Fig. 1: Dynamin plays key roles in TMEM16F-dependent PM expansion.
From: TMEM16F and dynamins control expansive plasma membrane reservoirs
A Representative recordings of PM area changes (%ΔCm) and PS scrambling in HEK293 cells with or without TMEM16F. All recordings at 37 °C, unless stated otherwise. Confocal images show extracellular binding of the fluorescent cationic peptide, K7r, used to track PS exposure by TMEM16F, n = 10. B Dynamin PM localization is disrupted by TMEM16F activation. Representative live-cell super-resolution airyscan confocal images of Dnm2-GFP in HEK-TMEM16F-null cells (bottom) and rescued with mTMEM16F (top) before and after Ca2+/ionomycin treatment. Images created from 1 μm z-stack near the adhesion surface. Composite results below show Dnm2 punctae loss for Dnm2 WT and K44A mutant but not K562E or when TMEM16F is absent, ionomycin: 5 µM; n = 6, 4, 7, 6, 4, resp. C Left, Ca2+ dialysis increases PM area (%ΔCm) and excess PM (1 pF = 110 μm2*Cm)/(π*d2) in uninduced MEF Dnm TKO cells but is blocked when expression of all three Dnm isoforms is ablated (tamoxifen induction) as excess PM remains stable and elevated, n = 6. Right. Dnm2 expression can control basal excess PM. The resting excess basal PM area was determined without elevated Ca and at RT in Dnm TKO and HEK cells. Cells were either transfected with mCherry (EV) or co-transfected with mCherry and K44A or K562E Dnm2. Induced cells lacking Dnm had increased basal excess PM while cells overexpressing K44A but not K562E reduced excess membrane, n = 9, 8,11,42,33,20,11. D Composite results for PM expansion in MEF Dnm TKO and H1299 cells (7 µM free Ca), n = 10, 6, 6, 13, 4, 9, 9, 9, 5,18,12. E Micrographs and composite results for K7r binding (scrambling) in uninduced (top), induced (mid), and uninduced TMEM16F-null cells (bottom), n = 6, 6, 6, 6, 6, 4. F As in (B), representative images of punctae loss in Dnm2-rescue (WT, K44A, K562E, and WT/TMEM16F-null) in induced MEF Dnm TKO cells, n = 4 independent experiments. All data analyzed from the total number of independent cells (n) from a minimum of three experiments and expressed as mean ± s.e.m. Unpaired Student’s t-test used for comparing two groups. All Scale bars: 5 µm. Box plot displays median value and upper and lower quartiles with whiskers representing 10th/90th percentile.
