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Syndapin Regulates the RAP-1 GTPase to Control Endocytic Recycling via RHO-1 and Non-Muscle Myosin II.

Wilmer R Rodriguez-PolancoAnne NorrisAgustin B VelascoAdenrele M GleasonBarth D Grant
Published in: bioRxiv : the preprint server for biology (2023)
After endocytosis, many plasma membrane components are recycled via narrow-diameter membrane tubules that emerge from early endosomes to form recycling endosomes, eventually leading to their return to the plasma membrane. We previously showed that the F-BAR and SH3 domain Syndapin/PACSIN-family protein SDPN-1 is required in vivo for basolateral endocytic recycling in the C. elegans intestine. Here we sought to determine the significance of a predicted 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 we engineered into the SDPN-1 SH3 domain, or its binding site in the PXF-1 protein, interfere with recycling in vivo , as does loss of the PXF-1 target RAP-1. Rap-GTPases have been shown in several contexts to negatively regulate RhoA activity. Our results show that RHO-1/RhoA is enriched on SDPN-1 and RAP-1 positive endosomes in the C. elegans intestine, and 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 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, while depletion of nmy-1 suppressed sdpn-1 mutant recycling defects, indicating actomyosin contractility in controlling recycling endosome function.
Keyphrases
  • smooth muscle
  • binding protein
  • protein kinase
  • skeletal muscle
  • magnetic resonance imaging
  • diffusion weighted imaging
  • prefrontal cortex