Flagellar Adhesion Research Articles

수염녹두말속(Chlamydomonas) 단세포 녹조의 유성생식

이 논문은 다세포 녹색식물의 조상으로 생각되는 단세포인 수염녹두말속(Chlamydomonas) 녹조의 복잡한 유성생식 과정을 종합적으로 이해하기 위해서 기술되었다. 모델생물로 알려진 클래미도모나스 레인하르티(Chlamydomonas reinhardtii)의 유성생식 생활사는 배우자발생, 배우자 활성화, 세포융합, 접합자 성숙, 감수분열과 발아 등의 다섯 시기로 구별된다. 배우자발생은 서식지에서 질소가 결핍되어 시작된다. 그 결과, 교배형 유전자자리(<TEX>$MT^+$</TEX> 또는 <TEX>$MT^-$</TEX>)에 의해 조절된 두 가지 교배형(<TEX>$mt^+$</TEX> 및 <TEX>$mt^-$</TEX>)의 배우자들이 발생된다. 유성생식을 위한 두 번째 자극원인 청색광이 교배능력을 갖고 있지 않는 예비배우자 세포에 조사되면 교배능력을 가진 배우자로 분화된다. 배우자발생의 초기에, 두 배우자 세포들은 응집소(agglutin) 분자들을 합성한다. 즉, <TEX>$mt^+$</TEX> 응집소 및 <TEX>$mt^-$</TEX> 응집소는 상염색체에 있는 성응집(SAG1) 및 성접착(SAD1) 유전자들이 발현되어 각각 합성된다. 응집소들에 의해서 두 배우자 세포들의 편모가 접착되면 cAMP가 신호전달 경로를 작동시켜서 편모의 끝 부부을 활성화시킨다. cAMP의 신호에 반응하여 두 배우자들의 세포벽이 벗겨지고 2개의 편모 사이에서 각각의 교배구조(mating structure)가 발달한다. 교배구조의 원형질막에는 <TEX>$mt^+$</TEX> 및 <TEX>$mt^-$</TEX> 배우자-특이 융합 단백질인 Fus1 및 Hap2/Gcs1이 있다. 이 단백질들의 상호작용으로 두 교배구조가 접촉되면, 두 배우자 사이에 세포질이 연결되어 수정세관(fertilization tubule)이 발달한다. <TEX>$mt^+$</TEX> 및 <TEX>$mt^-$</TEX> 배우자들의 핵과 엽록체가 융합되면 2배체의 접합자가 형성된다. 그 후 배우자들의 특성은 사라지며 융합단백질들(Fus1 및 Hap2)이 분해되고 접합자-특이(zygote-specific) 유전자들이 활성화 된다. 접합자는 24시간에 걸쳐 두꺼운 세포벽을 가진 접합포자(zygospore)로 발달한다. 어린 접합자에서 교배 후 60분 이내에 <TEX>$mt^-$</TEX> 엽록체 DNA와 <TEX>$mt^+$</TEX> 미토콘드리아 DNA가 선택적으로 제거된다. 따라서 이 두 소기관들은 각각 <TEX>$mt^-$</TEX> 미토콘드리아 DNA와 <TEX>$mt^+$</TEX> 엽록체 DNA만 남아서 단친유전(uniparental inheritance)이 이루어진다. 적당한 조건이 되면 접합포자가 감수분열과 발아 과정을 거쳐 반수체 영양세포가 방출되어 새로운 세대가 다시 시작된다. The sexual reproduction of the unicellular green alga Chlamydomonas is reviewed for a comprehensive understanding of the complex processes. The sexual life cycle of C. reinhardtii is distinguished into five main stages: gametogenesis, gamete activation, cell fusion, zygote maturation, and meiosis and germination. Gametogenesis is induced by nitrogen starvation in the environment. C. reinhardtii has two mating types: mating type plus (<TEX>$mt^+$</TEX>) and mating type minus (<TEX>$mt^-$</TEX>), controlled by a single complex mating type locus (<TEX>$MT^+$</TEX> or <TEX>$MT^-$</TEX>) on linkage group VI. In the early gametogenesis agglutinins are synthesized. The <TEX>$mt^+$</TEX> and <TEX>$mt^-$</TEX> agglutinins are encoded by the autosomal genes SAG1 (Sexual AGglutination1) and SAD1 (Sexual ADhesion1), respectively. The agglutinins are responsible for the flagellar adhesion of the two mating type of gametes. The flagellar adhesion initiates a cAMP mediated signal transduction pathways and activates the flagellar tips. In response to the cAMP signal, mating structures between two flagella are activated. The <TEX>$mt^+$</TEX> and <TEX>$mt^-$</TEX> gamete-specific fusion proteins, Fus1 and Hap2/Gcs1, are present on the plasma membrane of the two mating structures. Contact of the two mating structures leads to develop a fertilization tubule forming a cytoplasmic bridge between the two gametes. Upon fusion of nuclei and chloroplasts of <TEX>$mt^+$</TEX> and <TEX>$mt^-$</TEX> cells, the zygotes become zygospores. It is notable that the young zygote shows uniparental inheritance of chloroplast DNA from the <TEX>$mt^+$</TEX> parent and mitochondrial DNA from the <TEX>$mt^-$</TEX> parent. Under the favorable conditions, the zygospores divide meiotically and germinate and then new haploid progenies, vegetative cells, are released.

Estimation of the adhesive force distribution for the flagellar adhesion of Escherichia coli on a glass surface

The effects of the presence or absence of microbial flagella and the microbial motility on the colloidal behaviors of microbial cells were quantitatively evaluated. The microbial cell attachment and detachment processes on a glass surface were observed directly using a parallel-plate flow chamber. Wild-type, flagellar paralyzed, and nonflagellated Escherichia coli strains were used as model microbial cells. In the cell attachment tests, the microbial adhesion rate in a 160mM NaCl solution was approximately 10 times higher than that in a 10mM solution, for all E. coli strains. The colloidal behavior of the microbial cells agreed well with the predictions of the DLVO theory. In addition, the microbial flagella and motility did not significantly affect the cell attachment, regardless of the existence of a potential barrier between the cell and the glass substratum. In the cell detachment tests, the cumulative number of microbial cells detached from the glass substratum with increasing flow rate was fit well with the Weibull distribution function. The list of strains arranged in order of increasing median drag force required to remove them was nonflagellated strain, flagellar paralyzed strain, and wild-type strain. These results indicated that the flagella and the flagellar motility inhibited the cell detachment from the glass substratum. Furthermore, a large external force would likely be required to inhibit the microbial adhesion in the early stage of the biofilm formation.

Regulation of Flagellar Biogenesis by a Calcium Dependent Protein Kinase in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a bi-flagellated green alga, is a model organism for studies of flagella or cilia related activities including cilia-based signaling, flagellar motility and flagellar biogenesis. Calcium has been shown to be a key regulator of these cellular processes whereas the signaling pathways linking calcium to these cellular functions are less understood. Calcium-dependent protein kinases (CDPKs), which are present in plants but not in animals, are also present in ciliated microorganisms which led us to examine their possible functions and mechanisms in flagellar related activities. By in silico analysis of Chlamydomonas genome we have identified 14 CDPKs and studied one of the flagellar localized CDPKs – CrCDPK3. CrCDPK3 was a protein of 485 amino acids and predicted to have a protein kinase domain at the N-terminus and four EF-hand motifs at the C-terminus. In flagella, CrCDPK3 was exclusively localized in the membrane matrix fraction and formed an unknown 20 S protein complex. Knockdown of CrCDPK3 expression by using artificial microRNA did not affect flagellar motility as well as flagellar adhesion and mating. Though flagellar shortening induced by treatment with sucrose or sodium pyrophosphate was not affected in RNAi strains, CrCDPK3 increased in the flagella, and pre-formed protein complex was disrupted. During flagellar regeneration, CrCDPK3 also increased in the flagella. When extracellular calcium was lowered to certain range by the addition of EGTA after deflagellation, flagellar regeneration was severely affected in RNAi cells compared with wild type cells. In addition, during flagellar elongation induced by LiCl, RNAi cells exhibited early onset of bulbed flagella. This work expands new functions of CDPKs in flagellar activities by showing involvement of CrCDPK3 in flagellar biogenesis in Chlamydomonas .

Flagellar adhesion inTrypanosoma bruceirelies on interactions between different skeletal structures present in the flagellum and in the cell body

The Trypanosoma brucei flagellum is an essential organelle anchored along the surface of the cell body through a specialized structure called the flagellum attachment zone (FAZ). Adhesion relies on the interaction of the extracellular portion of two transmembrane proteins, FLA1 and FLA1BP. Here, we identify FLAM3 as a novel large protein associated with the flagellum skeleton whose ablation inhibits flagellum attachment. FLAM3 does not contain transmembrane domains and its flagellar localization matches closely, but not exactly, that of the paraflagellar rod, an extra-axonemal structure present in the flagellum. Knockdown of FLA1 or FLAM3 triggers similar defects in motility and morphogenesis, characterized by the assembly of a drastically reduced FAZ filament. FLAM3 remains associated with the flagellum skeleton even in the absence of adhesion or a normal paraflagellar rod. However, the protein is dispersed in the cytoplasm when flagellum formation is inhibited. By contrast, FLA1 remains tightly associated with the FAZ filament even in the absence of a flagellum. In these conditions, the extracellular domain of FLA1 points to the cell surface. FLAM3 is essential for proper distribution of FLA1BP, which is restricted to the most proximal portion of the flagellum upon knockdown of FLAM3. We propose that FLAM3 is a key component of the FAZ connectors that link the axoneme to the adhesion zone, hence it acts in an equivalent manner to the FAZ filament complex, but on the side of the flagellum.

Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella.

To analyze the function of ciliary polycystic kidney disease 2 (PKD2) and its relationship to intraflagellar transport (IFT), we cloned the gene encoding Chlamydomonas reinhardtii PKD2 (CrPKD2), a protein with the characteristics of PKD2 family members. Three forms of this protein (210, 120, and 90 kD) were detected in whole cells; the two smaller forms are cleavage products of the 210-kD protein and were the predominant forms in flagella. In cells expressing CrPKD2–GFP, about 10% of flagellar CrPKD2–GFP was observed moving in the flagellar membrane. When IFT was blocked, fluorescence recovery after photobleaching of flagellar CrPKD2–GFP was attenuated and CrPKD2 accumulated in the flagella. Flagellar CrPKD2 increased fourfold during gametogenesis, and several CrPKD2 RNA interference strains showed defects in flagella-dependent mating. These results suggest that the CrPKD2 cation channel is involved in coupling flagellar adhesion at the beginning of mating to the increase in flagellar calcium required for subsequent steps in mating.

The de Novo Synthesis of GDP-fucose Is Essential for Flagellar Adhesion and Cell Growth in Trypanosoma brucei

The protozoan parasite Trypanosoma brucei causes human African sleeping sickness in sub-Saharan Africa. The parasite makes several essential glycoproteins, which has led to the investigation of the sugar nucleotides and glycosyltransferases required to synthesize these structures. Fucose is a common sugar in glycoconjugates from many organisms; however, the sugar nucleotide donor GDP-fucose was only recently detected in T. brucei, and the importance of fucose metabolism in this organism is not known. In this paper, we identified the genes encoding functional GDP-fucose biosynthesis enzymes in T. brucei and created conditional null mutants of TbGMD, the gene encoding the first enzyme in the pathway from GDP-mannose to GDP-fucose, in both bloodstream form and procyclic form parasites. Under nonpermissive conditions, both life cycle forms of the parasite became depleted in GDP-fucose and suffered growth arrest, demonstrating that fucose metabolism is essential to both life cycle stages. In procyclic form parasites, flagellar detachment from the cell body was also observed under nonpermissive conditions, suggesting that fucose plays a significant role in flagellar adhesion. Fluorescence microscopy of epitope-tagged TbGMD revealed that this enzyme is localized in glycosomes, despite the absence of PTS-1 or PTS-2 target sequences.

Changes in photoreceptor currents and their sensitivity to the chemoeffector tryptone during gamete mating in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii Dangeard generates photoreceptor currents (PCs) upon light excitation. These currents play a key role in the signal transduction chain for photomotility responses. We have previously found that inhibition of PCs by tryptone occurs only in gametes that display chemotaxis toward this agent, and is not observed in chemotactically insensitive vegetative cells. Here we show that the sensitivity to tryptone is characteristic of gametes of both mating types, and examine the influence of gamete mating on PCs and their sensitivity to tryptone. The amplitude of PCs increases after cell fusion, but the sensitivity of these currents to tryptone decreases upon flagellar adhesion and/or an increase in the intracellular cAMP concentration. Net chemotaxis toward tryptone is reduced in young zygotes compared to gametes. We conclude that gamete mating leads to rapid inactivation of a gamete-specific chemosensory system.

Signaling Cilia Structures

You may think of cilia primarily as structures specialized to confer motility, but nonmotile cells (virtually all vertebrate cells have a primary cilium) also use cilia for sensory functions. Receptors concentrated on the surface of cilia allow cells to efficiently sample their immediate environment, but it is unclear how such receptors function in the absence of structures that usually associate with them at the plasma membrane. Wang et al. addressed this issue in the biflagellated alga Chlamydomonas . These cells use sex-specific adhesion receptors on their flagella (flagella are essentially the same as cilia) during mating. In this system, the authors could take advantage of temperature-sensitive mutations in kinesin 2, an essential motor that functions in the intraflagellar transport (IFT) system. The IFT actively transports material from the cell body to the flagellum, using kinesin and dynein to move particles along microtubules. Interaction of the flagella of mating Chlamydomonas cells leads to activation of a protein tyrosine kinase, and Wang et al. used immunopurification and mass spectrometry to identify a cyclic GMP (cyclic guanosine monophosphate)-dependent protein kinase (CrPKG) as a target of the tyrosine kinase. Although the flagellum represents about 5% of the volume of the cell, it contained almost 50% of the CrPKG. CrPKG was activated by flagellar adhesion, and depletion of CrPKG by RNAi showed that the kinase was required for successful fertilization. Fractionation by centrifugation revealed that CrPKG became associated with a new particulate compartment after flagellar adhesion but that functional IFT was required for this effect. The authors also used antibodies to monitor components of the IFT machinery before and after adhesion, and further fractionation showed that the IFT components became associated, along with CrPKG, with detergent-sensitive complexes in a particulate fraction derived from cell extracts. Given the signal-dependent modification of the IFT components and their association with CrPKG, which behaved similarly, the authors propose that IFT not only functions to move signaling components from the cell body to the flagellum but also actively participates in signaling. Q. Wang, J. Pan, W. J. Snell, Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas . Cell 125, 549-562 (2006). [Online Journal]

Intraflagellar Transport Particles Participate Directly in Cilium-Generated Signaling in Chlamydomonas

Primary cilia are widely used for signal transduction during development and in homeostasis and are assembled and maintained by intraflagellar transport (IFT). Here, we have dissected the role of IFT in signaling within the flagella (structural and functional counterparts of cilia) of the biflagellated green alga Chlamydomonas. Using a conditional IFT mutant enables us to deplete the IFT machinery from intact, existing flagella. We identify a cGMP-dependent protein kinase (CrPKG) within flagella as the substrate of a protein tyrosine kinase activated by flagellar adhesion during fertilization. We demonstrate that flagellar adhesion stimulates association of CrPKG with a new flagellar compartment. Moreover, formation of the compartment requires IFT, and IFT particles themselves are part of the compartment. Our results lead to a model in which the IFT machinery is required not only for assembling cilia and flagella but also for organizing a signaling pathway within the organelles during cilium-generated signaling.

Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi.

The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding the surface protein amastin, we found that the amastin double-stranded RNA (dsRNA) was not efficiently degraded in either epimastigotes or amastigotes, and the level of amastin mRNA remained unchanged. We generated a strain of T. cruzi CL-Brener in which the T7 promoter and tetracycline operator could be used to maximize tetracycline-regulated dsRNA synthesis and constructed plasmids that direct dsRNA against four different T. cruzi endogenous genes (encoding beta-tubulin, GP72 (flagellar adhesion protein), ribosomal protein P0 and amastin) and an exogenously added gene (GFP; green fluorescent protein). After either stable or transient transfection of these plasmids into T. cruzi, the expected RNAi phenotype was not observed for any of the five genes, although the T. cruzi beta-tubulin RNAi plasmid did give the expected FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These data indicate that, similar to Leishmania, T. cruzi lacks one or more components necessary for the RNAi pathway and that these components will need to be engineered into T. cruzi, or compensated for, before RNAi can be used to study gene function in this organism.

Flagellar Adhesion between Mating Type Plus and Mating Type Minus Gametes Activates a Flagellar Protein-tyrosine Kinase 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.

Protein transport and signal transduction during fertilization in chlamydomonas.

Fertilization in Chlamydomonas begins with flagellar adhesion between mating type plus and mating type minus gametes and is consummated within minutes by zygote formation. Once fusion occurs, the newly merged gametes cease existence as distinct entities, and the diploid zygote immediately initiates transcription of zygote-specific genes. Accomplishing fertilization within such a short time requires the rapid and signaled movement of pre-existing membrane and cytoplasmic proteins between and within several cellular compartments. Generation within the adhering flagella of the initial signals for protein movement, as well as movement itself of at least one cytoplasmic protein from the cell body to the flagella, depend on the microtubule motor, kinesin-II and presumably on intraflagellar transport (IFT). Adhesion and fusion of the two gametes depend on a second translocation event, the movement of an adhesion/fusion protein onto the surface of a rapidly elongating, microvillous-like fusion organelle. Finally, the merging of the two separate gametes, each containing sex-specific proteins, into a single cell allows the formerly separate proteins to form new interactions that regulate zygote development. Two proteins - a nuclease and a homeodomain protein - which were present only in the plus gamete, are 'delivered' to the cytoplasm of the zygote during gamete fusion. The nuclease is selectively imported into the minus chloroplast, where it degrades the chloroplast DNA, thereby ensuring uniparental inheritance of plus chloroplast traits. The homeodomain protein binds with an as yet unidentified protein delivered by the minus gamete, and the new complex activates transcription of zygote-specific genes.

Kinesin II Hauls Signaling Cargo

The unicellular green alga Chlamydomonas uses a flagellum not only for locomotion, but also for presentation of flagellar adhesion molecules that promote interaction between gamete cells and initiate signaling events important to fertilization. The flagella lack the apparatus to make new proteins, so the cells use an intraflagellar transport system to transport particles containing proteins made in the cell body to the flagella. Such transport occurs constitutively, but regulated transport has also been described. For example, the Chlamydomonas aurora protein kinase (CALK) is transported to the flagellum only in response to signals generated when receptors on flagella of cells of opposite mating types interact. (Interestingly, aurora protein kinases are implicated in cell division in other systems.) Pan and Snell used mutants of the motor protein kinesin II to show that kinesin II is required for such activated transport of CALK to the flagellum. Kinesin II is also the motor responsible for the constitutive transport of proteins to flagella. However, effects of the kinesin II mutation in cells maintained at different temperatures could distinguish between the two roles of kinesin II, and the regulated transport of CALK appeared to result in association with the flagellar membrane whereas unregulated transport delivers proteins to a soluble cytoplasmic location. Schnapp reviews other evidence for active transport of signaling molecules, which in some cases involves interaction of motor proteins with scaffold proteins, allowing whole signaling complexes to be delivered to the appropriate site of action. J. Pan, W. J. Snell, Kinesin II and regulated intraflagellar transport of Chlamydomonas aurora protein kinase. J. Cell Sci. 116 , 2179-2186 (2003). [Abstract] [Full Text] B. J. Schnapp, Trafficking of signaling modules by kinesin motors. J. Cell Sci. 116 , 2125-2135 (2003). [Abstract] [Full Text]

Kinesin II Hauls Signaling Cargo

The unicellular green alga Chlamydomonas uses a flagellum not only for locomotion, but also for presentation of flagellar adhesion molecules that promote interaction between gamete cells and initiate signaling events important to fertilization. The flagella lack the apparatus to make new proteins, so the cells use an intraflagellar transport system to transport particles containing proteins made in the cell body to the flagella. Such transport occurs constitutively, but regulated transport has also been described. For example, the Chlamydomonas aurora protein kinase (CALK) is transported to the flagellum only in response to signals generated when receptors on flagella of cells of opposite mating types interact. (Interestingly, aurora protein kinases are implicated in cell division in other systems.) Pan and Snell used mutants of the motor protein kinesin II to show that kinesin II is required for such activated transport of CALK to the flagellum. Kinesin II is also the motor responsible for the constitutive transport of proteins to flagella. However, effects of the kinesin II mutation in cells maintained at different temperatures could distinguish between the two roles of kinesin II, and the regulated transport of CALK appeared to result in association with the flagellar membrane whereas unregulated transport delivers proteins to a soluble cytoplasmic location. Schnapp reviews other evidence for active transport of signaling molecules, which in some cases involves interaction of motor proteins with scaffold proteins, allowing whole signaling complexes to be delivered to the appropriate site of action.J. Pan, W. J. Snell, Kinesin II and regulated intraflagellar transport of Chlamydomonas aurora protein kinase. J. Cell Sci. 116, 2179-2186 (2003). [Abstract] [Full Text]B. J. Schnapp, Trafficking of signaling modules by kinesin motors. J. Cell Sci. 116, 2125-2135 (2003). [Abstract] [Full Text]

Kinesin-II Is Required for Flagellar Sensory Transduction during Fertilization inChlamydomonas

The assembly and maintenance of eucaryotic flagella and cilia depend on the microtubule motor, kinesin-II. This plus end-directed motor carries intraflagellar transport particles from the base to the tip of the organelle, where structural components of the axoneme are assembled. Here we test the idea that kinesin-II also is essential for signal transduction. When mating-type plus (mt+) and mating-type minus (mt-) gametes of the unicellular green alga Chlamydomonas are mixed together, binding interactions between mt+ and mt- flagellar adhesion molecules, the agglutinins, initiate a signaling pathway that leads to increases in intracellular cAMP, gamete activation, and zygote formation. A critical question in Chlamydomonas fertilization has been how agglutinin interactions are coupled to increases in intracellular cAMP. Recently, fla10 gametes with a temperature-sensitive defect in FLA10 kinesin-II were found to not form zygotes at the restrictive temperature (32 degrees C). We found that, although the rates and extents of flagellar adhesion in fla10 gametes at 32 degrees C are indistinguishable from wild-type gametes, the cells do not undergo gamete activation. On the other hand, fla10 gametes at 32 degrees C regulated agglutinin location and underwent gamete fusion when the cells were incubated in dibutyryl cAMP, indicating that their capacity to respond to the cAMP signal was intact. We show that the cellular defect in the fla10 gametes at 32 degrees C is a failure to undergo increases in cAMP during flagella adhesion. Thus, in addition to being essential for assembly and maintenance of the structural components of flagella, kinesin-II/intraflagellar transport plays a role in sensory transduction in these organelles.

Regulated Targeting of a Protein Kinase into an Intact Flagellum: AN AURORA/Ipl1p-LIKE PROTEIN KINASE TRANSLOCATES FROM THE CELL BODY INTO THE FLAGELLA DURING GAMETE ACTIVATION INCHLAMYDOMONAS

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.

Flagellar Adhesion between mt+ and mt−Chlamydomonas Gametes Regulates Phosphorylation of the mt+-specific Homeodomain Protein GSP1

During fertilization in Chlamydomonas, flagellar adhesion between mt(+) and mt(-) gametes induces a cAMP-dependent signal transduction pathway that prepares the gametes for cell fusion and zygote formation. Previously, our laboratory identified a homeodomain protein (GSP1) whose expression was restricted to the cell bodies of mt(+) gametes and whose transcript level was up-regulated during flagellar adhesion. In this report, we describe a new form of GSP1 that appears early during gamete interactions. Immunoblot analysis showed that in addition to the 120-kDa form of GSP1 normally present in mt(+) gametes, a 122-kDa form was detected when the cells were mixed with mt(-) gametes. The more slowly migrating form of GSP1 was detectable within minutes after gametes were mixed together, and its appearance did not require new protein synthesis. Thus, the 122-kDa form represents a post-translational modification of the pre-existing 120-kDa form of GSP1. Moreover, conversion to the 122-kDa form did not require cell fusion. Although the 120-kDa form was expressed 10 h after vegetative cells were transferred to gametic induction medium, the 122-kDa form was detected only after mt(+) gametes were induced to undergo the sexual signaling that accompanies fertilization. Incubation of mt(+) gametes with dibutyryl cAMP led to the appearance of the 122-kDa form of GSP1, and the cyclic nucleotide-dependent protein kinase inhibitor H-8 inhibited the adhesion-induced conversion. Incubation of GSP1 immunoprecipitated from signaling mt(+) gametes with alkaline phosphatase showed that the conversion was due to phosphorylation. The results indicate that flagellar adhesion induces a rapid, cAMP-dependent phosphorylation of the homeodomain protein GSP1 early during fertilization in Chlamydomonas.

The Chlamydomonas mating type plus fertilization tubule, a prototypic cell fusion organelle: isolation, characterization, and in vitro adhesion to mating type minus gametes.

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this approximately 3-microm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt-) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt- gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2-3 x 10(6) fertilization tubules/microg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt- gametes between the two flagella, the site of the mt- mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt- gametes.

SksC, a Fertilization-Related Protein Kinase inChlamydomonas,Is Expressed throughout the Cell Cycle and Gametogenesis, and a Phosphorylated Form Is Present in both Flagella and Cell Bodies

Fertilization in the biflagellated eukaryote,Chlamydomonas,is initiated by flagellar adhesion between gametes of opposite mating types. An early event in the signal transduction pathway induced by these cell-cell interactions is the rapid inactivation of a flagellar protein kinase that phosphorylates a 48 kDa flagellar protein. Molecular cloning and characterization indicated that the 48 kDa substrate, termed SksC, itself is a novel protein kinase. Here, we have determined that its transcript levels were unchanged during prolonged flagellar adhesion. Moreover, resynthesis of new flagellar proteins following deflagellation was not accompanied by increases in transcript levels of SksC, suggesting that expression of this soluble protein kinase might not be restricted to flagella. Immunoblot analysis indicated that expression of SksC was ubiquitous: this soluble protein was found in both flagella and cell bodies and was expressed throughout the cell cycle and gametogenesis. Immunoprecipitation experiments indicated that SksC was phosphorylated in both flagella and cell bodies. Thus, in addition to its potential role in fertilization, this novel protein kinase may play a role in other signaling events inChlamydomonas.

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

Within seconds after the flagella of mt+ and mt- Chlamydomonas gametes adhere during fertilization, their flagellar adenylyl cyclase is activated several fold and preparation for cell fusion is initiated. Our previous studies indicated that early events in this pathway, including control of adenylyl cyclase, are regulated by phosphorylation and dephosphorylation. Here, we describe a soluble, flagellar protein kinase activity that is regulated by flagellar adhesion. A 48-kDa, soluble flagellar protein was consistently phosphorylated in an in vitro assay in flagella isolated from nonadhering mt+ and mt- gametes, but not in flagella isolated from mt+ and mt- gametes that had been adhering for 1 min. Although the 48-kDa protein was present in the flagella isolated from adhering gametes, we demonstrate that its protein kinase was inactivated by flagellar adhesion. Immunoblot analysis and inhibitor studies indicate that the 48-kDa protein in nonadhering gametes is phosphorylated by a protein tyrosine kinase. In vivo experiments showing that the protein tyrosine phosphatase inhibitor sodium orthovanadate inhibits fertilization suggest that protein dephosphorylation may be required for signal transduction. The 48-kDa protein and its protein kinase may be among the first elements of a novel signalling pathway that couples interaction of flagellar adhesion molecules to gamete activation.