Imaging constitutive exocytosis with total internal reflection fluorescence microscopy.
Imaging constitutive exocytosis with total internal reflection fluorescence microscopy.
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DOI：
10.1083/jcb.149.1.23
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发表时间：
2000-04-03
影响因子：
7.8
通讯作者：
Simon, S M
中科院分区：
文献类型：
--
作者：
Schmoranzer, J;Goulian, M;Axelrod, D;Simon, S M
全内反射荧光显微镜已被应用于图像的最后阶段的组成性胞吐，这是融合的单个后高尔基载体与质膜。用绿色荧光蛋白标记的膜蛋白的使用允许以30帧/秒的时间分辨率跟踪融合的动力学。定量分析允许进行融合的载体很容易区分垂直于质膜移动的载体。载体进入质膜的平坦化被看作是荧光强度的总量、峰值和宽度的同时上升。该压平过程的持续时间取决于载体的尺寸，将小球形载体与大管状载体区分开。融合时膜蛋白向质膜的扩散是扩散性的。绘制单个细胞的许多融合位点揭示了在该系统中没有组成性胞吐的优选位点。
Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.