Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

Abstract

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca²⁺ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.

Keywords: RhoA; actin stress fibers; calcium signaling; cancer; mechanotransduction.

Fig. 1.

Piezo2 is required for force transmission and mechanotransduction in MDA-MB-231-BrM2 cells. (A) Quantitative real-time PCR (qPCR) of Piezo1 and -2 expression in parental and MDA-MB-231-BrM2 cells, n = 3. (B) qPCR of Piezo2 in shControl and shPiezo2 BrM2 cells, n = 3. (C) Representative whole-cell traces of mechanically activated (MA) inward cationic currents recorded at a holding potential of −80 mV from BrM2-shControl and BrM2-shPiezo2 cells following stimulation with a series of mechanical steps of 0.5 μm (Inset illustration). (D) Peak MA current density and (E) percentage of MA current inactivation in BrM2-shControl and BrM2-shPiezo2 cells. (F) Color map images of traction forces exerted by cells on 15-kPa hydrogels. (Scale bar, 20 µm.) (G) Mean traction forces obtained at 2 (shControl n = 103; shPiezo2 n = 110), 15 (shControl n = 51; shPiezo2 n = 38), and 30 kPa (shControl n = 72; shPiezo2 n = 56), and at 2 (shControl n = 51; shPiezo2 n = 41) and 30 kPa (shControl n = 27; shPiezo2 n = 25) in the absence of extracellular Ca²⁺. ***P < 0.001 shPiezo2 vs. shControl for each stiffness. ^###P < 0.001 shControl vs. shControl 0Ca²⁺. (H) Nuclear and YAP stainings of cells seeded on glass. White rectangles define the zoomed region. (Scale bar, 50 µm.) (I) Mean YAP nuclear/cytoplasmic signal ratio. The number of cells analyzed for each condition is indicated in each bar. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2.

Ca²⁺ entry via Piezo2 regulates the actin cytoskeleton and adhesions. (A) Representative maximal intensity projections of whole-cell Z scans of phalloidin-stained actin in cells treated as indicated. (B) Mean percentage of total cell area occupied by actin. (C) Paxillin staining of the basal planes of cells. (D) Rose plot of the angles of adhesions respective to the cell major axis. (Top) DMSO, (Bottom) 500 nM jasplakinolide. (Scale bar, 50 µm.) Red rectangles define the zoomed region. ***P < 0.001. (E) Immunofluorescence confocal microscopy images of Piezo2 and Paxillin. Note the strong reinforcement of Piezo2 signal at the cell boundary of BrM2-shControl cells and the almost total absence of Piezo2 signal in BrM2-shPiezo2 cells. The merge panel displays the colocalization between Piezo2 and Paxillin. Magnified views of the respective boxes drawn at the cell leading edges are shown on the Right with two combinations of colors to facilitate viewing by color-blinded people. Arrowheads marked colocalization of Piezo2 and Paxillin at FA of the leading edge in shPiezo2 cells.

Fig. 3.

The Piezo2-RhoA-mDia1 axis controls actin polymerization and mechanotransduction. (A) FRET/CFP emission ratio measurements of Raichu-RhoA sensor transfected BrM2 cells. (Scale bar, 25 µm.) (B) Normalized mean FRET/CFP ratio of the whole-cell areas measured in the presence and in the absence of extracellular Ca²⁺. (C) Western blot of total lysates obtained from shControl and shPiezo2 BrM2 cells and probed with anti-RhoA, anti-tubulin, and anti-GAPDH antibodies. (D and F) Representative maximal intensity projections of whole-cell Z scans of phalloidin-marked actin (D) or YAP (F) in cells transfected with the indicated plasmids. (E) Mean percentage of total cell area occupied by actin. (G) Mean YAP nuclear/cytoplasm ratio. Red rectangles define the zoomed region. (Scale bar, 50 µm.) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to shControl (B), empty vector shControl (E and G, Left) or empty vector shPiezo2 (E and G, Right). ^###P < 0.001 relative to empty vector shControl cells.

Fig. 4.

Fyn localization and calpain activity is altered in BrM2-shPiezo2 cells. (A) Immunofluorescence confocal microscopy images of phosphorylated FAK (pFAK, marker of FA) and Fyn. Contour of cells is marked by a blue line. Magnified views of the respective boxes drawn at the cell leading edges are shown on the Right with two combinations of colors to facilitate viewing by color-blinded people. Note the colocalization of pFAK and Fyn signals at the leading edge of BrM2-shControl cells that is absent in BrM2-shPiezo2 cells. (B) Quantification of the Fyn/pFAK colocalization using Manders’ coefficient. (C) Representative images of Western blots (Left) and quantification of Fyn normalized to GAPDH levels (Right). (D) Calpain activity (measured using a permeant nonfluorescent substrate of calpain that becomes fluorescent upon calpain-mediated cleavage) is reduced in vehicle-treated shPiezo2 cells compared with shControl cells. Removal of extracellular calcium (0Ca²⁺) reduced calpain activity, whereas the presence of the calcium ionophore ionomycin (1 μM) increased it. **P < 0.01, ***P < 0.001 relative to shControl vehicle. ^###P < 0.001 relative to vehicle shPiezo2 condition. N.S., not significant.

Fig. 5.

Piezo2 knockdown impairs brain metastasis-favoring functions. (A) Mean time needed to enter 3 µm-wide channels and (B) mean percentage of cells achieving it. (C) Mean speeds of cells migrating at different fibronectin-coating concentrations. (D) Images of fluorescent gelatin substrates. White lines define the cell contour. Red rectangles define the zoomed region. (Scale bar, 10 µm.) (E) Mean percentage of degraded area relative to the number of cells. (F) Dot blot of supernatants collected from three independent wells containing shControl and shPiezo2 BrM2 cells probed with anti-SERPIN2 B2 antibody. (G) SERPIN B2 secretion normalized to total SERPIN B2. *P < 0.05, **P < 0.01, ***P < 0.001.