Worm Breeder's Gazette 15(4): 22 (October 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.
Dept. of Zoology, 6270 University Blvd., Vancouver, BC, Canada, V6T 1Z4.
Myofilament lattice assembly begins at the plasma membrane in regions of
contact between adjacent muscle cells (Hresko et al., 1994, JCB
124:491-506). UNC-52 and integrin are key early components in the
assembly process and are required for organization of cytoskeletal
associated proteins such as vinculin and talin (Hresko et al., 1994;
Moulder et al., 1996, MBC 7:1181-1193). To further dissect the assembly
process, we asked whether integrin and other muscle components affect
the localization of UNC-52 in the basement membrane. We stained embryos
homozygous for Pat mutations (pat-2, pat-3, pat-4, pat-5, pat-11,
pat-12, unc-112, and deb-1) with antibodies to UNC-52 (GM1) and MHC A
(DM5.6).
In wild-type embryos, GM1 stains the basement membranes
associated with the pharynx, body wall, and anal muscles. In larvae and
adults, staining is especially intense over the dense bodies and M-lines
in the body wall muscles. Because the dense body/M-line staining is not
observed in embryos, we focused instead on the basement membrane
staining. Surprisingly, we found that none of these Pat mutants exhibit
defects in the localization of UNC-52. For example, the GM1 staining
pattern in pat-3(st564) mutant embryos appears to be quite normal, even
in arrested embryos. On this basis, we conclude that beta-integrin is
not required for the correct localization of UNC-52.
Similarly, Pat alleles of unc-112 have no affect on the
localization of UNC-52. We took advantage of a characteristic defect to
identify unc-112 homozygotes prior to the one and a half-fold stage.
When stained with GM1, a small break in each ventral muscle quadrant can
be seen in wild-type embryos. This break may correspond to the position
of the amphid commissure (C. R. Norris, I. A. Bazykina, E. M. Hedgecock,
and D. H. Hall, personal communication). In unc-112 mutants, this break
is noticeably larger than in wild-type embryos and can be used to
distinguish unc-112 homozygotes from their wild-type siblings.
Consequently, we were able to look at early unc-112 embryos and
establish that there are no defects in the initial localization of
UNC-52.
Some disorganization of UNC-52 was observed in the milder Pat
mutants, including myo-3 and pat-12. In these mutants, sections of the
dorsal muscle quadrants often pull away from the hypodermis, especially
in older embryos (Williams and Waterston, 1994, JCB 124:475-490). The
disorganization of UNC-52 in these mutants appears to be a secondary
consequence of muscle detachment, rather than a direct effect on UNC-52.
On the basis of these results, we conclude that UNC-52 localization is
not obviously disrupted by mutations in any of these muscle-affecting
genes, including pat-3 (beta-integrin; Gettner et al., 1995, JCB
129:1127-1141) and unc-112 (MIG-2; see abstract by Rogalski et al.).
Studies on focal adhesion assembly in tissue culture systems indicate
that extracellular matrix proteins are required for integrin clustering
and cytoskeletal association. However, we suspect that the accumulation
of UNC-52 over dense bodies and M-lines during post-embryonic
development may be dependent on integrin and/or other membrane
associated proteins. The assembly of mature adhesion structures
probably involves inductive interactions that remodel and reorganize
both the cytoskeleton and associated proteins, and the basement
membrane.