Dennis R Diener

Yale University, New Haven, CT, United States

Are you Dennis R Diener?

Claim your profile

Publications (37)242.43 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The ciliary tip has been implicated in ciliary assembly/disassembly and signaling, yet information on its protein composition is limited. Using comparative, quantitative proteomics based on the fact that tip proteins will be ca. twice as concentrated in half-length compared to full-length flagella, we have identified FAP256 as a tip protein in Chlamydomonas. FAP256 localizes to the tips of both central pair and outer doublet microtubules (MTs) and it remains at the tip during flagellar assembly and disassembly. Similarly, its vertebrate counterpart, CEP104, localizes on the distal ends of both centrioles of nondividing cells until the mother centriole forms a cilium and then localizes at the tip of the elongating cilium. A null mutant of FAP256 in Chlamydomonas and RNAi in vertebrate cells showed that FAP256/CEP104 is required for ciliogenesis in a high percentage of cells. In those cells that could form cilia, there were structural deformities at the ciliary tips.
    Journal of Cell Science 08/2013; · 5.88 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The release of membrane vesicles from the surface of cells into their surrounding environment is now recognized as an important pathway for the delivery of proteins to extracellular sites of biological function. Membrane vesicles of this kind, termed exosomes and ectosomes, are the result of active processes and have been shown to carry a wide array of biological effector molecules that can play roles in cell-to-cell communication and remodeling of the extracellular space [1-7]. Degradation of the extracellular matrix (ECM) through the regulated release of proteolytic enzymes is a key process for development, morphogenesis, and cell migration in animal and plant cells. Here we show that the unicellular alga Chlamydomonas achieves the timely degradation of its mother cell wall, a type of ECM, through the budding of ectosomes directly from the membranes of its flagella. Using a combination of immunoelectron microscopy, immunofluorescence microscopy, and functional analysis, we demonstrate that these vesicles, which we term ciliary ectosomes, act as carriers of the proteolytic enzyme necessary for the liberation of daughter cells following mitosis [8, 9]. Chlamydomonas has proven to be the key unicellular model for the highly conserved mechanisms of mammalian cilia, and our results suggest that cilia may be an underappreciated source of bioactive, extracellular membrane vesicles.
    Current biology: CB 04/2013; · 10.99 Impact Factor
  • Source
    Dataset: 4.cover
  • Source
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes. Interestingly, LC8 dimers bound in tandem to the N-terminal region of a spoke phosphoprotein, RS protein 3 (RSP3), that docks RSs to axonemes. LC8 enhanced the binding of RSP3 N-terminal fragments to purified axonemes. Likewise, the N-terminal fragments extracted from axonemes contained LC8 and putative spoke-docking proteins. Lastly, perturbations of RSP3's LC8-binding sites resulted in asynchronous flagella with hypophosphorylated RSP3 and defective associations between LC8, RSs, and axonemes. We propose that at the tip of flagella, an array of LC8 dimers binds to RSP3 in RS precursors, triggering phosphorylation, stalk base formation, and axoneme targeting. These multiple effects shed new light on fundamental questions about LC8-containing complexes and axoneme assembly.
    The Journal of Cell Biology 07/2012; 198(1):115-26. · 10.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The cilium serves as a cellular antenna by coordinating upstream environmental cues with numerous downstream signaling processes that are indispensable for the function of the cell. This role is supported by the revelation that defects of the cilium underlie an emerging class of human disorders, termed "ciliopathies." Although mounting interest in the cilium has demonstrated the essential role that the organelle plays in vertebrate development, homeostasis, and disease pathogenesis, the mechanisms regulating cilia morphology and function remain unclear. Here, we show that the target-of-rapamycin (TOR) growth pathway modulates cilia size and function during zebrafish development. Knockdown of tuberous sclerosis complex 1a (tsc1a), which encodes an upstream inhibitor of TOR complex 1 (Torc1), increases cilia length. In contrast, treatment of embryos with rapamycin, an inhibitor of Torc1, shortens cilia length. Overexpression of ribosomal protein S6 kinase 1 (S6k1), which encodes a downstream substrate of Torc1, lengthens cilia. Furthermore, we provide evidence that TOR-mediated cilia assembly is evolutionarily conserved and that protein synthesis is essential for this regulation. Finally, we demonstrate that TOR signaling and cilia length are pivotal for a variety of downstream ciliary functions, such as cilia motility, fluid flow generation, and the establishment of left-right body asymmetry. Our findings reveal a unique role for the TOR pathway in regulating cilia size through protein synthesis and suggest that appropriate and defined lengths are necessary for proper function of the cilium.
    Proceedings of the National Academy of Sciences 02/2012; 109(6):2021-6. · 9.74 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Intraflagellar transport (IFT) proteins are well established as conserved mediators of flagellum/cilium assembly and disassembly. However, data has begun to accumulate in support of IFT protein involvement in other processes elsewhere in the cell. Here, we used synchronous cultures of Chlamydomonas to investigate the temporal patterns of accumulation and localization of IFT proteins during the cell cycle. Their mRNAs showed periodic expression that peaked during S and M phase (S/M). Unlike most proteins that are synthesized continuously during G1 phase, IFT27 and IFT46 levels were found to increase only during S/M phase. During cell division, IFT27, IFT46, IFT72, and IFT139 re-localized from the flagella and basal bodies to the cleavage furrow. IFT27 was further shown to be associated with membrane vesicles in this region. This localization pattern suggests a role for IFT in cell division.
    PLoS ONE 01/2012; 7(2):e30729. · 3.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Radial spokes (RSs) are ubiquitous components in the 9 + 2 axoneme thought to be mechanochemical transducers involved in local control of dynein-driven microtubule sliding. They are composed of >23 polypeptides, whose interactions and placement must be deciphered to understand RS function. In this paper, we show the detailed three-dimensional (3D) structure of RS in situ in Chlamydomonas reinhardtii flagella and Tetrahymena thermophila cilia that we obtained using cryoelectron tomography (cryo-ET). We clarify similarities and differences between the three spoke species, RS1, RS2, and RS3, in T. thermophila and in C. reinhardtii and show that part of RS3 is conserved in C. reinhardtii, which only has two species of complete RSs. By analyzing C. reinhardtii mutants, we identified the specific location of subsets of RS proteins (RSPs). Our 3D reconstructions show a twofold symmetry, suggesting that fully assembled RSs are produced by dimerization. Based on our cryo-ET data, we propose models of subdomain organization within the RS as well as interactions between RSPs and with other axonemal components.
    The Journal of Cell Biology 11/2011; 195(4):673-87. · 10.82 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The unicellular alga Chlamydomonas can assemble two 10 μm flagella in 1 h from proteins synthesized in the cell body. Targeting and transporting these proteins to the flagella are simplified by preassembly of macromolecular complexes in the cell body. Radial spokes are flagellar complexes that are partially assembled in the cell body before entering the flagella. On the axoneme, radial spokes are "T" shaped structures with a head of five proteins and a stalk of 18 proteins that sediment together at 20S. In the cell body, radial spokes are partially assembled; about half of the radial spoke proteins (RSPs) form a 12S complex. In mutants lacking a single RSP, smaller spoke subassemblies were identified. When extracts from two such mutants were mixed in vitro the 12S complex was assembled from several smaller complexes demonstrating that portions of the stepwise assembly of radial spoke assembly can be carried out in vitro to elucidate the order of spoke assembly in the cell body.
    Cytoskeleton 06/2011; 68(7):389-400. · 2.87 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The radial spoke (RS)/central pair (CP) system in cilia and flagella plays an essential role in the regulation of force generation by dynein, the motor protein that drives cilia/flagella movements. Mechanical and mechanochemicl interactions between the CP and the distal part of the RS, the spokehead, should be crucial for this control; however, the details of interaction are totally unknown. As an initial step toward an understanding of the RS-CP interaction, we examined the protein-protein interactions between the five spokehead proteins (radial spoke protein (RSP)1, RSP4, RSP6, RSP9, and RSP10) and three spoke stalk proteins (RSP2, RSP5, and RSP23), all expressed as recombinant proteins. Three of them were shown to have physiological activities by electroporation-mediated protein delivery into mutants deficient in the respective proteins. Glutathione S-transferase pulldown assays in vitro detected interactions in 10 out of 64 pairs of recombinants. In addition, chemical crosslinking of axonemes using five reagents detected seven kinds of interactions between the RS subunits in situ. Finally, in the mixture of the recombinant spokehead subunits, RSP1, RSP4, RSP6, and RSP9 formed a 7-10S complex as detected by sucrose density gradient centrifugation. It may represent a partial assembly of the spokehead. From these results, we propose a model of interactions taking place between the spokehead subunits.
    Cytoskeleton 03/2011; 68(4):237-46. · 2.87 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Chlamydomonas reinhardtii is a model system for the biology of unicellular green algae. Chemically regulated promoters, such as the nickel-inducible CYC6 or the low CO₂-inducible CAH1 promoter, may prove useful for expressing, at precise times during its cell cycle, proteins with relevant biological functions, or complementing mutants in genes encoding such proteins. To this date, this has not been reported for the above promoters. We fused the CYC6 and CAH1 promoters to an HA-tagged RSP3 gene, encoding a protein of the flagellar radial spoke complex. The constructs were used for chemically regulated complementation of the pf14 mutant, carrying an ochre mutation in the RSP3 gene. 7 to 8% of the transformants showed cells with restored motility after induction with nickel or transfer to low CO₂ conditions, but not in non-inducing conditions. Maximum complementation (5% motile cells) was reached with very different kinetics (5-6 hours for CAH1, 48 hours for CYC6). The two inducible promoters drive much lower levels of RSP3 protein expression than the constitutive PSAD promoter, which shows almost complete rescue of motility. To our knowledge, this is the first example of the use of the CYC6 or CAH1 promoters to perform a chemically regulated complementation of a Chlamydomonas mutant. Based on our data, the CYC6 and CAH1 promoters should be capable of fully complementing mutants in genes whose products exert their biological activity at low concentrations.
    BMC Plant Biology 01/2011; 11:22. · 4.35 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule-membrane links there. Ultrastructural analysis revealed defects in these microtubule-membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule-membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.
    The Journal of Cell Biology 09/2010; 190(5):927-40. · 10.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cilia are necessary for normal tissue development and homeostasis and are generally present during interphase, but not in mitosis. The precise mechanism of premitotic ciliary loss has been controversial, with data supporting either sequential disassembly through the transition zone or, alternatively, a severing event at the base of the cilia. Here we show by live cell imaging and immunofluorescence microscopy that resorbing flagella of Chlamydomonas leave remnants associated with the mother cell wall. We postulated that the remnants are the product of severing of doublet microtubules between the basal bodies and the flagellar transition zone, thereby freeing the centrioles to participate in spindle organization. We show via TEM that flagellar remnants are indeed flagellar transition zones encased in vesicles derived from the flagellar membrane. This transition zone vesicle can be lodged within the cell wall or it can be expelled into the environment. This process is observable in Chlamydomonas, first because the released flagellar remnants can remain associated with the cell by virtue of attachments to the cell wall, and second because the Chlamydomonas transition zone is particularly rich with electron-dense structure. However, release of basal bodies for spindle-associated function is likely to be conserved among the eukaryotes.
    Cytoskeleton 07/2010; 67(7):425-30. · 2.87 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.
    The Journal of Cell Biology 10/2009; 187(1):135-48. · 10.82 Impact Factor
  • Source
    Kaiyao Huang, Dennis R Diener, Joel L Rosenbaum
    [Show abstract] [Hide abstract]
    ABSTRACT: The disassembly of cilia and flagella is linked to the cell cycle and environmental cues. We have found that ubiquitination of flagellar proteins is an integral part of flagellar disassembly. Free ubiquitin and the ubiquitin-conjugating enzyme CrUbc13 are detected in flagella, and several proteins are ubiquitinated in isolated flagella when exogenous ubiquitin and adenosine triphosphatase are added, suggesting that the ubiquitin conjugation system operates in flagella. Levels of ubiquitinated flagellar proteins increase during flagellar resorption, especially in intraflagellar transport (IFT) mutants, suggesting that disassembly products are labeled with ubiquitin and transported to the cell body by IFT. Substrates of the ubiquitin conjugation system include alpha-tubulin (but not beta-tubulin), a dynein subunit (IC2), two signaling proteins involved in the mating process, cyclic guanosine monophosphate-dependent kinase, and the cation channel polycystic kidney disease 2. Ubiquitination of flagellar proteins is enhanced early in mating, suggesting that ubiquitination also plays an active role in regulating signaling pathways in flagella.
    The Journal of Cell Biology 09/2009; 186(4):601-13. · 10.82 Impact Factor
  • Dennis Diener
    [Show abstract] [Hide abstract]
    ABSTRACT: Chlamydomonas reinhardtii is the organism in which intraflagellar transport (IFT) was first visualized and in which the composition of IFT particles was originally elucidated. As the universality of IFT among ciliated/flagellated cells was uncovered, the diversity of organisms used to study IFT has grown. Still, because of the ease of isolation of flagella from Chlamydomonas and the battery of temperature-sensitive mutants affecting IFT proteins and motors, this unicellular alga remains the principal model for biochemical studies of IFT motors and cargo; furthermore, the long, exposed flagella of this cell are ideally suited for observing IFT in real time with GFP-tagged components of IFT.
    Methods in cell biology 01/2009; 93:111-9. · 1.44 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Radial spokes are critical multisubunit structures required for normal ciliary and eukaryotic flagellar motility. Experimental evidence indicates the radial spokes are mechanochemical transducers that transmit signals from the central pair apparatus to the outer doublet microtubules for local control of dynein activity. Recently, progress has been made in identifying individual components of the radial spoke, yet little is known about how the radial spoke is assembled or how it performs in signal transduction. Here we focus on radial spoke protein 3 (RSP3), a highly conserved AKAP located at the base of the radial spoke stalk and required for radial spoke assembly on the doublet microtubules. Biochemical approaches were taken to further explore the functional role of RSP3 within the radial spoke structure and for control of motility. Chemical crosslinking, native gel electrophoresis, and epitope-tagged RSP3 proteins established that RSP3 forms a dimer. Analysis of truncated RSP3 proteins indicates the dimerization domain coincides with the previously characterized axoneme binding domain in the N-terminus. We propose a model in which each radial spoke structure is built on an RSP3 dimer, and indicating that each radial spoke can potentially localize multiple PKAs or AKAP-binding proteins in position to control dynein activity and flagellar motility.
    Cell Motility and the Cytoskeleton 04/2008; 65(3):238-48. · 4.19 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To analyze the function of ciliary polycystic kidney disease 2 (PKD2) and its relationship to intraflagellar transport (IFT), we cloned the gene encoding Chlamydomonas reinhardtii PKD2 (CrPKD2), a protein with the characteristics of PKD2 family members. Three forms of this protein (210, 120, and 90 kD) were detected in whole cells; the two smaller forms are cleavage products of the 210-kD protein and were the predominant forms in flagella. In cells expressing CrPKD2-GFP, about 10% of flagellar CrPKD2-GFP was observed moving in the flagellar membrane. When IFT was blocked, fluorescence recovery after photobleaching of flagellar CrPKD2-GFP was attenuated and CrPKD2 accumulated in the flagella. Flagellar CrPKD2 increased fourfold during gametogenesis, and several CrPKD2 RNA interference strains showed defects in flagella-dependent mating. These results suggest that the CrPKD2 cation channel is involved in coupling flagellar adhesion at the beginning of mating to the increase in flagellar calcium required for subsequent steps in mating.
    The Journal of Cell Biology 12/2007; 179(3):501-14. · 10.82 Impact Factor
  • DENNIS R. DIENER, BROWER R. BURCHILL, PAUL R. BURTON
    [Show abstract] [Hide abstract]
    ABSTRACT: The ultrastructure of the cortex beneath the fission furrow of dividing Stentor coeruleus was examined using scanning and transmission electron microscopy. During division, basal bodies, axonemes, and km fibers beneath the furrow were absorbed near the moving primordial oral apparatus, and a circumferential band of microtubules and filaments was formed at the base of the furrow. The location and orientation of this fibrous band suggest that it may be an important component of the cytokinetic machinery. Treatment with vinblastine sulfate (4 × 10-5 M) disrupted the circumferential microtubules and blocked division, which is consistent with this hypothesis.
    Journal of Eukaryotic Microbiology 04/2007; 30(1):83 - 90. · 2.16 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Intraflagellar transport (IFT) is a motility process operating between the ciliary/flagellar (interchangeable terms) membrane and the microtubular axoneme of motile and sensory cilia. Multipolypeptide IFT particles, composed of complexes A and B, carry flagellar precursors to their assembly site at the flagellar tip (anterograde) powered by kinesin, and turnover products from the tip back to the cytoplasm (retrograde) driven by cytoplasmic dynein. IFT is essential for the assembly and maintenance of almost all eukaryotic cilia and flagella, and mutations affecting either the IFT motors or the IFT particle polypeptides result in the inability to assemble normal flagella or in defects in the sensory functions of cilia. We found that the IFT complex B polypeptide, IFT27, is a Rab-like small G protein. Reduction of the level of IFT27 by RNA interference reduces the levels of other complex A and B proteins, suggesting that this protein is instrumental in maintaining the stability of both IFT complexes. Furthermore, in addition to its role in flagellar assembly, IFT27 is unique among IFT polypeptides in that its partial knockdown results in defects in cytokinesis and elongation of the cell cycle and a more complete knockdown is lethal. IFT27, a small G protein, is one of a growing number of flagellar proteins that are now known to have a role in cell-cycle control.
    Current Biology 03/2007; 17(3):193-202. · 9.49 Impact Factor

Publication Stats

2k Citations
242.43 Total Impact Points

Institutions

  • 1989–2013
    • Yale University
      • Department of Molecular, Cellular and Developmental Biology
      New Haven, CT, United States
  • 2011
    • Paul Scherrer Institut
      • Laboratory of Biomolecular Research
      Villigen, AG, Switzerland
  • 2007
    • University of Kansas
      Lawrence, Kansas, United States
    • University of New Haven
      New Haven, Connecticut, United States