The Experts below are selected from a list of 279 Experts worldwide ranked by ideXlab platform
Elizabeth F. Smith - One of the best experts on this subject based on the ideXlab platform.
-
PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate Ciliary Motility
Molecular Biology of the Cell, 2019Co-Authors: Erin E. Dymek, Jianfeng Lin, Mary E. Porter, Daniela Nicastro, Elizabeth F. SmithAbstract:We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in Ciliary assembly and Motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine Ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell Motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not Ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional Ciliary components that play essential roles in generating coordinated Ciliary beating.
-
the pcdp1 complex coordinates the activity of dynein isoforms to produce wild type Ciliary Motility
Molecular Biology of the Cell, 2011Co-Authors: Christen G Dipetrillo, Elizabeth F. SmithAbstract:Generating the complex waveforms characteristic of beating cilia requires the coordinated activity of multiple dynein isoforms anchored to the axoneme. We previously identified a complex associated with the C1d projection of the central apparatus that includes primary Ciliary dyskinesia protein 1 (Pcdp1). Reduced expression of complex members results in severe Motility defects, indicating that C1d is essential for wild-type Ciliary beating. To define a mechanism for Pcdp1/C1d regulation of Motility, we took a functional and structural approach combined with mutants lacking C1d and distinct subsets of dynein arms. Unlike mutants completely lacking the central apparatus, dynein-driven microtubule sliding velocities are wild type in C1d- defective mutants. However, coordination of dynein activity among microtubule doublets is severely disrupted. Remarkably, mutations in either outer or inner dynein arm restore Motility to mutants lacking C1d, although waveforms and beat frequency differ depending on which isoform is mutated. These results define a unique role for C1d in coordinating the activity of specific dynein isoforms to control Ciliary Motility.
-
the csc is required for complete radial spoke assembly and wild type Ciliary Motility
Molecular Biology of the Cell, 2011Co-Authors: Erin E. Dymek, Daniela Nicastro, Thomas Heuser, Elizabeth F. SmithAbstract:The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the Motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity. Our results not only indicate that the CSC is required for spoke assembly and wild-type Motility, but also provide evidence for heterogeneity among the radial spokes.
-
pcdp1 is a central apparatus protein that binds ca2 calmodulin and regulates Ciliary Motility
Journal of Cell Biology, 2010Co-Authors: Christen G Dipetrillo, Elizabeth F. SmithAbstract:For all motile eukaryotic cilia and flagella, beating is regulated by changes in intraCiliary calcium concentration. Although the mechanism for calcium regulation is not understood, numerous studies have shown that calmodulin (CaM) is a key axonemal calcium sensor. Using anti-CaM antibodies and Chlamydomonas reinhardtii axonemal extracts, we precipitated a complex that includes four polypeptides and that specifically interacts with CaM in high [Ca2+]. One of the complex members, FAP221, is an orthologue of mammalian Pcdp1 (primary Ciliary dyskinesia protein 1). Both FAP221 and mammalian Pcdp1 specifically bind CaM in high [Ca2+]. Reduced expression of Pcdp1 complex members in C. reinhardtii results in failure of the C1d central pair projection to assemble and significant impairment of Motility including uncoordinated bends, severely reduced beat frequency, and altered waveforms. These combined results reveal that the central pair Pcdp1 (FAP221) complex is essential for control of Ciliary Motility.
-
calcium regulation of Ciliary Motility analysis of axonemal calcium binding proteins
Methods in Cell Biology, 2009Co-Authors: Christen G Dipetrillo, Elizabeth F. SmithAbstract:Substantial data have contributed to a model in which the axonemal microtubules act as a scaffold for the assembly of molecules that form a signal transduction pathway that ultimately regulates dynein. We have also known for some time that for virtually all motile cilia and flagella, the second messenger, calcium, impacts upon these signaling pathways to modulate beating in response to extracellular cues. Yet we are only beginning to identify the axonemal proteins that bind this second messenger and determine their role in regulating dynein-driven microtubule sliding to alter the size and shape of Ciliary bends. Here, we review our current understanding of calcium regulation of Motility, emphasizing recent advances in the detection and characterization of calcium-binding proteins anchored to the axoneme.
Jun Tamaoki - One of the best experts on this subject based on the ideXlab platform.
-
effect of macrolide antibiotics on Ciliary Motility in rabbit airway epithelium in vitro
Journal of Pharmacy and Pharmacology, 2011Co-Authors: Kiyoshi Takeyama, Jun Tamaoki, A Chiyotani, Etsuko Tagaya, Kimio KonnoAbstract:— We have studied Ciliary beat frequency (CBF) of rabbit cultured tracheal epithelium by a photoelectric method in-vitro. Addition of erythromycin and roxithromycin increased CBF in a dose-dependent fashion, whereas clarithromycin was without effect. The rank order potency of macrolide was roxithromycin > erythromycin » clarithromycin. The roxithromycin-induced increase in CBF was not altered by propranolol, AA-861, or verapamil, but partially attenuated by indomethacin. Roxithromycin increased intracellular cAMP concentrations. These results suggest that certain macrolides can stimulate airway Ciliary Motility probably via prostaglandin- and cAMP-dependent regulatory pathways, which may affect mucoCiliary transport function in the respiratory tract.
-
adenosine a3 receptor mediated potentiation of mucoCiliary transport and epithelial Ciliary Motility
American Journal of Physiology-lung Cellular and Molecular Physiology, 2002Co-Authors: Manako Taira, Jun Tamaoki, Kazuyuki Nishimura, Junko Nakata, Mitsuko Kondo, Hisashi Takemura, Atsushi NagaiAbstract:To examine the effect of adenosine A3 receptor stimulation on airway mucoCiliary clearance, we measured transport of Evans blue dye in rabbit trachea in vivo and Ciliary Motility of epithelium by t...
-
vasopressin stimulates Ciliary Motility of rabbit tracheal epithelium role of v1b receptor mediated ca2 mobilization
American Journal of Respiratory Cell and Molecular Biology, 1998Co-Authors: Jun Tamaoki, S Takeuchi, Mitsuko Kondo, Hisashi Takemura, Atsushi NagaiAbstract:Arginine vasopressin (AVP) has recently been shown to exist in and to be released from airway epithelial cells, but the physiologic role of this hormone in airway epithelial function is unknown. To determine whether AVP affects Ciliary Motility, and if so, to elucidate the mechanism of action and the subtype of AVP receptors involved, we measured Ciliary beat frequency (CBF) and the intracellular Ca2+ concentration ([Ca2+]i) of cultured rabbit tracheal epithelium with a photoelectric method and the fura-2 fluorescence method, respectively. Addition of AVP caused a rapid increase in CBF, followed by a decline and a subsequent sustained response. The Ciliary stimulatory action was dose dependent, the maximal peak increase from the baseline CBF being 20.6 ± 4.7% (mean ± SE, P < 0.001), and this effect was reduced to 5.9 ± 2.0% by the V1 receptor antagonist OPC-21268 (P < 0.01), but not by the V2 receptor antagonist OPC-31260. The AVP-induced increase in CBF was not altered by the protein kinase A (PKA) inhib...
-
zizyphi fructus a constituent of antiasthmatic herbal medicine stimulates airway epithelial Ciliary Motility through nitric oxide generation
Experimental Lung Research, 1996Co-Authors: Jun Tamaoki, Mitsuko Kondo, Etsuko Tagaya, K Takemura, Kimio KonnoAbstract:The effects of Zizyphi fructus, a major constituent of Chinese anti-asthmatic herbal medicine, on Ciliary Motility and nitric oxide (NO) generation in canine cultured tracheal epithelium were studied by the microphoto-oscillation method and the specific amperometric method, respectively. Addition of Zizyphi fructus at 100 micrograms/mL rapidly increased Ciliary beat frequency (CBF), an effect that was inhibited by NG-nitro-L-arginine methylester (L-NAME) but not by NG-nitro-D-arginine methylester (D-NAME), and this inhibition was reversed by L-arginine but not by D-arginine. Immersion of the NO-selective electrode in the medium bathing tracheal epithelial cells showed a baseline current of 21.3-60.4 pA, which corresponded to NO concentration ([NO]) at 34.4 +/- 7.8 nM. Zizyphi fructus caused a concentration-dependent increase in [NO], the maximal increase from the baseline [NO] level and the concentration of Zizyphi fructus required to produce a half-maximal effect (EC50) being 107 +/- 14 nM (p < .001) and 7.2 +/- 2.9 micrograms/mL, respectively. These results suggest that Zizyphi fructus enhances airway Ciliary Motility and that this effect is exerted through the stimulation of epithelial NO generation.
-
role of no generation in beta adrenoceptor mediated stimulation of rabbit airway Ciliary Motility
American Journal of Physiology-cell Physiology, 1995Co-Authors: Jun Tamaoki, A Chiyotani, M Kondo, Kimio KonnoAbstract:To determine possible contribution of nitric oxide (NO) to the stimulatory action of beta-adrenoceptor agonist on Ciliary Motility, we measured Ciliary beat frequency (CBF) of rabbit cultured tracheal epithelial cells by photoelectric method and NO release by specific amperometric sensors for this molecule in vitro. Salbutamol increased CBF, an effect that was potentiated by superoxide dismutase. Pretreatment of cells with NG-nitro-L-arginine methyl ester (L-NAME) attenuated the salbutamol-induced increase in CBF, causing a rightward displacement of the concentration-response curve by 2-2.5 log units, whereas NG-nitro-D-arginine methyl ester had no effect. The inhibitory effect of L-NAME was reversed by L-arginine but not by D-arginine. Immersion of the NO-selective electrode in the medium containing epithelial cells detected baseline current of 4.6-14.5 pA, which was abolished by L-NAME. Salbutamol dose-dependently increased the concentration of NO in the medium, the maximal increase being 56.2 +/- 5.3 nM (mean +/- SE; P < 0.001). These results suggest that NO is spontaneously released by airway epithelium and that the enhanced release of this molecule may play a role in the beta-adrenoceptor-mediated stimulation of Ciliary Motility
Axel Schweickert - One of the best experts on this subject based on the ideXlab platform.
-
a novel serotonin secreting cell type regulates Ciliary Motility in the mucoCiliary epidermis of xenopus tadpoles
Development, 2014Co-Authors: Peter Walentek, Susanne Bogusch, Thomas Thumberger, Philipp Vick, Eamon Dubaissi, Tina Beyer, Martin Blum, Axel SchweickertAbstract:The embryonic skin of Xenopus tadpoles serves as an experimental model system for mucoCiliary epithelia (MCE) such as the human airway epithelium. MCEs are characterized by the presence of mucus-secreting goblet and multiciliated cells (MCCs). A third cell type, ion-secreting cells (ISCs), is present in the larval skin as well. Synchronized beating of MCC cilia is required for directional transport of mucus. Here we describe a novel cell type in the Xenopus laevis larval epidermis, characterized by serotonin synthesis and secretion. It is termed small secretory cell (SSC). SSCs are detectable at early tadpole stages, unlike MCCs and ISCs, which are specified at early neurulation. Subcellularly, serotonin was found in large, apically localized vesicle-like structures, which were entirely shed into the surrounding medium. Pharmacological inhibition of serotonin synthesis decreased the velocity of cilia-driven fluid flow across the skin epithelium. This effect was mediated by serotonin type 3 receptor (Htr3), which was expressed in ciliated cells. Knockdown of Htr3 compromised flow velocity by reducing the Ciliary Motility of MCCs. SSCs thus represent a distinct and novel entity of the frog tadpole MCE, required for Ciliary beating and mucus transport across the larval skin. The identification and characterization of SSCs consolidates the value of the Xenopus embryonic skin as a model system for human MCEs, which have been known for serotonin-dependent regulation of Ciliary beat frequency.
-
a novel serotonin secreting cell type regulates Ciliary Motility in the mucoCiliary epidermis of xenopus tadpoles
Development, 2014Co-Authors: Peter Walentek, Susanne Bogusch, Thomas Thumberger, Philipp Vick, Eamon Dubaissi, Tina Beyer, Martin Blum, Axel SchweickertAbstract:The embryonic skin of Xenopus tadpoles serves as an experimental model system for mucoCiliary epithelia (MCE) such as the human airway epithelium. MCEs are characterized by the presence of mucus-secreting goblet and multiciliated cells (MCCs). A third cell type, ion-secreting cells (ISCs), is present in the larval skin as well. Synchronized beating of MCC cilia is required for directional transport of mucus. Here we describe a novel cell type in the Xenopus laevis larval epidermis, characterized by serotonin synthesis and secretion. It is termed small secretory cell (SSC). SSCs are detectable at early tadpole stages, unlike MCCs and ISCs, which are specified at early neurulation. Subcellularly, serotonin was found in large, apically localized vesicle-like structures, which were entirely shed into the surrounding medium. Pharmacological inhibition of serotonin synthesis decreased the velocity of cilia-driven fluid flow across the skin epithelium. This effect was mediated by serotonin type 3 receptor ( Htr3 ), which was expressed in ciliated cells. Knockdown of Htr3 compromised flow velocity by reducing the Ciliary Motility of MCCs. SSCs thus represent a distinct and novel entity of the frog tadpole MCE, required for Ciliary beating and mucus transport across the larval skin. The identification and characterization of SSCs consolidates the value of the Xenopus embryonic skin as a model system for human MCEs, which have been known for serotonin-dependent regulation of Ciliary beat frequency.
Andrew P Jarman - One of the best experts on this subject based on the ideXlab platform.
-
Survey of the Ciliary Motility machinery of Drosophila sperm and ciliated mechanosensory neurons reveals unexpected cell-type specific variations: a model for motile ciliopathies
Frontiers in Genetics, 2019Co-Authors: Petra I. Zur Lage, Fay G. Newton, Andrew P JarmanAbstract:The motile cilium/flagellum is an ancient eukaryotic organelle. The molecular machinery of Ciliary Motility comprises a variety of cilium-specific dynein motor complexes along with other complexes that regulate their activity. Assembling the motors requires the function of dedicated ‘assembly factors’ and transport processes. In humans, mutation of any one of at least 40 different genes encoding components of the Motility apparatus causes Primary Ciliary Dyskinesia (PCD), a disease of defective Ciliary Motility. Recently, Drosophila has emerged as a model for motile cilia biology and motile ciliopathies. This is somewhat surprising as most Drosophila cells lack cilia, and motile cilia are confined to just two specialised cell types: the sperm flagellum with a 9+2 axoneme and the ciliated dendrite of auditory/proprioceptive (chordotonal, Ch) neurons with a 9+0 axoneme. To determine the utility of Drosophila as a model for motile cilia, we survey the Drosophila genome for Ciliary Motility gene homologues, and assess their expression and function. We find that the molecules of cilium Motility are well conserved in Drosophila. Most are readily characterised by their restricted cell-type specific expression patterns and phenotypes. There are also striking differences between the two motile ciliated cell types. Notably, sperm and Ch neuron cilia express and require entirely different outer dynein arm variants – the first time this has been clearly established in any organism. These differences might reflect the specialised functions for Motility in the two cilium types. Moreover, the Ch neuron cilia lack the critical two-headed inner arm dynein (I1/f) but surprisingly retain key regulatory proteins previously associated with it. This may have implications for other motile 9+0 cilia, including vertebrate embryonic nodal cilia required for left-right axis asymmetry. We discuss the possibility that cell-type specificity in Ciliary Motility machinery might occur in humans, and therefore underlie some of the phenotypic variation observed in PCD caused by different gene mutations. Our work lays the foundation for the increasing use of Drosophila as an excellent model for new motile Ciliary gene discovery and validation, for understanding motile cilium function and assembly, as well as understanding the nature of genetic defects underlying human motile ciliopathies.
-
Table_1_Survey of the Ciliary Motility Machinery of Drosophila Sperm and Ciliated Mechanosensory Neurons Reveals Unexpected Cell-Type Specific Variations: A Model for Motile Ciliopathies.XLSX
2019Co-Authors: Petra Zur Lage, Fay G. Newton, Andrew P JarmanAbstract:The motile cilium/flagellum is an ancient eukaryotic organelle. The molecular machinery of Ciliary Motility comprises a variety of cilium-specific dynein motor complexes along with other complexes that regulate their activity. Assembling the motors requires the function of dedicated “assembly factors” and transport processes. In humans, mutation of any one of at least 40 different genes encoding components of the Motility apparatus causes Primary Ciliary Dyskinesia (PCD), a disease of defective Ciliary Motility. Recently, Drosophila has emerged as a model for motile cilia biology and motile ciliopathies. This is somewhat surprising as most Drosophila cells lack cilia, and motile cilia are confined to just two specialized cell types: the sperm flagellum with a 9+2 axoneme and the ciliated dendrite of auditory/proprioceptive (chordotonal, Ch) neurons with a 9+0 axoneme. To determine the utility of Drosophila as a model for motile cilia, we survey the Drosophila genome for Ciliary Motility gene homologs, and assess their expression and function. We find that the molecules of cilium Motility are well conserved in Drosophila. Most are readily characterized by their restricted cell-type specific expression patterns and phenotypes. There are also striking differences between the two motile ciliated cell types. Notably, sperm and Ch neuron cilia express and require entirely different outer dynein arm variants—the first time this has been clearly established in any organism. These differences might reflect the specialized functions for Motility in the two cilium types. Moreover, the Ch neuron cilia lack the critical two-headed inner arm dynein (I1/f) but surprisingly retain key regulatory proteins previously associated with it. This may have implications for other motile 9+0 cilia, including vertebrate embryonic nodal cilia required for left-right axis asymmetry. We discuss the possibility that cell-type specificity in Ciliary Motility machinery might occur in humans, and therefore underlie some of the phenotypic variation observed in PCD caused by different gene mutations. Our work lays the foundation for the increasing use of Drosophila as an excellent model for new motile Ciliary gene discovery and validation, for understanding motile cilium function and assembly, as well as understanding the nature of genetic defects underlying human motile ciliopathies.
-
Survey of the Ciliary Motility Machinery of Drosophila Sperm and Ciliated Mechanosensory Neurons Reveals Unexpected Cell-Type Specific Variations: A Model for Motile Ciliopathies
Frontiers Media S.A., 2019Co-Authors: Petra Zur Lage, Fay G. Newton, Andrew P JarmanAbstract:The motile cilium/flagellum is an ancient eukaryotic organelle. The molecular machinery of Ciliary Motility comprises a variety of cilium-specific dynein motor complexes along with other complexes that regulate their activity. Assembling the motors requires the function of dedicated “assembly factors” and transport processes. In humans, mutation of any one of at least 40 different genes encoding components of the Motility apparatus causes Primary Ciliary Dyskinesia (PCD), a disease of defective Ciliary Motility. Recently, Drosophila has emerged as a model for motile cilia biology and motile ciliopathies. This is somewhat surprising as most Drosophila cells lack cilia, and motile cilia are confined to just two specialized cell types: the sperm flagellum with a 9+2 axoneme and the ciliated dendrite of auditory/proprioceptive (chordotonal, Ch) neurons with a 9+0 axoneme. To determine the utility of Drosophila as a model for motile cilia, we survey the Drosophila genome for Ciliary Motility gene homologs, and assess their expression and function. We find that the molecules of cilium Motility are well conserved in Drosophila. Most are readily characterized by their restricted cell-type specific expression patterns and phenotypes. There are also striking differences between the two motile ciliated cell types. Notably, sperm and Ch neuron cilia express and require entirely different outer dynein arm variants—the first time this has been clearly established in any organism. These differences might reflect the specialized functions for Motility in the two cilium types. Moreover, the Ch neuron cilia lack the critical two-headed inner arm dynein (I1/f) but surprisingly retain key regulatory proteins previously associated with it. This may have implications for other motile 9+0 cilia, including vertebrate embryonic nodal cilia required for left-right axis asymmetry. We discuss the possibility that cell-type specificity in Ciliary Motility machinery might occur in humans, and therefore underlie some of the phenotypic variation observed in PCD caused by different gene mutations. Our work lays the foundation for the increasing use of Drosophila as an excellent model for new motile Ciliary gene discovery and validation, for understanding motile cilium function and assembly, as well as understanding the nature of genetic defects underlying human motile ciliopathies
Daniela Nicastro - One of the best experts on this subject based on the ideXlab platform.
-
PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate Ciliary Motility
Molecular Biology of the Cell, 2019Co-Authors: Erin E. Dymek, Jianfeng Lin, Mary E. Porter, Daniela Nicastro, Elizabeth F. SmithAbstract:We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in Ciliary assembly and Motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine Ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell Motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not Ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional Ciliary components that play essential roles in generating coordinated Ciliary beating.
-
asymmetric distribution and spatial switching of dynein activity generates Ciliary Motility
Science, 2018Co-Authors: Jianfeng Lin, Daniela NicastroAbstract:INTRODUCTION Motile cilia and flagella are highly conserved, hairlike appendages of eukaryotic cells that propel the movement of cells or fluids. They play important roles in the normal development and health of many species, including humans. Flagellar beating is driven by the coordinated activities of multiple dynein isoforms that must be spatially and temporally regulated. Although the prevailing “switch-point” hypothesis posits that flagellar Motility results from periodic switching of spatially restricted, asymmetrical activation of dyneins, no direct evidence has been reported, and how the thousands of dyneins inside a flagellum work together to generate flagellar Motility remains elusive. RATIONALE Here we rapidly froze swimming sea urchin sperm cells and used cryo–electron tomography (cryo-ET) to image their beating flagella. Subtomogram averaging and classification analyses allowed us to identify and visualize the different activity states of individual dyneins and their regulators in situ. These conformational states were then mapped to their locations along the sinusoidal wave of the beating flagellum, for example, in relation to principal bend, reverse bend, or straight regions between bends. The results allowed us to elucidate the distinct roles played by various dyneins and to propose a model for the mechanism that underlies Ciliary and flagellar Motility. RESULTS The native three-dimensional structures of flagellar complexes were determined in situ with resolutions sufficient for identifying different activity states. Dyneins of immotile control flagella were found to be in post–power stroke conformations (unprimed, inactive states). By contrast, in beating flagella, most dyneins were in pre–power stroke conformations (primed, active states), with only a few dyneins in intermediate conformations. Moreover, for all outer dyneins, the intermediate and inactive conformations were only found in bent regions and were clustered on one side of the flagellum in a bend direction–dependent manner. For inner dyneins, certain isoforms (dyneins I1, a, d, and g) showed similar bend direction–dependent distribution patterns in bent regions of flagella, whereas the distribution patterns of other isoforms (dyneins b, c, and e) lacked obvious correlations with bending direction. Our results revealed three key tenets that are important for generating flagellar Motility: (i) The asymmetric distribution of dynein activity on opposite sides of the flagellum results in unidirectional bending, and (ii) the switching of dynein conformations between opposite sides causes the undulating waveform of beating flagella, both of which directly confirmed the switching aspect of the previously proposed switch-point hypothesis. (iii) In contrast to predictions, however, the findings also suggested the paradigm-shifting model that dyneins are active by default and that the asymmetry of dynein activity is driven by spatially restricted inhibition rather than activation of dyneins on alternating sides of the flagellum. This “switch-inhibition” mechanism was further supported by our analyses of a regulation-deficient Chlamydomonas mutant, which revealed that dyneins consumed adenosine triphosphate (ATP) and adopted pre–power stroke conformations, even though flagella were paralyzed. CONCLUSION Our comprehensive structural analysis combined with biochemical investigations provides an enhanced understanding of the distinct roles played by various dyneins and regulatory complexes in the Motility of cilia and flagella and suggests critical modifications to previous hypotheses regarding robust molecular mechanisms underlying flagellar Motility. Our study demonstrates that comparative cellular cryo-ET studies provide the conceptual framework and experimental tools to better understand molecular mechanisms and cellular functions.
-
The I1 dynein-associated tether and tether head complex is a conserved regulator of Ciliary Motility.
Molecular biology of the cell, 2018Co-Authors: Qian Wang, Jianfeng Lin, Nhan Phan, Paulina Urbanska, Ewa Joachimiak, Dorota Wloga, Daniela NicastroAbstract:Motile cilia are essential for propelling cells and moving fluids across tissues. The activity of axonemal dynein motors must be precisely coordinated to generate Ciliary Motility, but their regulatory mechanisms are not well understood. The tether and tether head (T/TH) complex was hypothesized to provide mechanical feedback during Ciliary beating because it links the motor domains of the regulatory I1 dynein to the Ciliary doublet microtubule. Combining genetic and biochemical approaches with cryoelectron tomography, we identified FAP44 and FAP43 (plus the algae-specific, FAP43-redundant FAP244) as T/TH components. WT-mutant comparisons revealed that the heterodimeric T/TH complex is required for the positional stability of the I1 dynein motor domains, stable anchoring of CK1 kinase, and proper phosphorylation of the regulatory IC138-subunit. T/TH also interacts with inner dynein arm d and radial spoke 3, another important Motility regulator. The T/TH complex is a conserved regulator of I1 dynein and plays an important role in the signaling pathway that is critical for normal Ciliary Motility.
-
the mia complex is a conserved and novel dynein regulator essential for normal Ciliary Motility
Journal of Cell Biology, 2013Co-Authors: Ryosuke Yamamoto, Daniela Nicastro, Maureen Wirschell, Laura A. Fox, Kangkang Song, Haru Aki Yanagisawa, Toshiki Yagi, Masafumi Hirono, Ritsu Kamiya, Winfield S. SaleAbstract:Axonemal dyneins must be precisely regulated and coordinated to produce ordered Ciliary/flagellar Motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo–electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 dynein and structurally appeared to connect with the nexin–dynein regulatory complex. In axonemes from mutants that lack both the outer dynein arms and the MIA complex, I1 dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 dynein. The MIA complex appears to regulate I1 dynein and possibly outer arm dyneins, which are both essential for normal Motility.
-
the csc is required for complete radial spoke assembly and wild type Ciliary Motility
Molecular Biology of the Cell, 2011Co-Authors: Erin E. Dymek, Daniela Nicastro, Thomas Heuser, Elizabeth F. SmithAbstract:The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the Motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity. Our results not only indicate that the CSC is required for spoke assembly and wild-type Motility, but also provide evidence for heterogeneity among the radial spokes.
