Imae, Rieko et al. (2011) International Worm Meeting "Intracellular PLA1 and acyltransferase, which are involved in C. elegans stem cell divisions, determine the sn-1 fatty acyl chain of phosphatidylinositol."

Kimura, Masako, Mitani, Shohei, Kage-Nakadai, Eriko, Inoue, Takao, Imae, Rieko, Arai, Hiroyuki

[International Worm Meeting, 2011]

Phosphatidylinositol (PI), an important constituent of membranes, contains stearic acid as the major fatty acid at the sn-1 position. This fatty acid is thought to be incorporated into PI through fatty acid remodeling by sequential deacylation and reacylation. However, the genes responsible for the reaction are unknown, and consequently, the physiological significance of the sn-1 fatty acid remains to be elucidated. Here, we identified acl-8, acl-9, acl-10, which are closely related to each other, and ipla-1 as strong candidates for genes involved in fatty acid remodeling at the sn-1 position of PI. In both ipla-1 mutants and acl-8 acl-9 acl-10 triple mutants of C. elegans, the stearic acid content of PI is reduced and asymmetric division of stem cell-like epithelial cells is defective. The defects in asymmetric division of these mutants are suppressed by a mutation of the same genes involved in intracellular retrograde transport, suggesting that ipla-1 and acl genes act in the same pathway. IPLA-1 and ACL-10 have phospholipase A1 and acyltransferase activity, respectively, both of which recognize the sn-1 position of PI as their substrate. We propose that the sn-1 fatty acid of PI is determined by ipla-1 and acl-8, -9, -10 and crucial for asymmetric divisions.

Rieko Imae et al. (2007) International Worm Meeting "Functional analysis of acl-10, a novel acyltransferase in C. elegans."

Shohei MItani, Naoko Hara, Keiko Gengyo-Ando, Takao Inoue, Rieko Imae, Hiroyuki Arai

[International Worm Meeting, 2007]

Alignment of amino acid sequences from phospholipids acyltransferases, such as glycerol-3-phosphate acyltransferase, acl-glycerol-3-phosphate acyltransferase and dihydroxyacetonephosphate acyltransferase, reveals four regions of strong homology that comprise a glycerolipid acyltransferase signature sequence. There are more than 10 genes containing the acyltransferase signature sequence in human genome, of which physiological functions and substrate specificities are largely unknown. We identified 14 genes containing the signature sequence from C. elegans genome sequences (acl-1~14), and generated mutants lacking these genes. Among the genes, acl-10 shows ~40% identity with the corresponding mammalian gene, but until recently, its functions have not been elucidated. In this study, we analyzed the physiological function of acl-10 using the mutants lacking this gene. The acl-10 deletion mutant, acl-10 (tm1045), exhibited dumpy morphology and severely uncoordinated movement at adult stage. In addition, acl-10 (tm1045) displayed hypersensitivity to the acetylcholine receptor agonist levamisole, suggesting that the signaling pathway leading to muscle contraction is activated in the mutants. These defects were fully rescued by acl-10 expression at L4 stage, indicating that acl-10 is required for muscle function rater than muscle formation. To further elucidate how acl-10 controls muscle contraction, we performed rescue experiments using tissue-specific promoters (unc-119p, myo-3p, dpy-7p). Interestingly, all these constructs could rescue the movement defects of acl-10 (tm1045). The hypersensitivity to levamisole was also rescued by neuronal or epithelial expression of acl-10, indicating that acl-10 functions in a cell nonautonomous manner. We are currently investigating the mechanism of acl-10 function in muscle contraction.

Rieko Imae et al. (2010) East Asia Worm Meeting "Intracellular PLA1 and acyltransferase, which are involved in C. elegans stem cell divisions, determine the sn-1 fatty acyl chain of phosphatidylinositol"

Takao Inoue, Rieko Imae, Masako Kimura, Shohei MItani, Hiroyuki Arai, Eriko Kage-Nakadai

[East Asia Worm Meeting, 2010]

Phosphatidylinositol (PI), an important constituent of membranes, contains stearic acid as the major fatty acid at the sn-1 position. This fatty acid is thought to be incorporated into PI through fatty acid remodeling by sequential deacylation and reacylation. However, the genes responsible for the reaction are unknown, and consequently, the physiological significance of the sn-1 fatty acid remains to be elucidated. Here, we identified acl-8, acl-9, acl-10, which are closely related to each other, and ipla-1 as strong candidates for genes involved in fatty acid remodeling at the sn-1 position of PI. In both ipla-1 mutants and acl-8 acl-9 acl-10 triple mutants of C. elegans, the stearic acid content of PI is reduced and asymmetric division of stem cell-like seam cells, is defective. The defects in asymmetric division of these mutants are suppressed by a mutation of the same genes involved in intracellular retrograde transport, suggesting that ipla-1 and acl genes act in the same pathway. IPLA-1 and ACL-10 have phospholipase A1 and acyltransferase activity, respectively, both of which recognize the sn-1 position of PI as their substrate. We propose that the sn-1 fatty acid of PI is determined by ipla-1 and acl-8, -9, -10 and crucial for asymmetric divisions.

Dejima, Katsufumi et al. (2019) International Worm Meeting "An endosome-resident zinc transporter is involved in negative regulation of systemic dsRNA spreading in C. elegans."

Suehiro, Yuji, Imae, Rieko, Dejima, Katsufumi, Mitani, Shohei

[International Worm Meeting, 2019]

Double-stranded RNA (dsRNA) induces post-transcriptional gene silencing in base-paring mechanisms, a phenomenon termed RNA interference (RNAi). In C. elegans, the silencing RNA is transported between cells, and RNAi occurs systemically. Previous studies implied several endomembrane proteins predicted to function in membrane trafficking in systemic RNAi. However, the molecular mechanisms that enable dsRNA export and import through membrane trafficking machineries are still poorly understood. We previously reported that RSD-3, the endosomal protein with phospholipid-binding domain (ENTH), is required for efficient import of silencing RNA, and the rsd-3 mutants displayed an incomplete defect in systemic RNAi. Here, from a genetic screen for mutations that suppress the defective RNAi phenotype of rsd-3 mutants, we identified a zinc transporter as a suppressor gene. This zinc transporter is widely expressed, acts in a cell nonautonomous manner, and is mainly localized to late endosome. The mutant itself shows the enhanced RNAi (Eri) phenotype. We tested whether this phenotype is caused by downregulation of endogenous RNAi or upregulation of exogenous RNAi, which are antagonistic. We found that certain genes targeted by the endogenous RNAi pathway were not affected in the mutants. Instead, its null mutants showed altered RAB-11 positive vesicle sizes in intestine and LMP-1 positive vesicle sizes in coelomocytes, suggesting its cell-context dependent roles in membrane trafficking. Epistasis analysis indicated that sid-1 and sid-2 but not sid-3 or sid-5 are essential for the Eri phenotype seen in the mutants. The C. elegans genome contains 28 zinc transporters. Interestingly, mutants for all other zinc transporter genes exhibited normal response to RNAi, suggesting a unique role of this transporter on RNAi. Our data revealed the zinc transporter as a novel cellular factor functioning in regulation of systemic RNAi.

Dejima, Katsufumi et al. (2021) International Worm Meeting "Analysis of endomembrane-resident zinc transporter mutants that suppress the systemic RNAi defects of rsd-3 mutant"

Suehiro, Yuji, Dejima, Katsufumi, Imae, Rieko, Mitani, Shohei

[International Worm Meeting, 2021]

Functional RNAs including double-stranded RNA (dsRNA) modulate gene expression in a sequence-dependent manner. In C. elegans, experimentally introduced dsRNA spreads over the body and leads to RNA silencing systemically. To date, several genes acting in systemic RNAi have been genetically identified. However, cellular pathways and molecules that mediate RNA transport between cells remain largely unknown. We previously reported that RSD-3/EpsinR is involved in import of silencing RNA. In this presentation, we show an analysis of suppressors for RNAi defects in the rsd-3 mutants. Through the analysis of the suppressors, we found that a zinc transporter functions as a negative regulator for dsRNA import. Several experiments support the idea that its zinc transport activity is required for negative regulation of systemic RNAi. However, while the C. elegans genome contains 28 zinc transporters, mutants for all other zinc transporter genes exhibited a normal response to RNAi, suggesting a unique role of this transporter on RNAi or genetic redundancy. Since disruption of the suppressor gene itself showed enhanced RNAi (Eri) phenotype, we also tested whether this phenotype is caused by downregulation of endogenous RNAi or upregulation of exogenous RNAi, which are antagonistic. We found that certain genes targeted by the endogenous RNAi pathway were not affected in the mutants, excluding the possibility that it acts in the core endogenous RNAi pathway. Instead, its null mutants showed altered RAB-11 positive vesicle sizes in intestine and LMP-1, RAB-7 and 2xFYVE positive vesicle sizes in coelomocytes. From these observations, we speculate that this zinc transporter would be involved in the regulation of membrane trafficking, which in turn negatively regulate exogenous RNAi in parallel or downstream of RSD-3. Our data revealed the zinc transporter as a novel cellular factor functioning in systemic RNAi.

The C. elegans Deletion Mutant Consortium et al. (2012) G3 (Bethesda) "large-scale screening for targeted knockouts in the Caenorhabditis elegans genome."

Pritchett, Cherilyn, Shen, Bin, Holmes, Jeff, Cobb, Beth, Allan, BJ, Gilchrist, Erin, Vishwanathan, Malani, Motohashi, Tomoko, Perkins, Jaryn, Lorch, Adam, Otori, Muneyoshi, Mullen, Greg, Frazee, Stephen, Wang, Tina, Cheung, Rene, Tardif, Angela, Jerebie, Daniela, Kwitkowski, Christine, Hay, Allison, Landsdale, Martin, Kobuna, Hiroyuki, Fernando, Lisa, Suehiro, Yuji, Edgley, Mark, Liu, Lucy, Robertson, James, Gengyo-Ando, Keiko, Maydan, Jason, Hunt, Rebecca Worsley, Flibotte, Stephane, Stokes, Ed, Rogula, Alexandra, Hori, Sayaka, Neil, Sarah, Kage-Nakadai, Eriko, Funatsu, Osamu, Miller, Angela, Machiyama, Etsuko, Fisher, Angela, Navaroli, Candice, Rummage, John, Au, Vinci, Adair, Ryan, Mckay, Sheldon, Moulder, Gary, Moerman, Donald, Lee, Norris, Henthorn, Diane, Zapf, Rick, Chaudhry, Iasha, Lau, Joanne, Osborn, Jamie, Imae, Rieko, Eng, Simon, Huang, Peter, Yoshina, Sawako, Hunt-Newbury, Rebecca, Veiga, Mariana, Taylor, Jon, Raymant, Greta, Stegeman, Greg, Rankin, Anna, Dadivas, Owen, Luck, Candy, Rezania, Nadereh, The C. elegans Deletion Mutant Consortium, Partridge, Mikhaela, Mitani, Shohei, Barstead, Robert

[G3 (Bethesda), 2012]

The nematode Caenorhabditis elegans is a powerful model system to study contemporary biological problems. This system would be even more useful if we had mutations in all the genes of this multicellular metazoan. The combined efforts of the C. elegans Deletion Mutant Consortium and individuals within the worm community are moving us ever closer to this goal. At present, of the 20,377 protein-coding genes in this organism, 6764 genes with associated molecular lesions are either deletions or null mutations (WormBase WS220). Our three laboratories have contributed the majority of mutated genes, 6841 mutations in 6013 genes. The principal method we used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, we have used array comparative genome hybridization (aCGH) to detect deletions across the entire coding part of the genome and massively parallel short-read sequencing to identify nonsense, splicing, and missense defects in open reading frames. As deletion strains can be frozen and then thawed when needed, these strains will be an enduring community resource. Our combined molecular screening strategies have improved the overall throughput of our gene-knockout facilities and have broadened the types of mutations that we and others can identify. These multiple strategies should enable us to eventually identify a mutation in every gene in this multicellular organism. This knowledge will usher in a new age of metazoan genetics in which the contribution to any biological process can be assessed for all genes.