Worm Breeder's Gazette 8(1): 30
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.
Previously, a rabbit anti-serum (anti-p107) raised to a 107,000 MW polypeptide was shown to react primarily with the dense-bodies of the bodywall musculature of C. elegans. As the anti-p107 sera and an antibody to chicken gizzard alpha-actinin cross-reacted with the heterologous antigens, the 107,000 MW polypeptide was presumed to be a nematode alpha-actinin analogue. On two-dimensional gels p107 separates into 2 sets of spots differing markedly in isoelectric point. After transfer of 2D-gels to nitrocellulose, anti-p107 reacts with both the acidic (one spot, pI=6.1) and basic (several spots centered at pH 6.6) species. The acidic species (p107-A) is most abundant in 0. 5% deoxycholate extracts whereas the basic species (p107-B), although less restricted, is more enriched in 0.6 M NaCl and 8 M urea extracts than the detergent fraction. Chromatography of deoxycholate extracts on DEAE-Sephacel and Sepharose-4B yields a p107 fraction 70-80% pure that on 2D-gels consists entirely of p107-A and actin. The differential extraction and fractionation properties of the two 107,000 MW species suggests they differ substantially. We have obtained at least partially-specific monoclonal antibodies (mAbs) that support this conclusion yet indicate both species are components of, or associated with, dense-bodies although differing subtly in their distributions. One mAb, MH23, reacts only with p107-B on transfers of 2D-gels loaded with equal amounts of p107-A and p107-B. The fluorescent staining pattern of MH23 is easily recognized as being distinct from that of the anti-p107 sera: MH23 dense-body staining appears flat or two-dimensional and there is more intense staining along some edges of the bodywall muscle cells where, based on sarcomere orientation, we expect thin (actin) filaments to terminate. With the anti-p107 sera, the finger-like contours of the dense-bodies are visible and there is proportionately less staining along cell boundaries. Two other mAbs are indistinguishable from MH23 by immunoflourescence and react with p107 on gel transfers but have not been tested yet for specificity on transfers of 2D-gels. Another class of mAbs, represented by MH35, have fluorescent staining distinct from MH23 and the anti-p107 sera. After reaction with MH35, the three-dimensional shapes of dense-bodies are evident as with anti-107 sera but there is no staining along cell boundaries. Images of worms reacted with both MH35 and MH23 are similar to those seen with the anti-p107 sera. On transfers of 2D-gels, MH35 (ascites fluid, 40 g/ml) reacted strongly with p107-A and weakly with p107-B. On an identical transfer incubated with MH35 culture supernatant, MH35 reacted with p107-A at a level considerably less than the ascites- grown mAb and not at all with p107-B. Miller et. al. have shown that the specificity of apparently isozyme-specific mAbs to different C. elegans myosin heavy chains depends critically upon Ab concentration. Although an analogous situation may apply to the reaction of MH35 with p107-A and p107-B, the result could also be an artifact of isoelectric focusing as neither p107 species focuses particularly well. We are currently trying to obtain better localization of antigens in cross-sections of plastic-embedded Norms and to determine the relationships of p107-A and p107-B with each other and vertebrate alpha-actinin and their possible actin-binding properties. The distributions of the two classes of mAbs also differ in the pharynx and the vulva muscles. One explanation of the different patterns of immunofluorescence in the bodywall musculature is that the probable anti-p107-B mAbs are reacting with a restricted portion of the dense- bodies, perhaps the basement membrane-proximal regions. Another mAb, MH25, has similar immunofluoresence to the anti-p107-B mAbs except there is also discontinuous staining of M-line regions. The antigen(s) recognized by MH25 may therefore be a reasonable candidate for a macromolecule(s) responsible for the association of the dense-bodies and M-lines with the underlying basement membrane seen in electron micrographs of worm fragments extracted in nonionic detergent and 0.6 M NaCl.