scholarly journals Localization of Calmodulin and Dynein Light Chain Lc8 in Flagellar Radial Spokes

2001 ◽  
Vol 153 (6) ◽  
pp. 1315-1326 ◽  
Author(s):  
Pinfen Yang ◽  
Dennis R. Diener ◽  
Joel L. Rosenbaum ◽  
Winfield S. Sale
Keyword(s):  
Light Chain ◽  
Crucial Role ◽  
Cross Linking ◽  
Dynein Light Chain ◽  
Flagellar Motility ◽  
Radial Spoke ◽  
Size And Shape ◽  
Radial Spokes ◽  
Chemical Cross Linking

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.

scholarly journals Dimeric Novel HSP40 Is Incorporated into the Radial Spoke Complex during the Assembly Process in Flagella

2005 ◽  
Vol 16 (2) ◽  
pp. 637-648 ◽  
Author(s):  
Chun Yang ◽  
Mark M. Compton ◽  
Pinfen Yang
Keyword(s):  
Protein Folding ◽  
Cell Body ◽  
Stable Complex ◽  
Assembly Process ◽  
Flagellar Motility ◽  
Radial Spoke ◽  
Precursor Complex ◽  
Radial Spokes

The radial spoke is a stable structural complex in the 9 + 2 axoneme for the control of flagellar motility. However, the spokes in Chlamydomonas mutant pf24 are heterogeneous and unstable, whereas several spoke proteins are reduced differentially. To elucidate the defective mechanism, we clone RSP16, a prominent spoke protein diminished in pf24 axonemes. Unexpectedly, RSP16 is a novel HSP40 member of the DnaJ superfamily that assists chaperones in various protein-folding-related processes. Importantly, RSP16 is uniquely excluded from the 12S spoke precursor complex that is packaged in the cell body and transported toward the flagellar tip to be converted into mature 20S axonemal spokes. Rather, RSP16, transported separately, joins the precursor complex in flagella. Furthermore, RSP16 molecules in vitro and in flagella form homodimers, a characteristic required for the cochaperone activity of HSP40. We postulate that the spoke HSP40 operates as a cochaperone to assist chaperone machinery at the flagellar tip to actively convert the smaller spoke precursor and itself into the mature stable complex; failure of the interaction between the spoke HSP40 and its target polypeptide results in heterogeneous unstable radial spokes in pf24.

scholarly journals Assembly of flagellar radial spoke proteins in Chlamydomonas: identification of the axoneme binding domain of radial spoke protein 3.

1993 ◽  
Vol 123 (1) ◽  
pp. 183-190 ◽  
Author(s):  
D R Diener ◽  
L H Ang ◽  
J L Rosenbaum
Keyword(s):  
Amino Acids ◽  
Binding Activity ◽  
Binding Domain ◽  
Flagellar Motility ◽  
Radial Spoke ◽  
Acid Protein ◽  
Radial Spokes ◽  
Carboxy Terminal ◽  
Amino Acid Protein

Radial spokes of the eukaryotic flagellum extend from the A tubule of each outer doublet microtubule toward the central pair microtubules. In the paralyzed flagella mutant of Chlamydomonas pf14, a mutation in the gene for one of 17 polypeptides that comprise the radial spokes results in flagella that lack all 17 spoke components. The defective gene product, radial spoke protein 3 (RSP3), is, therefore, pivotal to the assembly of the entire spoke and may attach the spoke to the axoneme. We have synthesized RSP3 in vitro and assayed its binding to axonemes from pf14 cells to determine if RSP3 can attach to spokeless axonemes. In vitro, RSP3 binds to pf14 axonemes, but not to wild-type axonemes or microtubules polymerized from purified chick brain tubulin. The sole axoneme binding domain of RSP3 is located within amino acids 1-85 of the 516 amino acid protein; deletion of these amino acids abolishes binding by RSP3. Fusion of amino acids 1-85 or 42-85 to an unrelated protein confers complete or partial binding activity, respectively, to the fusion protein. Transformation of pf14 cells with mutagenized RSP3 genes indicates that amino acids 18-87 of RSP3 are important to its function, but that the carboxy-terminal 140 amino acids can be deleted with little effect on radial spoke assembly or flagellar motility.

scholarly journals Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

2018 ◽  
Vol 19 (10) ◽  
pp. 2928 ◽  
Author(s):  
Winfried Roseboom ◽  
Madhvi Nazir ◽  
Nils Meiresonne ◽  
Tamimount Mohammadi ◽  
Jolanda Verheul ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Binding Interface ◽  
Tetrameric Structure ◽  
Z Ring ◽  
Cross Links ◽  
Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

scholarly journals The Flagellar Motility ofChlamydomonas pf25 Mutant Lacking an AKAP-binding Protein Is Overtly Sensitive to Medium Conditions

2006 ◽  
Vol 17 (1) ◽  
pp. 227-238 ◽  
Author(s):  
Chun Yang ◽  
Pinfen Yang
Keyword(s):  
Regulatory Proteins ◽  
Flagellar Motility ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Wide Range ◽  
Anchoring Protein ◽  
Radial Spokes ◽  
Dependent Protein ◽  
Cilia And Flagella

Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via a network of phosphoenzymes and regulatory proteins. Consistently, a Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes the cAMP-dependent protein kinase (PKA) holoenzyme by binding to the RIIa domain of PKA RII subunit. However, the highly conserved docking domain of PKA is also found in the N termini of several AKAP-binding proteins unrelated to PKA as well as a 24-kDa novel spoke protein, RSP11. Here, we report that RSP11 binds to RSP3 directly in vitro and colocalizes with RSP3 toward the spoke base near outer doublets and dynein motors in axonemes. Importantly, RSP11 mutant pf25 displays a spectrum of motility, from paralysis with flaccid or twitching flagella as other spoke mutants to wild-typelike swimming. The wide range of motility changes reversibly depending on the condition of liquid media without replacing defective proteins. We postulate that radial spokes use the RIIa/AKAP module to regulate ciliary and flagellar beating; absence of the spoke RIIa protein exposes a medium-sensitive regulatory mechanism that is not obvious in wild-type Chlamydomonas.

scholarly journals Identification of a Ca(2+)-binding light chain within Chlamydomonas outer arm dynein

1995 ◽  
Vol 108 (12) ◽  
pp. 3757-3764 ◽  
Author(s):  
S.M. King ◽  
R.S. Patel-King
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Dynein Light Chain ◽  
Binding Studies ◽  
Significant Similarity ◽  
Helix Loop Helix ◽  
Ef Hand ◽  
Chain Sequence

We describe here the molecular cloning of the M(r) 18,000 dynein light chain from the outer arm of Chlamydomonas flagella. In vivo, this molecule is directly associated with the gamma dynein heavy chain. Sequence analysis indicates that this light chain is a novel member of the calmodulin superfamily of Ca2+ binding regulatory proteins; this molecule is 42, 37 and 36% identical to calmodulin, centrin/caltractin and troponin C, respectively, and also shows significant similarity to myosin light chains. Although four helix-loop-helix elements are evident, only two conform precisely to the EF hand consensus and are therefore predicted to bind Ca2+ in vivo. In vitro Ca2+ binding studies indicate that this dynein light chain (expressed as a C-terminal fusion with maltose binding protein) has at least one functional Ca2+ binding site with an apparent affinity for Ca2+ of approximately 3 × 10(−5) M. Within the Chlamydomonas flagellum, the transition from an assymmetric to a symmetric waveform (which implies an alteration in dynein activity) is mediated by an increase in intraflagellar Ca2+ from 10(−6) to 10(−1) M; this transition is altered in mutants that lack the outer arm. The data presented here suggest that a Ca(2+)-dependent alteration in the interaction of this dynein light chain with the motor containing heavy chain may affect outer arm function during flagellar reversal.

scholarly journals The dynein light chain protein Tda2 functions as a dimerization engine to regulate actin capping protein during endocytosis

2021 ◽  
pp. mbc.E21-01-0032
Author(s):  
Andrew K. Lamb ◽  
Andres N. Fernandez ◽  
Olve B. Peersen ◽  
Santiago M. Di Pietro
Keyword(s):  
Light Chain ◽  
Mammalian Cells ◽  
Dynein Light Chain ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Capping Protein ◽  
Actin Capping Protein ◽  
Actin Capping ◽  
Protein Recruitment

Clathrin- and actin-mediated endocytosis is a fundamental process in eukaryotic cells. Previously, we discovered Tda2 as a new yeast dynein light chain that works with Aim21 to regulate actin assembly during endocytosis. Here, we show Tda2 functions as a dimerization engine bringing two Aim21 molecules together using a novel binding surface different than the canonical dynein light chain ligand binding groove. Point mutations on either protein that diminish the Tda2-Aim21 interaction in vitro cause the same in vivo phenotype as TDA2 deletion showing reduced actin capping protein recruitment and increased filamentous actin at endocytic sites. Remarkably, chemically induced dimerization of Aim21 rescues the endocytic phenotype of TDA2 deletion. We also uncovered a capping protein interacting motif in Aim21, expanding its function to a fundamental cellular pathway and showing such motif exists outside mammalian cells. Furthermore, specific disruption of this motif causes the same deficit of actin capping protein recruitment and increased filamentous actin at endocytic sites as AIM21 deletion. Thus, the data indicates the Tda2-Aim21 complex functions in actin assembly primarily through capping protein regulation. Collectively, our results provide a mechanistic view of the Tda2-Aim21 complex and its function in actin network regulation at endocytic sites.

scholarly journals Identification of a Novel Leucine-rich Repeat Protein as a Component of Flagellar Radial Spoke in the Ascidian Ciona intestinalis

2003 ◽  
Vol 14 (2) ◽  
pp. 774-785 ◽  
Author(s):  
Potturi Padma ◽  
Yuhkoh Satouh ◽  
Ken-ichi Wakabayashi ◽  
Akiko Hozumi ◽  
Yuji Ushimaru ◽  
...  
Keyword(s):  
Polyclonal Antibody ◽  
Light Chain ◽  
Dynein Light Chain ◽  
Leucine Rich Repeat ◽  
Radial Spoke ◽  
Testis Cdna Library ◽  
Proteomics Approach ◽  
Testis Cdna ◽  
Lrr Protein ◽  
Leucine Rich Repeat Protein

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 ascidianCiona 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 inChlamydomonas 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.

Preparation of Carboxymethyl Chitosan Microspheres and Their Application in Hemostasis

2015 ◽  
Vol 11 (6) ◽  
pp. 660-667 ◽  
Author(s):  
Lu Liu ◽  
Qi Lv ◽  
Qingyun Zhang ◽  
Hui Zhu ◽  
Wei Liu ◽  
...  
Keyword(s):  
Hepatic Injury ◽  
Carboxymethyl Chitosan ◽  
Water Soluble ◽  
Cross Linking ◽  
Clotting Time ◽  
Hemostatic Agents ◽  
Hemostatic Effect ◽  
Injury Model ◽  
Chemical Cross Linking

AbstractObjectiveChitosan (CS) is currently used as a hemostatic agent in emergencies and in military settings. However, its application is limited owing to its poor hydrophilia at neutral pH. Carboxymethyl chitosan (CMCS) is an important, water-soluble derivative of CS. In this study, we prepared CS and CMCS microspheres (CSMs and CMCSMs, respectively) and evaluated their hemostatic effect.MethodsTo prepare the microspheres of various sizes, we used the emulsion cross-linking technique. CMCSMs were also loaded with etamsylate (DIC). Clotting time in vitro and in a hepatic injury model was examined to evaluate the hemostatic effect.ResultsCMCSMs swelled more and clotted faster than did CSMs. CMCSMs loaded with DIC had no effect on hemostasis.ConclusionsBoth increasing material hydrophilicity and expanding the contact area promoted clotting, whereas chemical cross-linking hampered it because of decreased swelling. CMCSMs are promising candidates for the production of effective hemostatic agents. (Disaster Med Public Health Preparedness. 2017;11:660–667)

scholarly journals An outer arm dynein light chain acts in a conformational switch for flagellar motility

2009 ◽  
Vol 186 (2) ◽  
pp. 283-295 ◽  
Author(s):  
Ramila S. Patel-King ◽  
Stephen M. King
Keyword(s):  
Light Chain ◽  
Dominant Negative ◽  
Motor Domain ◽  
Swimming Velocity ◽  
Dynein Light Chain ◽  
Flagellar Motility ◽  
Negative Effects ◽  
Independent Manner ◽  
Conformational Switch ◽  
Mutant Forms

A system distinct from the central pair–radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the γ heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this γ HC–LC1–microtubule ternary complex functions as a conformational switch to control outer arm activity.

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

2012 ◽  
Vol 23 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Cynthia F. Barber ◽  
Thomas Heuser ◽  
Blanca I. Carbajal-González ◽  
Vladimir V. Botchkarev ◽  
Daniela Nicastro
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Flagellar Motility ◽  
Three Dimensional Structure ◽  
Radial Spoke ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Axonemal Dynein ◽  
Subtomogram Averaging

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.

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