Live-cell microscopy demonstrates preferential granule fusion near liprin-α1 clusters at the β cell–ECM interface. (A) Live-cell two-photon imaging of isolated β cells expressing GFP–liprin-α1 (green) bathed in SRB (red). Cells were stimulated with 16.7 mM glucose for 15 min to induce granule fusion, identified in time and space, and marked with yellow dots. (B) A kymograph showing fluorescence over time along a line (indicated in white in Fig. 5 A) shows five example granule fusion events as small sudden bright flashes of SRB, also shown as sudden peaks in SRB intensity within a region of interest drawn over the fusing granules. Granule fusion sites overlap with regions of cell occupied by liprin-α1 clusters. (C) Frequency of granule fusion events in relation to distance from their nearest liprin-α1 neighbor. Measurements represent center-to-edge distances (center of a granule fusion event to edge of nearest GFP–liprin-α1 cluster) (three separate β cell clusters imaged, from three animals). (D) Percentage of granule fusion events contacting GFP–liprin-α1 plotted as a histogram, where contact is defined as a granule fusion event with any degree of colocalization with GFP–liprin-α1 (n = 3 animals, paired two-tailed Student’s t test). All data are shown as mean ± SEM.