Facilitation of Endosomal Recycling by an IRG Protein Homolog Maintains Apical Tubule Structure in Caenorhabditis elegans

Abstract

Determination of luminal diameter is critical to the function of small single-celled tubes. A series of EXC proteins, including EXC-1, prevent swelling of the tubular excretory canals in Caenorhabditis elegans. In this study, cloning of exc-1 reveals it to encode a homolog of mammalian IRG proteins, which play roles in immune response and autophagy and are associated with Crohn's disease. Mutants in exc-1 accumulate early endosomes, lack recycling endosomes, and exhibit abnormal apical cytoskeletal structure in regions of enlarged tubules. EXC-1 interacts genetically with two other EXC proteins that also affect endosomal trafficking. In yeast two-hybrid assays, wild-type and putative constitutively active EXC-1 binds to the LIM-domain protein EXC-9, whose homolog, cysteine-rich intestinal protein, is enriched in mammalian intestine. These results suggest a model for IRG function in forming and maintaining apical tubule structure via regulation of endosomal recycling. KEYWORDS tubulogenesis; trafficking; endosomes; IRG; immunity-related GTPase S MALL tubules are fundamental structures found in a wide range of tissues in multicellular organisms (Lubarsky and Krasnow 2003;Sigurbjornsdottir et al. 2014). Luminal diameter regulation occurs after initial tubule formation; mutations exist in a range of species in which tubes form initially, but cannot maintain their luminal diameter, and change shape over times ranging from minutes for some Caenorhabditis elegans mutants to decades for some forms of Schwann cell degradation (Patzko and Shy 2012). The mechanisms of maintenance of tube diameter as animals age and grow are still relatively unknown. Mechanosensitive channels on primary cilia projecting into the lumen regulate luminal diameter in multicellular tubes such as blood vessels, nephrons, and biliary ducts (Ware et al. 2011;Martinac 2014), but narrower single-celled tubules such as those of myelinating Schwann cells do not express cilia on their luminal surface (Yoshimura and Takeda 2012).The C. elegans excretory canal cell provides a tractable genetic model for investigating maintenance of luminal diameter in a seamless single-celled tube (Buechner 2002;Sundaram and Buechner 2016). The excretory canal cell is born in midembryogenesis, forms a hollow lumen, and extends both leftward and rightward to the lateral surface, where each side branches and extends canals both anteriorward and posteriorward to form an "H"-shaped structure (Chitwood and Chitwood 1974;Sulston et al. 1983) ( Figure 1A). Once formed, the excretory canals must continue to grow along with the animal. The diameter is tightly controlled, as the lumens taper toward their closed-tip distal ends and expand as the animal ages (Nelson et al. 1983;Sundaram and Buechner 2016). The luminal membrane is surrounded by a thick terminal web similar in appearance to that of intestinal cells and is rich in vacuolar ATPase (Nelson et al. 1983;Oka et al. 1997). Posterior canal extends full-length to the tip of the animal and is of normal diameter, indicat...

Mechanism of Stx17 recruitment to autophagosomes via IRGM and mammalian Atg8 proteins

“…Although immunity-related GTPases are usually considered to be a prolific family of proteins in vertebrates, lower species may not always have recognizable or annotated homologues. Therefore, the IRGM-based ARP complex described in our study may not be the only recruiter/chaperone to facilitate Stx17 delivery to autophagosomes, although Caenorhabditis elegans has an IRGM-like protein, EXC-1, thus far implicated only in endosomal recycling ( Grussendorf et al, 2016 ).…”

Tubular Excretory Canal Structure Depends on Intermediate Filaments EXC-2 and IFA-4 in Caenorhabditis elegans

“…Several exc genes (exc-1, exc-5, and exc-9) with knockout canal phenotypes of large cysts also exhibit characteristic overexpression phenotypes that rescue the canal lumen diameter, while shortening canal length, and show epistatic genetic interactions (Tong and Buechner 2008;Mattingly and Buechner 2011;Grussendorf et al 2016). We therefore looked at overexpression phenotypes of exc-2 and ifa-4.…”

“…single-cell excretory duct cell (Jones and Baillie 1995;Mancuso et al 2012;Gill et al 2016;Pu et al 2017) and the multicellular intestine (Bossinger et al 2004;Zhang et al 2011;Carberry et al 2012;Khan et al 2013;Zhu et al 2015;Geisler et al 2016). In the canal cell, proteins implementing tubule structure comprise apical cytoskeletal elements (McKeown et al 1998;Praitis et al 2005;Khan et al 2013;Kolotuev et al 2013;Shaye and Greenwald 2015), vesicular trafficking and exocyst proteins (Tong and Buechner 2008;Mattingly and Buechner 2011;Armenti et al 2014;Lant et al 2015;Grussendorf et al 2016), and ion and lipid transporters (Berry et al 2003;Khan et al 2013), among others.…”

“…While most of the original exc genes have been cloned (Grussendorf et al 2016;Sundaram and Buechner 2016), mutations in the exc-2 gene cause particularly severe canal defects. In these mutants, the canal length is shortened by over one-half, the animals accumulate multiple cysts in the canals, and are sensitive to growth at low osmolarity (Buechner et al 1999).…”

Terminal web and vesicle trafficking proteins mediate nematode single-cell tubulogenesis

“…All of the three encoded proteins have close human homologues: CRIP1 increases metastatic transformation and invasiveness in cell culture (Cousins and Lanningham-Foster, 2000;He et al, 2017;Li et al, 2017;Zhang et al, 2018); IRGM is involved with autophagy and pathogenic responses (Howard et al, 2011;Kumar et al, 2018;Pilla-Moffett et al, 2016); and FGD genes are loci of the developmental diseases Aarskog-Scott syndrome and Charcot-Marie-Tooth disease type 4H (Delague et al, 2007;Gao et al, 2001;Stendel et al, 2007). Mutants in these nematode genes show similar cystic defects in the structure of the canal apical surface as well as defects in vesicle transport: buildup of RAB-5-labeled vesicles and loss of RME-1-labeled vesicles in areas of cystic dilation (Grussendorf et al, 2016;Mattingly and Buechner, 2011). In addition, overexpression of any of these three genes results in a normal-diameter lumen (apical surface) folded up within a severely shortened canal.…”