Inducible cell fusion receptor analysis of isolated plasma membrane fractions of the myxomycete Didymium iridis

Abstract

Data are presented which focus on changes that occur in the cell surface glycoproteins of the myxomycete Didymium iridis, which permit cell fusion to occur in previously induced cells. It was therefore necessary to develop a reliable method for the isolation of pure plasma membrane fraction, prior to the characterization of mating and fusion factors. Methods of chromatographic analysis of isolated plasma membrane fraction of pre-fusion (uninduced) versus fusion competent (induced) myxamoeba were carried out. Previous studies have shown that fusion between competent myxamoeba of compatible mating types can be blocked by the lectin concanavalin A. Concanavalin preferentially binds glycoproteins thus indicating the biochemical character of this fusion receptor (Yemma and Soltis, 1988). Yemma and Perry (1985) provided evidence indicating that compatible cells of Didymium iridis must become competent to fuse either by attaining a critical cell density or by being induced into competency by an already fusion-competent population. Induction is initiated by a diffusible organic molecule produced by cells of the same mating type (self-induction) having attained a critical density of 1 x 10 E+05 cells/milliliter. Cells most suceptible to induction are in the G phase of interphase in the cell cycle (Yemma and Stroh, 1991).

Isolation of the plasma membrane of cells was carried out by modification of a technique by Barden et al. (1983), utilizing density gradient centrifugation in a linear sucrose gradient. This technique, which is a hybrid of isopycnic and rate zonal centrifugations, produced a highly pure membrane fraction. Location of the isolated plasma membrane fraction(s) was done by enzymatic assay of the marker enzyme 5-nucleotidase. The major plasma membrane fraction was found to be located in the 40% (w/v) sucrose gradient at a mean sucrose density of 44.5% (w/v). Purity of membrane fractions was determined by screening for the presence (or absence) of marker enzymes for membranes other than the plasma membrane.

It was initially hypothesized that a specific membrane fusion receptor would be present on induced cells or cells made competent to fuse, and therefore absent on uninduced cells. HPLC analysis of the membrane glycoproteins of these two respective isolated plasma membrane fractions should produce spectra upon analysis, indicative of this phenomenon. Results of HPLC analysis did indeed demonstrate differences in the plasma membrane glycoprotein profiles of induced versus uninduced cells. Evidence presented indicates that during the induction period prior to fusion (Yemma and Perry, 1985), the plasma membrane undergoes dynamic changes in which membrane fusion factors are made active through conformational changes of specific membrane proteins which occur on the membrane surface. The conformational changes that occur would account for these differences in HPLC analysis of membrane glycoproteins in induced versus uninduced plasma membrane fractions. Future studies will seek to characterize membrane glycoprotein changes which allow cells to become competent to fuse.