Edwards K A, Demsky M, Montague R A, Weymouth N, Kiehart D P
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Dev Biol. 1997 Nov 1;191(1):103-17. doi: 10.1006/dbio.1997.8707.
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 labeling 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.
膜突蛋白、埃兹蛋白和根蛋白（MER）是皮质肌动蛋白细胞骨架以及丝状伪足和微绒毛等膜性突起的组成成分。它们的C末端尾巴包含一个预测为螺旋状的延伸区域、一个肌动蛋白结合结构域以及参与自我缔合的一个或多个区域。我们通过将编码水母绿色荧光蛋白（GFP）的序列与编码果蝇唯一的MER同源物膜突蛋白[膜突蛋白样基因产物，先前称为D17 MER样蛋白；爱德华兹等人，1994年，《美国国家科学院院刊》91，4589]和Dmoesin[麦卡特尼和费洪，1996年，《细胞生物学杂志》133，843]的C末端的序列连接起来，构建了一种体内荧光肌动蛋白结合蛋白（GFP-膜突蛋白）。生成了在hsp70启动子控制下表达这种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的细胞形状变化。热休克后，所有体细胞组织都会产生高水平的稳定融合蛋白。GFP-膜突蛋白定位于皮质肌动蛋白细胞骨架，为上皮形态发生过程中的细胞形状和模式提供了一个强大的体内标记。该蛋白还会在伪足、微绒毛、轴突、齿状突起、边缘细胞突起和其他膜性突起中高度富集，这可能是通过与内源性膜突蛋白以及肌动蛋白结合实现的。我们表明GFP-膜突蛋白可用于在活体标本中检测这些动态结构的发育和行为。我们观察到一个明亮的绿色荧光、可能富含肌动蛋白的极性细胞喙，它插入形成中的卵孔，并似乎维持一个精子通过的开口，围绕此开口形成卵壳。我们还证实了在侧表皮前缘存在一条富含肌动蛋白的收缩环，并对其在背侧闭合过程中迁移时的行为进行了动态分析。对胚胎、幼虫和蛹的观察表明，GFP-膜突蛋白也可用于标记发育中的神经系统，并且将是活体组织形态发生过程中动态细胞行为的一个良好通用标记，还证明了将亚细胞定位信号与GFP融合极大地增加了其作为细胞标记的效用。
