Duleh Steve N, Collins Jeannie T B, Pope Robert K
Indiana University South Bend 1700 Mishawaka Avenue, South Bend, IN 46634, USA.
J Electron Microsc (Tokyo). 2005 Dec;54(6):519-28. doi: 10.1093/jmicro/dfi070. Epub 2006 Jan 13.
Rac1B, a small GTP-binding protein in Dictyostelium discoideum, is involved in regulation of the actin cytoskeleton. Scanning electron microscopy revealed distinctive phenotypes for the wild-type, constitutively active, constitutively inactive and overexpressing cell lines. Immunofluorescence showed constitutively active Rac1B localized to lamellipodia and sites of cell-to-cell contact. In contrast, constitutively inactive Rac1B was homogeneously distributed throughout the cell. Phalloidin staining demonstrated that active Rac1B co-localizes with F-actin. Amoebae expressing mutant Rac1B exhibited defects in endocytosis, cytokinesis and multicellular development. Overexpression of wild-type Rac1B positively affected fluid-phase endocytosis, whereas expression of either constitutively active or inactive forms of Rac1B inhibited endocytic rates. The greatest defects in cytokinesis were observed in amoebae producing constitutively active Rac1B or overexpressing wild-type Rac1B. These cells were severely multinucleated and divided by traction-mediated cytofission when placed onto a solid surface. Cells expressing mutant Rac1B were unable to form viable fruiting bodies. Elucidating the role of Rac1B in filamentous actin dynamics will lead to a better understanding of cell adhesion, development and cell motility.
Rac1B是一种盘基网柄菌中的小GTP结合蛋白，参与肌动蛋白细胞骨架的调节。扫描电子显微镜揭示了野生型、组成型激活型、组成型失活型和过表达细胞系的独特表型。免疫荧光显示组成型激活的Rac1B定位于片状伪足和细胞间接触位点。相比之下，组成型失活的Rac1B在整个细胞中均匀分布。鬼笔环肽染色表明活性Rac1B与F-肌动蛋白共定位。表达突变型Rac1B的变形虫在胞吞作用、胞质分裂和多细胞发育方面表现出缺陷。野生型Rac1B的过表达对液相胞吞作用有积极影响，而组成型激活或失活形式的Rac1B的表达均抑制胞吞速率。在产生组成型激活Rac1B或过表达野生型Rac1B的变形虫中观察到胞质分裂的最大缺陷。这些细胞严重多核，当放置在固体表面时通过牵引介导的细胞分裂进行分裂。表达突变型Rac1B的细胞无法形成存活的子实体。阐明Rac1B在丝状肌动蛋白动力学中的作用将有助于更好地理解细胞粘附、发育和细胞运动。
