Cryoelectron tomography of radial spokes in cilia and flagella

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.     

Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity

Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.     

Flagellar radial spokes contain a Ca2+-stimulated nucleoside diphosphate kinase

The radial spokes are required for Ca(2+)-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca(2+)-independent manner, whereas IQ2 and IQ3 show Ca(2+)-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca(2+). This Ca(2+)-responsive enzyme, which accounts for approximately 45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca(2+)-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes.     

Structure and organization of radial spokes in cilia of Tetrahymena pyriformis.

The structure and organization of radial spokes, the principal components between each of the peripheral doublet microtubules and the central sheath which surrounds the central pair of microtubules have been described in Tetrahymena pyriformis cilia. The radial spokes are grouped in triplets and are attached to the A-microtubule of each peripheral doublet at intervals of 200/280/360 A, the 200 A spacing being most distal to the base of the cilium. The radial spoke triplets are organized in the axoneme in a double helix with a pitch of 4,680 A. A method for determining the helical disposition by correcting for doublet sliding is presented.     

A new spin on protein dynamics

Site-directed spin labeling is a general method for investigating structure and conformational switching in soluble and membrane proteins. It will also be an important tool for exploring protein backbone dynamics. A semi-empirical analysis of nitroxide sidechain dynamics in spin-labeled proteins reveals contributions from fluctuations in backbone dihedral angles and rigid-body (collective) motions of alpha helices. Quantitative analysis of sidechain dynamics is sometimes possible, and contributions from backbone modes can be expressed in terms of relative order parameters and rates. Dynamic sequences identified by site-directed spin labeling correlate with functional domains, and so nitroxide scanning could provide an efficient strategy for identifying such domains in high-molecular weight proteins, supramolecular complexes and membrane proteins.     

Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes

Genetic and in vitro analyses have revealed that radial spokes play a crucial role in regulation of ciliary and flagellar motility, including control of waveform. However, the mechanisms of regulation are not understood. Here, we developed a novel procedure to isolate intact radial spokes as a step toward understanding the mechanism by which these complexes regulate dynein activity. The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes. Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14. Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8. Analyses of flagellar mutants and chemical cross-linking studies demonstrated calmodulin and LC8 form a complex located in the radial spoke stalk. We postulate that calmodulin, located in the radial spoke stalk, plays a role in calcium control of flagellar bending.     

Entropy and information in flagellar axoneme cybernetics: a radial spokes integrative function

Radial spokes and the consequences of their relationships with the central apparatus seem to play a very important role in the regulation of axonemal activity. We modeled their behavior and observed that it appears to differ in the cilium and the flagellum with respect to the development of bending as a function of time. Specifically, our calculation raises the question of the real function of the radial spokes in the regulation of the axoneme, because a given curvature of the flagellar axoneme may correspond to two opposite of their tilts. The stable nil/low amplitude shear points that we had characterized along the flagellum allowed us to describe their axoneme as a series of modules [Cibert, 2002: Cell Motil. Cytoskeleton 51:89-111]. We observed that a nil/low shearing point moves along each module during beating when a new bend is created at the base of the flagellum [Cibert, 2001: Cell Motil. Cytoskeleton 49:161-175]. We propose that the structural gradients of isoforms of tubulin could be basic verniers that act as structural references for the axonemal machinery during the beating. This allowed us to interpret the axonemal organization as a segmented structure, that could be analyzed according to the complexion(1) theory and Shannon's information theory, which associate entropy and probability in the creation of information. The important consequence of this interpretation is that regulation of the axonemal machinery appears to be due to the upstream and downstream cross-talk between the axonemal segments that do not involve any dedicated integrative structure but depend on the energy level of the entire length of each module.     

The three-dimensional arrangement of radial spokes in the flagella of Chlamydomonas reinhardii

The flagella of Chlamydomonas reinhardii have been studied by electron microscopy using a critical point drying technique. Details of the three-dimensional arrangement of the axoneme are to a great extent preserved by the use of this method, and the radial spokes have been shown to form a double spiral along the flagellar shaft. The axial displacement of paired groups on adjacent doublet microtubules is 22.2 nm, and the pitch of each helix is 200 nm. It is probable that the paired groups of spokes are the remains of triplets in the in vivo organelle.     

Proteomic characterization of sperm radial spokes identifies a novel spoke protein with an ubiquitin domain

Radial spokes are T-shaped protein complexes important for the regulation of axonemal dyneins in eukaryotic cilia and flagella. Using a functional proteomics approach, we identified six spoke proteins in sperm flagella of the ascidian Ciona intestinalis. Many of the domain/motif structures in spoke proteins are commonly found in flagella of both Ciona sperm and Chlamydomonas, but interestingly they often distribute over non-orthologous protein components. A novel 116 kDa protein named CMUB116 has both an ubiquitin domain and an IQ motif. It has orthologs in vertebrates, but not in Chlamydomonas. Furthermore, the results obtained by immunological analysis provide strong indication that CMUB116 is located at the stalk of radial spokes, where it is associated with MORN40.

Structured summary

MINT-7148244: CMUB116 (genbank_protein_gi:BAH59277) and MORN40 (genbank_protein_gi:BAH59284) colocalize (MI:0403) by cosedimentation (MI:0027)MINT-7148179: Ci-RSP3 (uniprotkb:Q8T898) physically interacts (MI:0915) with tubulin alpha (uniprotkb:Q8MVT7), LRR37 (uniprotkb:Q8T896), CMUB116 (genbank_protein_gi:BAH59277), Ci-SRP4/6 (genbank_protein_gi:BAH59283), AK58 (genbank_protein_gi:BAM59278), tubulin beta (genbank_protein_gi:XP_002130315), NDK/DPY26 (genbank_protein_gi:BAH59279), MORN40 (genbank_protein_gi:BAH59284), ARM37 (genbank_protein_gi:BAH59280), NDK/DPY26 (genbank_protein_gi:NP_001161489), LC8 (genbank_protein_gi:BAH59282) and Ci-RSP9 (genbank_protein_gi:NP_001154962) by anti bait coimmunoprecipitation (MI:0006)MINT-7148272: Ci-RSP3 (uniprotkb:Q8T898) and MORN40 (genbank_protein_gi:BAH59284) colocalize (MI:0403) by cosedimentation (MI:0027)     

Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella

Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo-electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named "radial spoke 3 stand-in," which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.     

Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components

The fine structure, protein composition, and roles in flagellar movement of specific axonemal components were studied in wild-type Chlamydomonas and paralyzed mutants pf-14, pf-15A, and pf-19. Electron microscope examination of the isolated axoneme of pf-14 showed that it lacks the radial spokes but is otherwise structurally normal. Comparison of isolated axonemes of wild type and pf-14 by sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the mutant is missing a protein of 118,000 mol wt; this protein is apparently a major component of the spokes. Pf-15A and pf-19 lack the central tubules and sheath; axonemes of these mutants are missing three high molecular weight proteins which are probably components of the central tubule-central sheath complex. Under conditions where wild-type axonemes reactivated, axonemes of the three mutants remained intact but did not form bends. However, mutant and wild-type axonemes underwent identical adenosine triphosphate-induced disintegration after treatment with trypsin; the dynein arms of the mutants are therefore capable of generating interdoublet shearing forces. These findings indicated that both the radial spokes and the central tubule-central sheath complex are essential for conversion of interdoublet sliding into axonemal bending. Moreover, because axonemes of pf-14 remained intact under reactivating conditions, the nexin links alone are sufficient to limit the amount of interdoublet sliding that occurs. The axial periodicities of the central sheath, dynein arms, radial spokes, and nexin links of Chlamydomonas were determined by electron microscopy using the lattice-spacing of crystalline catalase as an internal standard. Some new ultrastructural details of the components are described.     

Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella

The substructure of the components of the axoneme interior--the inner dynein arms, the radial spokes, and the central pair/projection complex--was analyzed for Chlamydomonas. Tetrahymena, Strongelocentrotus, and Mnemiopsis using the quick-freeze, deep-etch technique. The inner arms are shown to resemble the outer arms in overall molecular organization, but they are disposed differently on the microtubule and have two distinct morphologies--dyads with two heads and triads with three. The dyads associate with spokes S3 and S2; the triads associate with S1. The spokes form a three-start right-handed helix with a 288-nm rise; the central pair makes a shallow left-handed twist. The spoke heads are shown to be made up of four major subunits; two bind to the spoke shaft and two bind to a pair of central-sheath projections.     

A new sensitive radial diffusion method for microdetermination of alpha-amylase

A rapid, convenient, and efficient hybridization method for the determination of virus-specific RNAs in Py virus-infected cells is described. The method involves carrying out the hybridization of viral RNAs present in the RNA isolated from infected cells directly on nitrocellulose filters carrying denatured, immobilized Py-DNA (15 μl RNA solution/25-mm² filter). Under optimal conditions quantitative hybridization of viral RNA sequences is obtained within 24 h. The efficiency of hybridization is increased significantly when RNA fragmented by alkali under carefully controlled conditions is used.     

Dimeric heat shock protein 40 binds radial spokes for generating coupled power strokes and recovery strokes of 9 + 2 flagella

T-shape radial spokes regulate flagellar beating. However, the precise function and molecular mechanism of these spokes remain unclear. Interestingly, Chlamydomonas reinhardtii flagella lacking a dimeric heat shock protein (HSP) 40 at the spokehead-spokestalk juncture appear normal in length and composition but twitch actively while cells jiggle without procession, resembling a central pair (CP) mutant. HSP40(-) cells begin swimming upon electroporation with recombinant HSP40. Surprisingly, the rescue doesn't require the signature DnaJ domain. Furthermore, the His-Pro-Asp tripeptide that is essential for stimulating HSP70 adenosine triphosphatase diverges in candidate orthologues, including human DnaJB13. Video microscopy reveals hesitance in bend initiation and propagation as well as irregular stalling and stroke switching despite fairly normal waveform. The in vivo evidence suggests that the evolutionarily conserved HSP40 specifically transforms multiple spoke proteins into stable conformation capable of mechanically coupling the CP with dynein motors. This enables 9 + 2 cilia and flagella to bend and switch to generate alternate power strokes and recovery strokes.     

VS-oscilloscope: A new tool to parameterize tree radial growth based on climate conditions

It is generally assumed in dendroecological studies that annual tree-ring growth is adequately determined by a linear function of local or regional precipitation and temperature with a set of coefficients that are temporally invariant. However, various researchers have maintained that tree-ring records are the result of multivariate, often nonlinear biological and physical processes. To describe critical processes linking climate variables with tree-ring formation, the process-based tree-ring Vaganov–Shashkin model (VS-model) was successfully used. However, the VS-model is a complex tool requiring a considerable number of model parameters that should be re-estimated for each forest stand. Here we present a new visual approach of process-based tree-ring model parameterization (the so-called VS-oscilloscope) which allows the simulation of tree-ring growth and can be easily used by researchers and students. The VS-oscilloscope was tested on tree-ring data for two species (Larix gmeliniiand Picea obovata) growing in the permafrost zone of Central Siberia. The parameterization of the VS-model provided highly significant positive correlations (p < 0.0001) between simulated growth curves and original tree-ring chronologies for the period 1950–2009. The model outputs have shown differences in seasonal tree-ring growth between species that were well supported by the field observations. To better understand seasonal tree-ring growth and to verify the VS-model findings, a multi-year natural field study is needed, including seasonal observation of the thermo-hydrological regime of the soil, duration and rate of tracheid development, as well as measurements of their anatomical features.     

Animal locomotion: a new spin on bat flight

Biologists and engineers have long struggled to understand the hovering flight of insects, birds, and bats. The enormous diversity of these groups would suggest they fly using a variety of mechanisms, but a new study shows that hovering bats use the same aerodynamic mechanisms as do moths and other insects.     

A new membrane probing steroidal spin label: synthesis and applications

The applicability of a new steroidal spin label, 3-oxo-androstan-17 beta-yl-(2",2",6",6"-tetramethyl-N-oxyl) piperidyl butan-1',4'-dioate, in studying the phase transition properties of model membrane L-alpha-dipalmitoyl phosphatidyl choline (DPPC) in the presence and absence of drugs has been explored. Its synthesis and characterization has been described herein. Besides, the localization of this spin label in lipid liposomes has been studied using electron spin resonance (ESR), differential scanning calorimetry (DSC) and 1H and 31P NMR spectroscopic techniques. The label has also been used to study the permeability of epinephrine into membrane. The results show that the spin label has a good potential as a spin probe in the study of biomembranes.     

ChromoWheel: a new spin on eukaryotic chromosome visualization

ChromoWheel is an Internet browser application for generating whole-genome illustrations. It can be used to depict chromosomes, genes and relations between chromosomal loci. The circular layout of chromosomes is advantageous for showing relationships between different chromosomes, as the connecting line never crosses over a chromosome. All graphical image components are in the vector-based format Scalable Vector Graphics, which are highly scaleable and admit user interaction. ChromoWheel can either be run with user-provided data in the generic SFS format, or as a browser front-end for precompiled genomic data.     

A new spin label method for the measurement of erythrocyte internal microviscosity

A new spin-label method for the measurement of the internal microviscosity of erythrocyte is presented. The spin label used is 2,2',5,5'-tetramethyl-3-maleimidopyrrolidinyl-N-oxyl (MAL-5) which penetrates inside the red blood cell and binds covalently on cytoplasmic glutathione. After washing off the external label, 98% of the electron paramagnetic signal is due to the labelled glutathione. This signal allows one to measure the rotational correlation time of the label. A calibration curve established with spin-labelled glutathione in sucrose solutions of increasing viscosity is used to convert the measured rotation times into viscosity units. This method avoids the use of unphysiological salts like potassium ferricyanide, and permits the study of red blood cells in various suspension media. In normal human subjects, the mean value of microviscosity is 4.45 +/- 0.16 mPa . s at 20 degrees C in isotonic saline (25 subjects) and 6 +/- 0.25 mPa . s in plasma. The variations of microviscosity as a function of the osmolarity of the medium are explained according to a theoretical model taking into account the variations of the red blood cell volume and the viscometric properties of haemoglobin.