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.

A PINCH of LIM domains in UNC-97 et al.

Oliver Hobert1, Donald Moerman2, Yanxia Liu1, Gary Ruvkun1

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