Kochubey Olexiy, Majumdar Amitabha, Klingauf Jurgen
Department of Membrane Biophysics, Max-Planck Institute for Biophysical Chemistry, D-37077 Goettingen, Germany.
Traffic. 2006 Dec;7(12):1614-27. doi: 10.1111/j.1600-0854.2006.00492.x. Epub 2006 Oct 2.
Clathrin-mediated endocytosis (CME) is essential for maintaining many basic cellular processes. We monitored the dynamics of clathrin in live Drosophila melanogaster hemocytes overexpressing clathrin light chain fused to enhanced green fluorescent protein (EGFP) using evanescent wave microscopy. Membrane-associated clathrin-coated structures (CCS) constitutively appeared at the peripheral filopodial membrane, moved centripetally while growing in intensity, before being eventually endocytosed within a few tens of seconds. This directed CCS traffic was independent of microtubules but could be blocked by latrunculin A. Taking advantage of available mutants of Drosophila, we expressed clathrin-EGFP in wasp and shibire mutant backgrounds to study the role of actin and dynamin in CCS dynamics and CME in hemocytes. We show that actin plays an essential role in CME in these cells, and that actin and dynamin act at the same stage, but independent of each other. Drosophila melanogaster hemocytes proved to be a promising model system to uncover the molecular events during CME in combining live-cell imaging and genetic analysis.
网格蛋白介导的内吞作用（CME）对于维持许多基本细胞过程至关重要。我们使用倏逝波显微镜监测了过表达与增强型绿色荧光蛋白（EGFP）融合的网格蛋白轻链的活体黑腹果蝇血细胞中网格蛋白的动态变化。膜相关的网格蛋白包被结构（CCS）持续出现在外周丝状伪足膜上，向心移动的同时强度增加，最终在几十秒内被内吞。这种定向的CCS运输独立于微管，但可被拉春库林A阻断。利用果蝇的现有突变体，我们在黄蜂和发动蛋白突变体背景中表达网格蛋白-EGFP，以研究肌动蛋白和发动蛋白在血细胞中CCS动态变化和CME中的作用。我们表明，肌动蛋白在这些细胞的CME中起关键作用，并且肌动蛋白和发动蛋白在同一阶段起作用，但彼此独立。事实证明，结合活细胞成像和遗传分析，黑腹果蝇血细胞是揭示CME过程中分子事件的一个有前景的模型系统。
