Limor Broday et al. (2002) European Worm Meeting "A novel LIM domain protein (Y105E8A.6) that was isolated as an interactor protein of RNF-5, is localized to muscular focal adhesions"

Benjamin Podbilewicz, Irina Kolotuev, Ze'ev Ronai, Anindita Bhoumik, Limor Broday

[European Worm Meeting, 2002]

Search for proteins in C.elegans that share homology within the RING domain of MDM2/BRCA1/Cbl led to the identification of RNF-5, a 26 kDa RING finger protein with C- terminal tail that may acquire membrane anchoring properties. RNF-5 was found to be important for the growth and development of C. elegans and exhibits intrinsic E3 ligase activity (Broday et al., submitted). Yeast 2-hybrid screen identified the C. elegans LIM protein Y105E8A.6 as a partner of RNF-5. As the first step to study the functional importance of this interaction, we characterized the pattern of expression of the LIM interactor using transgenic animals harboring extrachromosomal arrays of GFP fusion constructs. We demonstrate that this novel LIM domain protein is expressed in body wall muscles at dense bodies and along the M lines. In the vulval muscles the protein is also localized at sites of muscle attachment to the hypodermis. Expression was detected in the anal depressor muscle and in two arms that extend from muscle cells to the nerve ring. In addition to the expression in dense bodies and M-lines, the fusion LIM::GFP is localized to the nuclei of muscle cells and to the cell cytoplasm. These observations suggest a role for this LIM domain protein in focal adhesion assembly or maintenance. In order to follow the in-vivo interaction between RNF-5 and its interactor LIM protein, and their putative role in the regulation of the integrity of the basement membrane connecting the myofibrils and the hypodermis, we are currently performing over-expression experiments of RNF-5 under the heat shock promoter and rnf-5(RNAi) analysis, both are carried out on the background of the LIM::GFP expressing animals.

Limor Broday et al. (2003) International Worm Meeting "The RING finger protein RNF-5 is expressed in epithelial cells junctions and muscle dense bodies, and interacts with the LIM domain protein unc-95"

Zeev Ronai, Irina Kolotuev, Benjamin Podbilewicz, Anindita Bhoumik, Limor Broday

[International Worm Meeting, 2003]

The search for proteins in C. elegans sharing homology within the RING domain of MDM2/BRCA1/Cbl led to the identification of RNF-5, a 26 kDa RING finger protein that has a C-terminal tail characteristic of a membrane anchoring domain. Immunohistochemistry revealed that RNF-5 is expressed in the spermathecal septate junctions, excretory pore, gonadal sheath cells, and in muscle dense bodies. Overexpression of RNF-5 under the heat shock promoter damages the hypodermal-cuticle attachment sites and results in growth inhibition and lethality. A yeast 2-hybrid screen identified the C. elegans LIM protein Y105E8A.6 as RNF-5 associated protein. Y015E8A.6 was found to be co-localized with RNF-5 in the muscle dense bodies. Sequence analysis and rescue experiments have revealed that the previously identified unc-95 gene (Zengel and Epstein, 1980) encodes to this LIM domain protein, Y105E8A.6. The unc-95(su33) allele harbors an amber mutation which results in a truncated protein that lacks the LIM domain. Biochemical studies in mammalian cells suggest a role for RNF-5 as an E3 ligase in the regulation of the UNC-95 protein.

Limor Broday et al. (2003) International Worm Meeting "The small ubiquitin like modifier (SUMO) is required for gonadal and uterine-vulval morphogenesis in C. elegans."

Irina Kolotuev, Limor Broday, Christine Didier, Anindita Bhoumik, Zeev Ronai, Benjamin Podbilewicz

[International Worm Meeting, 2003]

SUMO alters the subcellular distribution and function of its substrates. The role of SUMO in organ development remains largely elusive. We show that smo-1 deletion mutant of the Caenorhabditis elegans homologue of SUMO1 develops into sterile adult with abnormal somatic gonad, germline and vulva. SMO-1::GFP is expressed in the somatic reproductive system. smo-1 animals lack a vulval-uterine connection as a result of impaired ventral uterine cell differentiation and anchor cell fusion, a phenotype typical of lin-11 mutants. LIN-11 transcription factor is sumoylated and smo-1 animals exhibit reduced nucleo-cytoplasmic expression of LIN-11::GFP. Thus, our results identify the reproductive system as the principal SUMO target during postembryonic development and highlight LIN-11 as a representative substrate whose sumoylation is associated with the formation of a functional vulval-uterine connection.