[Show abstract][Hide abstract] ABSTRACT: During the assembly and maintenance of cilia, precursor proteins need to be transported from the cell body into the organelle. Intraflagellar transport (IFT) is assumed to be the predominant protein transport pathway in cilia, but it remains largely unknown how ciliary proteins use IFT to reach their destination sites in the cilium and whether the amount of cargo transported by IFT is regulated.
Single-particle imaging showed that DRC4, a structural protein of the axoneme, moves in association with IFT particles inside Chlamydomonas reinhardtii cilia. IFT is required for DRC4 transport both into and within the cilium. DRC4 cargoes dissociate from IFT trains at the tip as well as at various sites along the length of the cilium. Unloaded DRC4 diffuses before docking at its axonemal assembly site. In growing cilia, DRC4 transport by IFT was strongly increased over the steady-state level, and the frequency decreased linearly with the increasing ciliary length. The frequency of DRC4 transport was similarly elevated in short growth-arrested cilia and remained high even when the amount of DRC4 available in the cell body was reduced.
DRC4 is a bona fide cargo of IFT. Incompletely assembled cilia trigger an increase in the amount of DRC4 cargo transported by IFT particles, and DRC4 transport is downregulated as cilia approach their steady-state length. We propose a model in which ciliary length is controlled by regulating the amount of cargo transported by IFT particles.
Full-text · Article · Dec 2013 · Current biology: CB
[Show abstract][Hide abstract] ABSTRACT: The axonemal microtubules of cilia/flagella act as a scaffold for assembly of the protein complexes that ultimately regulate dynein activity to control the size and shape of ciliary bends. Despite our general understanding of the contribution of microtubule sliding to ciliary and flagellar motility, many questions regarding the regulation of dynein remain unanswered. For example, we know that the second messenger calcium plays an important role in modulating dynein activity in response to extracellular cues, but it remains unclear how calcium-binding proteins anchored to the axoneme contribute to this regulation. Recent work has focused on determining the identity and specific functions of these axonemal calcium-binding proteins. Here, we review our current knowledge of calcium-mediated motility and highlight key experiments that have substantially aided our understanding of calcium signaling within the axoneme.
No preview · Article · Mar 2013 · Methods in enzymology
[Show abstract][Hide abstract] ABSTRACT: Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ-ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo-electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.
Full-text · Article · Jun 2012 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: Virtually all motile eukaryotic cilia and flagella have a '9+2' axoneme in which nine doublet microtubules surround two singlet microtubules. Associated with the central pair of microtubules are protein complexes that form at least seven biochemically and structurally distinct central pair projections. Analysis of mutants lacking specific projections has indicated that each may play a unique role in the control of flagellar motility. One of these is the C1d projection previously shown to contain the proteins FAP54, FAP46, FAP74 and FAP221/Pcdp1, which exhibits Ca(2+)-sensitive calmodulin binding. Here we report the isolation and characterization of a Chlamydomonas reinhardtii null mutant for FAP46. This mutant, fap46-1, lacks the C1d projection and has impaired motility, confirming the importance of this projection for normal flagellar movement. Those cells that are motile have severe defects in phototaxis and the photoshock response, underscoring a role for the C1d projection in Ca(2+)-mediated flagellar behavior. The data also reveal for the first time that the C1d projection is involved in the control of interdoublet sliding velocity. Our studies further identify a novel C1d subunit that we term C1d-87, give new insight into relationships between the C1d subunits, and provide evidence for multiple sites of calmodulin interaction within the C1d projection. These results represent significant advances in our understanding of an important but little studied axonemal structure.
Preview · Article · May 2012 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: For all eukaryotic cilia the basal bodies provide a template for the assembly of the doublet microtubules, and intraflagellar transport provides a mechanism for transport of axonemal components into the growing cilium. What is not known is how the central pair of microtubules is nucleated or how their associated polypeptides are assembled. Here we report that the Chlamydomonas pf19 mutation results in a single amino acid change within the p60 catalytic subunit of katanin, and that this mutation prevents microtubule severing activity. The pf19 mutant has paralyzed flagella that lack the central apparatus. Using a combination of mutant analysis, RNAi-mediated reduction of protein expression and in vitro assays, we demonstrate that the p60 catalytic subunit of the microtubule severing protein katanin is required for central apparatus assembly in Chlamydomonas. In addition, we show that in Chlamydomonas the microtubule severing activity of p60 katanin is not required for stress-induced deflagellation or cell cycle progression as has been previously reported.
Preview · Article · Mar 2012 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Numerous studies have indicated that each of the seven projections associated with the central pair of microtubules plays a distinct role in regulating eukaryotic ciliary/flagellar motility. Mutants which lack specific projections have distinct motility phenotypes. For example, Chlamydomonas pf6 mutants lack the C1a projection and have twitchy, non-beating flagella. The C1a projection is a complex of proteins including PF6, C1a-86, C1a-34, C1a-32, C1a-18, and calmodulin. To define functional domains within PF6 and to potentially assign functions to specific C1a components, we generated deletion constructs of the PF6 gene and tested for their ability to assemble and rescue motility upon transformation of mutant pf6 cells. Our results demonstrate that domains near the carboxyl-terminus of PF6 are essential for motility and/or assembly of the projection. The amino terminal half of PF6 is not required for C1a assembly; however, this region is important for stability of the C1a-34, C1a-32, and C1a-18 sub-complex and wild-type beat frequency. Analysis of double mutants lacking the amino terminus of PF6 and outer dynein arms reveal that C1a may play a role in modulating both inner and outer dynein arm activity.
[Show abstract][Hide abstract] ABSTRACT: Generating the complex waveforms characteristic of beating cilia requires the coordinated activity of multiple dynein isoforms anchored to the axoneme. We previously identified a complex associated with the C1d projection of the central apparatus that includes primary ciliary dyskinesia protein 1 (Pcdp1). Reduced expression of complex members results in severe motility defects, indicating that C1d is essential for wild-type ciliary beating. To define a mechanism for Pcdp1/C1d regulation of motility, we took a functional and structural approach combined with mutants lacking C1d and distinct subsets of dynein arms. Unlike mutants completely lacking the central apparatus, dynein-driven microtubule sliding velocities are wild type in C1d- defective mutants. However, coordination of dynein activity among microtubule doublets is severely disrupted. Remarkably, mutations in either outer or inner dynein arm restore motility to mutants lacking C1d, although waveforms and beat frequency differ depending on which isoform is mutated. These results define a unique role for C1d in coordinating the activity of specific dynein isoforms to control ciliary motility.
Preview · Article · Dec 2011 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity. Our results not only indicate that the CSC is required for spoke assembly and wild-type motility, but also provide evidence for heterogeneity among the radial spokes.
Full-text · Article · May 2011 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: Presentations at the 2010 Conference on the Biology of Cilia and Flagella revealed new insights into the functions and assembly of cilia and highlighted their ever-expanding roles in development and disease.
No preview · Article · Jan 2011 · Science Signaling
[Show abstract][Hide abstract] ABSTRACT: Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite.
[Show abstract][Hide abstract] ABSTRACT: For all motile eukaryotic cilia and flagella, beating is regulated by changes in intraciliary calcium concentration. Although the mechanism for calcium regulation is not understood, numerous studies have shown that calmodulin (CaM) is a key axonemal calcium sensor. Using anti-CaM antibodies and Chlamydomonas reinhardtii axonemal extracts, we precipitated a complex that includes four polypeptides and that specifically interacts with CaM in high [Ca(2+)]. One of the complex members, FAP221, is an orthologue of mammalian Pcdp1 (primary ciliary dyskinesia protein 1). Both FAP221 and mammalian Pcdp1 specifically bind CaM in high [Ca(2+)]. Reduced expression of Pcdp1 complex members in C. reinhardtii results in failure of the C1d central pair projection to assemble and significant impairment of motility including uncoordinated bends, severely reduced beat frequency, and altered waveforms. These combined results reveal that the central pair Pcdp1 (FAP221) complex is essential for control of ciliary motility.
Preview · Article · May 2010 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: The radial spokes are a major structural feature of 9 + 2 axonemes and are crucial for flagellar beating. The enormous variety of research tools that Chlamydomonas offers a model system, such as radial spoke defective mutants and in vitro functional assays, as well as the unique biochemical properties of spoke components, have all contributed to our understanding of the radial spokes. This chapter reviews the composition, assembly, and possible functions of this macromolecular complex. Chlamydomonas mutants that are defective in radial spoke assembly have played a central role in studies of spoke composition and function. The observation presented in the genetics, supports the hypothesis that the direct interaction between the spoke heads and central pair projections is crucial for the motility of 9 + 2 cilia and flagella. For decades, the axonemal dynein arms have been the target of biochemical and functional analysis due in large part to the ease with which they can be extracted and purified from isolated flagella. Extraction along with composition and functional domains are explained in the biochemical characterization. A variety of approaches including genetic, structural, and functional studies have provided significant insights into understanding the function of the radial spokes. These observations provide strong evidence that regulation of dynein-driven microtubule sliding involves a signal transduction pathway that includes the radial spokes. An enormous amount of evidence indicates that the radial spokes play an important role in modulating flagellar motility.
[Show abstract][Hide abstract] ABSTRACT: For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)- binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which is located at the base of the spoke. In a microtubule sliding assay, the addition of antibodies generated against FAP91 to mutant axonemes with reduced dynein activity restores dynein activity to wild-type levels. These combined results indicate that the CaM- and spoke-associated complex mediates regulatory signals between the radial spokes and dynein arms.
Preview · Article · Dec 2007 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: One of the most surprising discoveries in cell biology in the past 5-10 years is the number of diverse human diseases that result from defects in ciliary assembly and/or motility, so-called ciliopathies (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125-148). The results presented by Lechtreck and Witman (see p. 473 of this issue) provide yet another example of how work in the model organism Chlamydomonas reinhardtii can reveal important insights into the underlying mechanisms of ciliary assembly/function and the diseases associated with defects in these organelles. By taking advantage of the wide array of experimental approaches C. reinhardtii offers, Lechtreck and Witman determined the precise axonemal location of hydin, a protein that, when mutated, causes hydrocephalus, and defined a unique role for hydin in ciliary motility.
Preview · Article · Mar 2007 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Kinesin-like calmodulin-binding protein, KCBP, is a novel member of the C-kinesin superfamily first discovered in flowering plants. This minus-end-directed kinesin exhibits Ca(2+)-calmodulin-sensitive motor activity in vitro and has been implicated in trichome morphogenesis and cell division. A homologue of KCBP is also found in the unicellular, biflagellate green alga Chlamydomonas reinhardtii (CrKCBP). Unlike plant cells, Chlamydomonas cells do not form trichomes and do not assemble a phragmoplast before cell division. To test whether CrKCBP is involved in additional microtubule-based processes not observed in plants, we generated antibodies against the putative calmodulin-binding domain and used these antibodies in biochemical and localization studies. In interphase cells CrKCBP primarily localizes near the base of the flagella, although surprisingly, a small fraction also localizes along the length of the flagella. CrKCBP is bound to isolated axonemes in an ATP-dependent fashion and is not a component of the dynein arms, radial spokes or central apparatus. During mitosis, CrKCBP appears concentrated at the centrosomes during prophase and metaphase. However, during telophase and cytokinesis CrKCBP co-localizes with the microtubules associated with the phycoplast. These studies implicate CrKCBP in flagellar functions as well as cell division.
[Show abstract][Hide abstract] ABSTRACT: Studies of flagellar motility in Chlamydomonas mutants lacking specific central apparatus components have supported the hypothesis that the inherent asymmetry of this structure provides important spatial cues for asymmetric regulation of dynein activity. These studies have also suggested that specific projections associated with the C1 and C2 central tubules make unique contributions to modulating motility; yet, we still do not know the identities of most polypeptides associated with the central tubules. To identify components of the C1a projection, we took an immunoprecipitation approach using antibodies generated against PF6. The pf6 mutant lacks the C1a projection and possesses flagella that only twitch; calcium-induced modulation of dynein activity on specific doublet microtubules is also defective in pf6 axonemes. Our antibodies specifically precipitated five polypeptides in addition to PF6. Using mass spectrometry, we determined the amino acid identities of these five polypeptides. Most notably, the PF6-containing complex includes calmodulin. Using antibodies generated against each precipitated polypeptide, we confirmed that these polypeptides comprise a single complex with PF6, and we identified specific binding partners for each member of the complex. The finding of a calmodulin-containing complex as an asymmetrically assembled component of the central apparatus implicates the central apparatus in calcium modulation of flagellar waveform.
Full-text · Article · Nov 2005 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Numerous studies have indicated that the central apparatus plays a significant role in regulating flagellar motility, yet
little is known about how the central pair of microtubules or their associated projections assemble. Several Chlamydomonas mutants are defective in central apparatus assembly. For example, mutant pf15 cells have paralyzed flagella that completely lack the central pair of microtubules. We have cloned the wild-type PF15 gene and confirmed its identity by rescuing the motility and ultrastructural defects in two pf15 alleles, the original pf15a mutant and a mutant generated by insertional mutagenesis. Database searches using the 798-amino-acid polypeptide predicted
from the complete coding sequence indicate that the PF15 gene encodes the Chlamydomonas homologue of the katanin p80 subunit. Katanin was originally identified as a heterodimeric protein with a microtubule-severing
activity. These results reveal a novel role for the katanin p80 subunit in the assembly and/or stability of the central pair
of flagellar microtubules.
[Show abstract][Hide abstract] ABSTRACT: Generating the complex waveforms characteristic of beating eukaryotic cilia and flagella requires spatial regulation of dynein-driven microtubule sliding. To generate bending, one prediction is that dynein arms alternate between active and inactive forms on specific subsets of doublet microtubules. Using an in vitro microtubule sliding assay combined with a structural approach, we determined that ATP induces sliding between specific subsets of doublet microtubules, apparently capturing one phase of the beat cycle. These studies were also conducted using high Ca2+ conditions. In Chlamydomonas, high Ca2+ induces changes in waveform which are predicted to result from regulating dynein activity on specific microtubules. Our results demonstrate that microtubule sliding in high Ca2+ buffer is also induced by dynein arms on specific doublets. However, the pattern of microtubule sliding in high Ca2+ buffer significantly differs from that in low Ca2+. These results are consistent with a 'switching hypothesis' of axonemal bending and provide evidence to indicate that Ca2+ control of waveform includes modulation of the pattern of microtubule sliding between specific doublets. In addition, analysis of microtubule sliding in mutant axonemes reveals that the control mechanism is disrupted in some mutants.
Full-text · Article · Jun 2004 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Tctex1 and Tctex2 were originally described in mice as putative distorters/sterility factors involved in the non-Mendelian
transmission of t haplotypes. Subsequently, these proteins were found to be light chains of both cytoplasmic and axonemal dyneins. We have
now identified a novel Tctex2-related protein (Tctex2b) within the Chlamydomonas flagellum. Tctex2b copurifies with inner arm I1 after both sucrose gradient centrifugation and anion exchange chromatography.
Unlike the Tctex2 homologue within the outer dynein arm, analysis of a Tctex2b-null strain indicates that this protein is
not essential for assembly of inner arm I1. However, a lack of Tctex2b results in an unstable dynein particle that disassembles
after high salt extraction from the axoneme. Cells lacking Tctex2b swim more slowly than wild type and exhibit a reduced flagellar
beat frequency. Furthermore, using a microtubule sliding assay we observed that dynein motor function is reduced in vitro. These data indicate that Tctex2b is required for the stability of inner dynein arm I1 and wild-type axonemal dynein function.
Preview · Article · Jun 2004 · Journal of Biological Chemistry