Abnormal basal-body number, location, and orientation in a striated fiber-defective mutant of Chlamydomonas reinhardtii

We describe a mutant of Chlamydomonas reinhardtii in which basal body associated striated fibers are absent or incomplete. Basal body spacing, angle, and relative rotational orientation are abnormal and extremely variable. Abnormal partitioning of cellular contents at cytokinesis is also evident. Mating, maintenance of flagellar length equality, and backward swimming response are normal. Genetic analysis indicates mutation of a new Mendelian gene--vfl-3--linked to the centromere of Chromosome VI.     

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.     

Extensive gene rearrangements in the chloroplast DNAs of Chlamydomonas species featuring multiple dispersed repeats

We have constructed a physical and gene map for the chloroplast DNA (cpDNA) of the unicellular green alga Chlamydomonas gelatinosa, a close relative of Chlamydomonas reinhardtii. At 285 kb, the C. gelatinosa cpDNA is 89 kb larger than its C. reinhardtii counterpart. The alterations in the order of 77 genes on the cpDNAs of these green algae are attributable to nine inversions and one event of expansion/contraction of the inverted repeat. These rearrangements are much more extensive than those previously reported between the cpDNAs of the closely related Chlamydomonas moewusii and Chlamydomonas pitschmannii. Because the divergence level of the C. gelatinosa and C. reinhardtii chloroplast-encoded large subunit rRNA gene sequences is equivalent to that of the corresponding C. moewusii and C. pitschmannii sequences, our results may suggest that, in the same period of time, there have been more numerous rearrangements in the lineage comprising C. gelatinosa and C. reinhardtii than in the lineage comprising C. moewusii and C. pitschmannii. Alternatively, given that substitution rates in chloroplast genes are not necessarily uniform across lineages, the extensive rearrangements between the C. gelatinosa and C. reinhardtii cpDNAs may reflect a longer divergence period for this pair of Chlamydomonas species compared to that for the C. moewusii/C. pitschmannii pair. We have also found that, like its C. reinhardtii homologue but unlike its C. moewusii and C. pitschmannii counterparts, the C. gelatinosa cpDNA features a large number of dispersed repeated sequences that are readily detectable by Southern blot hybridization with homologous fragment probes. Assuming that the two pairs of closely related Chlamydomonas species diverged at about the same time, these data suggest that the susceptibility of Chlamydomonas cpDNAs to rearrangements is correlated with the abundance of repeated sequences. Preliminary characterization of a 345-bp C. gelatinosa cpDNA region containing a repeated sequence by both DNA sequencing and Southern blot analysis has revealed no sequence homology between this region and the cpDNAs of C. reinhardtii and other Chlamydomonas species.      

Diurnal Variations in the Motility of Populations of Biflagellate Microalgae

The motility of microalgae has been studied extensively, particularly in model microorganisms such as Chlamydomonas reinhardtii. For this and other microalgal species, diurnal cycles are well known to control the metabolism, growth, and cell division. Diurnal variations, however, have been largely neglected in quantitative studies of motility. Here, we demonstrate using tracking microscopy how the motility statistics of C. reinhardtii are modulated by diurnal cycles. With nine independently inoculated cultures synchronized to the light-dark cycle at the exponential growth phase, we repeatedly observed that the mean swimming speed is greater during the dark period of a diurnal cycle. From this measurement, using a hydrodynamic power balance, we infer the mean flagellar beat frequency and conjecture that its diurnal variation reflects modulation of intracellular ATP. Our measurements also quantify the diurnal variations of the orientational and gravitactic transport of C. reinhardtii. We use this to explore the population-level consequences of diurnal variations of motility statistics by evaluating a prediction for how the gravitactic steady state changes with time during a diurnal cycle. Finally, we discuss the consequences of diurnal variations of microalgal motility in soil and pelagic environments.     

134 Cryopreservation of Chlamydomonas Reinhardtii (Chlorophyceae): A Cause of Low Viability at High Cell Density

Cryopreservation provides a convenient method for long term storage of living organisms. Current protocols allow the successful cryopreservation of a wide range of algae, although many strains remain recalcitrant to cryopreservation. Chlamydomonas reinhardtii , a species utilized in many molecular and biochemical studies, survives cryopreservation best at low cell density. We show that reduced viability at higher cell densities is caused by the accumulation of a substance released from C. reinhardtii into the culture medium during cryopreservation. A mutant strain of C. reinhardtii (cw10) with a greatly reduced cell wall did not release a substance inhibitory to wild type or cw10 C. reinhardtii during cryopreservation, and could be cryopreserved with the same viability regardless of cell density. The inhibitory substance is small (mw<1300), polar, heat-stable and organic. Chlamydomonas moewusii Gerloff and Chlamydomonas zebra Korschikov ex Pascher both produce substances that reduce the viability of cryopreserved C. reinhardtii . However, neither is affected by the inhibitory substance produced by themselves or C. rienhardtii. Pandorina morum (Müller) Bory and Volvox carteri f. nagariensis Iyengar are colonial Volvocalean algae related to C. reinhardtii that cannot be successfully cryopreserved. They both generate substances that inhibit C. reinhardtii during cryopreservation. The identification of the substance inhibitory to C. reinhardtii during cryopreservation should explain why this alga cryopreserves best at a low cell density, and may lead to protocols that facilitate the more successful cryopreservation of C. reinhardtii and related algae.     

Is the cell division cycle gated by a circadian clock? The case of Chlamydomonas reinhardtii

Circadian oscillators are known to regulate the timing of cell division in many organisms. In the case of Chlamydomonas reinhardtii, however, this conclusion has been challenged by several investigators. We have reexamined this issue and find that the division behavior of Chlamydomonas meets all the criteria for circadian rhythmicity: persistence of a cell division rhythm (a) with a period of approximately 24 h under free-running conditions, (b) that is temperature compensated, and (c) which can entrain to light/dark signals. In addition, a mutation that lengthens the circadian period of the phototactic rhythm similarly affects the cell division rhythm. We conclude that a circadian mechanism determines the timing of cell division in Chlamydomonas reinhardtii.     

Chlamydomonas sensory rhodopsins A and B: cellular content and role in photophobic responses

Two retinylidene proteins, CSRA and CSRB, have recently been shown by photoelectrophysiological analysis of RNAi-transformants to mediate phototaxis signaling in Chlamydomonas reinhardtii. Here we report immunoblot detection of CSRA and CSRB apoproteins in C. reinhardtii cells enabling assessment of the cellular content of the receptors. We obtain 9 x 10(4) CSRA and 1.5 x 10(4) CSRB apoprotein molecules per cell in vegetative cells of the wild-type strain 495, a higher value than that for functional receptor cellular content estimated previously from photosensitivity measurements and retinal extraction yields. Exploiting our ability to control the CSRA/CSRB ratio by transformation with receptor gene-directed RNAi, we report analysis of the CSRA and CSRB roles in the photophobic response of the organism by action spectroscopy with automated cell tracking/motion analysis. The results show that CSRA and CSRB each mediate the photophobic swimming response, a second known retinal-dependent photomotility behavior in C. reinhardtii. Due to the different light saturation and spectral properties of the two receptors, CSRA is dominantly responsible for photophobic responses, which appear at high light intensity.     

Seasonal analysis of Daphnia pulicaria swimming behavior

Our study documents individual swimming behavior of Daphnia pulicaria over a yearly cycle in a temperate lake. We collected D. pulicaria, a common freshwater zooplankton, from Lake Mendota on 10 dates between July 1994 and June 1995 from two depths, 2 m and 10 m. The Daphnia were rushed to the laboratory and video-taped as they swam in lake water under lake-ambient temperature and light conditions. Five-second swimming tracks of individual Daphnia were filmed and digitized using a motion analysis system. We measured average turning angle, swimming speed and sinking rate for each track. D. pulicaria swimming behavior varied over the annual cycle. We found significant differences in turning angle between depths and among months. Sinking rate and swimming speed were significantly different among months but not depths. Sinking rate and swimming speed were not significantly correlated with water temperature. Our results were contrary to Stokes' Law predictions, in that D. pulicaria had the slowest sinking speed in June, not in the winter when water temperatures were lowest and viscosity was highest. Body length was significantly correlated with all three swimming variables. We also studied swimming behavior in clonal populations of D. pulicaria in different concentrations of the alga, Chlamydomonas reinhardtii. D. pulicaria did not change swimming speed, turning angle or sinking rate over a range of food concentrations. Finally, swimming behavior in a D. pulicaria clone, tested at two temperatures in the laboratory, confirmed the results from our seasonal study; Daphnia did not sink as predicted by changes in viscosity.     

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.     

Molecular studies of linkage group XIX of Chlamydomonas reinhardtii: evidence against a basal body location

Linkage group XIX (also known as the UNI linkage group) in the green alga, Chlamydomonas reinhardtii, exhibits a number of unusual properties that have lead to the suggestion that it represents a basal body-associated chromosome. To begin a molecular analysis of this linkage group, we have identified DNA sequences from it and used them to determine the copy number of linkage group XIX within the cell. We find that linkage group XIX is present in the same copy number per cell as nuclear linkage groups in both haploid and diploid strains. We also find that the copy number of linkage group XIX is unchanged in mutants lacking basal bodies. We conclude that there is no convincing evidence that linkage group XIX localizes to the basal bodies of Chlamydomonas reinhardtii cells.     

A quasi-elastic light scattering and cinematographic investigation of motile Chlamydomonas reinhardtii.

Quasi-elastic light scattering and cinematographical techniques were used to investigate the motility of Chlamydomonas reinhardtii (wild type). It was found that quantitative information on the trajectory of motion was required for a meaningful interpretation of the autocorrelation functions. Two models for describing the oscillatory motion of the cell were developed; one based on the instantaneous forward-and-backward motion of the cell, and the other based on a sinusoidal perturbation to the average forward motion. Both models gave satisfactory agreement with the shape of the experimentally measured autocorrelation function, thus making it possible to use this measurement to determine mean progressive swimming velocities in a population of greater than 200 cells.     

Chloroplast DNA variation in Chlamydomonas and its potential application to the systematics of this genus

We have estimated the extent of chloroplast DNA (cpDNA) variation in three species of green algae belonging to the genus Chlamydomonas to determine if this variation could be used for taxonomic studies. The overall arrangement of sequences in the chloroplast genome of Chlamydomonas eugametos was compared with that of the closely related C. moewusii and that of the more distantly related C. reinhardtii. The results show that the chloroplast genomes of C. eugametos and C. moewusii are essentially co-linear and are highly homologous in sequence while those of C. eugametos and C. reinhardtii have been extensively rearranged and share a relatively low overall sequence homology. This wide range of chloroplast genome organization suggests that the analysis of cp-DNA variation will be useful for the classification of algae belonging to the Chlamydomonas genus.     

Kinetics of excitation trapping in intact Photosystem I of Chlamydomonas reinhardtii and Arabidopsis thaliana

We measured picosecond time-resolved fluorescence of intact Photosystem I complexes from Chlamydomonas reinhardtii and Arabidopsis thaliana. The antenna system of C. reinhardtii contains about 30-60 chlorophylls more than that of A. thaliana, but lacks the so-called red chlorophylls, chlorophylls that absorb at longer wavelength than the primary electron donor. In C. reinhardtii, the main lifetimes of excitation trapping are about 27 and 68 ps. The overall lifetime of C. reinhardtii is considerably shorter than in A. thaliana. We conclude that the amount and energies of the red chlorophylls have a larger effect on excitation trapping time in Photosystem I than the antenna size.     

Flagellar quiescence in Chlamydomonas: Characterization and defective quiescence in cells carrying sup-pf-1 and sup-pf-2 outer dynein arm mutations

Chlamydomonas reinhardtii can use their flagella for two distinct types of movement: swimming through liquid or gliding on a solid substrate. Cells switching from swimming to gliding motility undergo a reversible flagellar quiescence. This phenomenon appears to involve the outer dynein arms, since mutants having altered outer arm beta and gamma dyneins (sup-pf-1 and sup-pf-2) show a diminished ability to quiesce. Sup-pf-1 and sup-pf-2 were originally isolated as gain-of-function mutations that suppress the flagellar paralysis resulting from radial spoke or central pair defects. Defective quiescence is also a gain-of-function phenomenon, as cells completely lacking outer arm heavy chains show a normal quiescence phenotype. These data suggest that regulation of outer arm dynein activity is essential for flagellar quiescence and furthermore that regulation of quiescence involves a signal transduction pathway that shares elements with the radial spoke/central pair system.     

Helical swimming can provide robust upwards transport for gravitactic single-cell algae; a mechanistic model

In still fluid, many phytoplankton swim in helical paths with an average upwards motion. A new mechanistic model for gravitactic algae subject to an intrinsic torque is developed here, based on Heterosigma akashiwo, which results in upwards helical trajectories in still fluid. The resultant upwards swimming speed is calculated as a function of the gravitactic and intrinsic torques. Helical swimmers have a reduced upwards speed in still fluid compared to cells which swim straight upwards. However a novel result is obtained when the effect of fluid shear is considered. For intermediate values of shear and intrinsic torque, a new stable equilibrium solution for swimming direction is obtained for helical swimmers. This results in positive upwards transport in vertical shear flow, in contrast to the stable equilibrium solution for straight swimmers which results in downwards transport in vertical shear flow. Furthermore, for strong intrinsic torque, when there is no longer a stable orientation equilibrium, we show that the average downwards transport of helical swimmers in vertical shear flow is greatly suppressed compared to straight swimmers. We hypothesise that helical swimming provides robustness for upwards transport in the presence of fluid shearing motions.     

NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii

A precursor (pS) to the small subunit (S) of ribulose1-,5-bisphosphate carboxylase is the major product of cell-free protein synthesis directed by poly(A) containing RNA from Chlamydomonas reinhardtii. We present sequence data for in vitro-synthesized pS, for in vitro- synthesized S that in generated from pS by posttranslational incubation with a Chlamydomonas cell extract, and for in vitro-synthesized, mature S. We show that pS contains an NH2-terminal extension of 44 amino acid residues that is removed by cleavage at the correct site when pS is converted to S by an endoprotease present in the Chlamydomonas cell extract.     

Selective inhibition of flagellar activity in Chlamydomonas by nickel.

Flagellar activity in the biflagellate chlorophyte Chlamydomonas reinhardtii is selectively inhibited by Ni2+ or by treatment with Ca2+-chelating agents. Inhibitions of swimming speed, geotaxis, phototaxis, and pattern swimming result from qualitative and quantitative losses in the activity of individual flagella and in the coordination of activity between the 2 flagella of each cell. Addition of Ca2+ (a) prevents inhibition and (b) restores normal flagellar activity in inhibited cells. Mg2+ is partially effective in reversal of inhibition. Other ions do not cause similar inhibition or reversal of nickel inhibition. The characteristics of inhibition and reversal suggest that the primary target for nickel is a component of the flagellar apparatus, and that this component uses Ca2+ to perform its normal function in the regulation of flagellar activity. A 2nd target for nickel is a Ca-requiring process specific to phototaxis (and not involved in the photophobic response).     

A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii†

The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.