F-actin dynamics are known to control insulin secretion but the point of intersection with the stimulus-secretion cascade is unknown. Here, using multiphoton imaging of ß cells isolated from Lifeact-GFP transgenic mice, we show glucose stimulation does not cause global changes in subcortical F-actin. Instead, we observe spatially discrete and transient F-actin changes around each fusing granule. This F-actin remodelling is dependent on actin nucleation and is observed for granule fusion induced by either glucose or high potassium stimulation. Using GFP-labelled proteins we identify local enrichment of Arp3, dynamin and clathrin, all occurring after granule fusion, suggesting early recruitment of an endocytic complex to the fusing granules. Block of Arp2/3 activity with drugs or shRNA inhibit F-actin coating, traps granules at the cell membrane and reduces insulin secretion. Block of formin-mediated actin nucleation also blocks F-actin coating but has no effect on insulin secretion. We conclude that local Arp2/3 dependent actin nucleation at the sites of granule fusion plays an important role in post-fusion granule dynamics and in the regulation of insulin secretion.

Read more »