Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella . By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone . (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actinfilled fertilization tubule during activation . Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse . The imp-1 mutation is linked to the mating-type plus (mt + ) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome . (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse . The imp-11 mutation converted a wild-type minus cell into a pseudoplus strain ; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.Biological membranes do not ordinarily fuse with one another : the eucaryotic cell is filled with membrane systems and organelles that maintain their integrity . The important exceptions to this rule include endocytosis, the rough endoplasmic reticulum (RER) --* Golgi --* exocytosis transit, the fertilization of gametes, and the formation of such syncytia as myofibrils . In each case, the relevant membranes fuse in a highly specific fashion, and only in response to well defined physiological signals (e.g. receptor clustering, Ca" fluxes, or-possibly-the presence of a clathrin coat) .Fusion between gametic cells of Chlamydomonas reinhardi (reviewed in references 8 and 9) is particularly well suited to experimental analysis. (a) The discrete sector of the plasma membrane that is specialized for this event can be readily identified in either thin section or freeze-fracture replicas (2,7,13,23,24) . (b) Fusion specificity resides in this membrane sector : mating-type plus (mt+) gametes will fuse only with minus (mt) partners of the same species. (c) These membranes acquire the capacity to fuse in response to a defined physiological signal, namely, the adhesion of flagellar membranes (1,12,18,22,25). (d) Chlamydomonas can be readily manipulated genetically (4,20), offering the possibility of isolating nonfusing mutants. 378Previous studies from this laboratory (13, 24) defined several stages in the acquisition of fusion capacity . The present report exploits the ability of fixation in the presence of tannic acid and en bloc staining with uranyl acetate to reveal new features of th...

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Mutant strains of Chlamydomonas reinhardi that have lost their ability to undergo sexual agglutination via their flagellar tips have been induced to undergo zygotic cell fusion and meiosis, using a flagellar-directed antiserum. Genetic analysis of antiserum-mediated crosses involving five nonagglutinating mt  + mutant strains reveals the following: (1) None of the mutations is linked to the mt locus. (2) All of the mutations are "sex-limited," meaning that they can be carried and transmitted by, but not expressed in, mt  - cells. (3) Four of the mutations (imp-2, imp-5, imp-6, imp-7) are either allelic or closely linked to one another, with imp-8 defining a second locus.

Two independently isolated mutant strains, imp-10 and imp-12, were obtained by UV irradiation of wild-type mating-type minus (wt-). Each fails to agglutinate sexually with gametes of either mating type, but mating and zygote formation can be elicited by agglutinating either strain to wt+ gametes by means of anti-flagellar antiserum. Tetrad analysis of the resultant zygotes shows that both imp-10 and imp-12 are very closely linked to mt  -, with no recombinants observed. Diploid strains constructed between imp-10 or imp-12 and wt+ gametes are wt-, that is, they agglutinate and fuse like normal minus cells. Tetrad analysis of triploids from imp-10 diploid x wt+ haploid crosses shows that only imp-10 and wt+ products are recovered. A model is proposed to account for these results.