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Flagellar Adhesion between Mating Type Plus and Mating Type Minus Gametes Activates a Flagellar Protein-tyrosine Kinase during Fertilization in Chlamydomonas

2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês

10.1074/jbc.m303261200

1083-351X

Qian Wang, William J. Snell,

Reproductive Biology and Fertility

When Chlamydomonas gametes of opposite mating type are mixed together, flagellar adhesion through sex-specific adhesion molecules triggers a transient elevation of intracellular cAMP, leading to gamete activation in preparation for cell-cell fusion and zygote formation. Here, we have identified a protein-tyrosine kinase (PTK) activity that is stimulated by flagellar adhesion. We determined that the protein-tyrosine kinase inhibitor genistein inhibited fertilization, and that fertilization was rescued by dibutyryl cAMP, indicating that the genistein-sensitive step was upstream of the increase in cAMP. Incubation with ATP of flagella isolated from non-adhering and adhering gametes followed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies showed that adhesion activated a flagellar PTK that phosphorylated a 105-kDa flagellar protein. Assays using an exogenous protein-tyrosine kinase substrate confirmed that the activated PTK could be detected only in flagella isolated from adhering gametes. Our results indicate that stimulation of the PTK is a very early event during fertilization. Activation of the PTK was blocked when gametes underwent flagellar adhesion in the presence of the protein kinase inhibitor staurosporine, but not in the presence of the cyclic nucleotide-dependent protein kinase inhibitor, H8, which (unlike staurosporine) does not block the increases in cAMP. In addition, incubation of gametes of a single mating type in dibutyryl cAMP failed to activate the PTK. Finally, flagella adhesion between plus and minus fla10-1 gametes, which have a temperature-sensitive lesion in the microtubule motor protein kinesin-II, failed to activate the PTK at elevated temperatures. Our results show that kinesin-II is essential for coupling flagellar adhesion to activation of a flagellar PTK and cAMP generation during fertilization in Chlamydomonas. When Chlamydomonas gametes of opposite mating type are mixed together, flagellar adhesion through sex-specific adhesion molecules triggers a transient elevation of intracellular cAMP, leading to gamete activation in preparation for cell-cell fusion and zygote formation. Here, we have identified a protein-tyrosine kinase (PTK) activity that is stimulated by flagellar adhesion. We determined that the protein-tyrosine kinase inhibitor genistein inhibited fertilization, and that fertilization was rescued by dibutyryl cAMP, indicating that the genistein-sensitive step was upstream of the increase in cAMP. Incubation with ATP of flagella isolated from non-adhering and adhering gametes followed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies showed that adhesion activated a flagellar PTK that phosphorylated a 105-kDa flagellar protein. Assays using an exogenous protein-tyrosine kinase substrate confirmed that the activated PTK could be detected only in flagella isolated from adhering gametes. Our results indicate that stimulation of the PTK is a very early event during fertilization. Activation of the PTK was blocked when gametes underwent flagellar adhesion in the presence of the protein kinase inhibitor staurosporine, but not in the presence of the cyclic nucleotide-dependent protein kinase inhibitor, H8, which (unlike staurosporine) does not block the increases in cAMP. In addition, incubation of gametes of a single mating type in dibutyryl cAMP failed to activate the PTK. Finally, flagella adhesion between plus and minus fla10-1 gametes, which have a temperature-sensitive lesion in the microtubule motor protein kinesin-II, failed to activate the PTK at elevated temperatures. Our results show that kinesin-II is essential for coupling flagellar adhesion to activation of a flagellar PTK and cAMP generation during fertilization in Chlamydomonas. When mating type plus (mt+) 1The abbreviations used are: mt+ and mt–, mating type plus and minus, respectively; PTK, protein-tyrosine kinase; Bt2-cAMP, dibutyryl cAMP; PGT, poly((l-glutamic acid:l-tyrosine)EY4:1); IFT, intraflagellar transport; anti-Tyr(P), anti-phosphotyrosine antibody; H8, N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide. and mating type minus (mt–) Chlamydomonas gametes are mixed together, they adhere to each other via sex-specific adhesion molecules, the mt+ and mt– agglutinins, on their flagella surfaces and then undergo a series of events composing gamete activation that prepares the gametes for cell fusion. Gamete activation includes cell wall loss, movement of proteins (including agglutinins) from the cell body to the flagella, modification of the flagellar tip, phosphorylation of a gamete-specific homeodomain protein involved in activation of zygote-specific genes (1Zhao H. Lu M. Singh R. Snell W.J. Genes Dev. 2001; 15: 2767-2777Crossref PubMed Scopus (34) Google Scholar), activation of mating structures, and redistribution of the adhesion/fusion protein Fus1 onto the surface of the mt+ mating structure (Ref. 2Misamore M.J. Gupta S. Snell W.J. Mol. Biol. Cell. 2003; 14: 2530-2542Crossref PubMed Scopus (70) Google Scholar, reviewed in Ref. 3Pan J. Snell W.J. Curr. Opin. Microbiol. 2000; 3: 596-602Crossref PubMed Scopus (67) Google Scholar). Several years ago it was shown that one of the earliest biochemical events triggered by flagellar adhesion is a 10–20-fold increase in intracellular cAMP (4Pijst H.L.A. van Driel R. Janssens P.M.W. Musgrave A. van den Ende H. FEBS Lett. 1984; 174: 132-136Crossref Scopus (51) Google Scholar, 5Pasquale S.M. Goodenough U.W. J. Cell Biol. 1987; 105: 2279-2292Crossref PubMed Scopus (156) Google Scholar) brought about by adhesion-induced activation of a flagellar adenylyl cyclase (6Saito T. Small L. Goodenough U.W. J. Cell Biol. 1993; 122: 137-147Crossref PubMed Scopus (40) Google Scholar, 7Zhang Y.H. Ross E.M. Snell W.J. J. Biol. Chem. 1991; 266: 22954-22959Abstract Full Text PDF PubMed Google Scholar, 8Zhang Y. Snell W.J. J. Biol. Chem. 1993; 268: 1786-1791Abstract Full Text PDF PubMed Google Scholar, 9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar). Related experiments showing that addition of dibutyryl cAMP to gametes of a single mating type induced all of the events known to occur during flagellar adhesion has confirmed that cAMP is a key second messenger in Chlamydomonas fertilization (3Pan J. Snell W.J. Curr. Opin. Microbiol. 2000; 3: 596-602Crossref PubMed Scopus (67) Google Scholar, 5Pasquale S.M. Goodenough U.W. J. Cell Biol. 1987; 105: 2279-2292Crossref PubMed Scopus (156) Google Scholar). Consistent with a central role for cAMP in gamete activation, H8, an inhibitor of cAMP-dependent protein kinase A does not interfere with flagellar adhesion (5Pasquale S.M. Goodenough U.W. J. Cell Biol. 1987; 105: 2279-2292Crossref PubMed Scopus (156) Google Scholar) or the adhesion-induced rise in intracellular cAMP (9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar), but all of the downstream events that normally accompany the increase in cAMP are blocked by H8. In previous studies we have established that the protein kinase inhibitor staurosporine blocked flagellar adhesion-induced activation of the gamete-specific flagellar adenylyl cyclase (7Zhang Y.H. Ross E.M. Snell W.J. J. Biol. Chem. 1991; 266: 22954-22959Abstract Full Text PDF PubMed Google Scholar, 8Zhang Y. Snell W.J. J. Biol. Chem. 1993; 268: 1786-1791Abstract Full Text PDF PubMed Google Scholar, 9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar). More recently, we determined that the microtubule motor protein, kinesin-II, also is essential for the flagellar adhesion-induced increase in cAMP (10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar). We found that fla10-1 gametes, which have a mutation in the kinesin-II gene (11Huang B. Rifkin M.R. Luck D.J. J. Cell Biol. 1977; 72: 67-85Crossref PubMed Scopus (103) Google Scholar, 12Lux F.G.D. Dutcher S.K. Genetics. 1991; 128: 549-561Crossref PubMed Google Scholar, 13Walther Z. Vashishtha M. Hall J.L. J. Cell Biol. 1994; 126: 175-188Crossref PubMed Scopus (188) Google Scholar), exhibited wild type levels of flagellar adhesion, but failed to undergo gamete activation (10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar, 14Piperno G. Mead K. Henderson S. J. Cell Biol. 1996; 133: 371-379Crossref PubMed Scopus (111) Google Scholar). Our studies showed that the fla10 gametes were blocked at the step that couples agglutinin interactions to activation of adenylyl cyclase. Here, we have investigated the very early events that occur following flagellar adhesion to identify a flagellar protein kinase activity stimulated by adhesion, and we examined the role of kinesin-II in protein kinase activation. We show that incubation of gametes in the protein-tyrosine kinase inhibitor, genistein, does not interfere with flagellar adhesion, but inhibits the gamete activation that follows as assessed by gamete fusion. The block can be overcome by incubation of the gametes in dibutyryl cAMP, implicating a protein-tyrosine kinase in the pathway that connects flagellar adhesion to activation of adenylyl cyclase. Biochemical experiments demonstrate that immediately after flagellar adhesion is initiated, a flagellar protein-tyrosine kinase (PTK) is activated whose substrate is a soluble, ∼105-kDa flagellar protein. Our data indicate that flagellar adhesion per se, and not the downstream events, including protein kinase A activation and gamete fusion that normally accompany flagellar adhesion, is sufficient to activate the PTK. Moreover, microtubule-based motility and intraflagellar transport are connected to regulation of the PTK, because the PTK is not activated during flagellar adhesion between fla10-1 mt+ and mt– gametes, which have a lesion in the microtubule motor protein, kinesin-II. Chemicals and Reagents—Anti-phosphotyrosine antibody (anti-Tyr(P)) 4G10 was obtained from Upstate Biotechnology (Lake Placid, NY), HEPES, N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide (H8), staurosporine, dibutyryl cyclic AMP (Bt2-cAMP), cAMP, ATP, papaverine, and genistein were obtained from Sigma. All other chemicals were reagent grade. The ECL Western blotting kit was from Amersham Biosciences. Cells and Cell Culture—Chlamydomonas reinhardtii strains 21gr (mt+) (CC-1690), 6145c (mt–) (CC-1691), fus1-1 (mt+) (CC-1158) (all available from the Chlamydomonas Genetics Center, Duke University), and strains fla10-1 (4930 3–4, mt+) and fla10-1 (4930 6–2, mt–) (provided by Dr. Susan Dutcher, Washington University, St. Louis) were cultured vegetatively in liquid culture with aeration and gametic cells were obtained as described previously (15Snell W.J. J. Cell Biol. 1976; 68: 48-69Crossref PubMed Scopus (92) Google Scholar). To activate gametes of a single mating type, cells were incubated with 15 mm Bt2-cAMP and 150 μm papaverine in N-free medium for ∼30 min. Assessments of gamete activation as determined by assaying cell wall loss and zygote formation as determined by formation of quadriflagellated cells were carried out as described previously (10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar). For the experiments at 32 °C, wild-type gametes and fla10-1 mutant gametes were incubated with illumination in a water bath with constant aeration. Isolation of Flagella—Flagella were isolated by use of the pH shock method of Witman et al. (16Witman G.B. Carlson K. Berliner J. Rosenbaum J.L. J. Cell Biol. 1972; 54: 507-539Crossref PubMed Scopus (333) Google Scholar) with minor modifications as follows. Gametes (typically ∼6 liters, at 5 × 106 cells per ml in N-free medium) were harvested by centrifugation at 3,000 × g for 3 min and resuspended in 5% sucrose in 20 mm HEPES, pH 7.2. The cells were placed on ice and the flagella were immediately detached by rapidly reducing the pH to 4.5 using 0.5 m acetic acid with constant stirring. Deflagellation, which usually occurred within 15 s, was confirmed using phase-contrast microscopy and the cells were neutralized with 0.5 m KOH. Cell bodies were removed by centrifugation through a 15-ml cushion of 25% sucrose in 20 mm HEPES, pH 7.2, in 50-ml conical tubes at 3,000 × g for 9 min. The remaining cell bodies were removed from the top layer by centrifugation through a second 25% sucrose, 20 mm HEPES layer. Flagella were harvested from the overlying supernatant by centrifugation in an HB-4 rotor at 8,000 rpm for 15 min. The resulting pellet was thoroughly resuspended to a concentration of ∼3 mg/ml protein in 5% sucrose, 20 mm HEPES, pH 7.2, containing a 1/100 dilution of the protease inhibitor mixture for plant cells from Sigma (catalogue number P9599). The suspension was divided into small portions, frozen in liquid nitrogen, and stored at –70 °C. For fractionation, flagella (∼300 μg of protein in 100 μl 5% sucrose, 20 mm HEPES, pH 7.2) isolated from mixed plus and minus gametes and frozen as described above were thawed on ice and centrifuged at 13,000 rpm (Sigma 1–15K refrigerated table top centrifuge, rotor number 12132) for 20 min to yield the freeze/thaw supernatant fraction and the disrupted, sedimented flagella. The sedimented flagella were resuspended in 100 μl of buffer A that contained 0.1% Nonidet P-40, 20 mm HEPES, pH 7.2, 50 mm KCl, 10 mm MgCl2, 1 mm EDTA, and 2 mm dithiothreitol, kept on ice for 10 min, and centrifuged again as above to yield the 0.1% Nonidet P-40-soluble fraction. The sedimented material was resuspended by pipetting and sonication in 100 μl of buffer A. Protein concentration was determined with the Coomassie Blue protein assay reagent of Pierce using crystalline bovine serum albumin as standard. Assays for Protein-tyrosine Kinase Activity—Flagellar PTK activity was assayed in vitro using both endogenous substrates and an exogenously added PTK substrate. For assays using endogenous flagellar proteins as substrates, samples (5 μl) of whole flagella (∼3 μg/μl protein) or flagellar fractions (∼0.3–3.0 μg/μl protein) in 5% sucrose, 20 mm HEPES buffer were mixed with 5 μl of 2× PTK buffer (20 mm HEPES, pH 7.2, 10 mm MgCl2, 2 mm dithiothreitol, 1 mm EDTA, 50 mm KCl, 2 mm ATP, 0.2% Nonidet P-40, 0.4 mm orthovanadate, 20 mm β-glycerolphosphate, and 2% Sigma plant protease inhibitor mixture) at 23 °C for the times indicated in the figure legends. Samples were prepared for SDS-PAGE by addition of 5 μl of 4× SDS-PAGE sample buffer containing 0.25 m Tris, pH 6.8, 40% glycerol, 8% SDS, 0.1% bromphenol blue, and 0.4 m dithiothreitol. After boiling for 5 min, the samples were cooled on ice and loaded onto 8% mini-slab gels. Electrophoresis was performed at 48 mA for 1.5 h as described previously (17Wilson N.F. O'Connell J.S. Lu M. Snell W.J. J. Biol. Chem. 1999; 274: 34383-34388Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). To assay for PTK activity using an exogenous substrate, we used the dot-blot method of Allis et al. (18Allis C.D. Chicoine L.G. Glover C.V. White E.M. Gorovsky M.A. Anal. Biochem. 1986; 159: 58-66Crossref PubMed Scopus (9) Google Scholar) with the peptide poly((l-glutamic acid:l-tyrosine)EY4:1) (PGT) as substrate. The freeze/thaw supernatants of flagella (10 μl in 20 mm HEPES, pH 7.2, buffer containing 0.3 μg/μl protein) isolated from mt+ gametes alone, mt– gametes alone, and from adhering mt+ and mt– gametes were incubated with 10 μl of PTK buffer that contained 0.3 mg/ml PGT (Sigma) for 0–30 min at room temperature. Reactions were terminated by addition of 10 μl of stop reagent (0.25 m EDTA + 0.25 m EGTA, pH 8.0) and 5-μl portions were spotted onto a nitrocellulose membrane (Protran™ B85, Schleicher & Schuell Inc., Keene, NH) followed by processing to detect phosphotyrosine by use of the method described below for immunoblotting. Immunoblot Analysis—After SDS-PAGE, protein was transferred to a polyvinylidene fluoride membrane (Immobilon-P, Millipore, Bedford, MA) in buffer containing 25 mm Tris, pH 8.3, and 192 mm glycine at 100 V for 1 h. The membrane was blocked with 3% bovine serum albumin in 1× TBST (20 mm Tris, pH 7.6, 137 mm NaCl, 0.05% Tween 20) (19Hulen D. Baron A. Salisbury J. Clarke M. Cell Motil. Cytoskeleton. 1991; 18: 113-122Crossref PubMed Scopus (79) Google Scholar) for 1 h at room temperature and incubated with a 1:1000 dilution of anti-Tyr(P) 4G10 in blocking solution overnight at 4 °C. At the end of the incubation, membranes were washed three times with 1× TBST for about 20 min and incubated for 30 min with horseradish peroxidase in blocking solution. The membrane was washed and developed with ECL Western blotting reagents as described by the manufacturer (Amersham Biosciences). In some experiments, proteins were detected after immunoblotting by staining the polyvinylidene difluoride membrane with Coomassie Brilliant Blue R-250 for ∼5 min, followed by destaining in methanol:acetic acid:water (5:1:5). To determine whether protein-tyrosine kinase activity was required during flagellar adhesion-induced gamete activation in Chlamydomonas, we used the PTK inhibitor, genistein. We pretreated mt+ and mt– gametes in genistein for 30 min, mixed the cells together in the continued presence of the inhibitor, and assessed zygote formation as a measure of gamete activation. Phase-contrast and bright field microscopy (not shown) indicated that the inhibitor had little effect on flagellar adhesion, with 80–90% of the cells forming the large, swirling clumps of adhering cells that typify cell-cell adhesion in this highly motile organism. On the other hand, the inhibitor had a significant effect on gamete fusion, an event that only activated gametes can undergo, blocking fusion by 30% at 10 μg/ml and 87% at 20 μg/ml (Fig. 1). To determine whether the genistein blocked cell fusion per se, or if the block was upstream of the adhesion-dependent increase in cAMP, gametes that had been incubated in genistein subsequently were incubated in dibutyryl cAMP and papaverine, a treatment known to induce gamete activation (5Pasquale S.M. Goodenough U.W. J. Cell Biol. 1987; 105: 2279-2292Crossref PubMed Scopus (156) Google Scholar) and then mixed together. As shown in Fig. 1, the Bt2-cAMP treatment overcame the genistein inhibition of fusion. The reversal of inhibition by incubation of the gametes in dibutyryl cAMP was consistent with the idea that the genistein-sensitive step in the signaling pathway was upstream of the increase in cAMP induced by flagellar adhesion. These results implicated a PTK activity early during flagellar adhesion-induced gamete activation. A Protein-tyrosine Kinase That Phosphorylates a 105-kDa Protein Is Activated by Flagellar Adhesion—To test for activation of PTKs induced by flagellar adhesion, we isolated flagella from mt+ gametes alone and mt– gametes alone, and from mt+ and mt– gametes that were undergoing flagellar adhesion and gamete activation. Each of the flagellar samples was incubated for 10 min in a protein kinase assay buffer containing 1 mm ATP and 0.1% Nonidet P-40 followed by SDS-PAGE and immunoblotting with anti-Tyr(P). As shown in Fig. 2 (left panel), a tyrosine-phosphorylated protein of ∼105 kDa was readily and consistently detected in the adhering sample (mt+ and mt–) and was not present in flagella isolated from mt+ or mt– gametes alone (mt+, mt–). The middle panel in Fig. 2 shows the immunoblot stained with Coomassie Blue. The presence of approximately equal amounts of tubulin, which are the two strongly stained bands at ∼50 kDa, documented the comparable loading of the lanes. The right panel in Fig. 2 shows results from a control experiment in which the detergent-soluble, matrix fraction (see below) of flagella isolated from adhering gametes was assayed for PTK in the absence and the presence of ATP. The phosphorylated 105-kDa protein was detected only when the assay was carried out in the presence of ATP. These results suggested that a flagellar PTK that phosphorylated a 105-kDa flagellar protein was activated soon after mt+ and mt– gametes were mixed together and underwent flagellar adhesion. Fig. 3A shows a time course for the in vitro phosphorylation assay. Samples prepared from flagella isolated from mt+ gametes alone (mt+), from mt– gametes alone (mt–), and from adhering mt+ and mt– gametes that had been mixed together for 10 min (mt+ & mt–) were incubated as above in the protein kinase assay buffer for the indicated times followed by SDS-PAGE and immunoblotting with anti-Tyr(P). Phosphorylated 105-kDa protein was not detected in any of the samples at 0 min and did not appear at any time in the flagellar samples from the mt+ or mt– gametes alone. In the flagellar samples isolated from adhering mt+ and mt– gametes (mt+ & mt–), phosphorylated 105-kDa protein was detectable within 2 min after the incubation with ATP began and continued to increase during the 15-min assay. To determine whether the results were because of activation of a PTK or, for example, a change in accessibility of the substrate, we used an exogenous protein-tyrosine kinase substrate, polyglutamine tyrosine (4:1) (PGT) to assay for an adhesion-induced increase in PTK activity. Samples from flagella isolated from mt+ gametes alone, mt– gametes alone, and from adhering mt+ and mt– gametes were incubated with protein kinase buffer that included PGT. At the indicated times, samples were spotted onto nitrocellulose paper, and after several washes, the blot was processed to detect phosphotyrosine with the 4G10 antibody used for immunoblotting. As shown in Fig. 3B, there was substantial phosphorylation of the peptide substrate only in samples from adhering gametes (mt+ & mt–; PGT). Control assays, which did not contain PGT, carried out with flagellar samples from adhering gametes displayed a low level of activity (mt+ & mt–; no PGT), which presumably was because of phosphorylation of the endogenous 105-kDa substrate. The control, non-adhering mt+ and mt– samples (mt+, mt–) showed only a low level of activity that was much less than that exhibited by the samples from the adhering cells. Thus, experiments using endogenous and exogenous substrates indicated that flagellar adhesion activated a flagellar PTK. We investigated the time during fertilization when the PTK was activated. To do this, mt+ and mt– gametes were mixed together and at various times after mixing, the flagella were detached and isolated and samples were assayed for the presence of the active PTK. As shown in Fig. 4A while PTK activity was high in flagella isolated from gametes that had been mixed for 3 min or more, the PTK activity was detectable in flagella isolated from gametes that had been mixed for only 30 s. These results indicated that PTK activation took place very soon after gametes adhered to each other, most likely before gamete fusion, for example, which begins to take place 3–5 min after mixing. To further examine the properties of the PTK and its 105-kDa protein substrate, we identified the flagellar compartment in which the PTK and the substrate were localized. For fractionation, the flagella were disrupted by freezing and thawing and the sample was centrifuged to yield the freeze/thaw supernatant (matrix fraction). The sedimented flagella with their disrupted membranes (20Zhang Y. Snell W.J. Methods Cell Biol. 1995; 47: 459-465Crossref PubMed Scopus (4) Google Scholar, 21Cole D.G. Diener D.R. Himelblau A.L. Beech P.L. Fuster J.C. Rosenbaum J.L. J. Cell Biol. 1998; 141: 993-1008Crossref PubMed Scopus (704) Google Scholar) were further fractionated by detergent extraction and centrifugation and equivalent portions of each fraction were assayed for PTK activity. As shown in Fig. 4B, phosphorylation of the 105-kDa protein was detected only in the starting sample of whole flagella and in the initial freeze/thaw supernatant from the disrupted flagella. The results indicated that both the protein kinase and its substrate were released from flagella by freezing and thawing. Activation of the Flagellar PTK Occurs at a Very Early Step during Gamete Interactions—We used the protein kinase inhibitors staurosporine and H8 to examine the stage during flagellar adhesion and gamete activation that the PTK was activated. Previously, we showed that while flagellar adhesion was unaffected by these inhibitors, gamete activation was blocked, albeit at different steps by each inhibitor. The staurosporine-sensitive block is upstream of activation of adenylyl cyclase, as cAMP levels fail to increase when gametes undergo flagellar adhesion in the presence of staurosporine (9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar). We have also shown that H8, an inhibitor of cyclic nucleotide-dependent protein kinases, does not block the flagellar adhesion-induced increase in cAMP, but inhibits steps in gamete activation, including cell wall release and cell-cell fusion, that are down-stream of activation of adenylyl cyclase (9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar). To test the effects of the inhibitors on activation of the PTK, we preincubated mt+ and mt– gametes in each inhibitor and mixed the cells together in the continued presence of the inhibitors. Examination by light microscopy indicated that, as previously reported (9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar), flagellar adhesion occurred normally between mt+ and mt– gametes in the presence of each of the inhibitors, but cell fusion was blocked (not shown). Flagella were isolated from mt+ and mt– gametes that were mixed together in the presence and absence of each of the inhibitors as well as from control, non-treated, adhering cells and assayed for the PTK activity. As shown in Fig. 5A, the PTK became activated in flagella of control, adhering cells (C) and in flagella isolated from gametes adhering in the presence of H8, whereas activation of the PTK did not occur in flagella isolated from gametes that had undergone flagellar adhesion in the presence of staurosporine (St). These results indicated that activation of the PTK required a staurosporine-sensitive step and suggested that the PTK was activated at an early stage during gamete activation, down-stream of agglutinin interactions and flagellar adhesion and upstream of the appearance of cAMP. In an independent test of the results indicating that activation of the PTK was upstream of cyclic nucleotide-activated processes, including gamete fusion, we used fus1-1 mt+ gametes, which are capable of undergoing flagellar adhesion and gamete activation, but are unable to fuse (22Goodenough U.W. Hwang C. Martin H. Genetics. 1976; 82: 169-186Crossref PubMed Google Scholar, 23Goodenough U. Armbrust E. Campbell A. Ferris P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 21-44Crossref Scopus (47) Google Scholar). fus1-1 mt+ gametes were mixed with wild type mt– gametes and, 3 min after mixing, flagella were isolated and subjected to the in vitro PTK assay. As expected, the gametes underwent flagellar adhesion and gamete activation as evidenced by cell wall loss, but they did not fuse to form quadriflagellated zygotes (not shown). On the other hand, the in vitro assay showed that the PTK was activated (Fig. 5B). Thus, consistent with the H8 experiment above, flagellar adhesion in the absence of gamete fusion was sufficient to activate the PTK (Fig. 5B). It was also possible that activation of the PTK did not require flagellar adhesion per se, but was downstream of the increase in cAMP and involved a cAMP-induced event that was not sensitive to H8. We tested this idea by assaying for the PTK activity in flagella isolated from gametes of a single mating type that were activated by incubation in dibutyryl cAMP and papaverine. The requirement for flagellar adhesion to initiate many of the cellular events that prepare gametes for cell fusion can be bypassed by incubating gametes of a single mating type in the cell-permeable cAMP analogue, Bt2-cAMP and the phosphodiesterase inhibitor, papaverine (5Pasquale S.M. Goodenough U.W. J. Cell Biol. 1987; 105: 2279-2292Crossref PubMed Scopus (156) Google Scholar). We incubated gametes of a single mating type in Bt2-cAMP for 30 min, confirmed that they had become activated as determined by an assay for cell wall loss (not shown), and then assessed activation of the PTK. As shown in Fig. 5C, flagellar samples from adhering gametes exhibited the expected phosphorylation of the 105-kDa protein, whereas flagella isolated from mt+ gametes or mt– gametes alone activated by Bt2-cAMP failed to show active PTK in the in vitro assay. These results indicated that activation of the PTK required flagellar adhesion and was upstream of the appearance of cAMP. As a further test for a possible role for cAMP in activating the PTK, we assessed whether inclusion of cAMP in the PTK assay would stimulate the PTK activity in flagellar samples isolated from mt+ or mt– gametes alone. As shown in Fig. 5C, inclusion of cAMP in the assay of flagella isolated from gametes of a single mating type did not lead to activation of the PTK. The PTK Is Not Activated during Flagellar Adhesion in fla10-1 Gametes, Which Contain a Lesion in the Microtubule Motor Protein Kinesin-II—Previously, we showed that coupling of flagellar adhesion to the increase in cAMP was disrupted in gametes of the fla10-1 mutant (10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar, 14Piperno G. Mead K. Henderson S. J. Cell Biol. 1996; 133: 371-379Crossref PubMed Scopus (111) Google Scholar), which express a kinesin-II with a temperature-sensitive defect in the 90-kDa motor subunit FLA10 because of a single amino acid substitution in the motor domain (13Walther Z. Vashishtha M. Hall J.L. J. Cell Biol. 1994; 126: 175-188Crossref PubMed Scopus (188) Google Scholar). The defective kinesin-II is expressed in fla10-1 gametes, but at much lower levels than in wild type cells. When fla10-1 cells are grown at room temperature they are fully flagellated and exhibit IFT. On the other hand when grown at elevated temperature (32 °C), the cells do not have flagella (13Walther Z. Vashishtha M. Hall J.L. J. Cell Biol. 1994; 126: 175-188Crossref PubMed Scopus (188) Google Scholar, 21Cole D.G. Diener D.R. Himelblau A.L. Beech P.L. Fuster J.C. Rosenbaum J.L. J. Cell Biol. 1998; 141: 993-1008Crossref PubMed Scopus (704) Google Scholar, 24Kozminski K.G. Beech P.L. Rosenbaum J.L. J. Cell Biol. 1995; 131: 1517-1527Crossref PubMed Scopus (443) Google Scholar). In cells grown at room temperature and shifted to 32 °C, IFT particle movement ceases after about 40 min and within 1–2 h, the flagella begin to resorb (12Lux F.G.D. Dutcher S.K. Genetics. 1991; 128: 549-561Crossref PubMed Google Scholar, 21Cole D.G. Diener D.R. Himelblau A.L. Beech P.L. Fuster J.C. Rosenbaum J.L. J. Cell Biol. 1998; 141: 993-1008Crossref PubMed Scopus (704) Google Scholar, 24Kozminski K.G. Beech P.L. Rosenbaum J.L. J. Cell Biol. 1995; 131: 1517-1527Crossref PubMed Scopus (443) Google Scholar). For our experiments, fla10-1 mt+ and fla10-1 mt– gametes cultured at 21 °C were shifted to 32 °C for 40 min, mixed together, and their flagella were isolated 10 min after mixing. Flagella also were isolated from adhering mt+ fla10-1 gametes and mt– fla10-1 gametes that were mixed together at 21 °C, from wild type gametes mixed together at 21 °C, and from wild gametes shifted to 32 °C for 40 min before being mixed together for 3 min. As shown in Fig. 6, when analyzed by the in vitro assay, flagellar samples from wild type gametes mixed at 32 °C showed a slight reduction in ability to phosphorylate the 105-kDa protein compared with flagellar samples isolated from wild type gametes mixed at 21 °C. On the other hand, although phosphorylation of the 105-kDa protein was similar to wild type levels in assays of the 21 °C fla10-1 samples, phosphorylated 105-kDa protein was not detected in assays of the 32 °C fla10-1 samples. The bottom panel of Fig. 6, which is a loading control, shows the same samples probed with an antibody against a 48-kDa Chlamydomonas protein (25Kurvari V. Zhang Y.H. Luo Y.X. Snell W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 39-43Crossref PubMed Scopus (11) Google Scholar, 26Zhang Y. Luo Y. Emmett K. Snell W.J. Mol. Biol. Cell. 1996; 7: 515-527Crossref PubMed Scopus (6) Google Scholar), and documents equivalent protein loading. Thus, coupled with their failure to undergo increases in cAMP during flagellar adhesion at elevated temperature (10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar), fla10-1 gametes also failed to show flagellar adhesion-induced activation of the PTK. Previous studies on the molecular events regulated by flagellar adhesion during fertilization in Chlamydomonas indicated that flagellar protein kinases are activated at an early step after adhesion between mt+ and mt– flagella, before the adhesion-induced increase in cAMP (7Zhang Y.H. Ross E.M. Snell W.J. J. Biol. Chem. 1991; 266: 22954-22959Abstract Full Text PDF PubMed Google Scholar, 8Zhang Y. Snell W.J. J. Biol. Chem. 1993; 268: 1786-1791Abstract Full Text PDF PubMed Google Scholar, 9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar, 20Zhang Y. Snell W.J. Methods Cell Biol. 1995; 47: 459-465Crossref PubMed Scopus (4) Google Scholar, 26Zhang Y. Luo Y. Emmett K. Snell W.J. Mol. Biol. Cell. 1996; 7: 515-527Crossref PubMed Scopus (6) Google Scholar). In addition, we recently reported that in cells with a defect in the microtubule motor protein kinesin-II, gamete activation was blocked at a step downstream of flagellar adhesion but upstream of the adhesion-induced increase in cAMP (10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar). Here, we identified a protein-tyrosine kinase activity that was regulated by flagellar adhesion per se, and not by the downstream events that accompany flagellar adhesion such as increased levels of cAMP, activation of cyclic nucleotide-dependent protein kinases, or cell-cell fusion. Initial experiments with the PTK inhibitor genistein implicated PTK activity in fertilization downstream of flagellar adhesion and upstream of the increase in cAMP (Fig. 1). Use of in vitro PTK assays revealed a PTK activity that was present in flagella isolated from adhering gametes and that was nearly undetectable in flagella isolated from gametes of either mating type that had not been mixed with gametes of the opposite mating type (and therefore had not undergone flagellar adhesion). Importantly, flagellar adhesion was sufficient to activate the PTK, but not in gametes with a lesion in the microtubule motor protein, kinesin-II. A working model consistent with these data is that flagellar adhesion leads to a rapid stimulation of PTK activity in a pathway involving kinesin-II-dependent intraflagellar transport. PTK activation is followed, in turn, by activation of the adenylyl cyclase and gamete activation (Fig. 7). For detection of the activated PTK in these experiments, it was essential to incubate the flagellar samples with ATP before carrying out SDS-PAGE and immunoblotting. As shown in Fig. 2, when ATP was left out of the in vitro assay, phosphorylated 105-kDa protein was not detected; similarly, in the experiments to examine the relationship between the extent of phosphorylation and assay time, no phosphorylation of the 105-kDa protein was detected in the 0 time point sample. Previous experiments from our laboratory and others (7Zhang Y.H. Ross E.M. Snell W.J. J. Biol. Chem. 1991; 266: 22954-22959Abstract Full Text PDF PubMed Google Scholar, 9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar, 26Zhang Y. Luo Y. Emmett K. Snell W.J. Mol. Biol. Cell. 1996; 7: 515-527Crossref PubMed Scopus (6) Google Scholar, 27Bloodgood R.A. Exp. Cell Res. 1992; 198: 228-236Crossref PubMed Scopus (21) Google Scholar) on protein kinase activity in Chlamydomonas have shown that flagella contain high levels of protein phosphatase activity, and the omission of phosphatase inhibitors in the in vitro assay used in the current experiments led to much reduced levels of phosphorylation (not shown). Use of the exogenous PTK substrate PGT was important as an independent confirmation of PTK activation and also should be useful for future experiments to identify and further characterize this adhesion-regulated PTK. Several independent approaches indicated that activation of the PTK was a very early event during gamete activation: activation required flagellar adhesion, occurred within seconds after gametes of opposite mating type were mixed together, and took place at the same time or earlier than the increases in levels of cAMP shown previously (5Pasquale S.M. Goodenough U.W. J. Cell Biol. 1987; 105: 2279-2292Crossref PubMed Scopus (156) Google Scholar, 10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar). The results strongly indicated that PTK activation was not dependent on increases in cAMP or cell fusion and was induced by flagellar adhesion per se. Thus, fus1-1 mutant gametes, which are capable of flagellar adhesion but not gamete fusion, showed adhesion-dependent activation of the PTK. Similarly, when gametes underwent flagellar adhesion in the presence of the inhibitor of cyclic nucleotide-dependent protein kinases, H8, gamete activation was blocked as assessed by the absence of cell wall loss and gamete fusion, but the PTK still was activated. And, while incubation of gametes of a single mating type in dibutyryl cAMP bypasses the requirement for flagellar adhesion and induces the cellular changes that normally accompany flagellar adhesion, the PTK was not activated in such artificially activated cells. In addition, inclusion of cAMP in the in vitro assay for PTK activity in flagella isolated from gametes of a single mating type did not lead to activation of the PTK. Whereas the evidence is compelling that PTK activation is not downstream of activation of adenylyl cyclase and that regulation of both activities is tightly coupled to flagellar adhesion, determining whether PTK activation is upstream of activation of the flagellar adenylyl cyclase or in a parallel signaling pathway remains to be achieved. Activation of the PTK (Fig. 4) and increases in cAMP (5Pasquale S.M. Goodenough U.W. J. Cell Biol. 1987; 105: 2279-2292Crossref PubMed Scopus (156) Google Scholar, 9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar, 10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar) are the earliest biochemical or cellular events that have been demonstrated after flagellar adhesion is initiated, occurring within seconds after cells are mixed together. Activation of the PTK (Fig. 5) and the increase in cAMP and gamete activation are blocked when cells undergo flagellar adhesion in staurosporine (9Zhang Y. Snell W.J. J. Cell Biol. 1994; 125: 617-624Crossref PubMed Scopus (31) Google Scholar), suggesting that the activity of one or more protein kinases is essential for activation of the PTK and the adenylyl cyclase. Finally, adhesion-induced stimulation of the PTK (Fig. 6), increases in cAMP and gamete activation (10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar) fail in fla10-1 gametes, which have a lesion in the microtubule motor protein kinesin-II (13Walther Z. Vashishtha M. Hall J.L. J. Cell Biol. 1994; 126: 175-188Crossref PubMed Scopus (188) Google Scholar). The simplest model that explains the data is that a single signaling pathway is initiated by flagellar adhesion. Because cAMP-regulated events are down-stream of the PTK, it is likely that the PTK is upstream of adenylyl cyclase. In addition, all of the cellular events characterized to date that compose gamete activation, including flagellar tip activation, movement of agglutinin molecules from the cell body to the flagella, cell wall loss, and activation of mating structures are downstream of activation of adenylyl cyclase. That is, all these cellular responses can be induced by incubation of gametes of a single mating type in dibutyryl cAMP. Therefore, if the PTK is part of a signaling pathway that is independent of cAMP, the events induced by that putative pathway have not yet been reported. The observation that fla10-1 gametes at elevated temperature fail to activate the PTK (Fig. 6) is the second demonstration that kinesin-II plays a direct or indirect role in adhesion-induced signaling in Chlamydomonas (10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar, 28Pan J. Snell W.J. J. Cell Sci. 2003; 116: 2179-2186Crossref PubMed Scopus (24) Google Scholar). Microtubule motor proteins have been implicated in other signal transduction pathways in Chlamydomonas (10Pan J. Snell W.J. Mol. Biol. Cell. 2002; 13: 1417-1426Crossref PubMed Scopus (58) Google Scholar, 28Pan J. Snell W.J. J. Cell Sci. 2003; 116: 2179-2186Crossref PubMed Scopus (24) Google Scholar) (reviewed in Refs. 3Pan J. Snell W.J. Curr. Opin. Microbiol. 2000; 3: 596-602Crossref PubMed Scopus (67) Google Scholar, 29Pan J. Miisamore M.J. Wang W. Snell W.J. Traffic. 2003; 4: 452-459Crossref PubMed Scopus (16) Google Scholar, and 30Rosenbaum J.L. Witman G.B. Nat. Rev. Mol. Cell. Biol. 2002; 3: 813-825Crossref PubMed Scopus (1213) Google Scholar) and it will be interesting to determine the role of kinesin-II function in coupling agglutinin interactions during flagellar adhesion to gamete activation. For example, kinesin-II-dependent movement of IFT particles might be required for maintaining proper levels of key signaling proteins in the flagella. Or, binding between mt+ and mt– agglutinin molecules might trigger conformational changes in the interacting molecules that induce them to associate with kinesin-II or IFT particles as an early step in signal transduction. The results presented here should now make it possible to continue a detailed dissection of the molecular pathways that underlie cell-cell adhesion-induced signal transduction during fertilization in Chlamydomonas. We thank Dr. Susan Dutcher (Washington University, St. Louis, MO) for providing the fla10-1 mutant strains. We also thank our colleagues Drs. Fred Grinnell and Melanie Cobb for helpful discussions.