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Winfield S Sale - One of the best experts on this subject based on the ideXlab platform.
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Ciliary Motility: Regulation of Axonemal Dynein Motors.
Cold Spring Harbor perspectives in biology, 2017Co-Authors: Rasagnya Viswanadha, Winfield S Sale, Mary E. PorterAbstract:Ciliary motility is crucial for the development and health of many organisms. Motility depends on the coordinated activity of multiple dynein motors arranged in a precise pattern on the outer doublet microtubules. Although significant progress has been made in elucidating the composition and organization of the dyneins, a comprehensive understanding of dynein regulation is lacking. Here, we focus on two conserved signaling complexes located at the base of the Radial Spokes. These include the I1/f inner dynein arm associated with Radial Spoke 1 and the calmodulin- and Spoke-associated complex and the nexin-dynein regulatory complex associated with Radial Spoke 2. Current research is focused on understanding how these two axonemal hubs coordinate and regulate the dynein motors and ciliary motility.
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fap206 is a microtubule docking adapter for ciliary Radial Spoke 2 and dynein c
Molecular Biology of the Cell, 2015Co-Authors: Krishna Kumar Vasudevan, Winfield S Sale, Erin E. Dymek, Elizabeth F. Smith, Paulina Urbanska, Ewa Joachimiak, Lea M Alford, Kangkang Song, Todd M Hennessey, Dorota WlogaAbstract:Radial Spokes are conserved macromolecular complexes that are essential for ciliary motility. Little is known about the assembly and functions of the three individual Radial Spokes, RS1, RS2, and R...
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the chlamydomonas mutant pf27 reveals novel features of ciliary Radial Spoke assembly
Cytoskeleton, 2013Co-Authors: Lea M Alford, Susan K Dutcher, Alexa L Mattheyses, Emily L Hunter, Huawen Lin, Winfield S SaleAbstract:To address the mechanisms of ciliary Radial Spoke assembly, we took advantage of the Chlamydomonas pf27 mutant. The Radial Spokes that assemble in pf27 are localized to the proximal quarter of the axoneme, but otherwise are fully assembled into 20S Radial Spoke complexes competent to bind Spokeless axonemes in vitro. Thus, pf27 is not defective in Radial Spoke assembly or docking to the axoneme. Rather, our results suggest that pf27 is defective in the transport of Spoke complexes. During ciliary regeneration in pf27, Radial Spoke assembly occurs asynchronously from other axonemal components. In contrast, during ciliary regeneration in wild-type Chlamydomonas, Radial Spokes and other axonemal components assemble concurrently as the axoneme grows. Complementation in temporary dikaryons between wild-type and pf27 reveals rescue of Radial Spoke assembly that begins at the distal tip, allowing further assembly to proceed from tip to base of the axoneme. Notably, rescued assembly of Radial Spokes occurred independently of the established proximal Radial Spokes in pf27 axonemes in dikaryons. These results reveal that 20S Radial Spokes can assemble proximally in the pf27 cilium but as the cilium lengthens, Spoke assembly requires transport. We postulate that PF27 encodes an adaptor or modifier protein required for Radial Spoke – IFT interaction.
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control of axonemal inner dynein arms
Dyneins#R##N#Structure Biology and Disease, 2012Co-Authors: Juyeon Hwang, Winfield S Sale, Emily L Hunter, Maureen WirschellAbstract:Abstract In this chapter, we focus on the assembly, organization, and regulation of the axonemal inner dynein arms and their role in ciliary/flagellar motility. In contrast to the outer dynein arms, which are homogeneous, there are seven major and four minor species of inner dynein arms. Each inner dynein arm is distinct in composition and targeted to a precise and unique location within the 96-nm axonemal repeat. These conclusions are based on biochemical and ultrastructural analysis of Chlamydomonas mutants lacking subsets of dyneins. The motility phenotypes of these Chlamydomonas mutants also revealed the importance of the inner dynein arms for control of the size and shape of the axonemal bend, features of motility referred to as waveform. We review data revealing that second messengers, including calcium and cyclic nucleotides, can control ciliary motility by modulation of dynein activity. In particular, we focus on I1/f dynein and its regulation by a conserved phosphorylation pathway that includes signals from the central pair, Radial Spoke, and a network of axonemal kinases and phosphatases that are physically located in the axoneme.
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building a Radial Spoke flagellar Radial Spoke protein 3 rsp3 is a dimer
Cytoskeleton, 2008Co-Authors: Maureen Wirschell, Pinfen Yang, Chun Yang, Joel L Rosenbaum, Dennis R. Diener, Feifei Zhao, Anne R Gaillard, Winfield S SaleAbstract: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.
Pinfen Yang - One of the best experts on this subject based on the ideXlab platform.
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the structure and symmetry of the Radial Spoke protein complex in chlamydomonas flagella
Journal of Cell Science, 2020Co-Authors: Pinfen Yang, Dennis R. Diener, E Poghosyan, Ioan Iacovache, L Faltova, Alexander Leitner, Ruedi Aebersold, Benoit ZuberAbstract:The Radial Spoke is a key element in a transducer apparatus controlling the motility of eukaryotic cilia. The transduction biomechanics is a long-standing question in cilia biology. The Radial Spoke has three regions – a Spoke head, a bifurcated neck and a stalk. While the neck and the stalk are asymmetric, two-fold symmetry of the head has remained controversial. In this work we used single particle cryo-EM analysis to generate 3D structure of the whole Radial Spoke at unprecedented resolution. We show the head region at 15A resolution and confirm two-fold symmetry. Using distance constraints generated by crosslinking mass spectrometry we locate two components, RSP2 and 4 at the head/neck regions. Our biophysical analysis of isolated RSPs 4, 9, and 10 affirmed their oligomeric state. Our results enabled us to redefine the boundaries of the regions and propose a model of organization of the Radial Spoke component proteins.
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the structure and symmetry of Radial Spoke protein complex in chlamydomonas flagella
Journal of Cell Science, 2020Co-Authors: Pinfen Yang, E Poghosyan, Ioan Iacovache, L Faltova, Alexander Leitner, D R Diener, Ruedi Aebersold, Benoit ZuberAbstract:The Radial Spoke is a key element in a transducer apparatus controlling the motility of eukaryotic cilia. The transduction biomechanics is a long-standing question in cilia biology. The Radial Spoke has three regions - a Spoke head, a bifurcated neck and a stalk. While the neck and the stalk are asymmetric, two-fold symmetry of the head has remained controversial.In this work we used single particle cryo-EM analysis to generate 3D structure of the whole Radial Spoke at unprecedented resolution. We show the head region at 15A resolution and confirm two-fold symmetry. Using distance constraints generated by crosslinking mass spectrometry we locate two components, RSP2 and 4 at the head/neck regions. Our biophysical analysis of isolated RSPs 4, 9, and 10 affirmed their oligomeric state. Our results enabled us to redefine the boundaries of the regions and propose a model of organization of the Radial Spoke component proteins.
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general and specific promotion of flagellar assembly by a flagellar nucleoside diphosphate kinase
Molecular Biology of the Cell, 2017Co-Authors: Xiaoyan Zhu, Dennis R. Diener, Stephen M King, E Poghosyan, Yi Liu, Radhika Gopal, Kristine S Ciruelas, Yousif Maizy, Takashi Ishikawa, Pinfen YangAbstract:Nucleoside diphosphate kinases (NDKs) play a central role in diverse cellular processes using the canonical NDK activity or alternative mechanisms that remain poorly defined. Our study of dimeric NDK5 in a flagellar motility control complex, the Radial Spoke (RS), has revealed new modalities. The flagella in Chlamydomonas ndk5 mutant were paralyzed, albeit only deficient in three RS subunits. RS morphology appeared severely changed in averaged cryo-electron tomograms, suggesting that NDK5 is crucial for the intact Spokehead formation as well as RS structural stability. Intriguingly, ndk5's flagella were also short, resembling those of an allelic Spoke-less mutant. All ndk5's phenotypes were rescued by expressions of NDK5 or a mutated NDK5 lacking the canonical kinase activity. Importantly, the mutated NDK5 that appeared fully functional in ndk5 cells elicited a dominant-negative effect in wild-type cells, causing paralyzed short flagella with hypophosphorylated, less abundant, but intact RSs, and accumulated hypophosphorylated NDK5 in the cell body. We propose that NDK5 dimer is an RS structural subunit with an additional mechanism that uses cross-talk between the two NDK monomers to accelerate phosphorylation-related assembly of RSs and entire flagella.
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molecular tools for studying the Radial Spoke
Methods in Enzymology, 2013Co-Authors: Xiaoyan Zhu, Yi Liu, Priyanka Sivadas, Anjali Gupta, Pinfen YangAbstract:Abstract Studies of cilia and flagella often entail biochemical analysis of axonemal complexes that either associate with the nine outer doublet microtubules or the two singlet microtubules in the 9 + 2 axoneme. Each complex contains multiple subunits, a few of which are ubiquitous vital proteins, while many are novel with prevalent domains that remain to be characterized. Investigation of axoneme biochemistry will continue providing insights into flagellar biology as well as molecular complexes in general. Yet, the complicated contents and extensive molecular interactions pose significant challenges in experimentation. As such, most biochemical studies remain limited to dynein motors and often require extensive training and expensive equipment. The rapid accumulation of high-throughput database and versatile research tools has now lessened the obstacles significantly. Here, we describe the strategies and methods that were used to circumvent some of the common difficulties to characterize the Radial Spoke in Chlamydomonas axoneme, some of which were tailored to students with little research experience. They could be adapted for the study of many other axonemal complexes and for classroom settings as well.
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a flagellar a kinase anchoring protein with two amphipathic helices forms a structural scaffold in the Radial Spoke complex
Journal of Cell Biology, 2012Co-Authors: Priyanka Sivadas, Jennifer M Dienes, Martin St Maurice, William D Meek, Pinfen YangAbstract:A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar Radial Spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA Spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs.
Ritsu Kamiya - One of the best experts on this subject based on the ideXlab platform.
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Radial Spoke proteins of chlamydomonas flagella
Journal of Cell Science, 2006Co-Authors: Pinfen Yang, Winfield S Sale, Chun Yang, Dennis R. Diener, Takahiro Kohno, Gregory J Pazour, Jennifer M Dienes, Nathan S Agrin, Stephen M King, Ritsu KamiyaAbstract:The Radial Spoke is a ubiquitous component of '9+2' cilia and flagella, and plays an essential role in the control of dynein arm activity by relaying signals from the central pair of microtubules to the arms. The Chlamydomonas reinhardtii Radial Spoke contains at least 23 proteins, only 8 of which have been characterized at the molecular level. Here, we use mass spectrometry to identify 10 additional Radial Spoke proteins. Many of the newly identified proteins in the Spoke stalk are predicted to contain domains associated with signal transduction, including Ca2+-, AKAP- and nucleotide-binding domains. This suggests that the Spoke stalk is both a scaffold for signaling molecules and itself a transducer of signals. Moreover, in addition to the recently described HSP40 family member, a second Spoke stalk protein is predicted to be a molecular chaperone, implying that there is a sophisticated mechanism for the assembly of this large complex. Among the 18 Spoke proteins identified to date, at least 12 have apparent homologs in humans, indicating that the Radial Spoke has been conserved throughout evolution. The human genes encoding these proteins are candidates for causing primary ciliary dyskinesia, a severe inherited disease involving missing or defective axonemal structures, including the Radial Spokes.
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an axonemal dynein particularly important for flagellar movement at high viscosity implications from a new chlamydomonas mutant deficient in the dynein heavy chain gene dhc9
Journal of Biological Chemistry, 2005Co-Authors: Toshiki Yagi, Itsushi Minoura, Akiko Fujiwara, Ryo Saito, Takuo Yasunaga, Masafumi Hirono, Ritsu KamiyaAbstract:Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 Radial Spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor.
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identification of a novel leucine rich repeat protein as a component of flagellar Radial Spoke in the ascidian ciona intestinalis
Molecular Biology of the Cell, 2003Co-Authors: Potturi Padma, Ritsu Kamiya, Yuhkoh Satouh, Kazuo Inaba, Kenichi Wakabayashi, Akiko Hozumi, Yuji UshimaruAbstract:Axonemes are highly organized microtubule-based structures conserved in many eukaryotes. In an attempt to study axonemes by a proteomics approach, we selectively cloned cDNAs of axonemal proteins by immunoscreening the testis cDNA library from the ascidian Ciona intestinalis by using an antiserum against whole axonemes. We report here a 37-kDa protein of which cDNA occurred most frequently among total positive clones. This protein, named LRR37, belongs to the class of SDS22+ leucine-rich repeat (LRR) family. LRR37 is different from the LRR outer arm dynein light chain reported in Chlamydomonas and sea urchin flagella, and thus represents a novel axonemal LRR protein. Immunoelectron microscopy by using a polyclonal antibody against LRR37 showed that it is localized on the tip of the Radial Spoke, most likely on the Spoke head. The LRR37 protein in fact seems to form a complex together with Radial Spoke protein 3 in a KI extract of the axonemes. These results suggest that LRR37 is a component of the Radial Spoke head and is involved in the interaction with other Radial Spoke components or proteins in the central pair projection.
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vigorous beating of chlamydomonas axonemes lacking central pair Radial Spoke structures in the presence of salts and organic compounds
Cytoskeleton, 2000Co-Authors: Toshiki Yagi, Ritsu KamiyaAbstract:Flagella of Chlamydomonas mutants lacking the central pair of microtubules or Radial Spokes do not beat; however, axonemes isolated from these mutants were found to display vigorous bending movements in the presence of ATP and various salts, sugars, alcohols, and other organic compounds. For example, about 15% of the total axonemes isolated from pf18, a mutant lacking the central pair, displayed beating in the presence of 10 mM MgSO(4) and 0.2 mM ATP at about 22 Hz, while none beat with the same concentration of ATP and or = 25 mM MgSO(4). The beat frequency and waveform of beating pf18 axonemes were similar to those of wild type axonemes beating under the same conditions. Similarly, 10-50% of the axonemes beat in the presence of 0.5 M sucrose, 2.0 M glycerol, or 1.7 M[10% (v/v)] ethanol. The appearance of motility did not correlate with the change in axonemal ATPase; however, these substances at those concentrations commonly increased the amplitude of nanometer-scale oscillation (hyper-oscillation) in pf18 axonemes, as well as the extent of ATP-induced sliding disintegration of protease-treated axonemes. Axonemes of double mutants lacking both the central pair and various subspecies of inner-arm dynein also beat at increased MgSO(4) concentrations, but axonemes lacking outer-arm dynein in addition to the central pair did not beat. These and other observations suggest that small molecules perturb the regulation of microtubule sliding through some change in water activity or osmotic stress. Axonemes must have an intrinsic ability to beat without the central pair/Radial Spokes under a variety of non-physiological solution conditions, as long as the outer dynein arms are present. Apparently, the major function of the central pair/Radial Spoke structures is to restore this activity under physiological conditions.
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ca2 dependent waveform conversion in the flagellar axoneme of chlamydomonas mutants lacking the central pair Radial Spoke system
Cytoskeleton, 1997Co-Authors: Kenichi Wakabayashi, Toshiki Yagi, Ritsu KamiyaAbstract:Chlamydomonas flagella undergo a striking waveform conversion from an asymmetrical ciliary type to a symmetrical flagellar type when the cell is stimulated by intense light and the Ca2+ concentration within the flagellum is increased above approximately 10(-6) M. To see whether the central-pair/Radial Spoke system is needed for this conversion as suggested by previous studies, we examined the effect of Ca2+ on the reactivated axonemes of the mutants lacking the central pair (pf18) or the Radial Spokes (pf14). Although the flagella of these mutants are paralyzed in vivo, demembranated axonemes can be reactivated to beat under certain nucleotide conditions such as in the presence of low concentrations ( =10(-5) M Ca2+. Although the waveform is different between the mutants and the wild type, the Ca2+ concentration at which the waveform conversion occurred was similar. These results indicate that the central pair/Radial Spoke system is not essential for the waveform conversion.
J L Porter - One of the best experts on this subject based on the ideXlab platform.
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high yield inertial confinement fusion target design for a z pinch driven hohlraum
Physics of Plasmas, 1999Co-Authors: J H Hammer, Scott C. Wilks, Marcel Tabak, J D Lindl, G. B. Zimmerman, D. S. Bailey, Anju Toor, P W Rambo, J L PorterAbstract:Calculations are presented for a high yield inertial fusion design, employing indirect drive with a double-ended z-pinch-driven hohlraum radiation source. A high current (∼60 MA) accelerator implodes z pinches within an enclosing hohlraum. Radial Spoke arrays and shine shields isolate the capsule from the pinch plasma, magnetic field, and direct x-ray shine. Our approach places minimal requirements on z-pinch uniformity and stability, usually problematic due to magneto-Rayleigh–Taylor instability. Large inhomogeneities of the pinch and Spoke array may be present, but the hohlraum adequately smooths the radiation field at the capsule. Simultaneity and reproducibility of the pinch x-ray output to better than 7% are required, however, for good symmetry. Recent experiments suggest a pulse shaping technique, through implosion of a multishell z pinch. X-ray bursts are calculated and observed to occur at each shell collision. A capsule absorbing 1 MJ of x rays at a peak drive temperature of 210 eV is found to ha...
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High yield inertial confinement fusion target design for a z-pinch-driven hohlraum
Physics of Plasmas, 1999Co-Authors: J H Hammer, Scott C. Wilks, Marcel Tabak, J D Lindl, G. B. Zimmerman, D. S. Bailey, Anju Toor, P W Rambo, J L PorterAbstract:Calculations are presented for a high yield inertial fusion design, employing indirect drive with a double-ended z-pinch-driven hohlraum radiation source. A high current (∼60 MA) accelerator implodes z pinches within an enclosing hohlraum. Radial Spoke arrays and shine shields isolate the capsule from the pinch plasma, magnetic field, and direct x-ray shine. Our approach places minimal requirements on z-pinch uniformity and stability, usually problematic due to magneto-Rayleigh–Taylor instability. Large inhomogeneities of the pinch and Spoke array may be present, but the hohlraum adequately smooths the radiation field at the capsule. Simultaneity and reproducibility of the pinch x-ray output to better than 7% are required, however, for good symmetry. Recent experiments suggest a pulse shaping technique, through implosion of a multishell z pinch.X-ray bursts are calculated and observed to occur at each shell collision. A capsule absorbing 1 MJ of x rays at a peak drive temperature of 210 eV is found to have adequate stability and to produce 400 MJ of yield. A larger capsule absorbs 2 MJ with a yield of 1200 MJ.
Kazuo Inaba - One of the best experts on this subject based on the ideXlab platform.
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Western blots of cilia and flagella in C. intestinalis using several antibodies against axonemal proteins.
2015Co-Authors: Alu Konno, Kogiku Shiba, Chunhua Cai, Kazuo InabaAbstract:Branchial cilia (BC) and sperm flagella (SF) were separated by SDS-PAGE and immunoblotted with antibodies against several axonemal structures. Both IC2 and IC3 are intermediate chains of the outer arm dynein. LRR-LC and Tctex2-LC are light chains of the outer arm dynein. Ap58 is a protein involved in the anchoring of the outer arm dynein. Both IC98 (ortholog of Chlamydomonas IC110) and IC116 (ortholog of Chlamydomonas IC140) are intermediate chains of two-headed inner arm f/I1 dynein. RSP3, AxHsp40, LRR37 and MORN40 are components of the Radial Spoke. PF16 is a protein of the central pair apparatus. PKAR2, Calaxin is a Ca2+-binding protein that regulates the outer arm dynein. ARM94 and Tctex2-LC are known to be phosphorylated or dephosphorylated at the activation of sperm motility. ODF3 is a component of the outer dense fiber of mammalian sperm. Both TD27 and TD01 are uncharacterized proteins associated with flagellar axonemes. Tektin 3 is a structural protein in the axoneme (see text).
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molecular characterization of Radial Spoke subcomplex containing Radial Spoke protein 3 and heat shock protein 40 in sperm flagella of the ascidian ciona intestinalis
Molecular Biology of the Cell, 2004Co-Authors: Yuhkoh Satouh, Potturi Padma, Toshifusa Toda, Nori Satoh, Hiroyuki Ide, Kazuo InabaAbstract:Members of the heat-shock protein (HSP)40 regulate the protein folding activity of HSP70 proteins and help the functional specialization of this molecular chaperone system in various types of cellular events. We have recently identified Hsp40 as a component of flagellar axoneme in the ascidian Ciona intestinalis, suggesting a correlation between Hsp40 related chaperone system and flagellar function. In this study, we have found that Ciona 37-kDa Hsp40 is extracted from KCl-treated axonemes with 0.5 M KI solution and comigrates with Radial Spoke protein (RSP)3 along with several proteins as a complex through gel filtration and ion exchange columns. Peptide mass fingerprinting with matrix-assisted laser desorption ionization/time of flight/mass spectrometry revealed that other proteins in the complex include a homolog of sea urchin Spokehead protein (homolog of RSP4/6), a membrane occupation and recognition nexus repeat protein with sequence similarity with meichroacidin, and a functionally unknown 33-kDa protein. A Spoke head protein, LRR37, is not included in the complex, suggesting that the complex constructs the stalk of Radial Spoke. Immunoelectron microscopy indicates that Hsp40 is localized in the distal portion of Spoke stalk, possibly at the junction between Spoke head and the stalk.
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molecular architecture of the sperm flagella molecules for motility and signaling
Zoological Science, 2003Co-Authors: Kazuo InabaAbstract:Sperm motility is generated by a highly organized, microtubule-based structure, called the axoneme, which is constructed from approximately 250 proteins. Recent studies have revealed the molecular structures and functions of a number of axonemal components, including the motor molecules, the dyneins, and regulatory substructures, such as Radial Spoke, central pair, and other accessory structures. The force for flagellar movement is exerted by the sliding of outer-doublet microtubules driven by the molecular motors, the dyneins. Dynein activity is regulated by the Radial Spoke/central pair apparatus through protein phosphorylation, resulting in flagellar bend propagation. Prior to fertilization, sperm exhibit dramatic motility changes, such as initiation and activation of motility and chemotaxis toward the egg. These changes are triggered by changes in the extracellular ionic environment and substances released from the female reproductive tract or egg. After reception of these extracellular signals by specific ion channels or receptors in the sperm cells, intracellular signals are switched on through tyrosine protein phosphorylation, Ca2+, and cyclic nucleotide-dependent pathways. All these signaling molecules are closely arranged in each sperm flagellum, leading to efficient activation of motility.
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identification of a novel leucine rich repeat protein as a component of flagellar Radial Spoke in the ascidian ciona intestinalis
Molecular Biology of the Cell, 2003Co-Authors: Potturi Padma, Ritsu Kamiya, Yuhkoh Satouh, Kazuo Inaba, Kenichi Wakabayashi, Akiko Hozumi, Yuji UshimaruAbstract:Axonemes are highly organized microtubule-based structures conserved in many eukaryotes. In an attempt to study axonemes by a proteomics approach, we selectively cloned cDNAs of axonemal proteins by immunoscreening the testis cDNA library from the ascidian Ciona intestinalis by using an antiserum against whole axonemes. We report here a 37-kDa protein of which cDNA occurred most frequently among total positive clones. This protein, named LRR37, belongs to the class of SDS22+ leucine-rich repeat (LRR) family. LRR37 is different from the LRR outer arm dynein light chain reported in Chlamydomonas and sea urchin flagella, and thus represents a novel axonemal LRR protein. Immunoelectron microscopy by using a polyclonal antibody against LRR37 showed that it is localized on the tip of the Radial Spoke, most likely on the Spoke head. The LRR37 protein in fact seems to form a complex together with Radial Spoke protein 3 in a KI extract of the axonemes. These results suggest that LRR37 is a component of the Radial Spoke head and is involved in the interaction with other Radial Spoke components or proteins in the central pair projection.
