A monoclonal antibody that blocks adhesion of Chlamydomonas mt+ gametes.

Abstract

Abstract. During the mating reaction (fertilization) in the biflagellated alga, Chlamydomonas reinhardtii, mt* and mt-gametes adhere to each other via their flagella and subsequently fuse to form quadriflagellated zygotes. In the studies reported here, we describe a monoclonal antibody directed against an mt + flagellar surface molecule. The antibody blocks the adhesiveness of mt + gametes, isolated mt ÷ flagella, and detergent extracts thereof. It has no effect on mt-gametes. Cyanogen bromide-activated Sepharose beads derivatized with the antibody bind only mt + gametes; mt-gametes and mt ÷ and mt-vegetative cells are unreactive with the derivatized beads. The interaction of mt + gametes with the beads is dynamic and cells continuously bind, detach, and rebind to the beads. Surprisingly, antibody-derivatized beads that have been incubated with mt + gametes acquire the ability to bind mr-gametes. Moreover, extraction of the preincubated beads with detergents releases active mt + adhesion molecules. The evidence suggests that binding of the antibody to the flagellar surface adhesion molecules causes their release from the flagellar surface, possibly mimicking the normal mechanism of flagellar de-adhesion.W HEN gametes of opposite mating types of the biflagellated alga Chlamydomonas reinhardtii are mixed together, they adhere to each other via their flagella and form large clumps of aggregating cells. Within these clumps, pairs of cells form that eventually fuse to become quadriflagellated zygotes. Several laboratories have been interested in the initial adhesive interaction. Recently Goodenough, Heuser, Adair, and co-workers identified high molecular mass glycoproteins rich in hydroxyproline that are involved in the adhesive interaction (1, 2, 4, 9, 10, 16). Saito and Matsuda (35, 37) obtained similar results using different purification methods, and van den Ende's laboratory has identified similar high molecular mass agglutinin molecules in C. eugametos (20,33, 45).Our laboratory has been interested in the molecular details of the adhesive interaction as well as a specific de-adhesion mechanism that we have characterized (39,(40)(41)(42)44). We showed that as cells adhere to each other they continuously inactivate the adhesiveness of each other's flagella (42, 44). Replacement of these lost adhesive sites requires protein synthesis (36, 37, 42) and protein glycosylation (29, 39, 48); Cooper et al. showed that hydroxylation of proline was also required for replacement of these lost adhesion sites (10). Similar results have been shown for Chlamydomonas eugametos (32,33, 45,(47)(48)(49). To more fully study this adhesion-induced loss of flagellar adhesion sites and to identify molecules responsible for adhesion we have begun to use an immunological approach. In earlier unpublished studies we had attempted to prepare rabbit polyclonal antibodies against flagella or flagellar surface fractions that would specifically block adhesiveness of mating type plus (mr+) 1 or mating type minus (mr-) gametes. To date, all of th...

Cell body and flagellar agglutinins in Chlamydomonas reinhardtii: the cell body plasma membrane is a reservoir for agglutinins whose migration to the flagella is regulated by a functional barrier.

“…This loss of agglutinins is similar to the loss that occurs when mt + gametes bind to mAb 2b40-derivatized Sepharose beads. Cells bind to the beads, but then rapidly de-adhere, leaving their agglutinins on the antibodyderivatized beads (Snell et al, 1986). Together, these results indicate that interaction of the antibody with agglutinin leads to rapid release of the agglutinins from the flagella.…”

“…Production and characterization of the mAb 2b40, which blocks adhesion of mt÷ gametes, was previously described (Snell et al, 1986). Ascites fluids were prepared from pristane-primed mice injected intraperitoneally with mAb 2b40 hybridoma cells.…”

Signal transduction in the sexual life of Chlamydomonas

“…First, adhesion causes a deactivation of the agglutinins [74,88] by an unknown mechanism [40,87,90]. Recruitment of new agglutinins is required to maintain flagellar adhesiveness [40].…”

“…Many pharmacological agents, including La 2 +, Cd 2+ , lidocaine, trifluoroperizine (TFP), W7, colchicine and amiprophos-methyl (APM) block the adhesion-induced accumulation of cAMP (and therefore the mating responses) without blocking adhesion [13,33,65,90]. The Goodenough lab [33,76] refers to these agents as 'upstream inhibitors' because they prevent the coupling of adhesion to cAMP accumulation but do not interfere with cellular responses if dibutryl-cAMP is supplied.…”

Antibodies to flowering-plant sperm

“…In sea urchins, an egg receptor for sperm has been identified (Foltz et al 1993). In ChIamydomonas reinhardtii, membrane molecules are responsible for adhesion (Snell et al 1986). These provide experimental models for our work.…”