Fig. 3: Manipulations of PLs that mimic scrambling can trigger PM expansion without Ca or TMEM16F activation.

A Sequestration of inner leaflet anionic PLs induces modest PM expansion. Dnm TKO cells with cytoplasmic buffer supplemented as follows: 0 ATP, 0 Ca, and 10 mM EGTA or EDTA, as indicated; PA Peptide 10 or 1 µM, and 1 mM orthovanadate (VO₄). PM expansion induced by PA-binding peptide is significantly reduced in induced Dnm TKO cells lacking dynamins (*p = 0.008; **p < 0.001; ^#p = 0.020). Supplementation of cytoplasm with albumin or albumin/PL (0.2/0.2 mM) mix in Dnm TKO cells (right). The anionic PL, PS, and albumin alone do not induce PM expansion; however, supplementation with neutral PC induced PM expansion in the absence of Ca and TMEM16F activation. Expansion is blocked in induced Dnm TKO cells lacking dynamins, n = 11,11,5,5,5,8,7,6,6,6,8,7. B Composite results of BHK cells in Ca-free (10 mM EGTA) conditions over 10 min during cytoplasmic dialysis with Dnm1 and Dnm2 antibodies, dynamin inhibitory peptide (DIP) 100 µM, Dynab nanobody, and relevant controls (brain-natriuretic peptide and Dnm 1 antibody, FITC-labeled goat secondary antibody (*p = 0.003), n = 17,9,8,8,7,6,8,9. C Working model for the physiological function of dynamin-constricted PM compartments. (1) Membrane tension activates Ca-influx via mechanosensitive cation channels of multiple types. (2) A local elevation of cytoplasmic Ca activates TMEM16F and PL scrambling with loss of anionic PLs (yellow) and gain of neutral PLs (blue) in the inner monolayer. (3) In response to PL changes, dynamin constrictions (GREEN) relax and compartments open, allowing the sequestered membrane to alleviate lateral PM stress. All data were analyzed from the total number of independent cells (n) from a minimum of two experiments and expressed as mean ± s.e.m. Unpaired Student’s t-test was used for comparing two groups.