Discipline of Physiology, School of Medical Sciences, Charles Perkins Centre, University of Sydney, Camperdown 2006, Australia.
School of Biomedical Sciences, University of Queensland, St Lucia 4072, Australia.
J Cell Sci. 2020 Mar 30;133(6):jcs236794. doi: 10.1242/jcs.236794.
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 that 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 2 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 inhibits 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.
肌动蛋白动态变化已知可以控制胰岛素分泌，但与刺激-分泌级联的交汇点尚不清楚。在这里，我们使用 Lifeact-GFP 转基因小鼠分离的β细胞的多光子成像，表明葡萄糖刺激不会引起皮质下 F-肌动蛋白的全局变化。相反，我们观察到每个融合颗粒周围存在空间离散和瞬时的 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，并且在葡萄糖或高钾刺激诱导的颗粒融合中都观察到。使用 GFP 标记的蛋白质，我们确定了 Arp3、dynamin 2 和网格蛋白的局部富集，所有这些都发生在颗粒融合之后，表明早期将内吞复合物募集到融合颗粒。用药物或 shRNA 阻断 Arp2/3 活性会抑制 F-肌动蛋白涂层，将颗粒固定在细胞膜上，并减少胰岛素分泌。阻断形成素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，颗粒融合部位的局部 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起着重要作用。
