RacB regulates cytoskeletal function in Dictyostelium spp.
RacB regulates cytoskeletal function in Dictyostelium spp.
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DOI：
10.1128/ec.2.3.474-485.2003
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发表时间：
2003-06-01
期刊：
影响因子：
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通讯作者：
Knecht, DA
中科院分区：
文献类型：
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作者：
Lee, E;Seastone, DJ;Knecht, DA
到目前为止，已鉴定出14个哺乳动物Rac蛋白的同源物。目前尚不清楚这些基因中的每一个是否都具有独特的功能，或者它们在肌动蛋白细胞骨架组织中扮演多大程度的多余角色。为了研究racB的特殊功能，我们有条件地表达了该蛋白的野生型(WT-racB)、显性负性(N17-racB)和结构性激活(V12-racB)版本。诱导后，表达V12-racB的细胞停止生长，脱离表面，并形成许多球状表面突起，而过度表达WT-racB的细胞在表面变平。相反，过度表达N17-racB的细胞没有表现出任何明显的形态异常。在V12-racB细胞中看到的表面突起似乎是肌动蛋白驱动的突起，因为它们富含F-肌动蛋白，并且可以被细胞松弛素A抑制。V12-racB细胞中的突起不需要肌球蛋白11活性，这与应激下野生型细胞形成的气泡不同。最后，我们研究了野生型和突变型racB表达的功能后果。在所有表达racB蛋白的细胞系中，吞噬、内吞和液体相外排的速率都降低了，但表达V12-racB的细胞的下降幅度最大。根据这些结果，我们得出结论，与Rho家族的其他成员一样，racB可以诱导肌动蛋白聚合，但激活的结果似乎与迄今研究的其他Dictyostelialrae蛋白不同，导致细胞发生不同的形态和功能变化。
Thus far, 14 homologues of mammalian Rac proteins have been identified in Dictyostelium. It is unclear whether each of these genes has a unique function or to what extent they play redundant roles in actin cytoskeletal organization. To investigate the specific function of RacB, we have conditionally expressed wildtype (WT-RacB), dominant negative (N17-RacB), and constitutively activated (V12-RacB) versions of the protein. On induction, cells expressing V12-RacB stopped growing, detached from the surface, and formed numerous spherical surface protrusions while cells overexpressing WT-RacB became flattened on the surface. In contrast, cells overexpressing N17-RacB did not show any significant morphological abnormalities. The surface protrusions seen in V12-RacB cells appear to be actin-driven protrusions because they were enriched in F-actin and were inhibitable by cytochalasin A treatment. The protrusions in V12-RacB cells did not require myosin 11 activity, which distinguishes them from blebs formed by wild-type cells under stress. Finally, we examined the functional consequences of expression of wild-type and mutant RacB. Phagocytosis, endocytosis, and fluid phase efflux rates were reduced in all cell lines expressing RacB proteins but the greatest decrease was observed for cells expressing V12-RacB. From these results, we conclude that like other members of the Rho family, RacB induces polymerization of actin but the consequences of activation appear to be different from other Dictyostelium Rae proteins so far investigated, resulting in different morphological and functional changes in cells.