Fig. 4: Physiological proof of principle: mechanosensitive opening of TMEM16F-dependent PM reservoirs.
From: TMEM16F and dynamins control expansive plasma membrane reservoirs
A PM area changes in whole-cell recordings of Jurkat WT, TMEM16F-null, and BHK WT cells subjected to hypo-osmotic stimulation with 4 mM extracellular Ca. Below, representative traces for BHK cells, n = 10,9,7,7,5 independent cells from three experiments. B Annexin V-FITC labeling (top) to label surface PS exposure and bright-field overlay (bottom) of Jurkat WT and TMEM16F-null cells in isotonic and hypotonic solutions with 4 mM Ca. Sucrose was used to adjust osmolarity. Quantification below with percentage of AnnexinV positive cells (left) and fold increase in Ca-fluorescence (right) in cells loaded with X-Rhod1 (3 µM) Ca-indicator normalized to initial Ca values indicating that hypotonic-induced Ca-entry is independent of TMEM16F expression, n = 5,10,7,12,5,5,11 biologically independent samples. C Quantification of Cm changes during application of 4 mM Ca and PIEZO1 agonist Yoda1 in HEK-TMEM16F-null cells overexpressing PIEZO1 (right) and rescued with mTMEM16F (left) as well as WT HEK293 cells (middle), n = 13,7,8 independent cells from three experiments. D Representative recordings from (C) of Cm changes (red) conductance (blue) and inward current (pink) after Yoda1 application. All data expressed as mean ± s.e.m. Unpaired Student’s t-test was used for comparing two groups. Scale bars: 5 µm.
