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 β 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.

中文翻译：

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Arp2/3 使 F-肌动蛋白涂层成核，融合胰腺 β 细胞中的胰岛素颗粒，以控制胰岛素分泌。

已知 F-肌动蛋白动力学可控制胰岛素分泌，但与刺激-分泌级联的交叉点尚不清楚。在这里，使用从 Lifeact-GFP 转基因小鼠中分离的 β 细胞的多光子成像，我们发现葡萄糖刺激不会引起皮层下 F-肌动蛋白的整体变化。相反，我们观察到每个融合颗粒周围的空间离散和瞬时 F-肌动蛋白变化。这种 F-肌动蛋白重塑依赖于肌动蛋白成核，并且观察到由葡萄糖或高钾刺激诱导的颗粒融合。使用 GFP 标记的蛋白质，我们鉴定了 Arp3、动力蛋白和网格蛋白的局部富集，所有这些都发生在颗粒融合后，表明内吞复合物早期招募到融合颗粒中。用药物或 shRNA 阻断 Arp2/3 活性会抑制 F-肌动蛋白涂层，将颗粒捕获在细胞膜上并减少胰岛素分泌。阻断福尔明介导的肌动蛋白成核也会阻断 F-肌动蛋白涂层，但对胰岛素分泌没有影响。我们得出结论，颗粒融合位点的局部 Arp2/3 依赖性肌动蛋白成核在融合后颗粒动力学和胰岛素分泌调节中发挥重要作用。