GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in Drosophila
GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in Drosophila
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DOI：
10.1006/dbio.1997.8707
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发表时间：
1997-11-01
影响因子：
2.7
通讯作者：
Kiehart, DP
中科院分区：
文献类型：
--
作者：
Edwards, KA;Demsky, M;Kiehart, DP
Moesin， ezrin和radixin （MER）是皮质肌动蛋白细胞骨架和膜突（如丝状伪足和微绒毛）的组成部分。它们的c端尾部包含一个被预测为螺旋状的延伸区域、一个肌动蛋白结合域和一个参与自结合的区域。我们设计了一种体内荧光肌动蛋白结合蛋白（GFP-moe），通过将编码水母绿色荧光蛋白（GFP）的序列与编码唯一果蝇MER同源物moesin （moesin样基因产物，以前称为D17 MER样蛋白）的c端序列连接起来；Edwards et al., 1994, Proc. Natl。学会科学。[j].中国生物医学工程学报，1997,18(2):444 - 444。在hsp70启动子的控制下，产生了表达该融合蛋白的转基因果蝇，并用于分析不同发育阶段和组织形态发生过程中细胞形状的变化。热休克后，所有体细胞组织都产生高水平的稳定融合蛋白。GFP-moe定位于皮质肌动蛋白细胞骨架，在上皮形态发生过程中为细胞形状和模式提供了强有力的体内标记。该蛋白也在假足、微绒毛、轴突、小齿、边缘细胞突起和其他膜突起中高度富集，可能与内源性moesin和actin结合。我们表明，GFP-moe可以用来检查这些动态结构的发展和行为在活的标本。我们观察到一个明亮的绿色荧光，可能是富含肌动蛋白，极细胞的喙插入形成的微孔中，似乎为精子通道保持一个开口，绒毛膜在其周围形成。我们还证实了在外侧表皮前缘存在一个富含肌动蛋白的荷包线，并对其在背侧闭合期间的迁移行为进行了动态分析。对胚胎、幼虫和蛹的观察表明，GFP-moe也可用于标记发育中的神经系统，并将成为活组织形态发生过程中动态细胞行为的良好通用标记物，并证明亚细胞定位信号与GFP的融合大大增加了其作为细胞标记物的效用。(C) 1997学术出版社。
Moesin, ezrin, and radixin (MER) are components of the cortical actin cytoskeleton and membrane processes such as filopodia and microvilli. Their C-terminal tails contain an extended region that is predicted to be helical, an actin binding domain, and a region(s) that participates in self-association. We engineered an in vivo fluorescent actin binding protein (GFP-moe) by joining sequences that encode the jellyfish green fluorescent protein (GFP) to sequences that encode the C-terminal end of the sole Drosophila MER homolog, moesin [Moesin-like gene product, referred to previously as the D17 MER-like protein; Edwards et al., 1994, Proc. Natl. Acad. Sci. USA 91, 4589], and Dmoesin [McCartney and Fehon, 1996, J. Cell Biol. 133, 843]. Transgenic flies expressing this fusion protein under control of the hsp70 promoter were generated and used for analysis of cell shape changes during morphogenesis of various developmental stages and tissues. Following heat shock, high levels of stable fusion protein are produced by all somatic tissues. GFP-moe localizes to the cortical actin cytoskeleton, providing a strong in vivo marker for cell shape and pattern during epithelial morphogenesis. The protein also becomes highly enriched in pseudopods, microvilli, axons, denticles, the border cell process, and other membrane projections, potentially by binding to endogenous moesin as well as actin. We show that GFP-moe can be used to examine the development and behavior of these dynamic structures in live specimens. We observe a bright green fluorescent, presumably actin-rich, polar cell proboscis that inserts itself into the forming micropyle and appears to maintain an opening for sperm passage around which the chorion is formed. We also confirm the existence of an actin-rich purse string at the leading edge of the lateral epidermis and provide a dynamic analysis of its behavior as it migrates during dorsal closure. Observations of embryos, larvae, and pupae show that GFP-moe is also useful for labelling the developing nervous system and will be a good general marker of dynamic cell behavior during morphogenesis in live tissues and demonstrate that fusion of a subcellular localization signal to GFP greatly increases its utility as a cell marker. (C) 1997 Academic Press.