Sato K et al. (2014) WormBook "C. elegans as a model for membrane traffic."

Sato M, Norris A, Sato K, Grant BD

[WormBook, 2014]

The counterbalancing action of the endocytosis and secretory pathways maintains a dynamic equilibrium that regulates the composition of the plasma membrane, allowing it to maintain homeostasis and to change rapidly in response to alterations in the extracellular environment and/or intracellular metabolism. These pathways are intimately integrated with intercellular signaling systems and play critical roles in all cells. Studies in Caenorhabditis elegans have revealed diverse roles of membrane trafficking in physiology and development and have also provided molecular insight into the fundamental mechanisms that direct cargo sorting, vesicle budding, and membrane fisson and fusion. In this review, we summarize progress in understanding membrane trafficking mechanisms derived from work in C. elegans, focusing mainly on work done in non-neuronal cell-types, especially the germline, early embryo, coelomocytes, and intestine.

Sloan, Dillon E. et al. (2020) MicroPubl Biol

Bembenek, Joshua N., Sloan, Dillon E.

[MicroPubl Biol, 2020]

Caveolins are integral membrane proteins responsible for the formation of caveolae, invaginations of the plasma membrane linked to various disease states (Parton et al. 2020). In C. elegans, there are two caveolin proteins, CAV-1 and CAV-2. The cav-1 gene shares homology with all three mammalian caveolin genes (Tang et al. 1997). C. elegans CAV-1 protein does not appear to form caveolae, but a double-knockout mutant of cav-1 and cav-2 affects egg laying, and knockdown of cav-1 affects locomotion in a dynamin mutant background (Parker et al. 2007, Kirkham et al. 2008, Sato et al. 2008). Based on exogenous expression, CAV-1::GFP is known to localize to cortical granules and the plasma membrane in oocytes and the early embryo, to the plasma membrane in later embryos, as well as to the neuromuscular system in larvae and adult worms (Sato et al. 2006, Bembenek et al. 2007, Parker et al. 2007).

Jaspersen SL et al. (2009) Cell "Pushing and pulling: microtubules mediate meiotic pairing and synapsis."

Jaspersen SL, Hawley RS

[Cell, 2009]

Meiotic pairing in the nematode Caenorhabditis elegans is facilitated by chromosomal sites known as pairing centers that are tethered to the nuclear envelope. Sato et al. (2009) and Penkner et al. (2009) provide insight into how proteins linking pairing centers and the microtubule cytoskeleton mediate homolog pairing and restrict synapsis to homologous pairs of chromosomes.

Sato M et al. (2008) Tanpakushitsu Kakusan Koso "[C. elegans, an amenable model system for the study of membrane trafficking in living animals]."

Sato M, Sato K

[Tanpakushitsu Kakusan Koso, 2008]

Juanico, Iris Y et al. (2021) MicroPubl Biol

Gong, Ting, McNally, Francis J, Juanico, Iris Y, Meyer, Christina M, McCarthy, John E

[MicroPubl Biol, 2021]

RMD-1 was discovered as a cytoplasmic regulator of microtubule dynamics in C. elegans and several highly homologous proteins were inferred from the C. elegans and human genome sequences (Oishi et al., 2007). One of the human homologs, RMD-3/PTPIP51, has an N-terminal extension relative to other RMDs that targets it to the mitochondrial outer membrane and which binds the endoplasmic reticulum (ER) protein VAPB (De Vos et al., 2012). This interaction mediates tethering of mitochondria to the ER in human cells (Stoica et al., 2014). None of the C. elegans RMDs have sequence homology with the N-terminal extension found on human RMD-3/PTPIP51, however, C. elegans RMD-2 has an N-terminal extension relative to the other RMDs and this extension has potential to form a transmembrane helix as determined by TMpred release 25 (https://embnet.vital-it.ch/software/TMPRED_form.html). RMD-2 is present in sperm whereas RMD-3 and RMD-6 are highly enriched in sperm (Ma et al., 2014). Paternal mitochondria are eventually degraded in the embryo (Sato and Sato, 2011). However, they are maintained, along with other sperm contents, at the opposite end of the zygote from the female meiotic spindle during meiosis (Fig. 1). We hypothesized that RMD-2, 3 and 6 on paternal mitochondria bind to the VAPB homolog, VPR-1, on maternal ER at fertilization to tether the sperm contents at the future posterior end of the zygote.

Ken Sato et al. (2004) East Coast Worm Meeting "C. elegans rme-3 encodes a clathrin heavy chain required for embryogenesis and neuro-muscular function"

Chih-Hsiung Chen, Ken Sato, Barth D Grant, Miyuki Sato

[East Coast Worm Meeting, 2004]

To study the molecular mechanisms of receptor-mediated endocytosis in C. elegans , we developed a system to assess the efficiency of receptor-mediated endocytosis of yolk proteins by oocytes. We created a transgenic strain that expresses a GFP fusion gene with VIT-2 , encoding the lipid binding yolk protein YP170. YP170-GFP is expressed in the intestinal cells and secreted into the pseudocoelom. In wild type worms, YP170-GFP is taken up by oocytes via receptor-mediated endocytosis. Using this assay, we isolated mutants that fell into 11 complementation groups and named these genes rme for r eceptor- m ediated e ndocytosis defective. One of these, the rme-3 (b1025) mutant, showed a temperature sensitive defect in yolk uptake and ts embryonic lethality. Cloning revealed that rme-3 (b1025) is a temperature sensitive allele of clathrin heavy chain (CHC), the first temperature sensitive CHC mutant in a higher eukaryote. Interestingly, rme-3 (b1025) also shows severe defects in neuro-muscular function. After incubation at 25 ! (inverted exclamation mark) C for 24h, rme-3 (b1025) worms picked into liquid can thrash for a few minutes, but become almost completely paralyzed 3-5 min later, suggesting a progressive loss of synaptic function. rme-3 (b1025) also shows a moderate defect in pharyngeal pumping. A weaker CHC allele recovered in our screen, rme-3 (b1024) , also shows similar but weaker defects. rme-3 (b1025) is aldicarb-resistant and levamisole-sensitive, suggesting rme-3 functions pre-synaptically at neuro-muscular junctions. In the near future we plan on performing a detailed ultra-structural analysis of the synapses in the rme-3 mutants to determine the exact nature of these defects.

Sato, Manami et al. (2015) International Worm Meeting "Expression pattern of C. elegans Y RNA homologs."

Ushida, Chisato, Kihara, Shinya, Sato, Manami

[International Worm Meeting, 2015]

Y RNA is a small structured ncRNA of about 100 nt in length. This RNA binds to Ro60 protein, which is a target of autoimmune disease antibody in patients with systemic lupus erythematosus and Sjogren's syndrome. Several lines of evidence suggest that the role of Y RNA and Ro60 function in the quality control of structured ncRNAs in cells under stress conditions. It is also indicated that vertebrate Y RNAs function in the initiation of DNA replication without Ro60. However, the molecular mechanisms of these functions and the contribution of Ro60/Y RNP to the autoimmune disease are still unclear. C. elegans genome encodes one Ro60 homolog (ROP-1) and 19 Y RNA homologs (1 CeY RNA and 18 sbRNAs). Other animals also have several Y RNA homologs, but C. elegans is the first example which has more than 5 Y RNA homologs encoded in the genome. Here we show the expression pattern and the cellular localization of these Y RNA homologs in C. elegans examined by the RNA fluorescent in situ hybridization (RNA-FISH). The signals of 14 homologs were detected in the intestinal cytoplasm. The signals of two other homologs were detected in the germ cytoplasm. The remaining three could not be detected, probably because they present in too low abundance to be detected by RNA-FISH. All 19 Y RNA homologs have the structural elements required for the binding of ROP-1. In other organisms, Ro60 binding stabilizes Y RNAs in cells. To know whether C. elegans Y RNA homologs also stabilized by the presence of ROP-1, we examined RNA-FISH of the Y RNA homologs against a mutant strain MQ470, which has a transposon insertion in the middle of the ROP-1 gene and lacks ROP-1 proteins in the cell. As expected, all Y RNAs examined so far decreased extensively. These were confirmed by northern hybridization. The results suggest that several C. elegans Y RNA homologs are expressed in a tissue-specific manner and most Y RNA homologs are stabilized by ROP-1 binding as well as those in other organisms.

Sato H et al. (2021) STAR Protoc "Simultaneous recording of behavioral and neural responses of free-moving nematodes C.elegans."

Iino Y, Fei X, Sato H, Kunitomo H, Hashimoto K

[STAR Protoc, 2021]

To reveal the neural mechanisms that control animal behavior, it is necessary to link the neural responses to behavioral changes and interpret them. We have developed a protocol to simultaneously record the behavior and neural activity of freely moving <i>C.elegans</i> by combining a microfluidic device and a tracking stage. Here we detail the protocol for the experiment, with an example of behavioral and neural responses of nematodes to salt concentration changes. For complete details on the use and execution of this protocol, please refer to Sato etal. (2021).

Ken Sato et al. (2003) International Worm Meeting "Receptor-mediated endocytosis in C. elegans: Characterization of the rme-3 and rme-4 mutants"

Ken Sato, Chih-Hsiung Chen, Barth Grant, Miyuki Sato

[International Worm Meeting, 2003]

Receptor-mediated endocytosis is required for general cellular functions such as nutrient uptake, recycling of membrane and membrane proteins. To study this mechanism in C. elegans, we developed an assay system to assess the receptor-mediated endocytosis of yolk proteins by oocytes. We created transgenic strains that express a GFP fusion gene with VIT-2, encoding the lipid binding yolk protein YP170. YP170-GFP is expressed in the intestinal cells and secreted into the pseudocoelom (body cavity). In wild type worms, YP170-GFP is taken up by oocytes via the receptor-mediated endocytosis. Using this assay, we isolated mutants that fell into 11 complementation groups. We named these genes rmefor receptor-mediated endocytosis defective. These mutants show a defect in yolk uptake by oocytes and accumulate YP170-GFP in the pseudocoelom. Recently we have focused our analysis on rme-3 and rme-4, two genes defined by the RME mutant screen. In addition to yolk uptake defects, rme-3 (b1025) mutants show a temperature sensitive growth defect and a weak defect in nonspecific-fluid phase uptake by coelomocytes (scavenger cells in the pseudocoelom). On the other hand, rme-4 (b1001) mutants are viable and show a strong defect in fluid phase uptake by coelomocytes. We mapped rme-3 (b1025) and rme-4 (b1001) to chromosome III and X, respectively. We will present current data about these mutants.

Sato M et al. (2008) EMBO J "Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein."

Sato K, Grant BD, Pant S, Sato M, Liou W, Harada A

[EMBO J, 2008]

Using Caenorhabditis elegans genetic screens, we identified receptor-mediated endocytosis (RME)-4 and RME-5/RAB-35 as important regulators of yolk endocytosis in vivo. In rme-4 and rab-35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME-4 and RAB-35 function downstream of clathrin, upstream of RAB-7, and act synergistically with recycling regulators RAB-11 and RME-1. We find that RME-4 is a conserved DENN domain protein that binds to RAB-35 in its GDP-loaded conformation. GFP-RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. GFP-RAB-35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme-4 mutants. We propose that RME-4 functions on coated pits and/or vesicles to recruit RAB-35, which in turn functions in the endosome to promote receptor recycling.