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. 2018 Mar 9;7:e32346. doi: 10.7554/eLife.32346

Figure 3. Catalytic activity of Mtmr2 is necessary to suppress Piezo2-mediated RA-MA currents.

Figure 3.

(a) Stimulus-current curves upon co-expression of Piezo2 with the catalytically inactive Mtmr2 C417S mutant in HEK293 cells compared to mock controls. Mtmr2 C417S overexpression slightly, but not significantly increased Piezo2 RA-MA currents especially at lower stimulus magnitudes (Piezo2 + mock: n = 17 cells; Piezo2 + Mtmr2 C417S: n = 18 cells). 2-way ANOVA reported a significant (P<0.0021) overall effect of Mtmr2 C417S overexpression on RA-MA currents, however a Holm-Sidak’s multiple comparisons test showed no significant difference between currents at individual stimulus magnitudes.). Of note, the displacement thresholds and inactivation time constant were unaffected upon overexpression with Mtmr2 C417S compared to mock (Supplementary file 1). (b–c) Representative images (b) and quantification (c) of PLA signal using antibodies against GST and myc to detect Piezo2-GST-IRES-GFP and Mtmr2 C417S-myc, respectively. PLA signal upon co-transfection of Piezo2-GST + Mtmr2 C417S-myc was indistinguishable from Piezo2-GST + Mtmr2 myc and significantly stronger than Piezo2-GST + mock. Cell boundaries are demarcated in yellow. Only cells with pronounced GFP signal (due to expression of Piezo2-GST-IRES-GFP) were considered for the analysis. Scale bar: 10 µm. Quantification of the number of the total area of PLA signal/total cell area (fraction of PLA-positive area) (c); p<0.0001 compared to Piezo2-GST + mock, Kruskal-Wallis test followed by Dunn’s Multiple Comparison Test; Piezo2-GST + mock: n = 75 cells; Piezo2-GST + Mtmr2-myc: n = 70 cells; Piezo2-GST + Mtmr2 C417S-myc: n = 70 cells.