Wheat germ agglutinin induces mating reactions in Chlamydomonas eugametos by cross-linking agglutinin-associated glycoproteins in the flagellar membrane.

Abstract

Abstract. Species-specific binding between the flagellar surfaces of mating types plus and minus (mt ÷ and mt-) gametes of Chlamydomonas eugametos is mediated by mating type-specific agglutinins. Their interaction triggers several mating responses that are necessary for cell fusion, such as flagellar twitching, flagellar tip activation, redistribution of agglutinin molecules to the flagellar tip (tipping), and mating structure activation. Earlier, we reported that a monoclonal antibody (mAb 66. 3) 107:177-189). Here we report that the lectin wheat germ agglutinin (WGA), which does not bind to the agglutinins, can also invoke all these mating reactions. We show, by immunofluorescence studies using anti-WGA and an agglutinin-specific monoclonal antibody (mAb 66.3), that WGA induces the redistribution of agglutinin to the flagellar tips of mt-gametes. Vice versa, when agglutinin tipping is induced by mAb 66.3, the WGA-binding glycoproteins are also tipped. Under the same conditions, the major flagellar glycoproteins are not redistributed, indicating that membrane transport is limited to a few components. We conclude that each agglutinin is associated with a WGA-binding glycoprotein. When cells lacking agglutinin or cells possessing inactive agglutinins are treated with WGA, mating responses are again elicited.The data suggest that clustering of agglutinincontaining complexes results in the production of intracellular signals, such as cAMP, and the coupling of the complex to a force generating system. In nature, the complexes are clustered via the agglutinins, but artificially they can be clustered by lectins or antibodies directed against other proteins in the complex.

The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellar membrane glycoproteins involves the dephosphorylation of a 60-kD phosphoprotein that binds to the major flagellar membrane glycoprotein.

“…Cross-linking of a population of flagellar membrane glycoproteins by antibodies or lectins induces their movement within the plane of the flagellar membrane (Bloodgood et al, 1986;Homan et al, 1988;Kooijman et al, 1989). Micromolar concentrations of free calcium ions are required in the extracellular medium for the directed movements of flageUar glycoproteins (Bloodgood and Salomonsky, 1990).…”

“…Cross-linking of the 350-kD glycoproteins with concanavalin A or specific anti-carbohydrate monoclonal antibodies induces a directed, energy-dependent redistribution of these glycoproteins within the plane of the flagellar membrane (Bloodgood et al, 1986) that is reminiscent of the "capping" of cell surface receptors observed on other systems (Schreiner and Unanue, 1976;Bourguignon and Bourguignon, 1984). Other Chlamydomonas flagellar membrane glycoproteins can also exhibit active, directed movements within the plane of flagellar membrane in response to antibody/lectin crosslinking (Homan et al, 1988;Kooijman et al, 1989) and/or flagella-flagella interactions during mating (Homan et al, 1987;Musgrave and van den Ende, 1987). These directed movements of flagellar membrane glycoproteins are dependent on the presence of micromolar concentrations of free calcium in the medium and are inhibited by calcium channel blockers and calmodulin antagonists (Bloodgood and Salomonsky, 1990).…”

Cell adhesion-dependent inactivation of a soluble protein kinase during fertilization in Chlamydomonas.

“…For example, much like receptor aggregation in other systems (Kornberg et al, 1991;Hynes, 1992;Johnson and Vaillancourt, 1994;Weiss and Littman, 1994), interactions between mt+ and mt-agglutinins might induce agglutinin aggregation in the plane of the membrane (Goodenough and Jurivich, 1978;Kooijman et al, 1989;Bloodgood, 1992b) thereby leading to inhibition of the adhesion-regulated protein kinase. Based on the results presented here our working model is that a nonphosphorylated form of the 48-kDa protein is required in the signalling pathway that couples agglutinin interactions to activation of adenylyl cyclase.…”

Flagellar adhesion-dependent regulation of Chlamydomonas adenylyl cyclase in vitro: a possible role for protein kinases in sexual signaling.

“…Because the agglutinin may not be a transmembrane protein (reviewed in Adair, 1985), coupling of agglutinin interactions to the adenylyl cyclase could require intermediate transmembrane components (Kooijman et al, 1989;van den Ende, 1992). This observation, along with the evidence cited above suggesting that G proteins do not play a role in this adhesion-induced signal transduction pathway, has compelled US to search for a novel mechanism of regulation of adenylyl ~clase in Chlamydomonas.…”

“…During fertilization, cell-cell contacts between sperm and egg initiate G protein signaled responses in both cell types Gaffe, 1990;Kopf, 1990;Ward and Kopf, 1993). Although several molecules involved in cell-cell adhesion have been identified, the molecular mechanisms coupling adhe-adhesion is followed by a rapid increase of intracellular cAMP (Pijst et al, 1984;Pasquale and Goodenough, 1987;Kooijman et al, 1990) leading to several cellular responses required for continued adhesion as well as for preparation of the interacting cells for fusion and zygote formation (Goodenough, 1989;Snell et al, 1989;Kooijman et al, 1989;Tomson et al, 1990). This specific, cell-cell adhesion and signaling system is unique to gametes, and many of the signaled responses that occur in the cell body can be induced by incubation of gametes of a single mating type in dibutyryl cAMP (rib-CAMP) l (Pasquale and Goodenough, 1987;Snell et al, 1989;Goodenough, 1989;Hunnicutt et al, 1990).…”