Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA.
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA; Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Curr Biol. 2023 Nov 20;33(22):4844-4856.e5. doi: 10.1016/j.cub.2023.09.051. Epub 2023 Oct 12.
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.
内吞作用后，许多质膜成分通过从早期内体中出现的膜小管进行再循环，从而形成再循环内体，最终导致它们返回质膜。我们之前曾表明，Syndapin/PACSIN 家族蛋白 SDPN-1 在体内对于 C. elegans 肠道的基底外侧内吞体再循环是必需的。在这里，我们记录了 SDPN-1 SH3 结构域与 PXF-1/PDZ-GEF1/RAPGEF2 中的靶序列之间的相互作用，PXF-1/PDZ-GEF1/RAPGEF2 是已知的 Rap-GTPase 的交换因子。我们发现，工程改造到 SDPN-1 SH3 结构域中的内源性突变，或其在 PXF-1 蛋白中的结合位点，会干扰体内的再循环，而 PXF-1 靶标 RAP-1 的缺失也是如此。在某些情况下，Rap-GTPase 负调控 RhoA 活性，这表明 Syndapin 有可能调节 RhoA。我们的结果表明，在 C. elegans 肠道中，RHO-1/RhoA 富集在 SDPN-1 和 RAP-1 阳性的内体上，而 SDPN-1 或 RAP-1 的缺失会提高肠道内体上的 RHO-1(GTP)水平。此外，我们发现 RHO-1 的耗竭抑制了 sdpn-1 突变体的再循环缺陷，这表明控制 RHO-1 活性是 SDPN-1 促进内吞体再循环的关键机制。RHO-1/RhoA 是众所周知的肌动球蛋白收缩周期的调节因子，尽管对非肌肉肌球蛋白 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 在内体上富集，两种非肌肉肌球蛋白 II 重链同工型似乎在起作用。nmy-2 的耗竭与 sdpn-1 突变体一样抑制再循环，而 nmy-1 的耗竭抑制 sdpn-1 突变体的再循环缺陷，表明肌动球蛋白收缩性控制再循环内体的功能。
