FLAGELLAR MEMBRANE ADHESION AND SEXUAL SIGNALING

相信细胞识别，粘附和随后的细胞反应

Cell recognition, adhesion, and the ensuing cellular responses are believed to be critical to embryological development and growth control in multicellular organisms. The long-term objective of the proposed research is to understand the mechanisms of sexual adhesion between flagellar membranes of Chlamydomonas reinhardi and to learn how such adhesion signals the cell to secrete enzymes, polymerize actin, prepare its plasma membranes for cell fusion, and modify the pattern of its organelle DNA inheritance. Exerpiments are based on our success in obtaining high-specific-activity preparations of mating-type plus (mt+) and mt- agglutinins by treating cells in vivo with 12.5 mM EDTA. Partial purification is effected by sucrose gradient centrifugation and gel filtration; affinity purification will be undertaken using monoclonal antibodies presently being generated against the partially purified material. Identification of agglutinin domains important for adhesion will be made using 4 approaches: 1) monoclonal antibodies will be sought that recognize mt-specific peptide determinants; 2) mutant and inactive agglutinins will be sought in 8 nonagglutinating mutant strains and in disadhered zygotic cells and, if found, will be compared with their wild-type counterparts; 3) an in vitro neutralization of the two agglutinin types will be exploited to detect peptides important for neutralization; and 4) cleavable crosslinking reagents will be used to couple adhering surfaces and identify interacting regions of the two agglutinin types. Identification of intrinsic membrane proteins with which agglutinins associate will be made via binding studies and agglutinin affinity chromatography, and antibodies will be prepared to probe their surface localization, and agglutinin location, using immunoelectron microscopy. Flagellar tip activation, a pronounced morphological response to adhesion which appears important for signal transmission, will be probed by immunoelectron microscopy and by biochemical extraction and analysis of the fibrous material that fills the activated tips. Attempts to induce tip activation without adhesion will also be made. Finally, we will analyze the genetic properties of existing mutants and continue to isolate and characterize new mutants.