Figure 7: Residues required for oligomerization and correct localization are necessary for STOML3 function.
From: Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch
(a) Stimulus–response curves for N2a cells overexpressing LifeAct-mCherry and fluorescently tagged STOML3 variants. Data were binned by stimulus magnitude and current amplitudes within each bin averaged for each cell and then averaged between cells, presented as mean±s.e.m; STOML3-V190P-mGFP (magenta squares; 172 measurements, 15 cells), STOML3-R90A-mGFP (grey squares; 218 measurements, 15 cells), STOML3-LR89,90EE-mGFP (green squares; 136 measurements, 13 cells), STOML3-P40S-mGFP (black squares; 142 measurements, 15 cells). Data from wtSTOML3 overexpression are re-plotted here for comparison (cyan squares) (b) Inverted epifluorescent images of STOML3 variants. Note that STOML3-V190P is localized similarly to wtSTOML3; STOML3-R90A and –LR89,90EE are localized in part to the membrane, but the vesicular fraction is lost and STOML3-P40S does not seem to be localized at the membrane nor in a vesicle pool (observations made on 15 cells/4 transfections). Scale bar 10 μm. (c) BiFC assays were used as a cell-based assay for oligomerization. In all cases wtSTOML3-VN was used as bait and as a control wtSTOML3-VC as prey. For experiments conducted on a single day, average slope of YFP signal development for the control was calculated and used to normalize all data. Data are displayed as mean±s.e.m. Oligomerization was significantly reduced when the STOML3-V190P-VC, STOML3-R90A-VC and LR89,90EE-VC variants were used as prey, in comparison with controls; Student’s t-test; ***P<0.001; n numbers indicate the number of transfections.
