Flagellar membrane agglutination and sexual signaling in the conditional GAM-1 mutant of Chlamydomonas.

Abstract

The temperature-sensitive gametogenesis-defective mutant, gain-1 is sex-limited, expressed only in mating type minus (rot-), and can sexually agglutinate but not fuse at the restrictive temperature (35~ with gametes of wild type (wt) mt +.Thin-section, freeze-cleave, and scanning electron microscopy reveal that the gain-1 phenotype is dependent on both the temperature at which the cells undergo nitrogen starvation (and therefore gamete formation) and the temperature at which the cells are maintained during the 12 h before mating. Under all conditions of gametogenesis at 35~ each gam-1 cell produces a normal-appearing membrane-associated mating structure that fails to activate in response to flagellar agglutination. Varying with the conditions of gametogenesis, on the other hand, are the agglutination and signaling properties of the gain-1 flagella. The two mutant phenotypes displayed by gain-1 have been denoted gam-l-I and gam-l-II. An agglutination reaction involving gam-I-I cells does not result in activation of the wt mt § mating structure. A more stable agglutination reaction, which can result in activation of the wt mt+ mating structure, is characteristic of gam-l-II cells, but because the gam-1 rot-mating structure still fails to activate, cell fusion is precluded. We conclude that the gain-1 mutation affects flagellar component(s) involved in establishing an effective, signal-generating agglutination reaction.KEY WORDS mutant temperature sensitivity flagellar membrane sexual agglutination signaling mating structureThe sexual cycle of the single-celled eukaryoteChlamydomonas reinhardi represents an easily controlled developmental process. Gametogenesis is initiated when the haploid cells are starved for nitrogen (11). Both mating types then develop the ability to agglutinate by their flagellar tips (6, 11), a reaction that occurs immediately when gametes of opposite mating types are mixed. During gamete formation, mating-type-specific mating structures also develop in association with the plasma membrane (2,6,9,14,17). These mating structures remain unactivated unless stimulated by some sort of sexual agglutination. The activated structure of mating-type plus (mt +) is a long, 74 J. CELL BIOLOGY ~ The Rockefeller University Press

Cyclic AMP enhances the sexual agglutinability of Chlamydomonas flagella.

“…Wild-type strains C. reinhardtii CC-620 (rat +) and CC-621 (rot-) and the mutants imp-I rat + (CC-462) (10,12) and gain-1 (CC-1693) (8) were used, as well as C. smithii mt + (CC-13'73) (2), all available from the Chlamydomonas Genetics Center, Duke University, Durham, NC. Plate gametes (19) were suspended in nitrogen-free high-salt minimal medium (NFHSM) I (19) for 1-2 h before use.…”

“…If C. smithff is first incubated in db-cAMP/IBMX and then mated, however, widespread agglutination is observed, and when the yield of chloroform-resistant zygotes (17) is analyzed, the treated gametes generate 100 times more zygotes than the controls. A second example is the mutant strain gam-1 mt-which, when allowed to differentiate into gametes in liquid medium at 35°C, usually agglutinates poorly and hence fails to either transmit or receive sexual signals (8). If such gametes are incubated in db-cAMP/IBMX at 35°C for 20 min, their adhesivity improves markedly and they agglutinate with plus gametes as avidly as wild-type minus controls.…”

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

“…Some are directly linked to the mt locus itself (l0, 20), so that certain genes will be found exclusively in mt + but not mt cells, and vice-versa. Others are carried by both haploid mating types but are limited in their expression to one or the other type (5,6,11,14); thus, for example, the "sex-limited" sag-1 locus (11) functions to specify plus flagellar agglutinability in haploid mt + cells, but is apparently not expressed in haploid mt cells. Because the haploid imp-11 mutant derives from wt -and can readily revert to wt -, we can assume that it carries an mt but not an mt + locus and can write its genotype as imp-11 mt -.…”

Mating Structure Differences Demonstrated by Freeze‐Fracture Analysis of Fusion‐Defective Chlamydomonas Gametes

“…Freeze-fracture electron microscopy has demonstrated that during activation of mt-mating structures, intramembrane particles (IMPs) migrate into the center of the mating structure [22]. It has been postulated that these particles may be involved in fusion between gametes [6,221. Results from other systems also suggest that IMPS may be involved in the fusion process; particle rosettes are seen prior to fusion of: (a) mucocysts and the plasma membrane in Tetrahymena [ 171; (b) trichocysts and the plasma membrane in Paramecium [2]; (c) pinocytotic vesicles in smooth muscle cells [ 121; and (d) the "particle-free area surrounded by membrane particles" that is seen in sperm acrosomes [21].…”

“…In order to clarify the role of the mt-mating structure particles, it would be useful to determine if any differences are manifested in the mating structures of fusion-defective mutants at the ultrastructural level, during the activation process. Therefore, in these studies we examine freeze-fracture replicas of wild type mt-and 3 conditional fusion-defective mutants [4, 7, 81, (all fusion-defective mutants so far produced have been sexlimited mutants expressed only in mt- [6]) and detail the differences found. A preliminary account of some of these results has previously appeared [Forest, C. L. & Ojakian, G. K. 1986.…”