PDF(Pubmed)
Abstract：
After endocytosis, many plasma membrane components are recycled via membrane tubules that emerge from early endosomes to form recycling endosomes, eventually leading to their return to the plasma membrane. We previously showed that Syndapin/PACSIN-family protein SDPN-1 is required in vivo for basolateral endocytic recycling in the C. elegans intestine. Here, we document an interaction between the SDPN-1 SH3 domain and a target sequence in PXF-1/PDZ-GEF1/RAPGEF2, a known exchange factor for Rap-GTPases. We found that endogenous mutations engineered into the SDPN-1 SH3 domain, or its binding site in the PXF-1 protein, interfere with recycling in vivo, as does the loss of the PXF-1 target RAP-1. In some contexts, Rap-GTPases negatively regulate RhoA activity, suggesting a potential for Syndapin to regulate RhoA. Our results indicate that in the C. elegans intestine, RHO-1/RhoA is enriched on SDPN-1- and RAP-1-positive endosomes, and the loss of SDPN-1 or RAP-1 elevates RHO-1(GTP) levels on intestinal endosomes. Furthermore, we found that depletion of RHO-1 suppressed sdpn-1 mutant recycling defects, indicating that control of RHO-1 activity is a key mechanism by which SDPN-1 acts to promote endocytic recycling. RHO-1/RhoA is well known for controlling actomyosin contraction cycles, although little is known about the effects of non-muscle myosin II on endosomes. Our analysis found that non-muscle myosin II is enriched on SDPN-1-positive endosomes, with two non-muscle myosin II heavy-chain isoforms acting in apparent opposition. Depletion of nmy-2 inhibited recycling like sdpn-1 mutants, whereas depletion of nmy-1 suppressed sdpn-1 mutant recycling defects, indicating that actomyosin contractility controls recycling endosome function.
摘要：
胞吞后,许多质膜成分通过从早期内体出现的膜小管再循环形成再循环内体，最终导致它们回到质膜。我们先前表明Syndapin/PACSIN家族蛋白SDPN-1在秀丽隐杆线虫肠中的基底外侧胞吞再循环在体内是必需的。这里,我们记录了SDPN-1SH3结构域和PXF-1/PDZ-GEF1/RAPGEF2靶序列之间的相互作用,PXF-1/PDZ-GEF1/RAPGEF2是Rap-GTPases的已知交换因子.我们发现内源性突变工程改造到SDPN-1SH3结构域，或其在PXF-1蛋白中的结合位点，干扰体内回收，PXF-1靶RAP-1的丢失也是如此。在某些情况下,Rap-GTP酶负调节RhoA活性，这表明Syndapin具有调节RhoA的潜力。我们的结果表明，在秀丽隐杆线虫的肠道中，RHO-1/RhoA富集在SDPN-1-和RAP-1-阳性内体上，SDPN-1或RAP-1的丢失会升高肠内体的RHO-1（GTP）水平。此外，我们发现RHO-1的消耗抑制了sdpn-1突变体的再循环缺陷，表明RHO-1活性的控制是SDPN-1促进胞吞再循环的关键机制。RHO-1/RhoA是众所周知的控制肌动球蛋白收缩周期，尽管关于非肌肉肌球蛋白II对内体的影响知之甚少。我们的分析发现，非肌肉肌球蛋白II在SDPN-1阳性内体上富集，两个非肌肉肌球蛋白II重链同工型明显相反。像sdpn-1突变体一样，nmy-2的耗尽抑制了循环利用，而nmy-1的消耗抑制了sdpn-1突变体的再循环缺陷，表明肌动球蛋白收缩性控制循环内体功能。
