We were the first to show that C. elegans RME-1 and mammalian mRME-1/EHD1 (with Fred Maxfield) are required for recycling endosome to plasma membrane transport. With our collaborator Lois Greene we also showed that proteins of the RME-1 family are ATPases and require ATP binding for homo-oligomerization and association with endosomes (Lee et al., 2005). We later identified several RME-1 interacting proteins that collaborate with RME-1 in this function, including the BAR-domain protein AMPH-1/Amphiphysin/BIN1 (Shi et al., 2007; Pant et al., 2009; Liu and Grant, 2015). Furthermore, with our collaborator Steve Caplan, we showed that mammalian Amph2/BIN1 is also required for recycling endosome function (Pant et al., 2009). Importantly, in collaboration with Chavela Carr we advanced this work by establishing a synthetic liposome and pure recombinant protein reconstitution system for these proteins. In vitro we found that pure recombinant RME-1 forms a distinctive spiral coat that tubulates liposomes, consistent with our proposal that RME-1/EHD family proteins function as part of the tubulation and fission machinery on endosomal tubules (Pant et al., 2009).

 
Figure 1

Fig 1. (A) One cell length of the C. elegans intestine within the intact animal showing RME-1-labeling of the tubular endocytic network in the basolateral cortex. (B) Immuno-gold labeling of endogenous RME-1 on tubulovesicular endosomes of the C. elegans intestine.

FIg 2

Fig 2. Live super-resolution image of tubular recycling endoscopes in the basal cortex double labeled with hTAC (GFP) cargo, and EHBP-1 (mCherry) that links the endosomes to F-actin.

Figure 3

Fig 3. Current model for SDPN-1 Syndapin and NMII-mediated tubular endosomal fission. We observed most tubule retraction away from the SDPN-1 enriched vesicle, suggestion a contractile force along the length of the tubule, while the negative-regulation of NMII function by SDPN-1-centered mechanisms suggests a role in restraining NMII activity near the vesicular domain. Such localized regulation could create a force imbalance at the tubule/vesicle junction, contribution to fission.

Our studies of rme-1 mutant phenotypes led to our discovery that the basolateral endocytic recycling system in the C. elegans intestine is organized as a tubular endocytic network, an extensively interconnected system of vesicles and tubules (Figs. 1-2). Such tubular membrane systems are recognized as a common cellular organization for recycling endosome organelles emanating from sorting endosomes, but the factors and mechanisms that control such systems were largely unknown. In addition to RME-1, our studies identified RAB-10 and its effectors CNT-1/ARF-GAP and EHBP-1 as essential endocytic recycling factors required to elaborate these tubular endosomal elements (Chen et al., 2006; Shi et al., 2010; Shi et al., 2012; Liu and Grant, 2015; Wang et al., 2016). Our findings in C. elegans preceded the general acceptance of Rab10-mediated processes as key to tubular endocytic network formation in mammals. We showed that RAB-10 recruits CNT-1 to regulate endosomal ARF-6, thereby controlling endosomal PI(4,5)P2 levels (Shi et al., 2012). We also discovered that the EHBP-1 protein follows to provide essential links between tubular PI(4,5)P2-enriched recycling endosomal membranes and the F-actin cytoskeleton, and with my former student Anbing Shi we showed that RAB-10-binding to EHBP-1 directly potentiates this activity (Shi et al., 2010; Wang et al., 2016). We further identified a countercurrent regulatory loop by which RAB-10 directly binds to RAB-5 GAP TBC-2 to down-regulate RAB-5 during recycling (Liu and Grant, 2015). This Rab conversion is essential, as we showed that if RAB-5 is not downregulated on recycling endosomes, recycling fails (Liu and Grant, 2015). Our work provided the first molecular framework for understanding an early endosome to recycling endosome GTPase cascade in any organism.

Most recently we gained new insights into the fundamental forces essential for the tubular endocytic network to function. Actin polymerization on endosomes has long been proposed to provide necessary membrane tension to promote fission, but a clear understanding of how actin contributes to tension, and how fission is regulated remain as large gaps in understanding. Our recent results provide surprising new evidence that non-muscle myosin II (NMII) functions with actin to regulate endocytic recycling (Fig. 3) (Rodriguez-Polanco et al., 2023). We further discovered a novel endosomal signaling hub centered on the Syndapin (SDPN-1) protein that regulates endosomal RhoA and NMII in this process, likely providing tension control during tubule fission (Rodriguez-Polanco et al., 2023).

Related Publications

Syndapin and GTPase RAP-1 control endocytic recycling via RHO-1 and non-muscle myosin II Rodriguez-Polanco WR, Norris A, Velasco AB, Gleason AM, Grant BD. Syndapin and GTPase RAP-1 control endocytic recycling via RHO-1 and non-muscle myosin II. Curr Biol. 2023. Epub 20231005. doi: 10.1016/j.cub.2023.09.051. PubMed PMID: 37832552. [PubMed]

RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes Wang P, Liu H, Wang Y, Liu O, Zhang J, Gleason A, Yang Z, Wang H, Shi A, Grant BD. RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes. PLoS Genet. 2016;12(6):e1006093. Epub 20160606. doi: 10.1371/journal.pgen.1006093. PubMed PMID: 27272733; PMCID: PMC4894640. [PubMed]

Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes Liu O, Grant BD. Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes. PLoS Genet. 2015;11(9):e1005514. Epub 20150922. doi: 10.1371/journal.pgen.1005514. PubMed PMID: 26393361; PMCID: PMC4578947. [PubMed]

RAB-10-GTPase-mediated regulation of endosomal phosphatidylinositol-4,5-bisphosphate Shi A, Liu O, Koenig S, Banerjee R, Chen CC, Eimer S, Grant BD. RAB-10-GTPase-mediated regulation of endosomal phosphatidylinositol-4,5-bisphosphate. Proc Natl Acad Sci U S A. 2012;109(35):E2306-15. Epub 20120806. doi: 10.1073/pnas.1205278109. PubMed PMID: 22869721; PMCID: PMC3435156. [PubMed]

EHBP-1 functions with RAB-10 during endocytic recycling in Caenorhabditis elegans Shi A, Chen CC, Banerjee R, Glodowski D, Audhya A, Rongo C, Grant BD. EHBP-1 functions with RAB-10 during endocytic recycling in Caenorhabditis elegans. Mol Biol Cell. 2010;21(16):2930-43. Epub 20100623. doi: 10.1091/mbc.E10-02-0149. PubMed PMID: 20573983; PMCID: PMC2921114. [PubMed]

AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling Pant S, Sharma M, Patel K, Caplan S, Carr CM, Grant BD. AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling. Nat Cell Biol. 2009;11(12):1399-410. Epub 20091115. doi: 10.1038/ncb1986. PubMed PMID: 19915558; PMCID: PMC2788952. [PubMed]

A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport Shi A, Pant S, Balklava Z, Chen CC, Figueroa V, Grant BD. A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport. Curr Biol. 2007;17(22):1913-24. Epub 20071108. doi: 10.1016/j.cub.2007.10.045. PubMed PMID: 17997305; PMCID: PMC2175126. [PubMed]

Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit Sato M, Sato K, Fonarev P, Huang CJ, Liou W, Grant BD. Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit. Nat Cell Biol. 2005;7(6):559-69. Epub 20050515. doi: 10.1038/ncb1261. PubMed PMID: 15895077; PMCID: PMC1398054. [PubMed]

ATP binding regulates oligomerization and endosome association of RME-1 family proteins Lee DW, Zhao X, Scarselletta S, Schweinsberg PJ, Eisenberg E, Grant BD, Greene LE. ATP binding regulates oligomerization and endosome association of RME-1 family proteins. J Biol Chem. 2005;280(17):17213-20. Epub 20050213. doi: 10.1074/jbc.M412751200. PubMed PMID: 15710626. [PubMed]