Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility

Mutations in Hydin cause hydrocephalus in mice, and HYDIN is a strong candidate for causing hydrocephalus in humans. The gene is conserved in ciliated species, including Chlamydomonas reinhardtii. An antibody raised against C. reinhardtii hydin was specific for an approximately 540-kD flagellar protein that is missing from axonemes of strains that lack the central pair (CP). The antibody specifically decorated the C2 microtubule of the CP apparatus. An 80% knock down of hydin resulted in short flagella lacking the C2b projection of the C2 microtubule; the flagella were arrested at the switch points between the effective and recovery strokes. Biochemical analyses revealed that hydin interacts with the CP proteins CPC1 and kinesin-like protein 1 (KLP1). In conclusion, C. reinhardtii hydin is a CP protein required for flagellar motility and probably involved in the CP-radial spoke control pathway that regulates dynein arm activity. Hydrocephalus caused by mutations in hydin likely involves the malfunctioning of cilia because of a defect in the CP.     

Reversible inhibition of Chlamydomonas flagellar surface motility

Chlamydomonas exhibits force transduction in association with its flagellar surface; this can be visualized by the saltatory movements of attached polystyrene microspheres. This flagellar surface motility has been quantitated by determining the percentage of attached microspheres in motion at the time of observation (60% in the case of control cells at 25 degrees C). A number of experimental treatments reversibly inhibit flagellar surface motility. These include an increase in sodium or potassium chloride concentration, a decrease in temperature, or a decrease in the free calcium concentration in the medium. Many of the conditions that result in inhibition of flagellar surface motility also result in an induction of flagellar resorption. Although both flagellar stability and flagellar surface motility are dependent on the availability of calcium, the two processes are separable; under appropriate conditions, flagellar surface motility can occur at normal levels on flagella that are resorbing. Inhibition of protein synthesis results in a gradual loss of both the binding of microspheres to the flagellum and the flagellar surface motility. After resumption of protein synthesis, both binding and movement return to control levels. The effect of the inhibition of protein synthesis is interpreted in terms of selective turnover of certain components within the intact flagellum, one or more of these components being necessary for the binding of the microspheres and their subsequent movement. If this turnover is inhibited by keeping the cells below 5 degrees C, the absence of protein synthesis no longer has an effect on microsphere attachment and motility, when measured immediately after warming the cells to 25 degrees C.     

Gene expression profiling of flagellar disassembly in Chlamydomonas reinhardtii

Flagella are sensory organelles that interact with the environment through signal transduction and gene expression networks. We used microarray profiling to examine gene regulation associated with flagellar length change in the green alga Chlamydomonas reinhardtii. Microarrays were probed with fluorescently labeled cDNAs synthesized from RNA extracted from cells before and during flagellar assembly or disassembly. Evaluation of the gene expression profiles identified >100 clones showing at least a twofold change in expression during flagellar length changes. Products of these genes are associated not only with flagellar structure and motility but also with other cellular responses, including signal transduction and metabolism. Expression of specific genes from each category was further characterized at higher resolution by using quantitative real-time PCR (qRT-PCR). Analysis and comparison of the gene expression profiles coupled to flagellar assembly and disassembly revealed that each process involves a new and uncharacterized whole-cell response to flagellar length changes. This analysis lays the groundwork for a more comprehensive understanding of the cellular and molecular networks regulating flagellar length changes.     

Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants.

Paralyzed flagellar mutants pf-1, pf-2, pf-7, and pf-18 of the green alga Chlamydomonas reinhardtii (Dangeard) were shown to store a significantly greater amount of starch than the motile wild type 137c+. The increase in starch storage was significant relative to protein, chlorophyll, and cell number. Analysis of average cell size revealed that the paralyzed mutants were larger than the wild type. This increase in storage molecule accumulation supports an inverse relationship between chemical energy storage and energy utilization for biomechanical/motile cellular functions. Chlamydomonas reinhardtii provides a useful model for studies of the role of cytoskeletal activity in the energy relationship and balance of organisms.     

Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.     

Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii

A series of conditional mutants of the algal, biflagellate Chlamydomonas reinhardtii with temperature-sensitive defects in flagellar assembly and function were isolated. The genetics and phenotypes of 21 mutants displaying a rapid alteration in flagellar function upon shift from the permissive (20 degrees C) to the restrictive (32 degrees C) temperatures are described. These mutants designated as "drop-down" or dd-mutants have been placed in four categories on the basis of their defective phenotypes: (a) dd-assembly mutants - the preformed flagella are resorbed at 32 degrees C and reassembly of flagella is inhibited; (b) dd-fragile flagella mutants - the flagella are lost by detachment at 32 degrees C, but can be reassembled; (c) dd-motility mutants - the flagella are retained at 32 degrees C, but are functionally defective; (d) dd-lethal mutants - display combined defects in flagellar function and cell growth. Tetrad analysis of the mutants back-crossed to wild-type, recombination analysis of intermutant crosses, and complementation tests in the construction of heterozygous diploid strains indicate that at least 14 nuclear genetic loci are represented among 21 mutants. The availability of temperature-sensitive mutations affecting the assembly and function of the flagellum suggests that the morphogenesis of this complex eukaryotic organelle is amenable to genetic dissection.     

Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress. Under favorable conditions, flagella are quickly regrown. To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems. After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospholipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress. Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation. Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.     

mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii.

Flagellar amputation in Chlamydomonas reinhardtii induces the accumulation of a specific set of RNAs, many of which encode flagellar proteins. We prepared a cDNA clone bank from RNA isolated from cells undergoing flagellar regeneration. From this bank, we selected clones that contain RNA sequences that display several different patterns of abundance regulation. Based on quantitation of the relative amounts of labeled, cloned cDNAs hybridizing to dots of RNA on nitrocellulose filters, the cloned sequences were divided into five regulatory classes: class I RNAs remain at constant abundance during flagellar regeneration; classes II, III, and IV begin to increase in abundance within a few minutes after deflagellation, reach maximal abundance at successively later times during regeneration, and return to control cell levels within 2 to 3 h; and class V RNA abundance decreases during flagellar regeneration. Alpha- and beta-tubulin mRNAs are included in regulatory class IV. The abundance kinetics of alpha-tubulin mRNAs differ slightly from those of beta-tubulin mRNAs. The availability of these clones makes possible studies on the mechanisms controlling the abundance of a wide variety of different RNA species during flagellar regeneration in Chlamydomonas.     

ATP-dependent regulation of flagellar adenylylcyclase in gametes of Chlamydomonas reinhardtii.

Adenylylcyclase activity in the flagella of gametes of Chlamydomonas reinhardtii was inhibited by prior incubation at or below 30 degrees C in the presence of ATP. This decrease did not occur in the absence of ATP, in the presence of the ATP analog 5'-adenylylimidodiphosphate (App(NH)p), or in the presence of ATP plus the protein kinase inhibitor staurosporine (2 microM). If ATP treatment was performed in the absence of an ATP-regenerating system, activity initially declined and subsequently recovered. Incubation of flagella at 45 degrees C in the absence of ATP or incubation at lower temperatures in the presence of either App(NH)p or staurosporine both increased adenylylcyclase activity (over 10-fold) and blocked subsequent ATP-dependent loss of activity at 30 degrees C. This heat-induced activation was prevented by the presence of ATP plus an ATP-regenerating system. Incubation of flagella with [gamma-32P]ATP followed by gel electrophoresis in sodium dodecyl sulfate indicated the presence of endogenous protein kinase and protein phosphatase activities. These data suggest that the flagellar adenylylcyclase in Chlamydomonas gametes is inhibited by phosphorylation and stimulated by dephosphorylation. This mechanism for regulating adenylylcyclase may underlie the rapid increase in cyclic AMP that is induced by flagellar adhesion during fertilization in Chlamydomonas.     

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.     

Bioflocculant produced by Chlamydomonas reinhardtii

Bioflocculants of Chlamydomonas reinhardtii were investigated under axenic conditions. C. reinhardtii was found to produce significant amounts of bioflocculants. Flocculating activity by C. reinhardtii began in the linear phase of growth and continued until the end of the stationary phase. The highest flocculating efficiency of the culture broth was 97.06%. The purified C. reinhardtii bioflocculant was composed of 42.1% (w/w) proteins, 48.3% carbohydrates, 8.7% lipids, and 0.01% nucleic acid. The optimum condition for bioflocculant production of C. reinhardtii was as follows: under temperature of 15°C to 25°C, pH 6–10 and illumination of 40–60 μmol photons m^?2 s^?1. The bioflocculants produced by C. reinhardtii showed maximum activity in pH ranges from 2 to 10. The flocculating activity was significantly enhanced by the addition of CaCl₂ as a co-flocculant at an optimal concentration of 4.5 mM.     

Chlamydomonas reinhardtii

Recombinant proteins have become more and more important for the pharmaceutical and chemical industry. Although various systems for protein expression have been developed, there is an increasing demand for inexpensive methods of large-scale production. Eukaryotic algae could serve as a novel option for the manufacturing of recombinant proteins, as they can be cultivated in a cheap and easy manner and grown to high cell densities. Being a model organism, the unicellular green alga Chlamydomonas reinhardtii has been studied intensively over the last decades and offers now a complete toolset for genetic manipulation. Recently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its ability for biotechnological applications.     

Calcium influx signals normal flagellar RNA induction following acid shock of Chlamydomonas reinhardtii

Acid shock of Chlamydomonas results in flagellar excision and induction of flagellar protein RNAs. The magnitude of flagellar RNA accumulations after flagellar excision by mechanical shear depends on the extracel]ular Ca2+ concentration. In this report, we demonstrate that the magnitude and duration of flagellar RNA accumulations are signaled by an acid shock-induced Ca2+ influx. RNA accumulations were greater in cells acid shocked in 500 µM CaCl2 than in 200 µM CaCl2, although the accumulation durations were similar. RNA accumulations of lower magnitude and shorter duration were observed in cells in Ca2+-containing buffer treated with CdCl2. RNA accumulations were of still lower magnitude and shorter duration in cells shocked in buffer without added CaCl2 than in cells shocked in 200 or 500 µM CaCl2 or in the presence of CdCl2. RNA accumulations similar to those in cells shocked in buffer without added CaCl2 were measured in cells following acid shock in buffer containing 200 µM CaCl2 and supplemented with neomycin, ruthenium red, or LaCl3. Acid shock of the adf-1 mutant resulted in RNA accumulations of shorter duration and lower magnitude than those measured in adf-1 cells stimulated by mechanical shear. These results are consistent with an hypothesis that acid shock generates two genetically and pharmacologically distinct signals governing flagellar RNA induction; the first signal is independent of a Ca2+ influx and flagellar excision and results in low magnitude accumulations of short duration, and the second is a consequence of a Ca2+ influx and results in accumulations of high magnitude and long duration.     

Defective temporal and spatial control of flagellar assembly in a mutant of Chlamydomonas reinhardtii with variable flagellar number

Wild-type Chlamydomonas reinhardtii carry two flagella per cell that are used for both motility and mating. We describe a mutant, vfl-1, in which the biflagellate state is disrupted such that the number of flagella per cell ranges from 0 to as many as 10. vfl-1 cells possess the novel ability to assemble new flagella throughout the G1 portion of the cell cycle, resulting in an average increase of about 0.05 flagella per cell per hour. Such uncoupling of the flagellar assembly cycle from the cell cycle is not observed in other mutants with abnormal flagellar number. Rather than being located in an exclusively apical position characteristic of the wild type, vfl-1 flagella can be at virtually any location on the cell surface. vfl-1 cells display abnormally wide variations in cell size, probably owing to extremely unequal cell divisions. Various ultrastructural abnormalities in the flagellar apparatus are also present, including missing or defective striated fibers and reduced numbers of rootlet microtubules. The pleiotropic defects observed in vfl-1 result from a recessive Mendelian mutation mapped to Chromosome VIII.     

Effects of calcium channel blockers on phototaxis and motility of Chlamydomonas reinhardtii

The effects of the four calcium channel blockers flunarizine, verapamil, diltiazem and nimodipine on motility and phototaxis of Chlamydomonas reinhardtio have been tested with a fully automated and computerized population system. Flunarizine inhibits motility transiently by causing the detachement of the flagella which, however, are regenerated during some hours. Phototaxis is inhibited to the same extent, but this is simply the result of the decreased motility and, hence, a non-specific effect. Verapamil causes also a detachement of the flagella with following regeneration, but in addition motility and phototaxis are inhibited by this drug to different extents, indicating the involvement of calcium channels in both processes. Diltiazem and nimodipine inhibit phototaxis without impairing motility, indicating that both processes are regulated in different ways. If diltiazem and nimodipine are applied simultaneously, no additive inhibitory effect can be observed. However, the combination of both blockers with verapamil causes and additive inhibitory effect as if verapamil is applied alone. By increasing the external calcium concentration from 10^-4 M to 10^-3 M the optimum of positive phototaxis is shifted to higher fluence rates. This shifting occurs also in the presence of channel blockers, but the strength of the positive reaction is influenced. These results point to the involvement of calcium channels in both phototaxis and motility, but simultaneously demonstrate the different sensitivity of the two processes to these drugs.Abbreviations DIL diltiazem (=benzothiazepine) - FLU flunarizine (=(E)-1-(bis-(4-fluorophenyl(methyl)-4-(3-phenyl-2-propenyl)piperazinex2HCl) - NIM nimodipine (=1,4-dihydropyridine) - VER verapamil (=diphenylalkylamine) CaM, calmodulin - PDE phosphodiesterase - DMSO dimethylsulfoxide     

Analysis of force generation during flagellar assembly through optical trapping of free-swimming Chlamydomonas reinhardtii

Many studies have used velocity measurements, waveform analyses, and theoretical flagella models to investigate the establishment, maintenance, and function of flagella of the biflagellate green algae Chlamydomonas reinhardtii. We report the first direct measurement of Chlamydomonas flagellar swimming force. Using an optical trap ("optical tweezers") we detect a 75% decrease in swimming force between wild type (CC124) cells and mutants lacking outer flagellar dynein arms (oda1). This difference is consistent with previous estimates and validates the force measurement approach. To examine mechanisms underlying flagella organization and function, we deflagellated cells and examined force generation during flagellar regeneration. As expected, fully regenerated flagella are functionally equivalent to flagella of untreated wild type cells. However, analysis of swimming force vs. flagella length and the increase in force over regeneration time reveals intriguing patterns where increases in force do not always correspond with increases in length. These investigations of flagellar force, therefore, contribute to the understanding of Chlamydomonas motility, describe phenomena surrounding flagella regeneration, and demonstrate the advantages of the optical trapping technique in studies of cell motility.     

Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii

Previously, we reported that flagellar excision in Chlamydomonas reinhardtii is mediated by an active process whereby microtubules are severed at select sites within the flagellar-basal body transition zone (Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751- 1760). At the time of flagellar excision, stellate fibers of the transition zone contract and displace the microtubule doublets of the axoneme inward. The resulting shear force and torsional load generated during inward displacement leads to microtubule severing immediately distal to the central cylinder of the transition zone. In this study, we have used a detergent-extracted cell model of Chlamydomonas that allows direct experimental access to the molecular machinery responsible for microtubule severing without the impediment of the plasma membrane. We present four independent lines of experimental evidence for the essential involvement of centrin-based stellate fibers of the transition zone in the process of flagellar excision: (a) Detergent-extracted cell models excise their flagella in response to elevated, yet physiological, levels of free calcium. (b) Extraction of cell models with buffers containing the divalent cation chelator EDTA leads to the disassembly of centrin-based fibers and to the disruption of transition zone stellate fiber structure. This treatment results in a complete loss of flagellar excision competence. (c) Three separate anti-centrin monoclonal antibody preparations, which localize to the stellate fibers of the transition zone, specifically inhibit contraction of the stellate fibers and block calcium-induced flagellar excision, while control antibodies have no inhibitory effect. Finally, (d) cells of the centrin mutant vfl-2 (Taillon, B., S. Adler, J. Suhan, and J. Jarvik. 1992. J. Cell Biol. 119:1613-1624) fail to actively excise their flagella following pH shock in living cells or calcium treatment of detergent-extracted cell models. Taken together, these observations demonstrate that centrin-based fiber contraction plays a fundamental role in microtubule severing at the time of flagellar excision in Chlamydomonas.     

Chemoresponses of Chlamydomonas reinhardtii

Cells of Chlamydomonas reinhardtii have been found to respond to chemicals in two ways: chemokinesis and chemotaxis. Several amino acids, fatty acids, and inorganic salts can stimulate these responses.