Figure 2. Functional Tests of Lipid-Binding Residues on TM9-TM10 Loop and Other Residues with Ca²⁺-Dependent Placements.

(A–F) Analysis of residues involved in Ca²⁺ binding is depicted (A)–(C), and (D)–(F) are for analysis of residues involved in lipid binding. Live imaging of TMEM16Fdependent Ca²⁺ influx (A and D), PS exposure (B and E), and GPMV generation (C and F) is shown. Time-lapse imaging of 500–1,000 cells with 10× magnification was performed to concurrently monitor GPMV formation and Ca²⁺ influx via Fluo-8 fluorescence. Time-lapse imaging of individual cells viewed with 603 magnification was performed to monitor PS exposure via pSIVA fluorescence. Data are represented as mean ± SEM.

(G–I) Scattered dot plots of time of onset of TMEM16F-dependent Ca²⁺ influx (G), PS exposure (H), and GPMV generation (I). Time of onset, which is the maximum of the second derivative of the curve (maximal acceleration), could not be determined for those time courses with a linear rather than sigmoidal rise. Data are represented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Statistical significance of all mutants as compared to TMEM16F wild-type (WT) are determined by one-way ANOVA followed by Holm-Šídák multiple comparisons test.

See also Figure S3 and Table S2.