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.

Using Monoclonal Antibodies to Study Sperm Membrane Dynamics

F. Pavalko, T. Roberts

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.