scholarly journals Regulation of Flagellar Dynein by Calcium and a Role for an Axonemal Calmodulin and Calmodulin-dependent Kinase

2002 ◽  
Vol 13 (9) ◽  
pp. 3303-3313 ◽  
Author(s):  
Elizabeth F. Smith
Keyword(s):  
Calcium Signaling ◽  
Signaling Pathway ◽  
Molecular Mechanism ◽  
Calcium Signal ◽  
Sliding Velocity ◽  
Flagellar Motility ◽  
Wild Type ◽  
Wild Type Level ◽  
Radial Spokes ◽  
Central Apparatus

Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonasmutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at pCa4, dynein activity in pf18 and pf15axonemes was restored to wild-type level. The calcium-induced increase in dynein activity in pf18 axonemes was inhibited by antagonists of calmodulin and calmodulin-dependent kinase II. Axonemes lacking the C1 central tubule (pf16) or lacking radial spoke components (pf14 and pf17) do not exhibit calcium-induced increase in dynein activity in pCa4 buffer. We conclude that calcium regulation of flagellar motility involves regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase II may mediate the calcium signal, and that the central apparatus and radial spokes are key components of the calcium signaling pathway.

scholarly journals PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

1996 ◽  
Vol 132 (3) ◽  
pp. 359-370 ◽  
Author(s):  
E F Smith ◽  
P A Lefebvre
Keyword(s):  
Protein Interactions ◽  
Insertional Mutagenesis ◽  
Flagellar Motility ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Central Pair ◽  
Central Apparatus ◽  
Immunofluorescence Labeling ◽  
Armadillo Repeats

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas

1996 ◽  
Vol 109 (7) ◽  
pp. 1899-1907 ◽  
Author(s):  
G. Habermacher ◽  
W.S. Sale
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Double Mutant ◽  
Sliding Velocity ◽  
Wild Type ◽  
Phosphatase Inhibitors ◽  
Radial Spokes ◽  
Gamma S ◽  
Type 1 Phosphatase

Physiological studies have demonstrated that flagellar radial spokes regulate inner arm dynein activity in Chlamydomonas and that an axonemal cAMP-dependent kinase inhibits dynein activity in radial spoke defective axonemes. These studies also suggested that an axonemal protein phosphatase is required for activation of flagellar dynein. We tested whether inhibitors of protein phosphatases would prevent activation of dynein by the kinase inhibitor PKI in Chlamydomonas axonemes lacking radial spokes. As predicted, preincubation of spoke defective axonemes (pf14 and pf17) with ATP gamma S maintained the slow dynein-driven microtubule sliding characteristic of paralyzed axonemes lacking spokes, and blocked activation of dynein-driven microtubule sliding by subsequent addition of PKI. Preincubation of spoke defective axonemes with the phosphatase inhibitors okadaic acid, microcystin-LR or inhibitor-2 also potently blocked PKI-induced activation of microtubule sliding velocity: the non-inhibitory okadaic acid analog, 1-norokadaone, did not. ATP gamma S or the phosphatase inhibitors blocked activation of dynein in a double mutant lacking the radial spokes and the outer dynein arms (pf14pf28). We concluded that the axoneme contains a type-1 phosphatase required for activation of inner arm dynein. We postulated that the radial spokes regulate dynein through the activity of the type-1 protein phosphatase. To test this, we performed in vitro reconstitution experiments using inner arm dynein from the double mutant pf14pf28 and dynein-depleted axonemes containing wild-type radial spokes (pf28). As described previously, microtubule sliding velocity was increased from approximately 2 microns/second to approximately 7 microns/second when inner arm dynein from pf14pf28 axonemes ws reconstituted with axonemes containing wild-type spokes. In contrast, pretreatment of inner arm dynein from pf14pf28 axonemes with ATP gamma S, or reconstitution in the presence of microcystin-LR, blocked increased velocity following reconstitution, despite the presence of wild-type radial spokes. We conclude that the radial spokes, through the activity of an axonemal type-1 phosphatase, activate inner arm dynein by dephosphorylation of a critical dynein component. Wild-type radial spokes also operate to inhibit the axonemal cAMP-dependent kinase, which would otherwise inhibit axonemal dynein and motility.

scholarly journals PF15p Is the Chlamydomonas Homologue of the Katanin p80 Subunit and Is Required for Assembly of Flagellar Central Microtubules

2004 ◽  
Vol 3 (4) ◽  
pp. 870-879 ◽  
Author(s):  
Erin E. Dymek ◽  
Paul A. Lefebvre ◽  
Elizabeth F. Smith
Keyword(s):  
Amino Acid ◽  
Significant Role ◽  
Insertional Mutagenesis ◽  
Flagellar Motility ◽  
Wild Type ◽  
Central Pair ◽  
Coding Sequence ◽  
Microtubule Severing ◽  
Central Apparatus

ABSTRACT Numerous studies have indicated that the central apparatus plays a significant role in regulating flagellar motility, yet little is known about how the central pair of microtubules or their associated projections assemble. Several Chlamydomonas mutants are defective in central apparatus assembly. For example, mutant pf15 cells have paralyzed flagella that completely lack the central pair of microtubules. We have cloned the wild-type PF15 gene and confirmed its identity by rescuing the motility and ultrastructural defects in two pf15 alleles, the original pf15a mutant and a mutant generated by insertional mutagenesis. Database searches using the 798-amino-acid polypeptide predicted from the complete coding sequence indicate that the PF15 gene encodes the Chlamydomonas homologue of the katanin p80 subunit. Katanin was originally identified as a heterodimeric protein with a microtubule-severing activity. These results reveal a novel role for the katanin p80 subunit in the assembly and/or stability of the central pair of flagellar microtubules.

scholarly journals The CSC connects three major axonemal complexes involved in dynein regulation

2012 ◽  
Vol 23 (16) ◽  
pp. 3143-3155 ◽  
Author(s):  
Thomas Heuser ◽  
Erin E. Dymek ◽  
Jianfeng Lin ◽  
Elizabeth F. Smith ◽  
Daniela Nicastro
Keyword(s):  
Electron Tomography ◽  
Structural Heterogeneity ◽  
Flagellar Motility ◽  
Wild Type ◽  
Localization Model ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Regulatory Complex ◽  
Health And Development ◽  
Cilia And Flagella

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ­ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.

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 The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes

2013 ◽  
Vol 24 (8) ◽  
pp. 1134-1152 ◽  
Author(s):  
Raqual Bower ◽  
Douglas Tritschler ◽  
Kristyn VanderWaal ◽  
Catherine A. Perrone ◽  
Joshua Mueller ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Distal Region ◽  
Proximal Region ◽  
Optimal Alignment ◽  
Flagellar Motility ◽  
Wild Type ◽  
Regulatory Complex ◽  
Conserved Region ◽  
Motile Cilia ◽  
Discrete Complex

The nexin–dynein regulatory complex (N-DRC) is proposed to coordinate dynein arm activity and interconnect doublet microtubules. Here we identify a conserved region in DRC4 critical for assembly of the N-DRC into the axoneme. At least 10 subunits associate with DRC4 to form a discrete complex distinct from other axonemal substructures. Transformation of drc4 mutants with epitope-tagged DRC4 rescues the motility defects and restores assembly of missing DRC subunits and associated inner-arm dyneins. Four new DRC subunits contain calcium-signaling motifs and/or AAA domains and are nearly ubiquitous in species with motile cilia. However, drc mutants are motile and maintain the 9 + 2 organization of the axoneme. To evaluate the function of the N-DRC, we analyzed ATP-induced reactivation of isolated axonemes. Rather than the reactivated bending observed with wild-type axonemes, ATP addition to drc-mutant axonemes resulted in splaying of doublets in the distal region, followed by oscillatory bending between pairs of doublets. Thus the N-DRC provides some but not all of the resistance to microtubule sliding and helps to maintain optimal alignment of doublets for productive flagellar motility. These findings provide new insights into the mechanisms that regulate motility and further highlight the importance of the proximal region of the axoneme in generating flagellar bending.

Adenine-based calcium signal pathway messengers: Synthesis and agonistic properties of cyclic ADP-ribose analogs

2008 ◽  
Vol 80 (8) ◽  
pp. 1821-1825
Author(s):  
Liangren Zhang ◽  
Zhenjun Yang ◽  
Andreas H. Guse ◽  
Lihe Zhang
Keyword(s):  
T Lymphocytes ◽  
Calcium Signaling ◽  
Molecular Mechanism ◽  
Calcium Release ◽  
Biological Activities ◽  
Signal Pathway ◽  
Calcium Signal ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Cyclic Adp Ribose

A series of cyclic ADP-ribose (cADPR) analogs, in which modifications mainly focused on riboses, was synthesized in order to explore the molecular mechanism of calcium release regulated by cADPR. Biological activities investigated in intact T-lymphocytes showed that the structurally simplified analogs, N1-ethoxymethyl-substituted cyclic inosine diphosphoribose (cIDPRE), N1,N9-diethoxymethyl-substituted cyclic inosine diphosphoribose (cIDPDE), and N1-ethoxymethyl-substituted cyclic adenosine diphosphoribose (cADPRE) in which the northern ribose or both northern and southern riboses were replaced by ether linkages are membrane-permeant and induce calcium release from intracellular stores. This research has provided novel molecules to probe cADPR-mediated calcium signaling and enlarges our knowledge of the structure-activity relationships of cADPR analogs.

scholarly journals Mutations in Hydin impair ciliary motility in mice

2008 ◽  
Vol 180 (3) ◽  
pp. 633-643 ◽  
Author(s):  
Karl-Ferdinand Lechtreck ◽  
Philippe Delmotte ◽  
Michael L. Robinson ◽  
Michael J. Sanderson ◽  
George B. Witman
Keyword(s):  
Fluid Transport ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Flagellar Motility ◽  
Wild Type ◽  
Central Pair ◽  
Ciliary Motility ◽  
Pair Defect ◽  
Radial Spokes ◽  
The Brain

Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility, and mice with Hydin defects develop lethal hydrocephalus. To determine if defects in Hydin cause hydrocephalus through a mechanism involving cilia, we compared the morphology, ultrastructure, and activity of cilia in wild-type and hydin mutant mice strains. The length and density of cilia in the brains of mutant animals is normal. The ciliary axoneme is normal with respect to the 9 + 2 microtubules, dynein arms, and radial spokes but one of the two central microtubules lacks a specific projection. The hydin mutant cilia are unable to bend normally, ciliary beat frequency is reduced, and the cilia tend to stall. As a result, these cilia are incapable of generating fluid flow. Similar defects are observed for cilia in trachea. We conclude that hydrocephalus in hydin mutants is caused by a central pair defect impairing ciliary motility and fluid transport in the brain.

scholarly journals Exploring the Molecular Mechanism of Astragali Radix-Curcumae Rhizoma against Gastric Intraepithelial Neoplasia by Network Pharmacology and Molecular Docking

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Yuejin Ji ◽  
Yajun Liu ◽  
Jingyi Hu ◽  
Cheng Cheng ◽  
Jing Xing ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Calcium Signaling ◽  
Signaling Pathway ◽  
Molecular Mechanism ◽  
Intraepithelial Neoplasia ◽  
Receptor Interaction ◽  
Ppi Network ◽  
Active Components ◽  
Drug Pair ◽  
Astragali Radix

Background. Astragali Radix-Curcumae Rhizoma (ARCR), a classic drug pair, has been widely used for the treatment of gastric intraepithelial neoplasia (GIN) in China. However, the underlying mechanisms of this drug pair are still unknown. Thus, elucidating the molecular mechanism of ARCR for treating GIN is imperative. Methods. The active components and targets of ARCR were determined from the TCMSP database, and the differentially expressed genes related to GIN were identified from the GSE130823 dataset. The protein-protein interaction (PPI) network and ARCR-active component-target-pathway network were constructed by STRING 11.0 and Cytoscape 3.7.2, respectively. In addition, a receiver operating characteristic curve (ROC) was conducted to verify the key targets, and enrichment analyses were performed using R software. Molecular docking was carried out to test the binding capacity between core active components and key targets. Results. 31 active components were obtained from ARCR, among which 22 were hit by the 51 targets associated with GIN. Gene Ontology (GO) functional enrichment analysis showed that biological process (BP), molecular function (MF), and cellular component (CC) were most significantly enriched in response to a drug, catecholamine binding, and apical part of the cell, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated ARCR against GIN through regulation of neuroactive ligand-receptor interaction, nitrogen metabolism, calcium signaling pathway, chemical carcinogenesis-receptor activation, drug metabolism, gap junction, and cancers. In the PPI network, 15 potential targets were identified, of which nine key targets were proven to have higher diagnostic values in ROC. Molecular docking revealed a good binding affinity of active components (quercetin, bisdemethoxycurcumin, and kaempferol) with the corresponding targets (CYP3A4, CYP1A1, HMOX1, DRD2, DPP4, ADRA2A, ADRA2C, NR1I2, and LGALS4). Conclusion. This study revealed the active components and molecular mechanism by which ARCR treatment is effective against GIN through regulating multipathway, such as neuroactive ligand-receptor interaction, nitrogen metabolism, and calcium signaling pathway.

scholarly journals DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

2015 ◽  
Vol 26 (15) ◽  
pp. 2788-2800 ◽  
Author(s):  
Junya Awata ◽  
Kangkang Song ◽  
Jianfeng Lin ◽  
Stephen M. King ◽  
Michael J. Sanderson ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Motor Domain ◽  
Flagellar Motility ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Regulatory Complex ◽  
And Function ◽  
Linker Domain ◽  
Distal Lobe

The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo–electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC–dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform.

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