Fig. 2.

AFM imaging of WT and mutant syt1(80-421) on supported lipid bilayers. (A) Cartoon depiction of the AFM experimental conditions. (B) Representative AFM topographical images of 1 µM WT syt1(80-421) on supported lipid bilayers (72% DOPC, 25% DOPS, 3% PIP₂) in the presence (1 mM) and absence (0.5 mM EGTA) of Ca²⁺. (C) Representative AFM topographical images of the syt1(80-421) mutants under the same conditions as the WT samples. The protein concentration was 1 µM in all cases except for the R398,399Q sample in which a concentration of 300 nM was used to better visualize ring formation. Pixel size: left column, 9.8 nm/px and right column, 2.0 nm/px. (D) Ring diameters for WT syt1(80-421) (green), K326,327A (purple), F349A (orange), and R398,399Q (yellow) in the absence of Ca²⁺ and of Juxta K (blue) in the presence of Ca²⁺. For the Juxta K sample in Ca²⁺, 41.6% of the observed structures were rings, while the remainder were particles. Additional C2B mutations in the Juxta K background abolished ring formation, so these samples were excluded from ring analysis. **** denotes P values < 0.0001 for Juxta K compared to all other conditions. Other statistical comparisons were not significantly different (ns). For WT, F349A, K326,327A, R398,399Q, and Juxta K, the number of rings measured were the following: 26, 13, 9, 10, and 5 for each condition from 3, 2, 2, 2, and 2 sets of analyses using independent SLBs, respectively. Error bars represent SEM.