Hiroshi Qadota et al. (2001) International C. elegans Meeting "Functions of intermediate filaments and their regulatory proteins during embryonic tissue formation"

Daisuke Tsuboi, Katsuhisa Kasuya, Makiko Inoue, Mutsuki Amano, Hiroshi Qadota, Kozo Kaibuchi

[International C. elegans Meeting, 2001]

Cytoskeletal rearrangement is indispensable for tissue formation. Function of intermediate filaments, one of the major cytoskeletal components, in tissue is remained to be clarified due to their difficulty in biochemical and cell biological handling. In this study, we try to examine dynamic function and regulation of intermediate filaments in C. elegans embryonic tissue formation. From C. elegans Genome Sequencing database, we found 11 cytoplasmic intermediate filament genes except one nuclear lamin. Eight genes are reported by Dodemont et al. (1994 ) and three genes are novel. From sequencing homology, these 11 intermediate gene products were revealed to be prototype of many types of mammalian cytoplasmic intermediate filaments. We also performed characterization of MH antibodies reported to be recognized intermediate filaments in C. elegans . Using these MH antibodies, we found that intermediate filaments are expressed from mid phase of embryogenesis, suggesting that intermediate filaments are involved in also embryonic tissue organization. We also found a possible regulatory molecule of intermediate filaments, GEI-4. We previously identified gex-2 and gex-3 genes as essential factors for tissue formation. We performed yeast two hybrid screening of interacting molecules with GEX-2 ( gei genes, gex interacting molecule), and obtained 6 genes including GEI-4. GEI-4 shared weak homology to trichohyalin, a mammalian intermediate filament interacting protein. We found interaction between GEI-4 and C. elegans intermediate filaments by yeast two-hybrid method. RNAi of gei-4 caused an embryonic lethality with disorganization of tissue morphology. Consistent with the interaction of GEI-4 with GEX-2, immunostaining analysis revealed that GEI-4 is colocalized with GEX-2, GEX-3, and many intermediate filaments at the peripheral region of almost all the cells. Terminal phenotypes of gei-4 , gex-2 , and gex-3 showed disorganization of intermediate filaments. Thus, these results suggest that dynamics of intermediate filaments, regulated by GEX-2/GEX-3/GEI-4 protein complex, is essential for embryonic tissue formation.

M Soto et al. (1999) International C. elegans Meeting "inx-1 is required for body morphogenesis and encodes C. elegans HEM-2, a putative Rac GTPase associated protein"

K Kaibuchi, C Mello, M Soto, K Kasuya, H Qadota

[International C. elegans Meeting, 1999]

The inx-1 and inx-3 mutants ( in testine on the e x terior) were found in maternal effect screens for mutations affecting embryonic gut development. Inx mutant embryos arrest development with their intestinal cells spread over the ventral surface of the embryo. Lineage studies show that the E cells gastrulate properly, however the hypodermal cells fail to migrate over and enclose the embryo and instead contract up into a tight cluster on the dorsal surface. inx-1 was cloned by testing candidate genes in a small genetic interval for the presence of a Tc1 insertion. One gene, named CeHEM-2 by the Genome Project, contains a Tc1 insertion in the inx-1 mutator induced allele zu196 . CeHEM-2 (RNAi) induces the inx-1 phenotype, further supporting the identity of inx-1 as a mutation in CeHEM-2. INX-1 is a highly conserved but novel protein. Using column chromatography, mammalian HEM-2 was found to be associated with Rac1, a small GTPase implicated in rearrangements of the actin cytoskeleton. INX-1 antibodies recognize the cytoplasm and cell cortex of many if not all cells in the C. elegans embryo, but may be enriched at hypodermal junctions. inx-1 and inx-3 also have zygotic phenotypes. The vulva of Inx worms forms properly (Wendy Hanna-Rose, personal communication), yet homozygotes become severely Egl leaking only a few eggs. This phenotype is reminiscent of the Egl defect of unc-73 , a guanine exchange factor which activates the Rac GTPase in vitro. UNC-73 is involved in the gonad independent mechanism for sex myoblast migrations. Two hybrid assays suggest that INX-1 interacts with CeSra-1, an effector of Rac1 GTPase in vertebrates. RNAi with CeSra-1 has the same embryonic phenotype as inx-1 (see abstract by Kasuya et al). We are investigating the model that INX-1,CeSra-1/INX-2 and perhaps inx-3 are effectors of a small GTPase in C. elegans. This pathway may reorganize the actin cytoskeleton to control cell shape changes necessary for the migration of the hypodermal cells in the embryo and migration of the sex myoblasts post-embryonically.

Kasuya K et al. (2000) Japanese Worm Meeting "A novel protein functions as a bridge between the GEX-2/GEX-3 complex and intermediate filaments in C. elegans."

Inoue M, Tsuboi D, Qadota H, Kasuya K, Kaibuchi K

[Japanese Worm Meeting, 2000]

Tissue morphogenesis is indispensable for living functions of multicellular organisms. We have previously reported that loss-of-function of gex-2 (Ce-Sra-1) or gex-3 (Ce-HEM-2) leads to 100% embryonic lethality with abnormal tissue morphogenesis and that these molecules are colocalized at the cell-cell contact sites of all tissue cells throughout embryogenesis (K. Kasuya et al., M. Soto et al., 12th international C. elegans meeting). Immunoprecipitation assay and a two-hybrid system revealed that GEX-2 and GEX-3 physically interact. These results suggest that GEX-2 and GEX-3 probably form a novel protein complex in vivo. GEX-2 and GEX-3 proteins have no significant functional domain. To clarify the molecular mechanisms of GEX-2/GEX-3 functions in tissue morphogenesis, we screened physically and functionally interacting molecules. At first, by a two-hybrid screening, we isolated 29 positive clones that included 6 genes. Next, we performed RNAi of 6 genes. Among them, disruption of W07B3.2 resulted in 100% lethal embryos, some of which showed exploder phenotype of hypodermis and others arrested at the comma-stage. Although both phenotypes were weaker than the Gex phenotype, the disorganization of molecular markers of muscle and intestine was also observed in W07B3.2(RNAi) embryos. The GEX-2/GEX-3-like localization pattern of W07B3.2 and the binding of W07B3.2 to GEX-2 and GEX-3 in the immunoprecipitation assay suggested that W07B3.2 functionally related to GEX-2 and GEX-3. W07B3.2 gene product has a repeated structure which is weakly homologous to trichohyalin and raises the possibility to interact with intermediate filaments (IFs). To examine whether W07B3.2 interacts with C. elegans IFs, we performed two-hybrid binding assay. As a result, some IFs interacted with W07B3.2. Co-localization of GEX-2, GEX-3, and W07B3.2 with some IFs suggested that these molecules form a large complex. RNAi of gex-2, gex-3, and W07B3.2 eventually caused disorganization of the IF structure. Taken together, we propose a model that W07B3.2 serves as a bridge between GEX-2/GEX-3 complex and intermediate filaments, and that the large complex essentially functions in tissue morphogenesis in C. elegans.

Qadota H et al. (2000) East Coast Worm Meeting "A novel protein functions as a bridge between the GEX-2/GEX-3 complex and intermediate filaments in C. elegans."

Inoue M, Kaibuchi K, Qadota H, Tsuboi D, Kasuya K

[East Coast Worm Meeting, 2000]

Tissue morphogenesis is indispensable for living functions of multicellular organisms. We have previously reported that loss-of-function of gex-2 (Ce-Sra-1) or gex-3 (Ce-HEM-2) leads to 100% embryonic lethality with abnormal tissue morphogenesis and that these molecules are localized at the cell-cell contact sites of all tissue cells throughout embryogenesis. (K. Kasuya et al., M. Soto et al., 12th international C. elegans meeting) Based on binding in a two-hybrid system and co-localization in immunostaining, GEX-2 and GEX-3 probably form a novel protein complex. GEX-2 and GEX-3 proteins have no significant functional domain. To clarify the molecular mechanisms of GEX-2/GEX-3 functions in tissue morphogenesis, we screened physically and functionally interacting molecules. At first, by a two-hybrid screening, we isolated 29 positive clones that included 6 genes. Next, we performed RNAi of 6 genes. Among them, disruption of W07B3.2 resulted in 100% lethal embryos, some of which showed exploder phenotype of hypodermis and others arrested at the comma-stage. Although both phenotypes were weaker than Gex phenotype, the disorganization of molecular markers of muscle and intestine were also observed in W07B3.2(RNAi) embryos. The GEX-2/GEX-3-like localization pattern of W07B3.2 and the binding of W07B3.2 to GEX-2 and GEX-3 in the immunoprecipitation assay suggested that W07B3.2 functionally related to GEX-2 and GEX-3. W07B3.2 gene product has a repeated structure which is weakly homologous to trichohyalin and raises the possibility to interact with intermediate filaments (IFs). To examine whether W07B3.2 interacts with C. elegans IFs, we performed two-hybrid binding assay. As a result, some IFs interacted with W07B3.2. Co-localization of GEX-2, GEX-3, and W07B3.2 with some IFs suggested that these molecules form a large complex. RNAi of gex-2, gex-3, and W07B3.2 eventually caused disorganization of the IF structure. Taken together, we proposed a model that W07B3.2 serves as a bridge between GEX-2/GEX-3 complex and intermediate filaments, and that the large complex essentially functions in tissue morphogenesis in C. elegans.