Schumacher Kirstin, Matz Magnus, Brüning Dennis, Baumann Knut, Rustenbeck Ingo
Institute of Pharmacology and Toxicology, University of Braunschweig, Braunschweig, Germany.
Traffic. 2015 May;16(5):493-509. doi: 10.1111/tra.12261. Epub 2015 Feb 24.
The pre-exocytotic behavior of insulin granules was studied against the background of the entirety of submembrane granules in MIN6 cells, and the characteristics were compared with the macroscopic secretion pattern and the cytosolic Ca(2+) concentration of MIN6 pseudo-islets at 22°C, 32°C and 37°C. The mobility of granules labeled by insulin-EGFP and the fusion events were assessed by TIRF microscopy utilizing an observer-independent algorithm. In the z-dimension, 40 mm K(+) or 30 mm glucose increased the granule turnover. The effect of high K(+) was quickly reversible. The increase by glucose was more sustained and modified the efficacy of a subsequent K(+) stimulus. The effect size of glucose increased with physiological temperature whereas that of high K(+) did not. The mobility in the x/y-dimension and the fusion rates were little affected by the stimuli, in contrast to secretion. Fusion and secretion, however, had the same temperature dependence. Granules that appeared and fused within one image sequence had significantly larger caging diameters than pre-existent granules that underwent fusion. These in turn had a different mobility than residence-matched non-fusing granules. In conclusion, delivery to the membrane, tethering and fusion of granules are differently affected by insulinotropic stimuli. Fusion rates and secretion do not appear to be tightly coupled.
在MIN6细胞中，以整个膜下颗粒为背景研究胰岛素颗粒的胞吐前行为，并将其特征与22°C、32°C和37°C下MIN6假胰岛的宏观分泌模式和胞质Ca(2+)浓度进行比较。利用独立于观察者的算法，通过全内反射荧光显微镜（TIRF显微镜）评估胰岛素-增强绿色荧光蛋白（insulin-EGFP）标记颗粒的移动性和融合事件。在z维度上，40 mM K(+)或30 mM葡萄糖可增加颗粒周转率。高K(+)的作用可迅速逆转。葡萄糖引起的增加更持久，并改变了随后K(+)刺激的效力。葡萄糖的效应大小随生理温度升高而增加，而高K(+)则不然。与分泌不同，刺激对x/y维度上的移动性和融合速率影响较小。然而，融合和分泌具有相同的温度依赖性。在一个图像序列中出现并融合的颗粒的笼形直径明显大于发生融合的预先存在的颗粒。反过来，这些颗粒与驻留匹配的未融合颗粒具有不同的移动性。总之，促胰岛素刺激对颗粒向膜的递送、拴系和融合的影响不同。融合速率和分泌似乎没有紧密耦合。
