Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants.

Abstract

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...

TheChlamydomonasFus1 Protein Is Present on the Mating TypeplusFusion Organelle and Required for a Critical Membrane Adhesion Event during Fusion withminusGametes

“…Because the mating structures are located at the bases of the two flagella on each interacting gamete, flagellar adhesion brings the activated fusion organelles into intimate contact, allowing them to adhere to each other (Goodenough et al, 1982) and leading rapidly to fusion of their plasma membranes. Immediately after mating structure fusion, a cytoplasmic bridge representing the combined mating structures joins the two gametes to each other.…”

“…The activated plus organelle, which is termed the fertilization tubule, is a more prominent, 0.5 ϫ 3 m microvillouslike organelle, filled with 60 -80 actin filaments. The mating structures of both types of gametes display an extracellular coat of material called fringe (Goodenough et al, 1982).…”

“…On the other hand, the fus1-1 fertilization tubule fails to fuse with the activated minus mating structure and the cells in such mixtures continue to agglutinate for days. Ultrastructural analysis of the fertilization tubules on fus1 gametes indicated that the organelles do not contain fringe (Goodenough et al, 1982). More recently, the FUS1 gene was identified by its unique presence in the mtϩ locus, the sex-restricted expression of its transcript and by its ability in transformation experiments to restore fusion competence to several mtϩ mutant strains with lesions in the FUS1 gene (Ferris et al, 1996).…”

“…The activated plus organelle, which is termed the fertilization tubule, is a more prominent, 0.5 ϫ 3 m microvillouslike organelle, filled with 60 -80 actin filaments. The mating structures of both types of gametes display an extracellular coat of material called fringe (Goodenough et al, 1982).Because the mating structures are located at the bases of the two flagella on each interacting gamete, flagellar adhesion brings the activated fusion organelles into intimate contact, allowing them to adhere to each other (Goodenough et al, 1982) and leading rapidly to fusion of their plasma membranes. Immediately after mating structure fusion, a cytoplasmic bridge representing the combined mating structures joins the two gametes to each other.…”

Origin of Female/Male Gender as Deduced by the Mating-Type Loci of the Colonial Volvocalean Greens

“…Sexual reproduction of the unicellular volvocalean C. reinhardtii is isogametic, with plus and minus mating types. The minus mating type of this species represents a "dominant sex" because occasionally produced diploid cells exhibit the minus mating phenotype (Ebersold 1967 ); when the minus mating type-determining gene MID is lacking, the phenotype of the sex changes from minus to plus , forming a fertilization tubule (Goodenough et al 1982 ). Therefore, mating type minus in C. reinhardtii can be considered a "derived type of sex."…”