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

Zhang, Yuhua; Snell, William J.

doi:10.1083/jcb.125.3.617

Cited by 37 publications

(49 citation statements)

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“…A flagellar membrane-associated protein kinase activity in nonactivated gametes inhibits the adenylyl cyclase (10). During gamete activation, agglutinin interactions relieve this inhibition and also stimulate a second protein kinase whose activity is required to activate adenylyl cyclase (11). Concomitantly, flagellar adhesion leads to the inhibition of a third protein kinase, whose substrate itself is yet another protein kinase (GenBank TM accession number U36196) (11,21).…”

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“…During gamete activation, agglutinin interactions relieve this inhibition and also stimulate a second protein kinase whose activity is required to activate adenylyl cyclase (11). Concomitantly, flagellar adhesion leads to the inhibition of a third protein kinase, whose substrate itself is yet another protein kinase (GenBank TM accession number U36196) (11,21). In addition to these protein kinase activities involved in the very early stages of gamete activation upstream of generation of cAMP, several protein kinases act downstream of cAMP.…”

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confidence: 99%

“…In addition to binding the flagella of gametes of opposite mating types to each other, these receptor/ligand-like interactions also initiate a complex series of events termed gamete activation. One of the earliest documented steps in gamete activation triggered by agglutinin interactions is activation of a gamete-specific flagellar adenylyl cyclase and generation of cAMP (7)(8)(9)(10)(11). As part of an intricate feedback mechanism required to maintain and enhance flagellar adhesiveness and to keep the cells bound to each other until cell-cell fusion occurs, cAMP levels increase in the cell bodies of the gametes of both mating types during flagellar adhesion and lead to increased flagellar adhesiveness (6,9,12,13).…”

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Regulated Targeting of a Protein Kinase into an Intact Flagellum

Pan

Snell

2000

Journal of Biological Chemistry

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In the green alga Chlamydomonas reinhardtii flagellar adhesion between gametes of opposite mating types leads to rapid cellular changes, events collectively termed gamete activation, that prepare the gametes for cell-cell fusion. As is true for gametes of most organisms, the cellular and molecular mechanisms that underlie gamete activation are poorly understood. Here we report on the regulated movement of a newly identified protein kinase, Chlamydomonas aurora/Ipl1p-like protein kinase (CALK), from the cell body to the flagella during gamete activation. CALK encodes a protein of 769 amino acids and is the newest member of the aurora/ Ipl1p protein kinase family. Immunoblotting with an anti-CALK antibody showed that CALK was present as a 78/80-kDa doublet in vegetative cells and unactivated gametes of both mating types and was localized primarily in cell bodies. In cells undergoing fertilization, the 78-kDa CALK was rapidly targeted to the flagella, and within 5 min after mixing gametes of opposite mating types, the level of CALK in the flagella began to approach levels normally found in the cell body. Protein synthesis was not required for targeting, indicating that the translocated CALK and the cellular molecules required for its movement are present in unactivated gametes. CALK was also translocated to the flagella during flagellar adhesion of nonfusing mutant gametes, demonstrating that cell fusion was not required for movement. Finally, the requirement for flagellar adhesion could be bypassed; incubation of cells of a single mating type in dibutyryl cAMP led to CALK translocation to flagella in gametes but not vegetative cells. These experiments document a new event in gamete activation in Chlamydomonas and reveal the existence of a mechanism for regulated translocation of molecules into an intact flagellum.Cell-cell interactions leading to fusion between gametes of opposite sexes during fertilization are complex processes that involve dramatic changes in each of the interacting gametes. In most multicellular organisms, interactions between adhesion molecules/receptors on the sperm plasma membrane and ligands on the egg surface activate poorly understood signaling pathways that bring about transformation of the sperm into a fusion competent, activated gamete (1). For example, the sperm surface is remodeled as a consequence of the acrosome reaction, an event that accompanies gamete activation, and previously existing adhesion molecules are mobilized to new sites on the sperm (2-4). The molecular mechanisms that underlie and regulate signal transduction and movement of molecules between different gamete compartments during gamete activation largely are unknown (2).As in multicellular organisms, gamete activation and fertilization in the unicellular green alga Chlamydomonas (5) depend on adhesion-induced signaling pathways and intercompartmental communication. At the completion of gametogenesis, during which asexually growing mtϩ and mtϪ vegetative cells differentiate into sexually competent cells, the resultin...

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Regulated Targeting of a Protein Kinase into an Intact Flagellum

Pan

Snell

2000

Journal of Biological Chemistry

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“…When wild-type mtϩ and mtϪ gametes are mixed, they initially adhere to each other via sex-specific cell adhesion molecules, agglutinins, on their flagella (Adair, 1985). Interactions between the mating type plus and mating type minus agglutinin molecules on the flagellar membranes activate a gamete-specific flagellar adenylyl cyclase and the resultant increases in intracellular cAMP lead to gamete activation (Pasquale and Goodenough, 1987;Saito et al, 1993;Zhang and Snell, 1994). Both of the gametes release their extracellular matrices (cell walls), they recruit additional agglutinins and a protein kinase from the cell body to the flagella, and they activate sex-specific mating structures at their apical ends, which are the sites for cell-cell fusion (Detmers et al, 1983;Wilson et al, 1997;Wilson and Snell, 1998;Pan and Snell, 2000b).…”

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

Misamore

Gupta

Snell³

2003

MBoC

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The molecular mechanisms of the defining event in fertilization, gamete fusion, remain poorly understood. The FUS1 gene in the unicellular, biflagellated green alga Chlamydomonas is one of the few sex-specific eukaryotic genes shown by genetic analysis to be essential for gamete fusion during fertilization. In Chlamydomonas, adhesion and fusion of the plasma membranes of activated mtϩ and mtϪ gametes is accomplished via specialized fusion organelles called mating structures. Herein, we identify the endogenous Fus1 protein, test the idea that Fus1 is at the site of fusion, and identify the step in fusion that requires Fus1. Our results show that Fus1 is a ϳ95-kDa protein present on the external surface of both unactivated and activated mtϩ gametes. Bioassays indicate that adhesion between mating type plus and mating type minus fusion organelles requires Fus1 and that Fus1 is functional only after gamete activation. Finally, immunofluorescence demonstrates that the Fus1 protein is present as an apical patch on unactivated gametes and redistributes during gamete activation over the entire surface of the microvillous-like activated plus mating structure, the fertilization tubule. Thus, Fus1 is present on mtϩ gametes at the site of cell-cell fusion and essential for an early step in the fusion process. INTRODUCTIONGamete fusion defines the beginning of a new organism in all sexual species. In most organisms, the events that precede fusion have been well characterized at the cellular level (Snell and White, 1996;Wassarman et al., 2001;Primakoff and Myles, 2002). Initial cell-cell interactions between sperm surface proteins and extracellular matrix molecules on the egg trigger activation of the sperm and exposure of previously cryptic regions of the plasma membrane proposed to be involved in gamete fusion. Once the sperm has made its way to the egg plasma membrane, the membranes of the two gametes interact more intimately, finally bringing about gamete fusion. Several sperm and egg molecules implicated in activation of the sperm have been identified, and we know much about the morphological events that accompany gamete fusion (Yanagimachi, 1994). On the other hand, little is known about the molecular mechanisms that underlie the late steps in fertilization, when the plasma membranes of the interacting gametes adhere and fuse (for review, see Primakoff and Myles, 2002). In the absence of any purely eukaryotic systems in which bona fide fusion proteins have been identified, the best models for fusion of the external plasma membrane of cells have come from studies of fusion of viruses with their eukaryotic target cells (White, 1996;Eckert and Kim, 2001). In these systems, a viral transmembrane surface protein interacts with a receptor in the host cell plasma membrane, leading to docking of the virus particle on the cell surface. Subsequent conformational changes in the interacting proteins finally lead to complete fusion (Dimitrov, 2000;Malashkevich et al., 2001;Mayer, 2001).A small number of eukaryotic genes has been sho...

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“…cAMP is an important signalling molecule during the sexual interaction between mt + and mt -gametes of the diflagellate green alga Chlamydomonas. Both in Chlamydomonas reinhardtii (Saito et al, 1993 ;Zhang & Snell, 1994) and in Chlamydomonas eugametos (Kooijman et al, 1990) the intracellular [cAMP] increases after the agglutination of compatible mating types. The elevated [cAMP] stimulates a sequence of mating responses, such as the excretion of serine proteases, cell wall breakdown and actin polymerization into a mating structure, that eventually lead to cell fusion.…”

Section: mentioning

confidence: 99%

Tansley Review No. 106

et al. 1999

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 For three decades, hypotheses relating to the occurrence and function of cyclic nucleotides in higher plants have been highly controversial. Although cyclic nucleotides had been shown to have key regulatory roles in animals and bacteria, investigations with higher plants in the 1970s and early 1980s were criticized on the basis of (i) a lack of specificity of effects apparently elicited by cyclic nucleotides, (ii) the equivocal identification of putative endogenous cyclic nucleotides and (iii) ambiguity in the identification of enzymes connected with cyclic nucleotide. More recent evidence based on more rigorous identification procedures has demonstrated conclusively the presence of cyclic nucleotides, nucleotidyl cyclases and cyclic nucleotide phosphodiesterases in higher plants, and has identified plant processes subject to regulation by cyclic nucleotides. Here we review the history of the debate, the recent evidence establishing the presence of these compounds and their role ; future research objectives are discussed.

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

Cited by 37 publications

References 46 publications

Regulated Targeting of a Protein Kinase into an Intact Flagellum

Regulated Targeting of a Protein Kinase into an Intact Flagellum

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

Tansley Review No. 106

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