Department of Biology and Biotechnology, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA 01609, USA.
Dev Biol. 2013 Jan 1;373(1):1-13. doi: 10.1016/j.ydbio.2012.09.017. Epub 2012 Sep 27.
Directed cell migration and process outgrowth are vital to proper development of many metazoan tissues. These processes are dependent on reorganization of the actin cytoskeleton in response to external guidance cues. During development of the nervous system, the MIG-10/RIAM/Lamellipodin (MRL) signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons. In C. elegans, mutations in the MRL family member gene mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or 'canals' of the excretory cell, which is required for osmoregulation in the worm. In addition, mig-10 mutant animals have recently been shown to have defects in clustering of vesicles at the synapse. To determine additional molecular partners of MIG-10, we conducted a yeast two-hybrid screen using isoform MIG-10A as bait and isolated Abelson-interactor protein-1 (ABI-1). ABI-1, a downstream target of Abl non-receptor tyrosine kinase, is a member of the WAVE regulatory complex (WRC) involved in the initiation of actin polymerization. Further analysis using a co-immunoprecipitation system confirmed the interaction of MIG-10 and ABI-1 and showed that it requires the SH3 domain of ABI-1. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncated outgrowth of the excretory cell canals, suggesting that the ABI-1/MIG-10 interaction is relevant in vivo. Cell autonomous expression of MIG-10 isoforms rescued both the neuronal migration and the canal outgrowth defects, showing that MIG-10 functions autonomously in the ALM neurons and the excretory cell. These results suggest that MIG-10 and ABI-1 interact physically to promote cell migration and process outgrowth in vivo. In the excretory canal, ABI-1 is thought to act downstream of UNC-53/NAV2, linking this large scaffolding protein to actin polymerization during excretory canal outgrowth. abi-1(RNAi) enhanced the excretory canal truncation observed in mig-10 mutants, while double mutant analysis between unc-53 and mig-10 showed no increased truncation of the posterior canal beyond that observed in mig-10 mutants. Morphological analysis of mig-10 and unc-53 mutants showed that these genes regulate canal diameter as well as its length, suggesting that defective lumen formation may be linked to the ability of the excretory canal to grow out longitudinally. Taken together, our results suggest that MIG-10, UNC-53, and ABI-1 act sequentially to mediate excretory cell process outgrowth.
定向细胞迁移和突起生长对于许多后生动物组织的正常发育至关重要。这些过程依赖于细胞骨架肌动蛋白的重排,以响应外部导向信号。在神经系统发育过程中,MIG-10/RIAM/Lamellipodin(MRL)信号蛋白被认为将位置信息从表面导向信号传递到肌动蛋白聚合机制,从而促进轴突的极化生长。在秀丽隐杆线虫中,MRL 家族成员基因 mig-10 的突变导致动物在轴突导向、神经元迁移以及排泄细胞突起或“通道”的生长方面存在缺陷,这是线虫渗透调节所必需的。此外,最近mig-10 突变体动物在突触小泡聚集方面也存在缺陷。为了确定 MIG-10 的其他分子伴侣,我们使用同工型 MIG-10A 作为诱饵进行了酵母双杂交筛选,并分离到了 Abelson 相互作用蛋白-1(ABI-1)。ABI-1 是 Abl 非受体酪氨酸激酶的下游靶标,是参与肌动蛋白聚合起始的 WAVE 调节复合物(WRC)的成员。使用共免疫沉淀系统的进一步分析证实了 MIG-10 和 ABI-1 的相互作用,并表明它需要 ABI-1 的 SH3 结构域。mig-10 和 abi-1 的单突变体表现出相似的 ALM 神经元不完全迁移和排泄细胞通道截断生长的表型,表明 ABI-1/MIG-10 相互作用在体内是相关的。MIG-10 同工型的细胞自主表达挽救了神经元迁移和通道生长缺陷,表明 MIG-10 在 ALM 神经元和排泄细胞中自主发挥作用。这些结果表明,MIG-10 和 ABI-1 相互作用以促进体内细胞迁移和突起生长。在排泄道中,ABI-1 被认为在 UNC-53/NAV2 下游发挥作用,将这种大支架蛋白与排泄道生长过程中的肌动蛋白聚合联系起来。abi-1(RNAi)增强了 mig-10 突变体中观察到的排泄道截断,而 unc-53 和 mig-10 之间的双突变体分析显示,在后侧通道的截断程度没有超过 mig-10 突变体的程度。对 mig-10 和 unc-53 突变体的形态分析表明,这些基因调节通道直径及其长度,表明管腔形成缺陷可能与排泄道纵向生长的能力有关。综上所述,我们的结果表明,MIG-10、UNC-53 和 ABI-1 依次作用以介导排泄细胞突起生长。