Worm Breeder's Gazette 8(1): 24
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.
We have been applying hybridoma technology to our research by generating monoclonal antibodies (MAb's) against C. elegans sperm. We have produced 10 mAb's in our lab and have obtained 2 others from Susie Strome at Boulder. Seven of these mAb's target antigens present on the cell surface. We have been purifying monoclonal antibodies from ascites fluid by DEAE Affi-Gel blue chromatography (supplied by Bio-Rad). Although yields have been lower than ideal (40-60%), these antibodies are free of all contaminants except mouse transferrin. Membrane lipid flow and directed movement of pseudopod surface components on sperm was previously reported by Roberts and Ward (J. Cell Bio. 92: 113-120). We have evidence that the movement of antigen-antibody complexes may be due to a continual flow of membrane lipid (see Bretscher, 1976, Nature 260: 21-23) from the tip of the pseudopod back toward the cell body. Bretscher's lipid flow model of membrane movement predicts that membrane components which diffuse slowly are displaced by the lipid stream whereas those which diffuse rapidly effectively escape lipid flow. Because the rate of diffusion is related to molecular weight, large complexes (e.g. antibody- crosslinked membrane proteins) would be swept along more rapidly than individual molecules (e.g.- unbound membrane proteins). We have tested this prediction by comparing clearance rates of various sized complexes form the cell pseudopod. On sperm, relatively small antigen- mAb complexes clear from the pseudopod surface slowly (2-3 min) while larger, cross-linked antigen-mAb-2 antibody complexes are cleared more rapidly (30-45 seconds). Using mAb's conjugated directly to colloidal gold particles (CGP), we have obtained evidence that new surface antigens are preferentially inserted at the tips of pseudopodial projections and then move rearward toward the cell body pseudopod junction (Pavalko and Roberts, C. elegans Meeting Abstracts, 1983). This observation predicts that there must be a pool of antigen in the cytoplasm which would be available for insertion into the membrane. Labelling thin sections of spermatozoa with CGP conjugates of mAb's 11, 63, and 56 (each of which bind to the cell surface in indirect immunofluorescence assays) reveals antigen on the surface (plasma membrane, exposed face of fused membranous organelles and MO contents). In addition, these antibodies label the cytoplasmic laminar membranes and the pseudopod cytoplasm. Many of the gold particles in the cytoplasm lie just beneath the plasma membrane suggesting that they are bound to antigens that were destined for insertion onto the surface. Because there are no vesicles in the cytoplasm, the mechanism which transports these antigens is unknown. We will be trying to characterize the antigens targeted by the mAb's and to solve the question of how they are shuttled around the cell.