Worm Breeder's Gazette 15(2): 40 (February 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Dept. of Molecular Biology, Mass. General Hospital, Boston, MA 02114|
|2||Dept. of Zoology, University of British Columbia, Vancouver, Canada|
LIM domains are Zn-finger like structures implicated in protein-protein interactions and occur in proteins with cytoskeletal and gene regulatory functions (1). The worm contains scores of LIM domain genes, some of which contain additional functional domains, such as the homeodomain, while others do not contain other recognizable functional domains. To elucidate the function of LIM proteins from the latter category, we took a candidate gene approach and found that unc-97, a gene that had been described and mapped to LGX between lon-2 and dpy-8 by Zengel and Epstein (2), encodes a protein containing five LIM domains (F14D12.2). We rescued unc-97(su110) with a genomic subclone containing only F14D12.2 and then sequenced the only allele of the gene, su110. We found a single alteration, a splice site mutation that is predicted to disrupt the C-terminal LIM domain. Since a four LIM domain polypeptide can still be made in su110 mutants, this allele may not exhibit the null phenotype for unc-97. UNC-97 is the worm orthologue of a human protein that contains an autosenescent antigen and was named PINCH, for !Particularly Interesting His-Cys-Rich Protein" (3; nomen est omen ?!). The predicted gene structure for unc-97 was confirmed by sequencing several EST clones obtained from Y.Kohara. We fused GFP to the C-terminus of the protein and used an upstream regulatory region that extended to the next upstream predicted gene to reveal a) the expression pattern and b) the subcellular localization of UNC-97. UNC-97 is expressed in body wall muscles, vulval muscles and touch neurons. A common theme of all these cell types is their attachment via the ECM to the hypodermis. In adult body wall muscles UNC-97 is located, along with the integrin complex, at cell attachment sites called dense bodies. Considering this subcellular localization we further characterized the unc-97 mutant phenotype. Using EM, Zengel and Epstein found shallow sarcomeres and disorganized thin filaments in body wall muscles of unc-97(su110) animals (2). Using the beta-integrin/PAT-3 monoclonal antibody MH25 provided by Bob Waterston, we found that the dense bodies do not for the most part show up as rows of discrete spots in unc-97 mutants, but instead appear primarily as diffuse stripes running parallel to the M-line. These observations are consistent with UNC-97 playing a role in determining the structural integrity of the dense bodies, perhaps through clustering integrin or other dense body associated proteins. Since UNC-97 is also expressed in touch neurons we are now examining the neuroanatomy of the touch neurons in unc-97 mutants using mec-7-GFP. Interestingly, UNC-97 shows a dramatic subcellular redistribution during muscle development. In contrast to its adult localization, UNC-97 is not localized to dense bodies in embryos and young larvae, but instead is localized exclusively to discrete spots in muscle nuclei. This represents the first example of a LIM protein, whose dual subcellular localization is under developmental control. The genome sequencing project has uncovered one other protein with 5 LIM domains, F07C6.1, that shows a striking similiarity to UNC-97 both in its domain organization and amino acid similarity (57%). A full-length protein-GFP fusion of this gene does not show any overlap with UNC-97 expression: F07C6.1-GFP reveals a very restricted expression pattern in a few head and ventral cord neurons and intestinal cells. In both these cell types it uniformly distributes throughout the cytoplasm and the nucleus. Thus, despite their sequence similarity, UNC-97 and F07C6.1 apparently play entirely different roles. (1) Dawid et al., 1995, C.R.Acad.Sci.III 318, 295-306 (2) Zengel and Epstein, 1980, Cell Motility 1, 73-97 (3) Rearden, 1994, Biochem.Biophys.Res.Comm.201, 1124-1131