Imaging clathrin dynamics in Drosophila melanogaster hemocytes reveals a role for actin in vesicle fission
Imaging clathrin dynamics in Drosophila melanogaster hemocytes reveals a role for actin in vesicle fission
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DOI：
10.1111/j.1600-0854.2006.00492.x
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发表时间：
2006-12-01
期刊：
影响因子：
4.5
通讯作者：
Klingauf, Jurgen
中科院分区：
文献类型：
--
作者：
Kochubey, Olexiy;Majumdar, Arnitabha;Klingauf, Jurgen
笼状蛋白介导的内吞作用(CME)对于维持许多基本的细胞过程是必不可少的。我们用消逝波显微镜监测了活的果蝇、高表达胞壁蛋白轻链的黑腹血细胞与增强型绿色荧光蛋白(EGFP)融合后胞质蛋白的动态变化。膜相关的胞膜蛋白涂层结构(CCS)在外周丝状胞膜上结构性地出现，向心移动，强度增大，最终在几十秒内被内吞。这种定向的CCS运输不依赖于微管，但可以被Latrunculin A阻断。我们利用已有的果蝇突变体，在黄蜂和Shibire突变背景中表达了clathrin-EGFP，以研究肌动蛋白和动力蛋白在CCS动力学和血细胞CME中的作用。我们证明了肌动蛋白在这些细胞的CME中扮演着重要的角色，并且肌动蛋白和动力蛋白在同一阶段起作用，但彼此独立。通过结合活细胞成像和遗传分析，果蝇血细胞被证明是一个很有希望的模型系统来揭示CME过程中的分子事件。
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.