Gliding defective mutant cell lines ofChlamydomonas moewusii exhibit alterations in a 240 kDa surface-exposed flagellar glycoprotein

Summary TheChlamydomonas flagellar surface exhibits a number of dynamic properties associated with whole cell locomotion and the mating process. In this report, we quantitate the ability of a series of gliding defective mutant cell lines (Lewin 1982) to move polystyrene microspheres along their flagellar surface and describe alterations in the flagellar surfaces of three of these cell lines (fg-2, fg-3 and fg-7). Although all three of these mutant cell strains exhibit less than 16% of the control level of microsphere movement, they differ from each other and the parental cell line (M 475) in the level of flagellar surface adhesiveness as judged by the binding of polystyrene microspheres. SDS-polyacrylamide gel analysis of purified whole flagella from the nongliding mutant cell strains and M 475 demonstrates a correlation between the amount of a surface exposed glycoprotein and the level of flagellar surface adhesiveness. This surface exposed glycoprotein binds the lectin concanavalin A and has an apparent molecular weight of 240 kDa. Strains with low levels of flagellar surface adhesiveness (fg-3 and fg-7) exhibit a low amount of this glycoprotein while the strain with a high level of adhesiveness (fg-2) has an elevated amount of this glycoprotein relative to the parental strain, suggesting that this 240 kDa glycoprotein may be responsible for the adhesive properties of the flagellar surface. Concanavalin A inhibits microsphere binding to the flagellar surface, suggesting that the carbohydrate component of the 240 kDa glycoprotein may be involved in flagellar surface adhesiveness. Biotinylation of surface-exposed flagellar proteins demonstrates differences in the surfaces of these mutant cell lines, especially in terms of the amount of surface labelling of the 240 kDa flagellar glycoprotein. A rabbit polyclonal antibody (designated P-19) that binds to the flagellar surface and recognizes the 240 kDa glycoprotein on Western blots confirms the altered level of this glycoprotein in the mutant cell lines. The results of these experiments suggest that the major flagellar glycoprotein ofC. moewusii may be involved in adhesion of polystyrene microspheres to the flagellar surface and presumably also in the adhesive interaction of the flagellar surface with a solid substrate, which is a necessary prerequisite for the expression of gliding motility.Abbreviations BSA bovine serum albumin - DAB 3,3-diaminobenzidine - HRP horseradish peroxidase - kDa kilodaltons - LBB lectin blot buffer - NHS-LC biotin sulfosuccinimidyl 6-(biotinamido) hexanoate - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis