Worm Breeder's Gazette 13(3): 52 (June 1, 1994)
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.
SPARC is a conserved extracellular matrix protein that is expressed and secreted by body wall and sex muscle cells in C. elegans (Schwarzbauer and Spencer (1993) Mol. Biol. Cell 4, 941-952). SPARC has four domains, an amino-terminal acidic calcium binding domain, a cysteine-rich domain II, an alpha-helical domain, and a carboxy-terminal EF-hand motif. Polyclonal antibodies were raised against a fusion protein containing maltose binding protein (MBP) plus the EF-hand domain IV. The anti-MBP-SPARC IV antiserum was used to characterize SPARC protein in nematode Iysates. Soluble fractions of wild type animals were prepared using either buffered SDS or a non-denaturing solution containing the non-ionic detergent NP40 .These Iysates were subjected to SDS-PAGE and SPARC was detected by immunoblotting with the anti-SPARC IV antiserum revealed by chemiluminescence.
SPARC protein could be detected in both SDS and NP40 lysates indicating that, like its mammalian homologs, it is not an insoluble component of the matrix. Under reducing conditions, SPARC migrated with an apparent molecular weight of 30 kD, close to the predicted size for a 247 residue protein. When non-reduced, SPARC shifted to a size of about 20 kD. This is due to a more compact structure conferred by the intra-chain disulfide bonds located in the cysteine-rich domain II and flanking the EF-hand in domain IV. To determine whether the nematode protein contains N-linked carbohydrate, lysates were treated with N-glycosidase F prior to immunoblotting. The increased electrophoretic mobility showed that SPARC is indeed glycosylated. These biochemical results demonstrate that nematode SPARC is structurally very similar to its mammalian counterparts.
The SPARC gene, ost-1 ,is near dpy-9 on LG IV. The anti-SPARC antibodies were used to determine whether normal levels of SPARC are present in dpy-9 ( e12 )nematodes. When compared to wild type, similar levels of SPARC mRNA and protein were present in this dpy-9 strain and the protein appeared normally disulfide-bonded and glycosylated. These results make it unlikely that the dpy-9 mutation is within the SPARC gene.
We have previously shown that over-expression of wild type SPARC in an N2 background results in an Unc phenotype with paralysis and body deformities, in particular a large protrusion of the vulva (Schwarzbauer and Spencer, ibid.). We were unable to generate transgenic lines by microinjection of the SPARC gene alone. However, we have recently prepared a transgenic line with a very similar phenotype by co-injecting the wild type SPARC gene plus the plasmid pRF4 containing rol-6 ( su1006 ).This transgenic line, called E10 ,shows the same deformities and paralysis as with the SPARC gene alone and does not exhibit the Rol-6 phenotype.
Because SPARC is expressed by body wall and sex muscle cells, we examined the E10 animals for defects in muscle structure. Adult hermaphrodites were fixed with paraformaldehyde, permeabilized with acetone followed by subtilisin (Nonet et al. (1993) Cell 73, 1291-1305), and stained with rhodamine-labeled phalloidin. All wild type vulval muscles showed strong filamentous actin staining along their lengths. A major defect was observed in the sex muscles of the E10 adults. Specifically, no actin staining could be seen in these muscles. Occasionally, there were small spots of staining localized to the vulval protrusion. Therefore, over-expression of SPARC by these cells causes disorganization of the muscle filaments. By analogy to SPARC's proposed modulatory effects on adhesion of mammalian cells, this vulval defect may be due to SPARC-mediated disruption of sex muscle cell adhesion at the uterus, the vulva, or the body wall.