was performed using a Mann-Whitney test. C) Graph from Transwell Boyden chamber migration assays of MEFs transfected with shRNA^Scramble and shRNA^TRPM4. Cells were stimulated with 10% v/v serum for 16 h. The bars represent the mean ± s.d. (n = 3; p<0.05 compared to control). *, significant difference (p<0.05) versus shRNA^Scramble controls. Statistical analysis was performed using a two-way ANOVA test. D) Graph from Transwell Boyden chamber migration assays of MEFs transfected with shRNA^Scramble and shRNA^TRPM4 and coexpressing Rac1(Q61L). Cells were stimulated with 10% v/v serum for 16 h. The bars represent the mean ± s.d. (n = 3; p<0.05 compared to control). *, significant difference (p<0.05) versus shRNA^Scramble/Mock controls. **, significant difference (p<0.05) versus shRNA^TRPM4/Mock controls. Statistical analysis was performed using a two-way ANOVA test.</p

TRPM4 promotes cellular contractility and wound healing.

<p>A) Three-dimensional (3D) invasion assay of TREx293-TRPM4 cells. TRPM4 expression was induced by adding 1 μg/mL Tetracycline to the media (n = 3, p<0.05 compared to control). *, significant difference (p<0.05) versus non-stimulated cell control. Statistical analysis was performed using a Mann-Whitney test. B) Fibroblasts migration from skin grafts (E) treated with DMSO or 20 μM 9-phenanthrol. Grafts were fixed 5 days after explant and labeled with Hoechst (blue). Scale bar: 1 mm. C) Quantification of the experiment shown in (B). The data correspond to cell counts from 3 independent experiments (13 and 12 explants for DMSO and 9-phenanthrol treatments, respectively). *, significant difference (p<0.05) versus DMSO controls. Statistical analysis was performed using a Mann-Whitney test. D) Three dimensional contraction assay of MEFs transfected with shRNA^Scramble and shRNA^TRPM4. Contraction was induced by incubating the immersed cells with 10% v/v serum for 48 h. The upper graph represents the collected data for 4 independent assays. *, significant difference (p<0.05) versus shRNA^Scramble controls. **, significant difference (p<0.05) versus shRNA^TRPM4. Statistical analysis was performed using a two-way ANOVA test. E) Frames (t = 0 and 14 min) from time lapse of untreated, DMSO and 9-phenanthrol treated wounds in zebrafish tails. Scale bar: 1 mm. F) Quantification of the wound closure experiments from (E) (n = 10 larvae per condition). Statistical analysis was performed using a two-way ANOVA test. G) Excisional cutaneous wounds were created using a 3 mm biopsy punch. Images from the time course of wound closure in the presence of DMSO (control) and 9-phenanthrol (n = 5 mice). Scale bar: 1.5 mm. H) Wound closure was monitored measuring the area of the wound on the indicated days post-wounding. Statistical analysis was performed using a two-way ANOVA test. I) Images of skin wounds at 3 days post-wounding. Bottom panels show magnifications of the areas marked in the upper panels. Arrowheads mark the epithelial tissue. Scale bar: 50 μm. J) Images of wounds at 5 days post-wounding. The dashed lines indicate the limit of the wound area. Scale bar: 500 μm.</p

TRPM4 regulates the number and size of focal adhesions.

<p>A) Representative immunoblot from MEFs subjected to shRNA-mediated TRPM4 knock down. Membranes were incubated with mouse anti-TRPM4 mAb, and anti-Grp75 mAb as a loading control. B) Graph of the densitometric analyses of three independent immunoblot experiments. Statistical analysis was performed using a Mann Whitney test. C) Immunofluorescence labeling of MEFs transfected with shRNA^Scramble, and shRNA^TRPM4. Cells were labeled with Hoechst (blue), and mouse anti-TRPM4 mAb (green); tRFP (red) was used as transfection marker. Scale bar corresponds to 10 μm. D) Immunofluorescence labeling of MEFs transfected with shRNA^Scramble and shRNA^TRPM4. Cells were labeled with Hoechst (blue), and mouse anti-vinculin mAb (green); tRFP (red) was used as transfection marker. Scale bar corresponds to 10 μm. Quantification of FA number (E) and areas (F) from the shRNA-transfected cells (n = 15 cells for shRNA^Scramble and n = 15 cells for shRNA^TRPM4 from 7 independent experiments). G) Immunofluorescence labeling of MEFs treated with DMSO (0.1% v/v) and 10 μM 9-phenanthrol. Cells were labeled with Hoechst (blue) and mouse anti-vinculin mAb (green). Scale bar corresponds to 10 μm. Graphs of FA number (H) and areas (I) are shown (20 cells per condition, n = 3, p<0.05). The number and areas of the FAs were analyzed using NIH/ImageJ software. The graphs correspond to mean ± standard deviation. Statistical analysis was performed using a Mann-Whitney test.</p

TRPM4 localizes to focal adhesions.

<p>A) Classification of putative TRPM4-associated proteins identified by LC-MS/MS. B) FA-related proteins identified as putative TRPM4 interacting proteins. C) TRPM4 localizes to FAs in MEFs. Cells were labeled with Hoechst (blue), mouse anti-TRPM4 mAb (green), and tRFP (red). D) Magnification of the section from C. E) TRPM4 (green) colocalizes with Focal Adhesion Kinase (FAK, magenta) in MEFs. F) Amplification of the region marked in E. G) Biochemical isolation of FA complexes (FA) from MEFs. The cell body fraction is labeled as CB. TRPM4 localizes to FAs in mouse pancreatic (H) and skin fibroblasts (I); Human Umbilical Vein Endothelial Cells (HUVEC) (J) and astrocytes (K). Scale bar: 5 μm.</p

TRPM4 regulates FA turnover.

<p>(A) Representative time-lapse imaging from MEFs coexpressing shRNA^Scramble or shRNA^TRPM4 with EGFP-Paxillin. Assembling FAs are marked with arrows. Arrowheads mark FAs undergoing turnover. Scale bar: 5 μm. Quantification of assembly (B) and disassembly rates (C) of FAs from live-cell time-lapse recordings of EGFP-Paxillin transfected MEFs. Statistical analysis was performed using a Mann-Whitney test from 7 independent experiments.</p