To address the mechanisms of ciliary radial spoke assembly, we took advantage of the Chlamydomonas pf27 mutant. The radial spokes that assemble in pf27 are localized to the proximal quarter of the axoneme, but otherwise are fully assembled into 20S radial spoke complexes competent to bind spokeless axonemes in vitro. Thus, pf27 is not defective in radial spoke assembly or docking to the axoneme. Rather, our results suggest that pf27 is defective in the transport of spoke complexes. During ciliary regeneration in pf27, radial spoke assembly occurs asynchronously from other axonemal components. In contrast, during ciliary regeneration in wild-type Chlamydomonas, radial spokes and other axonemal components assemble concurrently as the axoneme grows. Complementation in temporary dikaryons between wild-type and pf27 reveals rescue of radial spoke assembly that begins at the distal tip, allowing further assembly to proceed from tip to base of the axoneme. Notably, rescued assembly of radial spokes occurred independently of the established proximal radial spokes in pf27 axonemes in dikaryons. These results reveal that 20S radial spokes can assemble proximally in the pf27 cilium but as the cilium lengthens, spoke assembly requires transport. We postulate that PF27 encodes an adaptor or modifier protein required for radial spoke – IFT interaction.
cilia; flagella; axonemes; microtubules; radial spokes
Conditional mutants for flagellar assembly (fla) provide a useful tool to study intraflagellar transport (IFT) at the molecular level, and provide a unique set of tools to analyze cilia. The analysis of IFT phenotypes of fla mutants at the permissive temperature by a quantitative image analysis approach identified four distinct phases of the IFT cycle and directly demonstrated structural and functional remodeling of IFT particles at both axonemal extremities. In addition, the genetic analysis of fla mutants reveal interesting interactions among genes involved in flagellar assembly that help to provide information about the structure and function of IFT particles and their motors. This chapter provides protocols to isolate, characterize, and identify conditional Chlamydomonas flagellar assembly mutants and their genes and to test genetic interactions among proteins encoded by these genes.
Correct centriole/basal body positioning is required for numerous biological processes, yet how the cell establishes this positioning is poorly understood. Analysis of centriolar/basal body duplication provides a key to understanding basal body positioning and function. Chlamydomonas basal bodies contain structural features that enable specific triplet microtubules to be specified. Electron tomography of cultures enriched in mitotic cells allowed us to follow basal body duplication and identify a specific triplet at which duplication occurs. Probasal bodies elongate in prophase, assemble transitional fibers (TF) and are segregated with a mature basal body near the poles of the mitotic spindle. A ring of nine-singlet microtubules is initiated at metaphase, orthogonal to triplet eight. At telophase/cytokinesis, triplet microtubule blades assemble first at the distal end, rather than at the proximal cartwheel. The cartwheel undergoes significant changes in length during duplication, which provides further support for its scaffolding role. The uni1-1 mutant contains short basal bodies with reduced or absent TF and defective transition zones, suggesting that the UNI1 gene product is important for coordinated probasal body elongation and maturation. We suggest that this site-specific basal body duplication ensures the correct positioning of the basal body to generate landmarks for intracellular patterning in the next generation.
Chlamydomonas; basal bodies; uni1; tomography
Cilia/flagella and basal bodies/centrioles play key roles in human health and homeostasis. Among the organisms used to study these microtubule-based organelles, the green alga Chlamydomonas reinhardtii has several advantages. One is the existence of a temporary phase of the life cycle, termed the dikaryon. These cells are formed during mating when the cells fuse and the behavior of flagella from two genetically distinguishable parents can be observed. During this stage, the cytoplasms mix allowing for a defect in the flagella of one parent to be rescued by proteins from the other parent. This offers the unique advantage of adding back wild-type gene product or labeled protein at endogenous levels that can used to monitor various flagellar and basal body phenotypes. Mutants that show rescue and ones that fail to show rescue are both informative about the nature of the flagella and basal body defects. When rescue occurs, it can be used to determine the mutant gene product and to follow the temporal and spatial patterns of flagellar assembly. This review describes many examples of insights into basal body and flagellar proteins’ function and assembly that have been discovered using dikaryons and discusses the potential for further analyses.
flagella; cilia; basal bodies; intraflagellar transport; ciliary trafficking
The building of a cilium or flagellum requires molecular motors and associated
proteins that allow the relocation of proteins from the cell body to the distal
end and the return of proteins to the cell body in a process termed intraflagellar
transport (IFT). IFT trains are carried out by kinesin and back to the cell body
We used whole genome sequencing to identify the causative mutations for two
temperature-sensitive flagellar assembly mutants in Chlamydomonas and
validated the changes using reversion analysis. We examined the effect of these
mutations on the localization of IFT81, an IFT complex B protein, the cytoplasmic
dynein heavy chain (DHC1b), and the dynein light intermediate chain (D1bLIC).
The strains, fla18 and fla24, have mutations in kinesin-2 and
cytoplasmic dynein, respectively. The fla18 mutation alters the same
glutamic acid (E24G) mutated in the fla10-14 allele
(E24K). The fla18 strain loses flagella at 32?C more
rapidly than the E24K allele but less rapidly than the fla10-1
allele. The fla18 mutant loses its flagella by detachment rather than by
shortening. The fla24 mutation falls in cytoplasmic dynein and changes a
completely conserved amino acid (L3243P) in an alpha helix in the AAA5
domain. The fla24 mutant loses its flagella by shortening within 6 hours
at 32?C. DHC1b protein is reduced by 18-fold and D1bLIC is reduced by 16-fold at
21?C compared to wild-type cells. We identified two pseudorevertants
(L3243S and L3243R), which remain flagellated at 32?C.
Although fla24 cells assemble full-length flagella at 21?C, IFT81 protein
localization is dramatically altered. Instead of localizing at the basal body and
along the flagella, IFT81 is concentrated at the proximal end of the flagella. The
pseudorevertants show wild-type IFT81 localization at 21?C, but proximal end
localization of IFT81 at 32?C.
The change in the AAA5 domain of the cytoplasmic dynein in fla24 may
block the recycling of IFT trains after retrograde transport. It is clear that
different alleles in the flagellar motors reveal different functions and roles.
Multiple alleles will be important for understanding structure-function
Kinesin-2; Cytoplasmic dynein; IFT81; Ciliary assembly; IFT recycling; Whole genome sequencing
Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect. A comparison of polymorphisms in the imp3 strain and the other 15 strains allowed us to identify a deletion of the last three amino acids, Y313F314L315, in a protein phosphatase 2A catalytic subunit (PP2A3) in the imp3 strain. Introduction of a wild-type HA-tagged PP2A3 rescues the mutant phenotype, but mutant HA-PP2A3 at Y313 or L315 fail to rescue. Our immunoprecipitation results indicate that the Y313, L315, or YFLΔ mutations do not affect the binding of PP2A3 to the scaffold subunit, PP2A-2r. In contrast, the Y313, L315, or YFLΔ mutations affect both the stability and the localization of PP2A3. The PP2A3 protein is less abundant in these mutants and fails to accumulate in the basal body area as observed in transformants with either wild-type HA-PP2A3 or a HA-PP2A3 with a V310T change. The accumulation of HA-PP2A3 in the basal body region disappears in mated dikaryons, which suggests that the localization of PP2A3 may be essential to the mating process. Overall, our results demonstrate that the terminal YFL tail of PP2A3 is important in the regulation on Chlamydomonas mating.
Whole genome sequencing is a powerful tool to detect changes in genomic DNA. However, how to identify a causative mutation from over 20,000 changes remains a big challenge. For the unicellular green alga Chlamydomonas, we built a library that consists of over 2 million changes from 16 strains. A comparison of changes found in a mutant strain with a mating defect, imp3, to 15 other strains, leads to the identification of a three amino acid deletion in the catalytic subunit of a protein phosphatase 2A (PP2A3). The mating defect of imp3 is rescued by an HA-tagged PP2A3 gene. Introduction of the HA-tagged PP2A3 gene with various mutations in these three amino acids reveals that they play a key role in stabilizing and ensuring the proper localization of PP2A3. The ubiquitous enzyme PP2A is involved in diverse cellular processes. Our discovery that PP2A3 is involved in the Chlamydomonas mating signaling pathway, which also contains the polycystin2 homolog (PKD2), makes Chlamydomonas mating an excellent model to study ciliary/flagellar signaling. Since both PP2A and PKD2 play important roles in human health, further investigation of the relationship between these two proteins in Chlamydomonas will facilitate better understanding of their functions.
Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.
Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.
A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.
Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.
Cilia are microtubule based organelles that project from cells. Cilia are found on almost every cell type of the human body and numerous diseases, collectively termed ciliopathies, are associated with defects in cilia, including respiratory infections, male infertility, situs inversus, polycystic kidney disease, retinal degeneration, and Bardet-Biedl Syndrome. Here we show that Illumina-based whole-genome transcriptome analysis in the biflagellate green alga Chlamydomonas reinhardtii identifies 1850 genes up-regulated during ciliogenesis, 4392 genes down-regulated, and 4548 genes with no change in expression during ciliogenesis. We examined four genes up-regulated and not previously known to be involved with cilia (ZMYND10, NXN, GLOD4, SPATA4) by knockdown of the human orthologs in human retinal pigment epithelial cells (hTERT-RPE1) cells to ask whether they are involved in cilia-related processes that include cilia assembly, cilia length control, basal body/centriole numbers, and the distance between basal bodies/centrioles. All of the genes have cilia-related phenotypes and, surprisingly, our data show that knockdown of GLOD4 and SPATA4 also affects the cell cycle. These results demonstrate that whole-genome transcriptome analysis during ciliogenesis is a powerful tool to gain insight into the molecular mechanism by which centrosomes and cilia are assembled.
flagella; deflagellation; ZMYND10; NXN; SPATA4; GLOD4
Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects.
Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.
The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, fifty-four proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologues of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles and orthologues has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.
Chlamydomonas; Cilia; Dynein; Flagella; Microtubule
Because of the highly conserved nature of the ciliary axoneme, researchers studying the structure and function of cilia have used many different model systems. Each system has advantages and disadvantages, but all provide important information relevant to the understanding and treatment of the ciliopathies. For example, Chlamydomonas is easy to grow and amenable to rapid genetic manipulation and therefore is excellent for motility studies and studies of the structural components of the axoneme. However, this organism cannot be used to study developmental defects or physiological abnormalities that occur in higher organisms (e.g., mucociliary clearance). Human cilia have the advantage of being obtained directly from the tissue of interest but are obtainable only in limited quantities and are difficult to manipulate. Mouse models of ciliopathies are more difficult to study than Chlamydomonas but can be useful to elucidate more aspects of the human diseases. In this review, the overlap between the structure of primary and motile cilia is discussed, and recent advancements in our understanding of cilia structure and function using these three different model systems are presented. Potential therapeutic approaches, based on fundamental knowledge gained from work in these model systems, are also presented.
proteomics; suppressor screens; heterotaxy; congenital heart disease
Analysis of Chlamydomonas axonemes revealed that the protein phosphatase, PP2A, is localized to the outer doublet microtubules and is implicated in regulation of dynein-driven motility. We tested the hypothesis that PP2A is localized to the axoneme by a specialized, highly conserved 55-kDa B-type subunit identified in the Chlamydomonas flagellar proteome. The B-subunit gene is defective in the motility mutant pf4. Consistent with our hypothesis, both the B- and C-subunits of PP2A fail to assemble in pf4 axonemes, while the dyneins and other axonemal structures are fully assembled in pf4 axonemes. Two pf4 intragenic revertants were recovered that restore PP2A to the axonemes and re-establish nearly wild-type motility. The revertants confirmed that the slow-swimming Pf4 phenotype is a result of the defective PP2A B-subunit. These results demonstrate that the axonemal B-subunit is, in part, an anchor protein required for PP2A localization and that PP2A is required for normal ciliary motility.
Cilia; flagella; dynein; axonemes; protein phosphatases; microtubules
Whole-genome sequencing (WGS) provides a new platform for the identification of mutations that produce a mutant phenotype. We used Illumina sequencing to identify the mutational profile of three Chlamydomonas reinhardtii mutant strains. The three strains have more than 38,000 changes from the reference genome. NG6 is aflagellate and maps to 269 kb with only one nonsynonymous change; the V12E mutation falls in the FLA8 gene. Evidence that NG6 is a fla8 allele comes from swimming revertants that are either true or pseudorevertants. NG30 is aflagellate and maps to 458 kb that has six nonsynonomous changes. Evidence that NG30 has a causative nonsense allele in IFT80 comes from rescue of the nonswimming phenotype with a fragment bearing only this gene. This gene has been implicated in Jeune asphyxiating thoracic dystrophy. Electron microscopy of ift80-1 (NG30) shows a novel basal body phenotype. A bar or cap is observed over the distal end of the transition zone, which may be an intermediate in preparing the basal body for flagellar assembly. In the acetate-requiring mutant ac17, we failed to find a nonsynonymous change in the 676 kb mapped region, which is incompletely assembled. In these strains, 43% of the changes occur on two of the 17 chromosomes. The excess on chromosome 6 surrounds the mating-type locus, which has numerous rearrangements and suppressed recombination, and the changes extend beyond the mating-type locus. Unexpectedly, chromosome 16 shows an unexplained excess of single nucleotide polymorphisms and indels. Overall, WGS in combination with limited mapping allows fast and accurate identification of point mutations in Chlamydomonas.
intraflagellar transport (IFT); flagellar assembly; mating-type; basal bodies; mapping
Mutations in the IC138 regulatory subunit of I1 dynein alter dynein motor activity and the flagellar waveform but do not affect phototaxis.
I1 dynein, or dynein f, is a highly conserved inner arm isoform that plays a key role in the regulation of flagellar motility. To understand how the IC138 IC/LC subcomplex modulates I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles. bop5-3, bop5-4, and bop5-5 are null alleles, whereas bop5-6 is an intron mutation that reduces IC138 expression. I1 dynein assembles into the axoneme, but the IC138 IC/LC subcomplex is missing. bop5 strains, like other I1 mutants, swim forward with reduced swimming velocities and display an impaired reversal response during photoshock. Unlike mutants lacking the entire I1 dynein, however, bop5 strains exhibit normal phototaxis. bop5 defects are rescued by transformation with the wild-type IC138 gene. Analysis of flagellar waveforms reveals that loss of the IC138 subcomplex reduces shear amplitude, sliding velocities, and the speed of bend propagation in vivo, consistent with the reduction in microtubule sliding velocities observed in vitro. The results indicate that the IC138 IC/LC subcomplex is necessary to generate an efficient waveform for optimal motility, but it is not essential for phototaxis. These findings have significant implications for the mechanisms by which IC/LC complexes regulate dynein motor activity independent of effects on cargo binding or complex stability.
Sequence similarity based protein clustering methods organize proteins into families of similar sequences, a task that continues to be critical for automated protein characterization. However, many protein families cannot be automatically characterized further because little is known about the function of any protein in a family of similar sequences. We present a novel phylogenetic profile comparison (PPC) method called Automated Protein Annotation by Coordinate Evolution (APACE) that facilitates the automated characterization of proteins beyond their homology to other similar sequences. Our method implements a new approach for the normalization of similarity scores among multiple species and automates the characterization of proteins by their patterns of co-evolution with other proteins that do not necessarily share a similar sequence. We demonstrate that our method is able to recapitulate the topology of the latest, unresolved, composite deep eukaryotic phylogeny and is able to quantify the as yet unresolved branch lengths. We further demonstrate that our method is able to detect more functionally related proteins, given the same starting data, than existing methods. Finally, we demonstrate that our method can be successfully applied to much larger comparative genomic problem instances where existing methods often fail.
The essential coenzyme nicotinamide adenine dinucleotide (NAD+) plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD+. Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD+ is synthesized from aspartate (de novo synthesis), as in plants, or nicotinamide, as in mammals (salvage synthesis). The de novo pathway requires five different enzymes: L-aspartate oxidase (ASO), quinolinate synthetase (QS), quinolate phosphoribosyltransferase (QPT), nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT), and NAD+ synthetase (NS). Sequence similarity searches, gene isolation and sequencing of mutant loci indicate that mutations in each enzyme result in a nicotinamide-requiring mutant phenotype in the previously isolated nic mutants. We rescued the mutant phenotype by the introduction of BAC DNA (nic2-1 and nic13-1) or plasmids with cloned genes (nic1-1 and nic15-1) into the mutants. NMNAT, which is also in the de novo pathway, and nicotinamide phosphoribosyltransferase (NAMPT) constitute the nicotinamide-dependent salvage pathway. A mutation in NAMPT (npt1-1) has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with the npt1-1 mutation and any of the nic mutations are inviable. When the de novo pathway is inactive, the salvage pathway is essential to Chlamydomonas for the synthesis of NAD+. A homolog of the human SIRT6-like gene, SRT2, is upregulated in the NS mutant, which shows a longer vegetative life span than wild-type cells. Our results suggest that Chlamydomonas is an excellent model system to study NAD+ metabolism and cell longevity.
Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme. NAD+ is necessary for electron transfer in many metabolic reactions. NAD+ functions as a substrate for several enzymes, one of which is sirtuin, an enzyme involved in gene regulation and aging. NAD+ can be synthesized either from amino acids (de novo) or metabolites (salvage). Given the importance of NAD+, enzymes involved in NAD+ synthesis are targets for drug discovery. In the unicellular green alga Chlamydomonas we investigated both the de novo and salvage NAD+ biosynthetic pathways. Mutations in the plant-like de novo synthesis pathway lead to a nicotinamide-requiring phenotype. We identified an insertional mutation in the first enzyme in the mammal-like salvage pathway; it has no growth defect in cells with an active de novo synthesis pathway but causes lethality when the de novo synthesis pathway is inactive. Coupled with NAD+ biosynthesis, sirtuin is involved in NAD+ consumption. Our study links upregulation of a sirtuin gene with extended life span in the nic13-1 mutant strain, which has a defective de novo synthesis pathway and suggests that Chlamydomonas is an excellent genetic model to study NAD+ metabolism and cell longevity.
Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.
The availability of whole-genome sequences allows for the identification of the entire set of protein coding genes as well as their regulatory regions. This can be accomplished using multiple complementary methods that include ESTs, homology searches and ab initio gene predictions. Previously, the Genie gene-finding algorithm was trained on a small set of Chlamydomonas genes and shown to improve the accuracy of gene prediction in this species compared to other available programs. To improve ab initio gene finding in Chlamydomonas, we assemble a new training set consisting of over 2,300 cDNAs by assembling over 167,000 Chlamydomonas EST entries in GenBank using the EST assembly tool PASA.
The prediction accuracy of our cDNA-trained gene-finder, GreenGenie2, attains 83% sensitivity and 83% specificity for exons on short-sequence predictions. We predict about 12,000 genes in the version v3 Chlamydomonas genome assembly, most of which (78%) are either identical to or significantly overlap the published catalog of Chlamydomonas genes . 22% of the published catalog is absent from the GreenGenie2 predictions; there is also a fraction (23%) of GreenGenie2 predictions that are absent from the published gene catalog. Randomly chosen gene models were tested by RT-PCR and most support the GreenGenie2 predictions.
These data suggest that training with EST assemblies is highly effective and that GreenGenie2 is a valuable, complementary tool for predicting genes in Chlamydomonas reinhardtii.
The Chlamydomonas anterograde intraflagellar transport motor, kinesin-2, is isolated as a heterotrimeric complex containing two motor subunits and a nonmotor subunit known as kinesin-associated polypeptide or KAP. One of the two motor subunits is encoded by the FLA10 gene. The sequence of the second motor subunit was obtained by mass spectrometry and sequencing. It shows 46.9% identity with the Fla10 motor subunit and the gene maps to linkage group XII/XIII near RPL9. The temperature-sensitive flagellar assembly mutants fla1 and fla8 are linked to this kinesin-2 motor subunit. In each strain, a unique single point mutation gives rise to a unique single amino acid substitution within the motor domain. The fla8 strain is named fla8-1 and the fla1 strain is named fla8-2. The fla8 and fla10 alleles show a chromosome loss phenotype. To analyze this chromosome loss phenotype, intragenic revertants of fla8-1, fla8-2, and fla10-14 were generated. The analysis of the mutants and the revertants demonstrates the importance of a pocket in the amino terminus of these motor subunits for both motor activity and for a novel, dominant effect on the fidelity of chromosome segregation.
Improved methods of specimen preparation and dual-axis electron tomography
have been used to study the structure and organization of basal bodies in the
unicellular alga Chlamydomonas reinhardtii. Novel structures have
been found in both wild type and strains with mutations that affect specific
tubulin isoforms. Previous studies have shown that strains lacking
δ-tubulin fail to assemble the C-tubule of the basal body. Tomographic
reconstructions of basal bodies from the δ-tubulin deletion mutant
uni3-1 have confirmed that basal bodies contain mostly doublet
microtubules. Our methods now show that the stellate fibers, which are present
only in the transition zone of wild-type cells, repeat within the core of
uni3-1 basal bodies. The distal striated fiber is incomplete in this
mutant, rootlet microtubules can be misplaced, and multiflagellate cells have
been observed. A suppressor of uni3-1, designated tua2-6,
contains a mutation in α-tubulin. tua2-6; uni3-1 cells build
both flagella, yet they retain defects in basal body structure and in rootlet
microtubule positioning. These data suggest that the presence of specific
tubulin isoforms in Chlamydomonas directly affects the assembly and
function of both basal bodies and basal body-associated structures.
Centrioles and basal bodies are cylinders composed of nine triplet microtubule blades that play essential roles in the centrosome and in flagellar assembly. Chlamydomonas cells with the bld2-1 mutation fail to assemble doublet and triplet microtubules and have defects in cleavage furrow placement and meiosis. Using positional cloning, we have walked 720 kb and identified a 13.2-kb fragment that contains ε-tubulin and rescues the Bld2 defects. The bld2-1 allele has a premature stop codon and intragenic revertants replace the stop codon with glutamine, glutamate, or lysine. Polyclonal antibodies to ε-tubulin show peripheral labeling of full-length basal bodies and centrioles. Thus, ε-tubulin is encoded by the BLD2 allele and ε-tubulin plays a role in basal body/centriole morphogenesis.
To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning.
We have cloned the UNI3 gene in Chlamydomonas and find that it encodes a new member of the tubulin superfamily. Although Uni3p shares significant sequence identity with α-, β-, and γ-tubulins, there is a region of Uni3p that has no similarity to tubulins or other known proteins. Mutant uni3–1 cells assemble zero, one, or two flagella. Pedigree analysis suggests that flagellar number in uni3–1 cells is a function of the age of the cell. The uniflagellate uni3–1 cells show a positional phenotype; the basal body opposite the eyespot templates the single flagellum. A percentage of uni3–1 cells also fail to orient the cleavage furrow properly, and basal bodies have been implicated in the placement of cleavage furrows in Chlamydomonas. Finally when uni3–1 cells are observed by electron microscopy, doublet rather than triplet microtubules are observed at the proximal end of the basal bodies. We propose that the Uni3 tubulin is involved in both the function and cell cycle-dependent maturation of basal bodies/centrioles.