Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii

Chlamydomonas cells excise their flagella in response to a variety of experimental conditions (e.g., extremes of temperature or pH, alcohol or detergent treatment, and mechanical shear). Here, we show that flagellar excision is an active process whereby microtubules are severed at select sites within the transition zone. The transition zone is located between the flagellar axoneme and the basal body; it is characterized by a pair of central cylinders that have an H shape when viewed in longitudinal section. Both central cylinders are connected to the A tubule of each microtubule doublet of the transition zone by fibers (approximately 5 nm diam). When viewed in cross section, these fibers are seen to form a distinctive stellate pattern characteristic of the transition zone (Manton, I. 1964. J. R. Microsc. Soc. 82:279-285; Ringo. D. L. 1967. J. Cell Biol. 33:543-571). We demonstrate that at the time of flagellar excision these fibers contract and displace the microtubule doublets of the axoneme inward. We believe that the resulting shear force and torsional load act to sever the axonemal microtubules immediately distal to the central cylinder. Structural alterations of the transition zone during flagellar excision occur both in living cells and detergent-extracted cell models, and are dependent on the presence of calcium (greater than or equal to 10(-6) M). Immunolocalization using monoclonal antibodies against the calcium-binding protein centrin demonstrate the presence of centrin in the fiber-based stellate structure of the transition zone of wild-type cells. Examination of the flagellar autotomy mutant, fa-1, which fails to excise its flagella (Lewin, R., and C. Burrascano. 1983. Experientia. 39:1397-1398), demonstrates that the fa-1 lacks the ability to completely contract the fibers of the stellate structure. We conclude that flagellar excision in Chlamydomonas involves microtubule severing that is mediated by the action of calcium-sensitive contractile fibers of the transition zone. These observations have led us to question whether microtubule severing may be a more general phenomenon than previously suspected and to suggest that microtubule severing may contribute to the dynamic behavior of cytoplasmic microtubules in other cells.     

The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells

Monoclonal and polyclonal antibodies raised against algal centrin, a protein of algal striated flagellar roots, were used to characterize the occurrence and distribution of this protein in interphase and mitotic Chlamydomonas cells. Chlamydomonas centrin, as identified by Western immunoblot procedures, is a low molecular (20,000-Mr) acidic protein. Immunofluorescence and immunogold labeling demonstrates that centrin is a component of the distal fiber. In addition, centrin-based flagellar roots link the flagellar apparatus to the nucleus. Two major descending fibers extend from the basal bodies toward the nucleus; each descending fiber branches several times giving rise to 8-16 fimbria which surround and embrace the nucleus. Immunogold labeling indicates that these fimbria are juxtaposed to the outer nuclear envelope. Earlier studies have demonstrated that the centrin-based linkage between the flagellar apparatus and the nucleus is contractile, both in vitro and in living Chlamydomonas cells (Wright, R. L., J. Salisbury, and J. Jarvik. 1985. J. Cell Biol. 101:1903-1912; Salisbury, J. L., M. A. Sanders, and L. Harpst. 1987. J. Cell Biol. 105:1799-1805). Immunofluorescence studies show dramatic changes in distribution of the centrin-based system during mitosis that include a transient contraction at preprophase; division, separation, and re-extension during prophase; and a second transient contraction at the metaphase/anaphase boundary. These observations suggest a fundamental role for centrin in motile events during mitosis.     

Calmodulin Sensitivity of the Flagellar Membrane Adenylate Cyclase and Signaling of Motile Responses by cAMP in Gametes of Chlamydomonas reinhardtii

Cyclic AMP (cAMP) has been shown to be a primary signal of the agglutination-induced mating events of flagellar tip activation, cell wall loss, and mating structure activation in the unicellular alga Chlamydomonas reinhardtii (Pasquale and Goodenough, Cell Biol. 105 (1987), 2279–2293). The flagellar membrane adenylate cyclase of Chlamydomonas is here shown to be inhibited in vitro by EGTA, La³⁺, and trifluoperazine, and to be stimulated in the presence of calcium by incubation with exogenous calmodulin. Also, the motility of detergent-extracted models of Chlamydomonas is shown to be enhanced by cAMP. These observations suggest the hypothesis that the twitching motility characteristic of agglutinating Chlamydomonas gametes may be signaled by cAMP produced locally within the flagella by a calmodulin-sensitive adenylate cyclase.     

Receptor-Mediated Calcium Influx in Chlamydomonas reinhardtii

ABSTRACT. Alcian blue acts as a secretagogue and chemorepellent in a variety of unicellular eukaryotes. We report that alcian blue stimulates flagellar excision and induction of RNA encoding flagellar proteins in Chlamydomonas reinhardtii . Flagellar excision by alcian blue is dependent on extracellular Ca²⁺ and is blocked by La³⁺, ruthenium red, and neomycin, and so is similar to flagellar excision by acid shock. However, the adf-l mutant excises its flagella following alcian blue treatment, but not following acid shock, thus genetically distinguishing alcian-blue-induced excision from acid-shock-induced excision. Wild-type, but not adf-1, cells regrow their flagella in the continued presence of alcian blue. Wild-type cells that regrow flagella in the presence of alcian blue fail to excise their flagella in response to either increased concentrations of alcian blue or to acid shock. Alcian blue treatment of cells also induces RNA encoding flagellar components, but in a manner distinct from other means of stimulation. These results suggest that treating Chlamydomonas with the secretagogue alcian blue initiates a Ca²⁺ influx pathway and that prolonged treatment with alcian blue desensitizes the acid-shock-activated Ca²⁺ influx pathway to acid treatment. Alcian blue will thus be a useful excitatory ligand in future studies of receptor-mediated Ca²⁺ signaling in the Chlamydomonas flagellar regeneration system.     

Reversal of the posttranslational modification on Chlamydomonas flagellar alpha-tubulin occurs during flagellar resorption

We previously have shown that a posttranslational modification of alpha-tubulin takes place in the flagellum during Chlamydomonas flagellar assembly (L'Hernault, S. W., and J. L. Rosenbaum, 1983, J. Cell Biol., 97:258-263). In this report, we show that the posttranslationally modified alpha-3 tubulin is changed back to its unmodified alpha-1 precursor form during the microtubular disassembly that takes place during flagellar resorption. These data indicate that the addition and removal of a posttranslational modification on alpha-tubulin might be a control step in the assembly and disassembly of flagella.     

Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity

Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein.     

CrPP2C蛋白影响衣藻细胞鞭毛过渡区结构

目的:在莱茵衣藻细胞中构建并筛选鞭毛组装缺陷突变体,克隆缺陷基因,探索其对鞭毛组装的影响。方法:使用带有巴龙霉素(Paromomycin)抗性的基因片段随机插入衣藻细胞基因组中,通过性状筛选和基因序列分析获得与CrPP2C(Chlamydomonas reinhardtii type 2C protein phosphatase)基因相关的鞭毛异常突变体,根据突变体基本生物学性状和生化分析对CrPP2C基因的功能进行分析。结果:采用电转法成功获得衣藻细胞鞭毛缺陷相关突变体,部分细胞具有短鞭毛,部分细胞则不具有鞭毛;通过RESDA-PCR(restriction enzyme site-directed amplification PCR)对突变体基因序列分析,鞭毛缺陷性状由CrPP2C基因遭到破坏导致;把含有完整CrPP2C基因的重组质粒通过电转法导入突变体后,其鞭毛几乎恢复为野生型长度,并可检测到PP2C-HA融合蛋白的表达;观察鞭毛再生,突变体鞭毛只能再生为原有长度;使用药物处理使鞭毛缩短,突变体鞭毛能正常解聚;电镜检测突变体的鞭毛显微结构,发现过渡区的Y形结构缺陷。结论:CrPP2C基因的破坏导致鞭毛过渡区结构缺失,影响鞭毛组装过程,不组装鞭毛或组装短鞭毛。     

Intrinsic difference in beat frequency between the two flagella of Chlamydomonas reinhardtii

The flagellar beat frequency of the biflagellated green alga Chlamydomonas reinhardtii was measured by fast Fourier transform analysis of the light intensity fluctuation in microscope images of swimming cells. Live cells had a mean beat frequency of 48-53 Hz at 20 degrees C. However, detergent-extracted "cell models," when reactivated in the presence of 1 mM ATP, appeared to have two different beat frequencies of about 30 and 45 Hz. Measurements in cell models in which only one of the two flagella was beating indicated that the lower and higher frequencies most likely represented the beat frequency of the flagellum nearer to the eyespot (the cis-flagellum) and that of the flagellum farther from it (the trans-flagellum), respectively. In live cells also, the trans-flagellum beat at a frequency about 30% higher than that of the cis-flagellum when the cells were rendered uniflagellated by mechanical treatment, whereas both flagella beat at the frequency of the cis-flagellum under normal conditions. These observations suggest that the two flagella of Chlamydomonas have different intrinsic beat frequencies but that they are somehow synchronized when beating together on a live swimming cell.     

Uncoupling of Chlamydomonas flagellar gene expression and outgrowth from flagellar excision by manipulation of Ca2+

Chlamydomonas cells respond to certain environmental stimuli by shedding their flagella. Flagellar loss induces a rapid, transient increase in expression of a specific set of genes encoding flagellar proteins, and assembly of a new flagellar pair. While flagellar gene expression and initiation of flagellar outgrowth are normally tightly coupled to flagellar excision, our results demonstrate that these processes can be uncoupled by manipulating Ca2+ levels or calmodulin activity. In our experiments, wild-type cells were stimulated to excise their flagella using mechanical shearing, and at times after deflagellation, flagellar lengths were measured and flagellar mRNA abundance changes were determined by S1 nuclease protection analysis. When extracellular Ca2+ was lowered by addition of EGTA to cultures before excision, flagellar mRNA abundance changes and flagellar outgrowth were temporally uncoupled from flagellar excision. When extracellular Ca2+ was lowered immediately after excision or when calmodulin activity was inhibited with W-7, flagellar outgrowth was uncoupled from flagellar excision and flagellar mRNA abundance changes. Whenever events in the process of flagellar regeneration were temporally uncoupled, the magnitude of the flagellar mRNA abundance change was reduced. These results suggest that flagellar gene expression may be regulated by multiple signals generated from these events, and implicate Ca2+ as a factor in the mechanisms controlling flagellar regeneration.     

Basal bodies and associated structures are not required for normal flagellar motion or phototaxis in the green alga Chlorogonium elongatum

The interphase flagellar apparatus of the green alga Chlorogonium elongatum resembles that of Chlamydomonas reinhardtii in the possession of microtubular rootlets and striated fibers. However, Chlorogonium, unlike Chlamydomonas, retains functional flagella during cell division. In dividing cells, the basal bodies and associated structures are no longer present at the flagellar bases, but have apparently detached and migrated towards the cell equator before the first mitosis. The transition regions remain with the flagella, which are now attached to a large apical mitochondrion by cross-striated filamentous components. Both dividing and nondividing cells of Chlorogonium propagate asymmetrical ciliary-type waveforms during forward swimming and symmetrical flagellar-type waveforms during reverse swimming. High-speed cinephotomicrographic analysis indicates that waveforms, beat frequency, and flagellar coordination are similar in both cell types. This indicates that basal bodies, striated fibers, and microtubular rootlets are not required for the initiation of flagellar beat, coordination of the two flagella, or determination of flagellar waveform. Dividing cells display a strong net negative phototaxis comparable to that of nondividing cells; hence, none of these structures are required for the transmission or processing of the signals involved in phototaxis, or for the changes in flagellar beat that lead to phototactic turning. Therefore, all of the machinery directly involved in the control of flagellar motion is contained within the axoneme and/or transition region. The timing of formation and the positioning of the newly formed basal structures in each of the daughter cells suggests that they play a significant role in cellular morphogenesis.     

Experimental dissection of flagellar surface motility in chlamydomonas

Experiments have explored the possible relationships between the flagellar surface motility of chlamydomonas, visualized as translocation of polystyrene beads by paralyzed (pf) mutants (Bloodgood, 1977, J. Cell Biol. 15:983-989), and the capacity of gametic flagella to participate in the mating reaction. While vegetative and gametic flagella bind beads with equal efficiencies and are capable of transporting them along entire flagellar lengths, beads on vegetative flagella are primarily associated with the proximal half of the flagella whereas those of gametic flagella exhibit no such preference. This difference may relate to the "tipping" response of gametes during sexual flagellar agglutination (Goodenough and Jurivich, 1978, J. Cell Biol. 79:680-693). Colchicine, vinblastine, chymotrypsin, cytochalasins B and D, and anti-β-tubulin antiserum are all able to inhibit the binding of beads to the flagellar suface. Trysin digestion and an antiserum directed against whole chlamydomonas flagella have no effect on the ability of flagella to bind beads, but the beads remain immobile. These results suggest that at least two flagellar activities participate in surface motility: (a) bead binding, which may involve a tubulin-like component at the flagellar surface; and (b) bead translocation, which may depend on a second component (e.g. an ATPase) of the flagellar surface. Surface motility is shown to be distinct from gametic adhesiveness per se, but it may participate in concentrating dispersed agglutinins, in driving them toward the flagellar tips, and/or in generating a signal-to-fuse from the flagellar tips to the cell body. Directly supporting these concepts is the observation that bound beads remain immobilized at the flagellar tips during the "tip-locking" stage of pf x pf matings, and the observation that bound ligands such as antibody fail to be tipped by trypsinized flagella.     

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

Interactions between adhesion molecules, agglutinins, on the surfaces of the flagella of mt+ and mt- gametes in Chlamydomonas rapidly generate a sexual signal, mediated by cAMP, that prepares the cells for fusion to form a zygote. The mechanism that couples agglutinin interactions to increased cellular levels of cAMP is unknown. In previous studies on the adenylyl cyclase in flagella of a single mating type (i.e., non-adhering flagella) we presented evidence that the gametic form of the enzyme, but not the vegetative form, was regulated by phosphorylation and dephosphorylation (Zhang, Y., E. M. Ross, and W. J. Snell. 1991. J. Biol. Chem. 266:22954-22959; Zhang, Y., and W. J. Snell. 1993. J. Biol. Chem. 268:1786-1791). In the present report we describe studies on regulation of flagellar adenylyl cyclase during adhesion in a cell-free system. The results show that the activity of gametic flagellar adenylyl cyclase is regulated by adhesion in vitro between flagella isolated from mt+ and mt- gametes. After mixing mt+ and mt- flagella together for 15 s in vitro, adenylyl cyclase activity was increased two- to threefold compared to that of the non-mixed (non- adhering), control flagella. This indicates that the regulation of gametic flagellar adenylyl cyclase during the early steps of fertilization is not mediated by signals from the cell body, but is a direct and primary response to interactions between mt+ and mt- agglutinins. By use of this in vitro assay, we discovered that 50 nM staurosporine (a protein kinase inhibitor) blocked adhesion-induced activation of adenylyl cyclase in vitro, while it had no effect on adenylyl cyclase activity of non-adhering gametic flagella. This same low concentration of staurosporine also inhibited adhesion-induced increases in vivo in cellular cAMP and blocked subsequent cellular responses to adhesion. Taken together, our results indicate that flagellar adenylyl cyclase in Chlamydomonas gametes is coupled to interactions between mt+ and mt- agglutinins by a staurosporine- sensitive activity, probably a protein kinase.     

Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas

Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy.     

Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii

When Chlamydomonas cells are deflagellated by pH shock or mechanical shear the nucleus rapidly moves toward the flagellar basal apparatus at the anterior end of the cell. During flagellar regeneration the nucleus returns to a more central position within the cell. The nucleus is connected to the flagellar apparatus by a system of fibers, the flagellar roots (rhizoplasts), which undergo a dramatic contraction that coincides with anterior nuclear movement. A corresponding extension of the root system, back to its preshock configuration is observed as the nucleus retracts to a central position. Anterior displacement of the nucleus and flagellar root contraction require free calcium in the medium. Nuclear movement and flagellar root contraction and extension are not sensitive to inhibitors of protein synthesis (cycloheximide), or drugs that influence either microtubules (colchicine) or actin-based microfilaments (cytochalasin D). Detergent-extracted cell models contract and extend their flagellar roots and move their nuclei in response to alterations of free calcium levels in the medium. Cycles of nuclear movement in detergent-extracted models require ATP to potentiate the contractile mechanism for subsequent calcium-induced contraction. Flagellar root contraction and nuclear movement in Chlamydomonas may be causally related to signaling of induction of flagellar precursor genes or to the transport of flagellar precursors or their messages to sites of synthesis or assembly near the basal apparatus of the cell.     

Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine

Previous work has shown that the principal alpha-tubulin within Chlamydomonas reinhardtii flagellar axonemes differs from the major alpha-tubulin in the cell body. These two variants of alpha-tubulin are related to one another since posttranslational modification of the cell body form converts it to the axonemal form. When flagella are induced to assemble in the absence of de novo protein synthesis, tritiated acetate can be used to posttranslationally label alpha-tubulin in vivo, and under these conditions, no other flagellar polypeptides exhibit detectable labeling [L'Hernault, S. W., & Rosenbaum, J. L. (1983) J. Cell Biol. 97, 258-263]. In the present report, this labeling method has been used to provide material for chemical analysis of the tritiated moiety that is posttranslationally added to flagellar alpha-tubulin. This radioactivity was volatile after acid hydrolysis, suggesting that the posttranslational modification is the addition of neither an amino acid nor carbohydrate. Treatment of posttranslationally 3H-labeled alpha-tubulin with hydrazine yields radioactive acetylhydrazine, indicating that the moiety involved in posttranslational modification is an acetyl group. Analysis of complete proteolytic digests by thin-layer chromatography has revealed that this acetyl group is located on the epsilon-amino group of a flagellar alpha-tubulin lysine residue.     

A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation

We have isolated a nucleus-basal body complex from Chlamydomonas reinhardtii. The complex is strongly immunoreactive to an antibody generated against a major protein constituent of isolated Tetraselmis striata flagellar roots (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, J. Cell Biol., 99:962-970). Electrophoretic and immunoelectrophoretic analysis indicates that, like the Tetraselmis protein, the Chlamydomonas antigen consists of two acidic isoforms of approximately 20 kD. Indirect immunofluorescent staining of nucleus- basal body complexes reveals two major fibers in the connector region, one between each basal body and the nucleus. The nucleus is also strongly immunoreactive, with staining radiating around much of the nucleus from a region of greatest concentration at the connector pole. Calcium treatment causes shortening of the connector fibers and also movement of nuclear DNA towards the connector pole. Electron microscopic observation of negatively stained nucleus-basal body complexes reveals a cluster of approximately 6-nm filaments, suspected to represent the connector, between the basal bodies and nuclei. A mutant with a variable number of flagella, vfl-2-220, is defective with respect to the nucleus-basal body association. This observation encourages us to speculate that the nucleus-basal body union is important for accurate basal body localization within the cell and/or for accurate segregation of parental and daughter basal bodies at cell division. A physical association between nuclei and basal bodies or centrioles has been observed in a variety of algal, protozoan, and metazoan cells, although the nature of the association, in terms of both structure and function, has been obscure. We believe it likely that fibrous connectors homologous to those described here for Chlamydomonas are general features of centriole-bearing eucaryotic cells.     

Flagellar protein dynamics in Chlamydomonas

Cilia and flagella appear to be stable, terminal, microtubule-containing organelles, but they also elongate and shorten in response to a variety of signals. To understand mechanisms that regulate flagellar dynamics, Chlamydomonas cells with nongrowing flagella were labeled with (35)S, and flagella and basal body components were examined for labeled polypeptides. Maximal incorporation of label into the flagella occurred within 3 h. Twenty percent of the flagellar polypeptides were exchanged. These included tubulins, dyneins, and 80 other axonemal and membrane plus matrix polypeptides. The most stable flagellar structure is the PF-ribbon, which comprises part of the wall of each doublet microtubule and is composed of tubulin and three other polypeptides. Most (35)S was incorporated into the high molecular weight ribbon polypeptide, rib240, and little, if any, (35)S is incorporated into PF-ribbon-associated tubulin. Both wild-type (9 + 2) and 9 + 0 flagella, which lack central microtubules, exhibited nearly identical exchange patterns, so labeling is not due to turnover of relatively labile central microtubules. To determine if flagellar length is balanced by protein exchange, (35)S incorporation into disassembling flagella was examined, as was exchange in flagella in which microtubule assembly was blocked by colchicine. Incorporation of (35)S-labeled polypeptides was found to occur into flagellar axonemes during wavelength-dependent shortening in pf18 and in fla10 cells induced to shorten flagella by incubation at 33 degrees C. Colchicine blocked tubulin addition but did not affect the exchange of the other exchangeable polypeptides; nor did it induce any change in flagellar length. Basal bodies also incorporated newly synthesized proteins. These data reveal that Chlamydomonas flagella are dynamic structures that incorporate new protein both during steady state and as flagella shorten and that protein exchange does not, alone, explain length regulation.