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1.
Biosci Rep ; 40(12)2020 12 23.
Article in English | MEDLINE | ID: mdl-33200789

ABSTRACT

PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl transferase (αTAT1) and histone deacetylase 6 (HDAC6) in animal models and cell lines illustrate disparity in correlating tubulin acetylation status with stability of MT. Our earlier studies showed reduced acetyl tubulin in sperm of asthenozoospermic individuals. Our studies on rat sperm showed that on inhibition of HDAC6 activity, although tubulin acetylation increased, sperm motility was reduced. Studies were therefore undertaken to investigate the influence of tubulin acetylation/deacetylation on MT dynamicity in sperm flagella using rat and human sperm. Our data on rat sperm revealed that HDAC6 specific inhibitor Tubastatin A (T) inhibited sperm motility and neutralized the depolymerizing and motility debilitating effect of Nocodazole. The effect on polymerization was further confirmed in vitro using pure MT and recHDAC6. Also polymerized axoneme was less in sperm of asthenozoosperm compared to normozoosperm. Deacetylase activity was reduced in sperm lysates and axonemes exposed to T and N+T but not in axonemes of sperm treated similarly suggesting that HDAC6 is associated with sperm axonemes or MT. Deacetylase activity was less in asthenozoosperm. Intriguingly, the expression of MDP3 physiologically known to bind to HDAC6 and inhibit its deacetylase activity remained unchanged. However, expression of acetyl α-tubulin, HDAC6 and microtubule stabilizing protein SAXO1 was less in asthenozoosperm. These observations suggest that MAPs and threshold levels of MT acetylation/deacetylation are important for MT dynamicity in sperm and may play a role in regulating sperm motility.


Subject(s)
Asthenozoospermia/enzymology , Axoneme/enzymology , Flagella/enzymology , Histone Deacetylase 6/metabolism , Microtubule-Associated Proteins/metabolism , Protein Processing, Post-Translational , Sperm Motility , Spermatozoa/enzymology , Acetylation , Animals , Asthenozoospermia/pathology , Axoneme/drug effects , Axoneme/pathology , Case-Control Studies , Flagella/drug effects , Flagella/pathology , Histone Deacetylase 6/antagonists & inhibitors , Histone Deacetylase Inhibitors/pharmacology , Humans , Male , Rats, Sprague-Dawley , Sperm Motility/drug effects , Spermatozoa/drug effects , Spermatozoa/pathology , Tubulin/metabolism
2.
Nat Commun ; 5: 5081, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-25270598

ABSTRACT

Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis.


Subject(s)
Axoneme/enzymology , Axoneme/metabolism , Centrioles/metabolism , Cilia/metabolism , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Centrioles/enzymology , Cilia/enzymology , Cilia/genetics , Cullin Proteins/genetics , Cullin Proteins/metabolism , Humans , Proteasome Endopeptidase Complex/genetics
3.
Am J Respir Cell Mol Biol ; 51(6): 750-60, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24874272

ABSTRACT

Ciliary beating is important for effective mucociliary clearance. Soluble adenylyl cyclase (sAC) regulates ciliary beating, and a roughly 50-kD sAC variant is expressed in axonemes. Normal human bronchial epithelial (NHBE) cells express multiple sAC splice variants: full-length sAC; variants with catalytic domain 1 (C1) deletions; and variants with partial C1. One variant, sACex5v2-ex12v2, contains two alternative splices creating new exons 5 (ex5v2) and 12 (ex12v2), encoding a roughly 45-kD protein. It is therefore similar in size to ciliary sAC. The variant increases in expression upon ciliogenesis during differentiation at the air-liquid interface. When expressed in NHBE cells, this variant was targeted to cilia. Exons 5v2-7 were important for ciliary targeting, whereas exons 2-4 prevented it. In vitro, cytoplasmic sACex2-ex12v2 (containing C1 and C2) was the only variant producing cAMP. Ciliary sACex5v2-ex12v2 was not catalytically active. Airway epithelial cells isolated from wild-type mice revealed sAC-dependent ciliary beat frequency (CBF) regulation, analogous to NHBE cells: CBF rescue from HCO3(-)/CO2-mediated intracellular acidification was sensitive to the sAC inhibitor, KH7. Compared with wild type, sAC C2 knockout (KO) mice revealed lower CBF baseline, and the HCO3(-)/CO2-mediated CBF decrease was not inhibited by KH7, confirming lack of functional sAC. Human sACex5v2-ex12v2 was targeted to cilia and sACex2-ex12v2 to the cytoplasm in these KO mice. Introduction of the ciliary sACex5v2-ex12v2 variant, but not the cytoplasmic sACex2-ex12v2, restored functional sAC activity in C2 KO mice. Thus, we show, for the first time, a mammalian axonemal targeting sequence that localizes a sAC variant to cilia to regulate CBF.


Subject(s)
Adenylyl Cyclases/metabolism , Axoneme/enzymology , Cilia/enzymology , Adenylyl Cyclases/genetics , Alternative Splicing , Animals , Cilia/physiology , HEK293 Cells , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Mice, Knockout , Mucociliary Clearance , Protein Transport , Solubility
4.
Mol Biol Cell ; 25(1): 107-17, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24196831

ABSTRACT

Tubulin undergoes various posttranslational modifications, including polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase-like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a polyglutamylation complex specialized for regulation of ciliary motility.


Subject(s)
Axoneme/enzymology , Chlamydomonas reinhardtii/enzymology , Peptide Synthases/metabolism , Plant Proteins/metabolism , Amino Acid Sequence , Chlamydomonas reinhardtii/cytology , Conserved Sequence , Cytoplasm/enzymology , Enzyme Stability , Flagella/enzymology , Protein Binding , Protein Processing, Post-Translational , Protein Transport , Sequence Homology, Amino Acid , Tubulin/metabolism
5.
Biochemistry ; 52(47): 8501-9, 2013 Nov 26.
Article in English | MEDLINE | ID: mdl-24152136

ABSTRACT

When Chlamydomonas cells resorb their flagella, seven polypeptides become asymmetrically dimethylated (aDMA) on arginine residues. Tandem mass spectrometry has identified these as radial spoke proteins 1, 2, 5, and 6; tektin, a structural component of the outer doublets; and flagellar-associated protein 172 (FAP172) (coiled-coil domain containing protein 40 (CCDC40)) and FAP250 (CCDC65), which are associated with inner arm dynein and the nexin-dynein regulatory complex. The enzyme protein arginine methyl transferase 1 (PRMT1), which generates aDMA residues, is a component of the flagellar matrix; antibodies to PRMT1 label full-length flagella in a punctate pattern along the length of the axoneme. During resorption, PRMT1 localization becomes enhanced at the flagellar tip, which is the site of the net disassembly of the flagellar axoneme, and gel shift assays indicate PRMT1 is phosphorylated under resorbing conditions. These data are consistent with a model in which a resorption signal activates one or more protein kinases, resulting in the up-regulation of the components of a protein methylation pathway resident in flagella. Methylation results in axonemal instability and/or enhances the interaction of axonemal polypeptides with intraflagellar transport particles, which then move disassembled components to the cell body for degradation or recycling.


Subject(s)
Algal Proteins/metabolism , Axoneme/metabolism , Chlamydomonas/physiology , Cytoskeletal Proteins/metabolism , Flagella/metabolism , Plant Proteins/metabolism , Up-Regulation , Algal Proteins/chemistry , Arginine/metabolism , Axoneme/chemistry , Axoneme/enzymology , Chlamydomonas/cytology , Chlamydomonas/enzymology , Cytoskeletal Proteins/chemistry , Electrophoretic Mobility Shift Assay , Flagella/chemistry , Flagella/enzymology , Metamorphosis, Biological , Methylation , Microtubule Proteins/chemistry , Microtubule Proteins/metabolism , Molecular Weight , Peptide Mapping , Plant Proteins/chemistry , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Processing, Post-Translational , Protein Stability , Protein Transport , Protein-Arginine N-Methyltransferases/chemistry , Protein-Arginine N-Methyltransferases/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism
6.
Alcohol Clin Exp Res ; 37(4): 609-15, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23078267

ABSTRACT

BACKGROUND: Cilia are finger-like motor-driven organelles, which propel inhaled particles and mucus from the lung and airways. We have previously shown that brief alcohol exposure stimulates ciliary motility through an endothelial nitric oxide synthase (eNOS)-dependent pathway localized in the ciliary metabolon. However, the signaling molecules of the ciliary metabolon involved in alcohol-triggered ciliary beat frequency (CBF) stimulation upstream of eNOS activation remain unknown. METHODS: We hypothesized that brief alcohol exposure alters threonine and serine phosphorylation of proteins involved in stimulating CBF. Two-dimensional electrophoresis indicated both increases and decreases in the serine and threonine phosphorylation states of several proteins. One of the proteins identified was heat shock protein 90 (HSP90), which undergoes increased threonine phosphorylation after brief alcohol exposure. Because HSP90 has been shown to associate with eNOS in lung tissue, we hypothesized that HSP90 is a key component in alcohol-triggered eNOS activation and that these 2 proteins co-localize within the ciliary metabolon. RESULTS: Immunofluorescence experiments demonstrate that eNOS and HSP90 co-localize within basal bodies of the ciliary metabolon and partially translocate to the axoneme upon brief alcohol exposure. Pretreatment with geldanamycin, which disrupts HSP90 chaperone functions, prevented eNOS-HSP90 association and prevented the translocation of eNOS from the ciliary metabolon to the axoneme. Functional cilia motility studies revealed that geldanamycin blocked alcohol-stimulated ciliary motility in bovine bronchial epithelial cells and mouse tracheal rings. CONCLUSIONS: On the basis of the HSP90 localization with eNOS, alcohol activation of HSP90 phosphorylation, and geldanamycin's ability to inhibit HSP90-eNOS association, prevent eNOS translocation to the axoneme, and block alcohol-stimulated ciliary motility, we conclude that alcohol-induced cilia stimulation occurs through the increased association of HSP90 with eNOS. These data help further elucidate the mechanism through which brief alcohol exposure stimulates CBF.


Subject(s)
Axoneme/physiology , Cilia/physiology , Ethanol/administration & dosage , HSP90 Heat-Shock Proteins/physiology , Nitric Oxide Synthase Type III/physiology , Proteomics/methods , Animals , Axoneme/enzymology , Cattle , Cells, Cultured , Cilia/enzymology , Drug Delivery Systems , HSP90 Heat-Shock Proteins/metabolism , Mice , Mice, Inbred C57BL , Organ Culture Techniques
7.
Am J Pathol ; 181(2): 431-40, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22677421

ABSTRACT

Alcohol use disorders are associated with increased lung infections and exacerbations of chronic lung diseases. Whereas the effects of cigarette smoke are well recognized, the interplay of smoke and alcohol in modulating lung diseases is not clear. Because innate lung defense is mechanically maintained by airway cilia action and protein kinase C (PKC)-activating agents slow ciliary beat frequency (CBF), we hypothesized that the combination of smoke and alcohol would decrease CBF in a PKC-dependent manner. Primary ciliated bronchial epithelial cells were exposed to 5% cigarette smoke extract plus100 mmol/L ethanol for up to 24 hours and assayed for CBF and PKCε. Smoke and alcohol co-exposure activated PKCε by 1 hour and decreased both CBF and total number of beating cilia by 6 hours. A specific activator of PKCε, DCP-LA, slowed CBF after maximal PKCε activation. Interestingly, activation of PKCε by smoke and alcohol was only observed in ciliated cells, not basal bronchial epithelium. In precision-cut mouse lung slices treated with smoke and alcohol, PKCε activation preceded CBF slowing. Correspondingly, increased PKCε activity and cilia slowing were only observed in mice co-exposed to smoke and alcohol, regardless of the sequence of the combination exposure. No decreases in CBF were observed in PKCε knockout mice co-exposed to smoke and alcohol. These data identify PKCε as a key regulator of cilia slowing in response to combined smoke and alcohol-induced lung injury.


Subject(s)
Bronchi/pathology , Cilia/metabolism , Environmental Exposure , Epithelial Cells/enzymology , Ethanol/adverse effects , Protein Kinase C-epsilon/metabolism , Smoking/adverse effects , Animals , Axoneme/enzymology , Biocatalysis , Cattle , Enzyme Activation , Epithelial Cells/pathology , In Vitro Techniques , Mice , Mice, Knockout , Protein Transport
8.
Arch Biochem Biophys ; 510(2): 93-100, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21513695

ABSTRACT

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9+2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation/dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.


Subject(s)
Axoneme/enzymology , Cilia/enzymology , Conserved Sequence , Movement , Phosphoric Monoester Hydrolases/metabolism , Protein Kinases/metabolism , Animals , Axoneme/metabolism , Cilia/metabolism , Humans , Phosphoric Monoester Hydrolases/chemistry , Protein Kinases/chemistry
9.
J Cell Biol ; 186(6): 817-24, 2009 Sep 21.
Article in English | MEDLINE | ID: mdl-19752022

ABSTRACT

Experimental analysis of isolated ciliary/flagellar axonemes has implicated the protein kinase casein kinase I (CK1) in regulation of dynein. To test this hypothesis, we developed a novel in vitro reconstitution approach using purified recombinant Chlamydomonas reinhardtii CK1, together with CK1-depleted axonemes from the paralyzed flagellar mutant pf17, which is defective in radial spokes and impaired in dynein-driven microtubule sliding. The CK1 inhibitors (DRB and CK1-7) and solubilization of CK1 restored microtubule sliding in pf17 axonemes, which is consistent with an inhibitory role for CK1. The phosphatase inhibitor microcystin-LR blocked rescue of microtubule sliding, indicating that the axonemal phosphatases, required for rescue, were retained in the CK1-depleted axonemes. Reconstitution of depleted axonemes with purified, recombinant CK1 restored inhibition of microtubule sliding in a DRB- and CK1-7-sensitive manner. In contrast, a purified "kinase-dead" CK1 failed to restore inhibition. These results firmly establish that an axonemal CK1 regulates dynein activity and flagellar motility.


Subject(s)
Axoneme/enzymology , Casein Kinase I/metabolism , Cell Movement , Chlamydomonas reinhardtii/enzymology , Dyneins/metabolism , Flagella/enzymology , Animals , Axoneme/drug effects , Casein Kinase I/antagonists & inhibitors , Casein Kinase I/genetics , Cell Movement/drug effects , Chlamydomonas reinhardtii/drug effects , Chlamydomonas reinhardtii/genetics , Dichlororibofuranosylbenzimidazole/pharmacology , Flagella/drug effects , Isoquinolines/pharmacology , Marine Toxins , Microcystins/pharmacology , Mutation , Phosphoprotein Phosphatases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Recombinant Proteins/metabolism
10.
Dev Cell ; 16(6): 773-4, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19531345

ABSTRACT

Numerous posttranslational modifications alter surface-exposed residues of tubulin within stable microtubules. The significance of one modification, glycylation, characteristic of ciliary and flagellar microtubules, has been particularly elusive. Two groups now identify the glycylation enzymes and determine the developmental consequences of their depletion. Glycylation enzymes and those responsible for another modification, glutamylation, work in opposition to one another in modifying microtubules.


Subject(s)
Models, Biological , Protein Processing, Post-Translational , Tubulin/metabolism , Animals , Axoneme/enzymology , Axoneme/ultrastructure , Cilia/enzymology , Cilia/ultrastructure , Glutamic Acid/metabolism , Glycine/metabolism
11.
Dev Cell ; 16(6): 867-76, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19531357

ABSTRACT

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffer's vesicle and olfactory placode. We also show that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation.


Subject(s)
Cilia/enzymology , Glycine/metabolism , Peptide Synthases/metabolism , Protozoan Proteins/metabolism , Tetrahymena/enzymology , Tubulin/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Axoneme/drug effects , Axoneme/enzymology , Axoneme/ultrastructure , Body Patterning/drug effects , Cilia/drug effects , Cilia/ultrastructure , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/enzymology , Gene Knockdown Techniques , Genes, Dominant , Glutamic Acid/metabolism , Ligases/metabolism , Mutation/genetics , Oligonucleotides, Antisense/pharmacology , Sequence Homology, Amino Acid , Tetrahymena/cytology , Tetrahymena/drug effects , Tetrahymena/ultrastructure , Zebrafish/embryology
12.
Methods Cell Biol ; 92: 133-51, 2009.
Article in English | MEDLINE | ID: mdl-20409803

ABSTRACT

The purpose of this chapter is to review the methodology and advances that have revealed conserved signaling proteins that are localized in the 9+2 ciliary axoneme for regulating motility. Diverse experimental systems have revealed that ciliary and eukaryotic flagellar motility is regulated by second messengers including calcium, pH, and cyclic nucleotides. In addition, recent advances in in vitro functional studies, taking advantage of isolated axonemes, pharmacological approaches, and biochemical analysis of axonemes have demonstrated that otherwise ubiquitous, conserved protein kinases and phosphatases are transported to and anchored in the axoneme. Here, we focus on the functional/pharmacological, genetic, and biochemical approaches in the model genetic system Chlamydomonas that have revealed highly conserved kinases, anchoring proteins (e.g., A-kinase anchoring proteins), and phosphatases that are physically located in the axoneme where they play a direct role in control of motility.


Subject(s)
Axoneme/enzymology , Biological Assay/methods , Chlamydomonas/enzymology , Dyneins/metabolism , Flagella/enzymology , Phosphoric Monoester Hydrolases/metabolism , Phosphotransferases/metabolism , Algal Proteins/metabolism , Axoneme/chemistry , Biological Transport , Models, Biological , Mutation/genetics , Perfusion
13.
Eukaryot Cell ; 7(1): 154-61, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17981992

ABSTRACT

Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.


Subject(s)
Algal Proteins/genetics , Axoneme/enzymology , Chlamydomonas reinhardtii/enzymology , Dyneins/chemistry , Protozoan Proteins/chemistry , Algal Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cell Movement , Chlamydomonas reinhardtii/genetics , Cilia/metabolism , Conserved Sequence , Dyneins/genetics , Dyneins/metabolism , Flagella/genetics , Flagella/metabolism , Fluorescent Antibody Technique , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Immunoblotting , Mice , Molecular Sequence Data , Protein Subunits , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Rabbits , Sequence Homology, Amino Acid
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