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

Cáceres M, Ortiz L, Recabarren T, Romero A, Colombo A, Leiva-Salcedo E, Varela D, Rivas J, Silva I, Morales D, Campusano C, Almarza O, Simon F, Toledo H, Park KS, Trimmer JS, Cerda O - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile; Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California Davis, Davis, California, United States of America.

ABSTRACT
Cellular 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.

No MeSH data available.


Related in: MedlinePlus

TRPM4 regulates the number and size of focal adhesions.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 shRNAScramble, and shRNATRPM4. 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 shRNAScramble and shRNATRPM4. 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 shRNAScramble and n = 15 cells for shRNATRPM4 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.
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pone.0130540.g002: TRPM4 regulates the number and size of focal adhesions.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 shRNAScramble, and shRNATRPM4. 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 shRNAScramble and shRNATRPM4. 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 shRNAScramble and n = 15 cells for shRNATRPM4 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.

Mentions: To define the role of TRPM4 in FA function, we performed shRNA-based TRPM4 knockdown (Fig 2A and 2C) and measured the number of FAs. We found that TRPM4 knockdown caused a diminished number of FAs (Fig 2D and 2E). Moreover, we found that TRPM4 silencing caused a ~34% increase in FA size (Fig 2F). Interestingly, pharmacological inhibition of TRPM4 with 9-phenanthrol, a potent inhibitor of TRPM4, caused similar effects on the number of FAs (Fig 2G and 2H), as well as a ~13% increase in the average FA size (Fig 2I).


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

Cáceres M, Ortiz L, Recabarren T, Romero A, Colombo A, Leiva-Salcedo E, Varela D, Rivas J, Silva I, Morales D, Campusano C, Almarza O, Simon F, Toledo H, Park KS, Trimmer JS, Cerda O - PLoS ONE (2015)

TRPM4 regulates the number and size of focal adhesions.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 shRNAScramble, and shRNATRPM4. 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 shRNAScramble and shRNATRPM4. 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 shRNAScramble and n = 15 cells for shRNATRPM4 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.
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pone.0130540.g002: TRPM4 regulates the number and size of focal adhesions.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 shRNAScramble, and shRNATRPM4. 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 shRNAScramble and shRNATRPM4. 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 shRNAScramble and n = 15 cells for shRNATRPM4 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.
Mentions: To define the role of TRPM4 in FA function, we performed shRNA-based TRPM4 knockdown (Fig 2A and 2C) and measured the number of FAs. We found that TRPM4 knockdown caused a diminished number of FAs (Fig 2D and 2E). Moreover, we found that TRPM4 silencing caused a ~34% increase in FA size (Fig 2F). Interestingly, pharmacological inhibition of TRPM4 with 9-phenanthrol, a potent inhibitor of TRPM4, caused similar effects on the number of FAs (Fig 2G and 2H), as well as a ~13% increase in the average FA size (Fig 2I).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile; Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California Davis, Davis, California, United States of America.

ABSTRACT
Cellular 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.

No MeSH data available.


Related in: MedlinePlus