Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Traffic. 2018 Jun;19(6):436-445. doi: 10.1111/tra.12562. Epub 2018 Apr 24.
Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P ) by recruitment of a 5'-phosphatase strongly inhibited Ca -dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by α-toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site.
磷脂酰肌醇（PtdIns）在胞吐作用中发挥重要作用，被认为通过与 SNARE 蛋白 syntaxin 共同聚集形成质膜上的停靠受体来调节分泌颗粒的 docking。在这里，我们通过对活胰岛素分泌细胞中分泌颗粒释放部位的 EGFP 标记 PtdIns 标记物或 syntaxin-1 进行高分辨率全内反射成像来检验这一想法。在完整的细胞中，PtdIns 标记物均匀分布在质膜上，没有对颗粒 docking 位点的偏好。相比之下，syntaxin-1 被发现聚集在质膜中，主要位于停靠颗粒下方。我们还观察到 granule 到达 docking 位点时 syntaxin-1 的快速积累。通过募集 5'-磷酸酶急性耗尽质膜磷脂酰肌醇（4,5）二磷酸（PtdIns(4,5)P）强烈抑制 Ca2+依赖性胞吐作用，但对 docked 颗粒或 syntaxin-1 的分布和聚集没有影响。α-毒素或甲醛固定引起的细胞通透性会导致 PtdIns 标记物缓慢聚集，部分靠近停靠颗粒。总之，我们的数据表明 PtdIns(4,5)P 加速颗粒的引发，但挑战了 PtdIns 在分泌颗粒 docking 或 syntaxin-1 在释放部位聚集中的作用。
