\u3cem\u3eChlamydomonas Reinhardtii ODA5\u3c/em\u3e Encodes an Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Flagellar Adenylate Kinase: A Dissertation

The first type of dynein identified, axonemel dynein (Gibbons and Rowe, 1965), slides adjacent microtubules within the axoneme, generating the force necessary for ciliary and flagellar beating. The outer dynein arm is an important component of the flagellar axoneme, providing up to 60% of the force for flagellar motility. In the absence of the outer arm, cells swim with a slow-jerky motion at about 1/3rd the speed of wild-type cells, and the flagellar beat frequency is markedly reduced. Sixteen genes (ODA1-ODA16) have been identified that are required for outer arm assembly in Chlamydomonas reinhardtii. In addition, PF13, PF22, and FLA14 are required for outer dynein arm assembly, but their phenotypes are pleiotropic, suggesting that they affect additional flagellar components. Of the uncloned genes, ODA5, ODA8, and ODA10 are of particular interest because they do not encode subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC). Mutant alleles of these genes are unable to complement in temporary dikaryons, suggesting that the gene products interact with each other (Kamiya, 1988). Since the genes encoding all of the known components of the outer dynein arm and the ODA-DC have been characterized, it is of great interest to identify the gene products of these additional, uncloned ODA alleles. The first chapter provides an introduction to the Chlamydomonasflagellum, the dyneins in general, the outer dynein arm in particular, and mutations that impinge on the assembly and regulation of this important axonemal structure. The second chapter addresses the identification and isolation of genomic DNA containing the ODA5 gene. Utilizing a NIT1-tagged oda5-insertional mutant, I identified sequences flanking the site of the inserted NIT1 gene. These sequences were used to isolate wild-type genomic clones spanning the ODA5 gene. When transformed into the oda5 mutant, the wild-type clones rescued the mutant phenotype. These results demonstrated the successful isolation of the ODA5 gene. The third chapter describes the identification of the ODA5 gene and its corresponding cDNA. The rescuing genomic fragments were sequenced. Gene modeling was used to predict intron-exon splice sites. Primers to predicted exons were designed and used to obtain the ODA5 cDNA. The gene structure of Oda5 was analyzed and its predicted amino acid sequence deduced. Secondary structure predictions indicate that Oda5p is likely to contain a series of coiled-coil domains, followed by a poly-glycine sequence and a short, highly charged region. Northern analysis demonstrated that ODA5 gene expression is upregulated by deflagellation, a hallmark of many flagellar mRNAs. Data in CHAPTER IV further characterize the Oda5 protein and its association with the axoneme. Oda5p localizes to the flagellum, consistent with the enhancement in mRNA levels in response to deflagellation. Within the flagellum, Oda5p is an axonemal component that is released from the axoneme upon high salt extraction, as are the ODA-DC and the outer dynein arm. However, Oda5p does not associate with this super-complex in the high salt extract as determined by sucrose gradient sedimentation. Oda5p assembles onto the axoneme independently of the outer dynein arm and the ODA-DC,demonstrating it does not require these complexes for localization. Furthermore, Oda5p assembles onto the axoneme in the oda8, but not the oda10 mutant, demonstrating a role for the Oda10 protein in localization of Oda5p. These data provide the first biochemical evidence for an interaction between Oda5p and Oda10p. CHAPTER V reveals the discovery of a previously unrecognized phenotype exhibited in both oda5 and oda10 mutant strains: a defect in the assembly of a previously unknown flagellar adenylate kinase (AK). The protein levels of this flagellar AK are reduced in oda5 mutant axonemes, as determined by quantitative mass spectrometry. Direct enzymatic assays confirmed a reduction in AK activity in both oda5 and oda10 mutant axonemes, providing a second line of biochemical evidence supporting a complex containing Oda5p and OdalOp. The sequence of the flagellar AK gene and its cDNA were determined. CHAPTER VI details our efforts to identify the ODA10 gene. Genomic clones were isolated, which contain sequences at, or near, the ODA10 locus. Analysis of the genomic clones yielded no insights into the identity of the ODA10 gene. The inability of these clones to rescue the Oda10-motility phenotype indicates that these clones most likely do not contain an intact ODA10 gene. And lastly, CHAPTER VII discusses future experimentation that can be done based on the data provided by the current study

Novel LC8 Mutations Have Disparate Effects on the Assembly and Stability of Flagellar Complexes

LC8 functions as a dimer crucial for a variety of molecular motors and non-motor complexes. Emerging models, founded on structural studies, suggest that the LC8 dimer promotes the stability and refolding of dimeric target proteins in molecular complexes, and its interactions with selective target proteins, including dynein subunits, is regulated by LC8 phosphorylation, which is proposed to prevent LC8 dimerization. To test these hypotheses in vivo, we determine the impacts of two new LC8 mutations on the assembly and stability of defined LC8-containing complexes in Chlamydomonas flagella. The three types of dyneins and the radial spoke are disparately affected by dimeric LC8 with a C-terminal extension. The defects include the absence of specific subunits, complex instability, and reduced incorporation into the axonemal super complex. Surprisingly, a phosphomimetic LC8 mutation, which is largely monomeric in vitro, is still dimeric in vivo and does not significantly change flagellar generation and motility. The differential defects in these flagellar complexes support the structural model and indicate that modulation of target proteins by LC8 leads to the proper assembly of complexes and ultimately higher level complexes. Furthermore, the ability of flagellar complexes to incorporate the phosphomimetic LC8 protein and the modest defects observed in the phosphomimetic LC8 mutant suggest that LC8 phosphorylation is not an effective mechanism for regulating molecular complexes

The IDA3 adapter, required for intraflagellar transport of I1 dynein, is regulated by ciliary length

Axonemal dyneins, including inner dynein arm I1, assemble in the cytoplasm prior to transport into cilia by intraflagellar transport (IFT). How I1 dynein interacts with IFT is not understood. We take advantage of the Chlamydomonas reinhardtii ida3 mutant, which assembles the inner arm I1 dynein complex in the cytoplasm but fails to transport I1 into the cilium, resulting in I1 dynein-deficient axonemes with abnormal motility. The IDA3 gene encodes an ∼115-kDa coiled-coil protein that primarily enters the cilium during ciliary growth but is not an axonemal protein. During growth, IDA3, along with I1 dynein, is transported by anterograde IFT to the tip of the cilium. At the tip, IDA3 uncouples from IFT and diffuses within the cilium. IFT transport of IDA3 decreases as cilia lengthen and subsides once full length is achieved. IDA3 is the first example of an essential and selective IFT adapter that is regulated by ciliary length. </jats:p

Correction for \u3cem\u3eIC97 Is a Novel Intermediate Chain of I1 Dynein That Interacts with Tubulin and Regulates Interdoublet Sliding\u3c/em\u3e

Our goal is to understand the assembly and regulation of flagellar dyneins, particularly the Chlamydomonas inner arm dynein called I1 dynein. Here, we focus on the uncharacterized I1-dynein IC IC97. The IC97 gene encodes a novel IC without notable structural domains. IC97 shares homology with the murine lung adenoma susceptibility 1 (Las1) protein—a candidate tumor suppressor gene implicated in lung tumorigenesis. Multiple, independent biochemical assays determined that IC97 interacts with both α- and β-tubulin subunits within the axoneme. I1-dynein assembly mutants suggest that IC97 interacts with both the IC138 and IC140 subunits within the I1-dynein motor complex and that IC97 is part of a regulatory complex that contains IC138. Microtubule sliding assays, using axonemes containing I1 dynein but devoid of IC97, show reduced microtubule sliding velocities that are not rescued by kinase inhibitors, revealing a critical role for IC97 in I1-dynein function and control of dynein-driven motility

Regulation of dynein-driven microtubule sliding by the axonemal protein kinase CK1 in Chlamydomonas flagella

CK1 puts the brakes on dynein activity when added to purified axonemes in vitro, presumably to regulate how flagella bend

Comparative analysis of cryopreservation methods in Chlamydomonas reinhardtii

AbstractChlamydomonas is a model organism used for studies of many important biological processes. Traditionally, strains have been propagated on solid agar, which requires routine passaging for long-term maintenance. Cryopreservation of Chlamydomonas is possible, yet long-term viability is highly variable. Thus, improved cryopreservation methods for Chlamydomonas are an important requirement for sustained study of genetically defined strains. Here, we tested a commercial cryopreservation kit and directly compared it's effectiveness to a methanol-based method. We also tested thaw-back procedures comparing the growth of cells in liquid culture or on solid agar media. We demonstrated that methanol was the superior cryopreservation method for Chlamydomonas compared to the commercial kit and that post-thaw culture conditions dramatically affect viability. We also demonstrated that cryopreserved cells could be successfully thawed and plated directly onto solid agar plates. Our findings have important implications for the long-term storage of Chlamydomonas that can likely be extended to other algal species

Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase

Of the uncloned ODA genes required for outer dynein arm assembly in Chlamydomonas, ODA5 and ODA10 are of particular interest because they do not encode known subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC), and because genetic studies suggest their products interact. Beginning with a tagged oda5 allele, we isolated genomic and cDNA clones of the wild-type gene. ODA5 predicts a novel, 66-kDa coiled-coil protein. Immunoblotting indicates Oda5p is an axonemal component that assembles onto the axoneme independently of the outer arm and ODA-DC and is uniquely missing in oda5 and oda10 axonemes. Oda5p is released from the axoneme by extraction with 0.6 M KCl, but the soluble Oda5p does not cosediment with the outer dynein arm/ODA-DC in sucrose gradients. Quantitative mass spectrometry by using isotope coded affinity tagging revealed that a previously unidentified adenylate kinase is reduced 35–50% in oda5 flagella. Direct enzymatic assays demonstrated a comparable reduction in adenylate kinase activity in oda5 flagella, and also in oda10 flagella, but not in flagella of other oda mutants. We propose that Oda5p is part of a novel axonemal complex that is required for outer arm assembly and anchors adenylate kinase in proximity to the arm

Characterization of a new oda3 allele, oda3-6, defective in assembly of the outer dynein arm-docking complex in Chlamydomonas reinhardtii.

We have used an insertional mutagenesis approach to generate new C. reinhardtii motility mutants. Of 56 mutants isolated, one is a new allele at the ODA3 locus, called oda3-6. Similar to the previously characterized oda3 alleles, oda3-6 has a slow-jerky swimming phenotype and reduced swimming speed. The oda3-6 mutant fails to assemble the outer dynein arm motor and outer dynein arm-docking complex (ODA-DC) in the ciliary axoneme due to an insertion in the 5' end of the DCC1 gene, which encodes the DC1 subunit of the ODA-DC. Transformation of oda3-6 with the wild-type DCC1 gene rescues the mutant swimming phenotype and restores assembly of the ODA-DC and the outer dynein arm in the cilium. This is the first oda3 mutant to be characterized at the molecular level and is likely to be very useful for further analysis of DC1 function