Heaslip Aoife T, Nelson Shane R, Lombardo Andrew T, Beck Previs Samantha, Armstrong Jessica, Warshaw David M
University of Vermont, Department of Molecular Physiology and Biophysics, Health Sciences Research Facility, Burlington, Vermont, United States of America.
PLoS One. 2014 Oct 13;9(10):e109082. doi: 10.1371/journal.pone.0109082. eCollection 2014.
For pancreatic β-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 β-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited.
为了使胰腺β细胞在血糖升高时分泌胰岛素，保留在亚膜下空间的胰岛素颗粒必须被转运到质膜上的分泌位点。我们结合超分辨率STORM成像和活细胞TIRF显微镜技术，研究了INS-1β细胞中肌动蛋白和微管细胞骨架的组织和动力学如何促成这一过程。绿色荧光蛋白标记的胰岛素颗粒呈现出三种不同的运动模式（静止、扩散样和定向）。在基础的低葡萄糖条件下，扩散样运动占主导。在葡萄糖刺激后，未观察到肌动蛋白细胞骨架的总体重排，但出现了以下情况：1）微管聚合速率增加；2）扩散样运动速率增加；3）经历基于微管的定向运动的颗粒比例增加。通过药理学方法干扰肌动蛋白和微管细胞骨架，我们确定微管依赖性颗粒转运发生在亚膜下空间，并且在基础条件下，当需要抑制胰岛素分泌时，肌动蛋白细胞骨架会限制这种转运。
