Fig. 3.

The GUV assay demonstrates the C2 domain's ability to induce membrane budding. A: Experiments with POPC:POPE:POPS (60:20:20) GUVs were performed in triplicate with a minimum of 60 GUVs assessed per measurement to provide a quantitative representation of membrane curvature changes shown in B. B: Quantitative representation of GUV vesiculation induced by C2 domain and respective mutants. WT induced significant vesiculation of GUVs in the presence of 10 μM CaCl₂ compared with control experiments performed in 100 μM EGTA. M38A and M98A displayed some induction of GUV vesiculation and membrane reorganization but to a significantly lesser extent than WT. F35A/L39A and Y96A did not appreciably induce GUV vesiculation when compared with the control. C: The WT C2 domain and full-length cPLA₂α were assessed at 200 nM protein concentration for their ability to induce membrane curvature changes in the presence of 500 nM CaCl₂ to GUVs containing POPC:POPE:POPS (60:20:20). The WT C2 domain induced substantial vesiculation, whereas full-length cPLA₂α induced vesiculation and long tubule formation from GUVs. D: Quantification of vesiculation in control versus WT C2 experiments shown in C. The P value for each protein was determined in comparison to the control in C and D (ns, not significant; *P < 0.001; **P < 0.0001) using an unpaired Student t-test. Scale bars = 5 μm.