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.     

FLAGELLAR ELONGATION AND SHORTENING IN CHLAMYDOMONAS : The Use of Cycloheximide and Colchicine to Study the Synthesis and Assembly of Flagellar Proteins

Flagella can be removed from the biflagellate Chlamydomonas and the cells begin to regenerate flagella almost immediately by deceleratory kinetics. Under usual conditions of deflagellation, more than 98% of all flagella are removed. Under less drastic conditions, cells can be selected in which one flagellum is removed and the other left intact. When only one of the two flagella is amputated, the intact flagellum shortens by linear kinetics while the amputated one regenerates. The two flagella attain an equal intermediate length and then approach their initial length at the same rate. A concentration of cycloheximide which inhibits protein synthesis permits less than one-third of each flagellum to form when both flagella are amputated. When only one is amputated in cycloheximide, shortening proceeds normally and the degree of elongation in the amputated flagellum is greater than if both were amputated in the presence of cycloheximide. The shortening process is therefore independent of protein synthesis, and the protein from the shortening flagellum probably enters the pool of precursors available for flagellar formation. Partial regeneration of flagella occurs in concentrations of cycloheximide inhibitory to protein synthesis suggesting that some flagellar precursors are present. Cycloheximide and flagellar pulse-labeling studies indicate that precursor is used during the first part of elongation, is resynthesized at mid-elongation, and approaches its original level as the flagella reach their initial length. Colchicine completely blocks regeneration without affecting protein synthesis, and extended exposure of deflagellated cells to colchicine increases the amount of flagellar growth upon transfer to cycloheximide. When colchicine is applied to cells with only one flagellum removed, shortening continues normally but regeneration is blocked. Therefore, colchicine can be used to separate the processes of shortening and elongation. Radioautographic studies of the growth zone of Chlamydomonas flagella corroborate previous findings that assembly is occurring at the distal end (tip growth) of the organelle.     

Chlamydomonas shortens its flagella by activating axonemal disassembly, stimulating IFT particle trafficking, and blocking anterograde cargo loading

Almost all eukaryotic cells form cilia/flagella, maintain them at their genetically specified lengths, and shorten them. Here, we define the cellular mechanisms that bring about shortening of flagella prior to meiotic cell division and in response to environmental cues in the biflagellated green alga Chlamydomonas. We show that the flagellar shortening pathway is distinct from the one that enforces transient shortening essential for length control. During flagellar shortening, disassembly of the axoneme is stimulated over the basal rate, and the rate of entry into flagella of intraflagellar transport (IFT) particles is increased. Moreover, the particles entering the disassembling flagella lack cargo. Thus, flagellar shortening depends on the interplay between dynamic properties of the axoneme and the IFT machinery; a cell triggered to shorten its flagellum activates disassembly of the axoneme and stimulates entry into the flagellum of IFT particles possessing empty cargo binding sites available to retrieve the disassembled components.     

Intraflagellar transport (IFT) during assembly and disassembly of Chlamydomonas flagella

Intraflagellar transport (IFT) of particles along flagellar microtubules is required for the assembly and maintenance of eukaryotic flagella and cilia. In Chlamydomonas, anterograde and retrograde particles viewed by light microscopy average 0.12-microm and 0.06-microm diameter, respectively. Examination of IFT particle structure in growing flagella by electron microscopy revealed similar size aggregates composed of small particles linked to each other and to the membrane and microtubules. To determine the relationship between the number of particles and flagellar length, the rate and frequency of IFT particle movement was measured in nongrowing, growing, and shortening flagella. In all flagella, anterograde and retrograde IFT averaged 1.9 microm/s and 2.7 microm/s, respectively, but retrograde IFT was significantly slower in flagella shorter than 4 mum. The number of flagellar IFT particles was not fixed, but depended on flagellar length. Pauses in IFT particle entry into flagella suggest the presence of a periodic "gate" that permits up to 4 particles/s to enter a flagellum.     

Turnover of actin in Chlamydomonas flagella detected by fluorescence recovery after photobleaching (FRAP)

Recent indirect observations have suggested that various axonemal proteins in cilia and flagella of live cells undergo turnover independently of shortening or elongation of the axoneme. To gain direct evidence, here we examined using a FRAP (fluorescence recovery after photobleaching) technique whether actin, a subunit of inner arm dynein, is being turned over in Chlamydomonas flagella. Fluorescently labeled rabbit actin was introduced by electroporation into the cells of ida5oda1, a double mutant between oda1 lacking outer arm dynein and ida5 lacking several species of inner arm dyneins due to the absence of a conventional-type actin. In actin-loaded cells, flagella became motile and fluorescent due to incorporation of inner-arm dyneins containing the labeled actin. Cells were sandwiched between an agar layer and a coverslip so as to restrict flagellar movement. After a small portion of a flagellum was photobleached, the fluorescence intensity in the bleached area was monitored with a sensitive video camera. The fluorescence intensity in the photobleached region was found to recover 10-40% of the original level over several tens of minutes without changing its position. The time course and extent of the recovery varied greatly from one cell to another, suggesting that the turnover depends on cellular conditions. Western blot analysis indicated that 70-80% of flagellar actin was associated with the axoneme. Hence this experiment provides direct evidence that an axonemal component undergoes dynamic exchange in stationary flagella.     

Assembly of flagellar radial spoke proteins in Chlamydomonas: identification of the axoneme binding domain of radial spoke protein 3

Radial spokes of the eukaryotic flagellum extend from the A tubule of each outer doublet microtubule toward the central pair microtubules. In the paralyzed flagella mutant of Chlamydomonas pf14, a mutation in the gene for one of 17 polypeptides that comprise the radial spokes results in flagella that lack all 17 spoke components. The defective gene product, radial spoke protein 3 (RSP3), is, therefore, pivotal to the assembly of the entire spoke and may attach the spoke to the axoneme. We have synthesized RSP3 in vitro and assayed its binding to axonemes from pf14 cells to determine if RSP3 can attach to spokeless axonemes. In vitro, RSP3 binds to pf14 axonemes, but not to wild-type axonemes or microtubules polymerized from purified chick brain tubulin. The sole axoneme binding domain of RSP3 is located within amino acids 1-85 of the 516 amino acid protein; deletion of these amino acids abolishes binding by RSP3. Fusion of amino acids 1-85 or 42-85 to an unrelated protein confers complete or partial binding activity, respectively, to the fusion protein. Transformation of pf14 cells with mutagenized RSP3 genes indicates that amino acids 18-87 of RSP3 are important to its function, but that the carboxy-terminal 140 amino acids can be deleted with little effect on radial spoke assembly or flagellar motility.     

PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.     

Acetylcholine receptors in the central nervous system of Drosophila melanogaster.

Mating between gametes of the biflagellated unicellular green alga Chlamydomonas reinhardi consists of several events culminating in zygote formation. Initially, the cells agglutinate by their flagellar tips. This is followed by pairing, cell wall loss, and cell fusion. Here we report on the relationship between the length of the flagellum, and the cells' ability to agglutinate, undergo cell wall loss (as measured by medium carbohydrate accumulation), and to form zygotes. We found that deflagellated gametes regained the potential for sexual agglutination when the flagella had regenerated to less than 3 μm (compared to the full length flagella of approx. 11 μm), while medium carbohydrate appeared only after the flagella had reached an average length greater than 5 μm. By inhibiting flagellar regeneration with cycloheximide or colchicine, we determined that carbohydrate release is related to the length of the flagellum and not to the time after deflagellation. A flagellar length dependence similar to that of carbohydrate release was also observed when we measured the relationship between the gametes' ability to fuse and flagellar length.     

Polarity of flagellar assembly in Chlamydomonas.

During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.     

PF15p is the chlamydomonas homologue of the Katanin p80 subunit and is required for assembly of flagellar central microtubules

Numerous studies have indicated that the central apparatus plays a significant role in regulating flagellar motility, yet little is known about how the central pair of microtubules or their associated projections assemble. Several Chlamydomonas mutants are defective in central apparatus assembly. For example, mutant pf15 cells have paralyzed flagella that completely lack the central pair of microtubules. We have cloned the wild-type PF15 gene and confirmed its identity by rescuing the motility and ultrastructural defects in two pf15 alleles, the original pf15a mutant and a mutant generated by insertional mutagenesis. Database searches using the 798-amino-acid polypeptide predicted from the complete coding sequence indicate that the PF15 gene encodes the Chlamydomonas homologue of the katanin p80 subunit. Katanin was originally identified as a heterodimeric protein with a microtubule-severing activity. These results reveal a novel role for the katanin p80 subunit in the assembly and/or stability of the central pair of flagellar microtubules.     

Developmental studies on the loricate choanoflagellateStephanoeca diplocostata Ellis. V. The cytoskeleton and the effects of microtubule poisons

Summary InStephanoeca diplocostata microtubules are located in four positions namely: within the flagellar axoneme; just beneath the plasmalemma; associated with the silica deposition vesicles (SDVs) during early stages of costal strip deposition; and in the mitotic spindle. At the anterior end of the cell the 50–60 peripheral microtubules, which are organized more or less parallel to the long axis of the cell, converge around the base of the emergent flagellum. A short second flagellar base is positioned between the nucleus and the base of the emergent flagellum. Developing costal strips are located individually within SDVs in the peripheral cytoplasm. During the early stages of silica deposition each SDV is curved and subtended longitudinally on its concave side by two microtubules. When a costal strip has achieved sufficient rigidity to withstand bending the SDV-associated microtubules are depolymerized. Treatment of exponentially growing cells with sublethal concentrations of microtubule poisons, such as colchicine, podophyllotoxin, griseofulvin andVinca alkaloids depresses growth. Treatment with these drugs also affects the length and morphology of developing costal strips perhaps by interfering with the shaping and supporting functions of SDV-associated microtubules. Instead of being long and crescentic with a standard radius of curvature, costal strips of treated cells are usually short and misshapen, with irregular bends. After drug treatment, juveniles produced as a result of cell division do not develop flagella but can still assemble a lorica although it is usually misshapen. The role of microtubules and microfilaments in lorica production is discussed.     

Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a "high-load environment," we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters.     

Regulation of flagellar length in Chlamydomonas

Chlamydomonas reinhardtii has two apically localized flagella that are maintained at an equal and appropriate length. Assembly and maintenance of flagella requires a microtubule-based transport system known as intraflagellar transport (IFT). During IFT, proteins destined for incorporation into or removal from a flagellum are carried along doublet microtubules via IFT particles. Regulation of IFT activity therefore is pivotal in determining the length of a flagellum. Reviewed is our current understanding of the role of IFT and signal transduction pathways in the regulation of flagellar length.     

Polarity and asymmetry in the arrangement of dynein and related structures in the Chlamydomonas axoneme

Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer-inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella.     

Kinesin-like proteins in the flagella of Chlamydomonas

The flagella of the biflagellate unicellular alga Chlamydomonas have long been known to contain the microtubule-dependent motor protein dynein, but recent findings indicate they also contain multiple members of the kinesin superfamily. Two of these kinesin-like proteins are restricted to a single central-pair microtubule, raising the question of how proteins are targeted to specific microtubules within the flagellum. The kinesin-like proteins on the central-pair microtubules may cause the central-pair apparatus to rotate or twist during flagellar beating. Other kinesins within the flagellum may participate in movements associated with the flagellar membrane.     

Flagellar regeneration in Chlamydomonas reinhardtii: evidence that cycloheximide pulses induce a delay in morphogenesis.

The behaviour of a pool of flagellar precursors, assayed by the ability of cells to regenerate flagella in the absence of de novo protein synthesis, has been examined during organelle morphogenesis in the biflagellate alga Chlamydomonas. The results demonstrate that flagellar elongation can continue even when this pool is apparently empty and suggest that 2 sources of precursors are available to the regenerating flagella: those pre-existing in the cellular pool and those synthesized de novo. Further evidence for this was obtained by subjecting regenerating cells to pulses of cycloheximide. Cells exposed to this drug during the first 60 min post deflagellation formed only half-length (5-mum) flagella, whereas a pulse administered after this point allowed the formation of longer flagella and suggested that some de novo protein synthesis was required for the formation of full-length flagella, although it was not a prerequisite for the initiation of regeneration. In addition, it was found that, subsequent to the removal of the cycloheximide, flagellar regeneration did not recommence immediately, but was delayed for a period of approximately 45 min, irrespective of length of flagella formed prior to drug inhibition. The nature of this cycloheximide-induced delay is unclear and certain alternatives, based on the exhaustion of structural/regulatory components are considered. Although it is not possible to distinguish between these alternatives, tubulin is not the limiting component, since a pool of this protein is present when flagellar elongation is prevented by cycloheximide.     

Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50

Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca2+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.     

Twist (and rotation?) of central-pair microtubules in flagella ofPoterioochromonas

Summary The chrysoflagellate algaPoterioochromonas bears two unequal flagella. There is a short naked one and a long flagellum with mastigonemes. Ultrastructural investigation reveals that the centralpair microtubules in both flagella have no fixed position with respect to the flagellar base and root system, or the mastigoneme rows in the long flagellum. The central-pair microtubules are twisted several times along the length of the flagellum. This might indicate active or passive rotation of the central-pair microtubules during flagellar beat.     

An aurora kinase is essential for flagellar disassembly in Chlamydomonas

Cilia and flagella play key roles in development and sensory transduction, and several human disorders, including polycystic kidney disease, are associated with the failure to assemble cilia. Here, we show that the aurora protein kinase CALK in the biflagellated alga Chlamydomonas has a central role in two pathways for eliminating flagella. Cells rendered deficient in CALK were defective in regulated flagellar excision and regulated flagellar disassembly. Exposure of cells to altered ionic conditions, the absence of a centriole/basal body for nucleating flagellar assembly, cessation of delivery of flagellar components to their tip assembly site, and formation of zygotes all led to activation of the regulated disassembly pathway as indicated by phosphorylation of CALK and the absence of flagella. We propose that cells have a sensory pathway that detects conditions that are inappropriate for possession of a flagellum, and that CALK is a key effector of flagellar disassembly in that pathway.