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3-D architecture of growing flagella.
J. L. Hoog1, S. Lacomble1, E. O'Toole2, C. Bouchet-Marquis2, R. J. McIntosh2, A. Hoenger2, K. Gull1; 1University of Oxford, Oxford, United Kingdom, 2University of Colorado
Many cells have a cilium or flagellum, organelles that generate cell motility and important sensory and signaling organelles. Two of the most studied flagella with a canonical 9+2 structure are those of the green-algae Clamydomonas reinhardtii and the parasitic protozoa Trypanosoma brucei. C. reinhardtii has two flagella that are reabsorbed and shed prior to mitosis. T. brucei has a single flagellum for about half its cell cycle when a new flagellum starts growing along the pre-existing flagellum in preparation for mitosis. Thus, growing flagella can be found in recently divided C. reinhardtii and in T. brucei cells with two flagella. The large protein complexes containing the flagellar building blocks are delivered to the site of axonemal elongation in an evolutionary conserved process called intra flagellar transport (IFT). IFT transport is well studied, yet we know little about the order by which these structural components of the axoneme assemble into the growing flagellum. To examine the process of axoneme elongation, we studied the tips of growing flagella in C. reinhardtii and T. brucei using
electron tomography. The axoneme is disorganized during growth in long T. brucei flagella since associated proteins are not added at the same rate as MTs grow. We also show that membrane extension is not dependent on axonemal pushing.
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A novel distal appendage protein required for primary cilium formation. B. Tanos1, W-J. Wang1, R. Soni1, M. Tsou1; 1Cell Biology, MSKCC, New York, NY
Primary cilia (PC) are microtubule-based organelles that play important mechano- and chemosensory roles in eukaryotic cells, and whose function is compromised in a number of diseases including polycystic kidney disease, obesity, and certain forms of mental retardation. The basal body that functions in PC assembly derives from the older mother centriole, which can be distinguished from the younger mother by the acquisition of subdistal and distal appendages, which are required for cilia formation. Here we have identified CCDC41 as a component of the mother centriole. We found that CCDC41 is recruited to mother centrioles between late S and the beginning of G2 phases of the cell cycle, coincident with the recruitment of two other previously characterized appendage proteins, Cep164 and Odf2, but prior to the recruitment of a third appendage protein, centriolin. Furthermore, CCDC41 colocalizes with Cep164, a distal appendage protein, but not with Odf2 or Centriolin, both subdistal appendage proteins. Knockdown of CCDC41 resulted in a defect in primary cilia formation and length, and a failure to recruit Cep164 and Odf2, but had no effect on centriolin recruitment. We hypothesize that CCDC41 is recruited to mother centrioles during appendage formation in a complex with other appendage proteins that likely includes Cep164.
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The pf27 mutant is defective in radial spoke transport in the cilium.
L. M. Alford1, S. Dutcher2, W. Sale1; 1Emory Univ Sch Med, Atlanta, GA, 2Washington Univ Sch Med
Ciliary proteins are generated in the cell body and subsequently targeted to precise locations in the cilium. To address the mechanisms of assembly and transport, we focused on assembly of the radial spoke structure. The radial spoke first assembles as 12S precursor complexes in the cytoplasm. This is followed by IFT mediated transport in the ciliary compartment and docking of a fully assembled 20S radial spoke structure (Diener et al., 2011; Yang et al., 2005; Qin et al. 2004). We took advantage of the Chlamydomonas reinhardtii mutant pf27, which is deficient in ciliary radial spokes (Huang et al., 1981). Comparison of cytoplasmic extracts from pf27 and wild-type show no difference in composition of the 12S radial spoke precursor complex. Interestingly, the spokes that assemble in pf27 are localized to the proximal third of the axoneme, but otherwise are fully assembled into the mature 20S radial spoke complexes. Furthermore, 20S spoke complexes derived from wild-type axonemes are competent to bind pf27 axonemes in vitro. Thus, pf27 is apparently not defective in radial spoke assembly in the cytoplasm or docking of radial spokes to the axoneme. Rather, our results suggest that pf27 is defective in the transport of spoke complexes. To further test this idea, complementation in temporary dikaryons of wild-type and pf27 reveal rescue of radial spoke assembly from tip to base as previously described for other radial spoke mutants (Johnson and Rosenbaum, 1992). Additionally, Next-Generation high-throughput sequencing of pf27 reveals a mutation in a candidate flagellar associated protein (FAP) predicted to be a kinase. Consistent with this prediction, radial spoke proteins exhibit reduced phosphorylation in pf27 axonemes (Huang et al., 1981). Based on these results, we hypothesize that PF27 encodes a protein kinase required for facilitating interaction between the radial spoke precursor complex and the ciliary IFT
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A Novel Cytoplasmic Protein Necessary for Proper Assembly of Eukaryotic Flagella. B. W. Smith1, D. R. Mitchell2; 1SUNY Upstate Med Univ, Syracuse, NY
Nearly all organisms in the eukaryotic branch of life are dependent on motile (9+2) cilia. Proper function of motile cilia is essential for normal embryonic development, mucous clearance from the respiratory tract and reproduction. A breakdown of normal ciliary function can lead to Primary Ciliary Dyskinesia (PCD), which is characterized by respiratory disorders, male infertility and female subfertility as well as body plan defects. As cilia have multiple regulatory structures, including the central pair, radial spokes and the dynein regulatory complex, a defect with even a portion of any of these structures may result in PCD. Little is currently understood about how these structures are assembled, this is especially the case with the central pair. Using the model organism Chlamydomonas reinhardtii, we have isolated a new central pair mutant called uncoordinated 1(unc1). Unc1 was originally identified as nearly immotile, resulting from asymmetrically activating flagella that often stall mid-bend. Electron microscopy of unc1 axonemes shows that the C2b projection is missing in 95% of observed cross-sections, and of these about half are missing part or all of the C2 microtubule and its associated projections. Using available antibodies for central pair proteins, western blotting of unc1 axonemes reveals reductions in Klp1 and Hydin, while Cpc1 and PF6 levels are equal to that of wild-type, confirming that unc1 results in C2 specific defects. The UNC1 locus maps along LG V, and is part of a tandem duplication. Both UNC1 and its tandem duplicate are predicted subtilisin-like serine proteases. Knockdown of the tandem duplicate with an amiRNA construct results in a similar motility phenotype to unc1. Transformation of a epitope-tagged UNC1 construct restores motility with a normal waveform. Isolation of cell bodies, cytoplasmic extracts and flagella from rescued cells shows that UNC1-HA localizes to the cell body, supporting a potential cytoplasmic role for UNC1p in central pair pre-assembly processing events. Supported by NIGMS 44228 to DRM.
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Regulation of ciliary assembly through intron retention.
T. C. Boothby1, S. M. Wolniak1; 1Cell Biology and Molecular Genetics, University of Maryland, College Park, MD
The microspore of Marsilea vestita is a meiotic product that produces a male gametophyte. A series of nine divisions in precise planes generates a gametophyte comprising 7 sterile cells and 32 spermatozoids. Each spermatozoid possesses ~140 cilia. Gametophyte development is rapid and temporally precise (reaching completion in 11 h), spatially precise (cells and division planes are fixed within the microspore), and transcriptionally quiescent (the microspore stockpiles proteins and RNA, which are used to carry out spermatogenesis with no new RNA production). We employed deep sequencing (Solexa Illumina) and de novo transcriptome assembly (Trinity) to gain insights into how the gametophyte regulates the storage, processing and translation of stored RNA.
In silico analysis was used to generate a list of annotated ‘early’ transcripts predicted to contain retained introns. Many of these intron-containing transcripts encode proteins whose biological functions are required during late stages of development. A subset of these transcripts (e.g., PF16/SPAG6, PFK, IFT88/OSM-6, FAP234, FAP71, Shaggy/GSK3) has previously been found to be essential for ciliary/flagellar axoneme formation or are associated with axonemal structures. Early in gametophyte development, ~30% of all identified ciliary/flagellar axonemal transcripts contain retained introns. Comparisons of genomic and RNA sequences of ‘early’ intron-retaining ciliary transcripts reveals that these transcripts possess at least 1 intron embedded in the coding sequence both in the desiccated microspore and during early
gametophyte development, while other introns within and beyond the ORFs had been previously removed from the pre-mRNAs. In all cases examined, these retained introns are within the coding sequence of the transcript, code for a premature stop codon, and usually disrupt conserved encoded protein domains. Isoforms of these ‘early’ intron-containing transcripts were found at later stages of development to lack any introns, and this loss of intron retention results in an mRNA encoding for a full length, functional protein. We performed RNAi knockdowns of intron containing transcripts at the onset of development. Detectable anomalies in phenocopies were not observed until after the developmental time point when these transcripts are spliced, suggesting that the later spliced isoforms of these transcripts are developmentally essential, while the intron-containing transcripts might serve only as translationally/functionally incompetent precursors. While the proportion of ciliary/flagella axonemal transcripts found in our study to retain introns (8) constitutes only a small portion of the ciliome (i.e., 124 components in C. reinhardtii) it is important to note that several of these intron-retaining transcripts encode proteins essential for ciliary/axoneme formation and/or stability (e.g., SPAG6 and OSM-6). Our data suggest that the retention of introns may be a mechanism involved in temporally regulating the onset of translation of essential ciliary axonemal proteins and thus, forestall the assembly of cilia in the rapidly developing gametophyte until late in spermatid maturation. Supported by NSF grant 0842525 to S.M.W. 261
Whole genome transcriptome analysis identifies new cilia genes.
A. J. Albee1, A. L. Kwan2, G. D. Stormo1, S. K. Dutcher1; 1Genetics, Washington University School of Medicine, St. Louis, MO, 2Molecular and Cellular Technologies, Platform Technology and Science, GlaxoSmithKline, Collegeville, United Kingdom
Cilia are microtubule based organelles that project from a cell. Cilia are found on almost every cell type of the human body and numerous diseases are associated with defects in cilia including respiratory infections, male infertility, situs inversus, polycystic kidney disease, retinal degeneration, and Bardet-Biedl Syndrome. Mass spectrometry analysis has identified over 600 proteins that compose cilia. At the base of cilia lie basal bodies that template cilia and recruit proteins for ciliary assembly. To understand how cilia are formed, we used Illumina-based whole genome transcriptome analysis during flagellar growth in the biflagellate green alga Chlamydomonas reinhardtii. Chlamydomonas cells were deflagellated by pH shock and then sampled at 3, 10, 30, and 60 min during flagellar growth for whole transcriptome analysis. We identified over 1400 genes that were upregulated during flagellar growth. We also used phylogenetic profiling to identify genes that are conserved in organisms with basal bodies, triplet microtubules and motile cilia; we found 65 proteins that have coevolved in Chlamydomonas, humans, zebrafish and moss and are missing from land plants, fungi, and nematodes. We focus on those genes that are conserved among this set of ciliated organisms and a set of genes that are up-regulated during flagellar growth. To validate these genes, we used 4-5 different shRNA lentiviral constructs to knockdown each candidate gene in human retinal pigment epithelial cells (hTERT-RPE) stably expressing centrin-GFP, a basal body/centriole marker. We focused our analysis on five phenotypes: the percent ciliated cells, cilia length, numbers of basal bodies/centrioles, distance between basal bodies/centrioles, and cell cycle defects as determined by EdU staining. We show that knockdown of GLOD4, a glyoxylase implicated in retinal degeneration, is upregulated 7-fold following deflagellation in Chlamydomonas. Knockdown in RPE cells leads to decreased ciliation of cells and the severity of the ciliary phenotype is correlated to the GLOD4 mRNA levels. ZMYND10, a zinc finger protein containing an MYND domain, is upregulated 71 fold following deflagellation in Chlamydomonas. Knockdown in RPE cells leads to cell cycle defects without affecting cilia or basal body/centriole numbers. In total, we validated 22 of our predicted genes and a large proportion of these show
defects in at least one of the five phenotypic categories. Characterization of these genes helped us to gain insight into the molecular mechanism by which centrosomes and cilia are assembled. Research is funded by grants from the National Institutes of Health.
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A Forward Enhancer Screen in C. elegans to Identify Novel Ciliopathy Genes.
J. Pieczynski1, S. Masyukova2, D. Landis2, S. Henke2, C. Williams1, B. Yoder1,2; 1Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, 2Department of Genetics, University of Alabama at Birmingham
Cilia are signaling organelles and defects in cilia signaling or structure lead to diseases referred to as ciliopathies. Many ciliopathies including, Nephronopthisis (NPHP) and Meckel-Gruber Syndrome (MKS), are autosomal recessive disorders and can involve a series of mutations in multiple, overlapping genetic loci, most of which have yet to be identified. It has been hypothesized that the complex genetic program of ciliopathies leads to a spectrum of related phenotypes determined by the nature of the mutation and/or the combination of different mutations (mutational load) in the patient’s background. Genetic screens in C. elegans can be utilized to identify candidate genes that contribute to cilia dysfunction. The ciliated sensory neurons (CSNs) of C. elegans can be used to assess the mutational load of ciliopathy defects based on quantifiable assays including CSN regulated behaviors and the ability of CSNs to uptake lipophilic dye. Worms unable to take up dye are designated Dyf, for dye-filling defective. We previously demonstrated this phenomenon by crossing worms mutant for the homolog of the human ciliopathy gene NPHP4 (nphp4-/-) with worms mutant for a homolog of a human MKS gene (mks(x)-/-). Alone, nphp4-/- or mks(x)-/- have very mild or no Dyf phenotypes but when crossed, the resultant nphp4-/-;mks(x)-/- F2 generation display enhanced Dyf phenotypes as well
as behavioral phenotypes indicative of CSN defects. Using this nphp4-/- Dyf-based enhancer phenotype paradigm we performed chemical mutagenesis on nphp4-/- mutant worms to identify genes that enhance Dyf phenotypes and contribute to mutational load. After outcrossing, we identified nine independent worm strains with synergistic Dyf phenotypes on the nphp4-/- mutant background that were more severe than those found in nphp4-/- mutants alone. The enhanced Dyf phenotype in these strains segregated in ratios indicative of double recessive mutations. Bulk chromosomal analysis and non-complementation tests were used to map mutations and check for allelism. After mapping, we employed Illumina deep sequencing to identify causative genes/alleles. We have begun to analyze the function of new candidates in an effort to understand how mutations in these genes cause synergistic phenotypes with the NPHP4 mutation. Genes from this screen are likely to be strong candidates for novel human ciliopathy genes.
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Structural and functional transition of choroid plexus epithelial cilia revealed by proteomic analysis.
K. Narita1, H. Kozuka-Hata2, Y. Nonami3, H. Ao-Kondo2, M. Oyama2, T. Inoue3, S. Takeda1;
1Department of Anatomy and Cell Biology, Interdisciplinary Graduate School of Medicine &
Engineering, University of Yamanashi, Chuo, Japan, 2Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Japan, 3Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku, Japan
Mammalian cilia and flagella have been classified into three major subtypes depending on the function and axonemal structure, namely, motile 9+2 cilia, non-motile 9+0 primary cilia and motile 9+0 nodal cilia. In addition, whereas 9+2 cilia are expressed as many as hundreds, 9+0 primary and nodal cilia are usually solitary. One of the rare exceptions is the multiple 9+0
primary cilia expressed in choroid plexus epithelial cells (CPECs). We have previously reported that CPEC cilia modulate the production of cerebrospinal fluid (Narita et al., Traffic, 11:287-301, 2010). In the present study, we performed a proteomic analysis of CPEC cilia to elucidate their exceptional features in molecular basis. By LC-MS analysis of proteins from purified swine CPEC cilia, 868 proteins were identified as a CPEC ciliome, of which 419 were not shared with other ciliome datasets. Unexpectedly, we found several molecules implicated in ciliary motility in the CPEC ciliome. Real-time PCR also validated the expression of these molecules in mouse CPECs. Immunostaining for one of them, radial spoke head 9 homolog (Rsph9), demonstrated that the molecule localized to a subpopulation of CPEC cilia. Live imaging of choroid plexus tissue exhibited that some CPEC cilia could beat vigorously at the neonatal stage, whereas adult CPEC cilia were non-motile. When observed with high-speed video microscopy, the beating pattern appeared to be similar to that of typical 9+2 cilia of ependyma, though several parameters were different. The motility of CPEC cilia did not produce any directional planar flow, as assessed by the movement of fluorescent microbeads added to the medium. Transmission electron microscopy of P1 mouse choroid plexus revealed that the coexistence of 9+0 and 9+2 cilia. In conclusion, our proteomic analysis revealed unique structural and functional changes of CPEC cilia, which highlight the diversities of mammalian cilia. Based on the present study, we propose a new concept on ciliary subtype classification to deal with various atypical cilia systematically, with consideration to possible molecular mechanisms underlying the diversity. 264
Epsilon tubulin is essential for the formation and maintenance of basal bodies in Tetrahymena thermophila.
I. Ross1, C. Clarissa2, T. Giddings2, M. Winey2; 1MCD Biology, University of Colorado, Boulder, CO, 2MCD Biology, University of Colorado
Basal bodies and centrioles are conserved microtubule based organelles whose improper assembly leads to a number of diseases, including ciliopathies and cancer. Tubulin family members play integral roles in the function of these structures. I have identified the ε-tubulin gene in Tetrahymena thermophila and localized it through immunofluorescence to basal bodies. Immunoelectron microscopy has revealed that ε-tubulin localizes to the three main domains of the basal body: the proximal end, the distal end, and the microtubule scaffold. A complete knockout of ε-tubulin reveals that it is an essential gene required for the assembly and maintenance of the core triplet microtubule structure of basal bodies. Most recently we have identified possible novel binding partners of ε-tubulin that may shed insight into the mechanism of ε-tubulin’s function. Concurrently, a mutational analysis of ε-tubulin is underway, with the hope of establishing a structure function relationship for this protein. The results of this research have enhanced our current understanding of the function of ε-tubulin and will further shed light on its role at basal bodies.
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The two human centrin homologues in Tetrahymena have similar but distinct functions at basal bodies.
T. Vonderfecht1, M. W. Cookson1, T. H. Giddings1, C. Clarissa1, M. Winey1; 1Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO
Centrins are a ubiquitous family of small Ca2+ binding proteins at centrioles and basal bodies. Structurally, they consist of two domains tethered by a short linker with each domain containing a pair of EF hands, a Ca2+ binding motif. Centrins are grouped into two groups based on sequence similarity to the human centrins, Centrin 2 and Centrin 3, and analyses of components that make up centrioles or basal bodies in different species suggest that they
contain a centrin isoform from each group. The study of centrins has largely focused on those belonging to the human Centrin 2 group, while neglecting those in the human Centrin 3 group.