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.

Monoclonal Antibodies to Two Dense-Body Components

R. Francis, R.H. Waterston

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.