RESUMEN
Cilia generate three-dimensional waveforms required for cell motility and transport of fluid, mucus, and particles over the cell surface. This movement is driven by multiple dynein motors attached to nine outer doublet microtubules that form the axoneme. The outer and inner arm dyneins are organized into 96-nm repeats tandemly arrayed along the length of the doublets. Motility is regulated in part by projections from the two central pair microtubules that contact radial spokes located near the base of the inner dynein arms in each repeat. Although much is known about the structures and protein complexes within the axoneme, many questions remain about the regulatory mechanisms that allow the cilia to modify their waveforms in response to internal or external stimuli. Here, we used Chlamydomonas mbo (move backwards only) mutants with altered waveforms to identify at least two conserved proteins, MBO2/CCDC146 and FAP58/CCDC147, that form part of a L-shaped structure that varies between doublet microtubules. Comparative proteomics identified additional missing proteins that are altered in other motility mutants, revealing overlapping protein defects. Cryo-electron tomography and epitope tagging revealed that the L-shaped, MBO2/FAP58 structure interconnects inner dynein arms with multiple regulatory complexes, consistent with its function in modifying the ciliary waveform.
Asunto(s)
Axonema , Dineínas , Axonema/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Cilios/metabolismo , Proteínas/metabolismo , Flagelos/metabolismoRESUMEN
To identify proteins specific to the proximal ciliary axoneme, we used iTRAQ to compare short (~2 µm) and full-length (~11 µm) axonemes of Chlamydomonas. Known compoents of the proximal axoneme such as minor dynein heavy chains and LF5 kinase as well as the ciliary tip proteins FAP256 (CEP104) and EB1 were enriched in short axonemes whereas proteins present along the length of the axoneme were of similar abundance in both samples. The iTRAQ analysis revealed that FAP93, a protein of unknown function, and protein phosphatase 2A (PP2A) are enriched in the short axonemes. Consistently, immunoblots show enrichment of FAP93 and PP2A in short axonemes and immunofluorescence confirms the localization of FAP93 and enrichment of PP2A at the proximal axoneme. Ciliary regeneration reveals that FAP93 assembles continuously but more slowly than other axonemal structures and terminates at 1.03 µm in steady-state axonemes. The length of FAP93 assembly correlates with ciliary length, demonstrating ciliary length-dependent assembly of FAP93. Dikaryon rescue experiments show that FAP93 can assemble independently of IFT transport. In addition, FRAP analysis of GFP-tagged FAP93 demonstrates that FAP93 is stably anchored in axoneme. FAP93 may function as a scaffold for assembly of other specific proteins at the proximal axoneme.
RESUMEN
Ciliary motility requires the spatiotemporal coordination of multiple dynein motors by regulatory complexes located within the 96 nm axoneme repeat. Many organisms can alter ciliary waveforms in response to internal or external stimuli, but little is known about the specific polypeptides and structural organization of complexes that regulate waveforms. In Chlamydomonas, several mutations convert the ciliary waveform from an asymmetric, ciliary-type stroke to a symmetric, flagellar-type stroke. Some of these mutations alter subunits located at the inner junction of the doublet microtubule and others alter interactions between the dynein arms and the radial spokes. These and other axonemal substructures are interconnected by a network of poorly characterized proteins. Here we re-analyze several motility mutants (mbo, fap57, pf12/pacrg) to identify new components in this network. The mbo (move backwards only) mutants are unable to swim forwards with an asymmetric waveform. Proteomics identified more than 19 polypeptides that are missing or reduced in mbo mutants, including one inner dynein arm, IDA b. Several MBO2-associated proteins are also altered in fap57 and pf12/parcg mutants, suggesting overlapping networks. Two subunits are highly conserved, coiled coil proteins found in other species with motile cilia and others contain potential signaling domains. Cryo-electron tomography and epitope tagging revealed that the MBO2 complex is found on specific doublet microtubules and forms a large, L-shaped structure that contacts the base of IDA b that interconnects multiple dynein regulatory complexes and varies in a doublet microtubule specific fashion.
RESUMEN
Many axonemal proteins enter cilia and flagella on intraflagellar transport (IFT) trains, which move bidirectionally along the axonemal microtubules. Certain axonemal substructures including the radial spokes and outer dynein arms are preassembled in the cell body and transported as multisubunit complexes into flagella by IFT. Here, we used in vivo imaging to analyze the transport and assembly of DRC2 and DRC4, two core subunits of the nexin-dynein regulatory complex (N-DRC). Tagged DRC2 moved by IFT in mutants lacking DRC4 and vice versa, showing that they do not depend on each other for IFT. Simultaneous imaging of tagged DRC2 and DRC4, expressed from transgenes that rescue a corresponding double mutant, mostly showed transport on separate IFT trains, but occasional cotransports were also observed. The results demonstrate that DRC2 and DRC4 are transported largely independently of each other into flagella. These studies suggest that the N-DRC assembles onto the axoneme by the stepwise addition of subunits.
Asunto(s)
Chlamydomonas reinhardtii , Dineínas , Axonema/metabolismo , Transporte Biológico , Chlamydomonas reinhardtii/metabolismo , Cilios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Catión/metabolismoRESUMEN
The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the "functional subunits," namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.
Asunto(s)
Chlamydomonas/metabolismo , Dineínas/metabolismo , Flagelos/ultraestructura , Proteínas Asociadas a Microtúbulos/metabolismo , Chlamydomonas/genética , Chlamydomonas/ultraestructura , Estructura Cuaternaria de ProteínaRESUMEN
Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. IDA8 encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.
Asunto(s)
Proteínas Algáceas/metabolismo , Axonema/metabolismo , Cilios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Proteómica/métodos , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Axonema/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cilios/genética , Cilios/ultraestructura , Microscopía por Crioelectrón/métodos , Dineínas/genética , Tomografía con Microscopio Electrónico , Flagelos/genética , Flagelos/ultraestructura , Microscopía Fluorescente/métodos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mutación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Grabación de Cinta de Video/métodosRESUMEN
The nexin-dynein regulatory complex (N-DRC) plays a central role in the regulation of ciliary and flagellar motility. In most species, the N-DRC contains at least 11 subunits, but the specific function of each subunit is unknown. Mutations in three subunits (DRC1, DRC2/CCDC65, DRC4/GAS8) have been linked to defects in ciliary motility in humans and lead to a ciliopathy known as primary ciliary dyskinesia (PCD). Here we characterize the biochemical, structural, and motility phenotypes of two mutations in the DRC2 gene of Chlamydomonas Using high-resolution proteomic and structural approaches, we find that the C-terminal region of DRC2 is critical for the coassembly of DRC2 and DRC1 to form the base plate of N-DRC and its attachment to the outer doublet microtubule. Loss of DRC2 in drc2 mutants disrupts the assembly of several other N-DRC subunits and also destabilizes the assembly of several closely associated structures such as the inner dynein arms, the radial spokes, and the calmodulin- and spoke-associated complex. Our study provides new insights into the range of ciliary defects that can lead to PCD.
Asunto(s)
Proteínas Algáceas/fisiología , Axonema/fisiología , Chlamydomonas/fisiología , Cilios/fisiología , Glicoproteínas/fisiología , Proteínas Algáceas/genética , Chlamydomonas/genética , Glicoproteínas/genética , Mutación , ProteómicaRESUMEN
Intraflagellar transport (IFT) is essential for the elongation and maintenance of eukaryotic cilia and flagella. Due to the traffic jam of multiple trains at the ciliary tip, how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging. Using PhotoGate, we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella. Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains. Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains. Unlike dynein-1b, kinesin-II detaches from IFT trains at the tip and diffuses in flagella. As the flagellum grows longer, diffusion delays return of kinesin-II to the basal body, depleting kinesin-II available for anterograde transport. Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas.
Asunto(s)
Chlamydomonas/metabolismo , Flagelos/metabolismo , Dineínas/metabolismo , Cinesinas/metabolismo , Microscopía Fluorescente , Microscopía por Video , Transporte de ProteínasRESUMEN
Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.
Asunto(s)
Tipificación del Cuerpo/genética , Cilios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animales , Movimiento Celular/genética , Chlamydomonas/genética , Cilios/patología , Proteínas del Citoesqueleto , Citoesqueleto/genética , Modelos Animales de Enfermedad , Extremidades/crecimiento & desarrollo , Extremidades/patología , Predisposición Genética a la Enfermedad , Humanos , Síndrome de Kartagener/patología , Ratones , Microtúbulos/genética , Mutación , Tubo Neural/crecimiento & desarrollo , Tubo Neural/patología , Transducción de Señal/genéticaRESUMEN
The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms.
Asunto(s)
Cilios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Animales , Chlamydomonas/metabolismo , Microtúbulos/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of â¼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Axonema/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Plantas/metabolismo , Axonema/genética , Chlamydomonas reinhardtii/genética , Cilios/genética , Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Plantas/genéticaRESUMEN
Cryo-electron tomography (cryo-ET) has reached nanoscale resolution for in situ three-dimensional imaging of macromolecular complexes and organelles. Yet its current resolution is not sufficient to precisely localize or identify most proteins in situ; for example, the location and arrangement of components of the nexin-dynein regulatory complex (N-DRC), a key regulator of ciliary/flagellar motility that is conserved from algae to humans, have remained elusive despite many cryo-ET studies of cilia and flagella. Here, we developed an in situ localization method that combines cryo-ET/subtomogram averaging with the clonable SNAP tag, a widely used cell biological probe to visualize fusion proteins by fluorescence microscopy. Using this hybrid approach, we precisely determined the locations of the N and C termini of DRC3 and the C terminus of DRC4 within the three-dimensional structure of the N-DRC in Chlamydomonas flagella. Our data demonstrate that fusion of SNAP with target proteins allowed for protein localization with high efficiency and fidelity using SNAP-linked gold nanoparticles, without disrupting the native assembly, structure, or function of the flagella. After cryo-ET and subtomogram averaging, we localized DRC3 to the L1 projection of the nexin linker, which interacts directly with a dynein motor, whereas DRC4 was observed to stretch along the N-DRC base plate to the nexin linker. Application of the technique developed here to the N-DRC revealed new insights into the organization and regulatory mechanism of this complex, and provides a valuable tool for the structural dissection of macromolecular complexes in situ.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Dineínas/metabolismo , Tomografía con Microscopio Electrónico/métodos , Flagelos/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Algáceas/genética , Axonema/genética , Axonema/metabolismo , Axonema/ultraestructura , Western Blotting , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/fisiología , Dineínas/genética , Flagelos/genética , Flagelos/ultraestructura , Microscopía Fluorescente , Modelos Moleculares , Movimiento , Complejos Multiproteicos/química , Mutación , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.
Asunto(s)
Trastornos de la Motilidad Ciliar/genética , Glicoproteínas/genética , Síndrome de Kartagener/genética , Pez Cebra/genética , Animales , Chlamydomonas/genética , Cilios/genética , Análisis Mutacional de ADN/métodos , Dineínas/genética , Femenino , Humanos , Masculino , Mutación , Sistemas de Lectura Abierta , Planarias/genética , Proteoma/genéticaRESUMEN
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.
Asunto(s)
Dineínas Axonemales/metabolismo , Axonema/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Plantas/metabolismo , Nexinas de Clasificación/metabolismo , Secuencia de Aminoácidos , Dineínas Axonemales/genética , Secuencia Conservada , Elementos Transponibles de ADN , Flagelos/metabolismo , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas de Plantas/genética , Mapeo de Interacción de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
Primary ciliary dyskinesia (PCD) is characterized by dysfunction of respiratory cilia and sperm flagella and random determination of visceral asymmetry. Here, we identify the DRC1 subunit of the nexin-dynein regulatory complex (N-DRC), an axonemal structure critical for the regulation of dynein motors, and show that mutations in the gene encoding DRC1, CCDC164, are involved in PCD pathogenesis. Loss-of-function mutations disrupting DRC1 result in severe defects in assembly of the N-DRC structure and defective ciliary movement in Chlamydomonas reinhardtii and humans. Our results highlight a role for N-DRC integrity in regulating ciliary beating and provide the first direct evidence that mutations in DRC genes cause human disease.
Asunto(s)
Proteínas Algáceas/genética , Proteínas Portadoras/genética , Chlamydomonas , Cilios , Trastornos de la Motilidad Ciliar , Síndrome de Kartagener , Proteínas Asociadas a Microtúbulos/genética , Secuencia de Aminoácidos , Dineínas Axonemales/genética , Dineínas Axonemales/metabolismo , Dineínas Axonemales/ultraestructura , Axonema/genética , Axonema/metabolismo , Axonema/ultraestructura , Chlamydomonas/genética , Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Cilios/genética , Cilios/metabolismo , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/patología , Citoesqueleto/genética , Citoesqueleto/metabolismo , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/fisiopatología , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Mutación , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/ultraestructuraRESUMEN
To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120--defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.
Asunto(s)
Proteínas Algáceas/metabolismo , Dineínas/metabolismo , Flagelos/fisiología , Microtúbulos/fisiología , Proteínas Algáceas/genética , Animales , Axonema/metabolismo , Axonema/fisiología , Axonema/ultraestructura , Sitios de Unión , Southern Blotting , Western Blotting , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/fisiología , Dineínas/genética , Exones/genética , Flagelos/genética , Flagelos/metabolismo , Eliminación de Gen , Microscopía Electrónica , Microtúbulos/metabolismo , Mutación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Reacción en Cadena de la Polimerasa , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
How ciliary and flagellar motility is regulated is a challenging problem. The flagellar movement in Chlamydomonas reinhardtii is in part regulated by phosphorylation of a 138 kD intermediate chain (IC138) of inner arm dynein f (also called I1). In the present study, we found that the axoneme of mutants lacking dynein f lacks a novel protein having ankyrin repeat motifs, registered as FAP120 in the flagellar proteome database. FAP120 is also missing or decreased in the axonemes of bop5, a mutant that has a mutation in the structural gene of IC138 but assembles the dynein f complex. Intriguingly, the amounts of FAP120 in the axonemes of different alleles of bop5 and several dynein f-lacking mutants roughly parallel their contents of IC138. These results suggest a weak but stoichiometric interaction between FAP120 and IC138. We propose that FAP120 functions in the regulatoryprocess as part of a protein complex involving IC138. Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc.
Asunto(s)
Repetición de Anquirina , Chlamydomonas reinhardtii/metabolismo , Dineínas/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Axonema/metabolismo , Chlamydomonas reinhardtii/fisiología , Cilios/metabolismo , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Microscopía Fluorescente , Mutación , Unión Proteica , Proteínas Protozoarias/genéticaRESUMEN
Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21 degrees C, but cannot maintain them at 33 degrees C. Although the Kinesin-2 complex is present at both 21 and 33 degrees C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella.
Asunto(s)
Flagelos/fisiología , Cinesinas/biosíntesis , Cinesinas/metabolismo , Cinesinas/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico , Southern Blotting , Western Blotting , Centrómero/ultraestructura , Chlamydomonas/metabolismo , Cilios/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Epítopos/química , Ligamiento Genético , Cinesinas/química , Microscopía Fluorescente , Microscopía por Video , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Fenotipo , Estructura Terciaria de Proteína , ARN/química , Homología de Secuencia de Aminoácido , Temperatura , Factores de TiempoRESUMEN
The assembly of cilia and flagella depends on bidirectional intraflagellar transport (IFT). Anterograde IFT is driven by kinesin II, whereas retrograde IFT requires cytoplasmic dynein 1b (cDHC1b). Little is known about how cDHC1b interacts with its cargoes or how it is regulated. Recent work identified a novel dynein light intermediate chain (D2LIC) that colocalized with the mammalian cDHC1b homolog DHC2 in the centrosomal region of cultured cells. To see whether the LIC might play a role in IFT, we characterized the gene encoding the Chlamydomonas homolog of D2LIC and found its expression is up-regulated in response to deflagellation. We show that the LIC subunit copurifies with cDHC1b during flagellar isolation, dynein extraction, sucrose density centrifugation, and immunoprecipitation. Immunocytochemistry reveals that the LIC colocalizes with cDHC1b in the basal body region and along the length of flagella in wild-type cells. Localization of the complex is altered in a collection of retrograde IFT and length control mutants, which suggests that the affected gene products directly or indirectly regulate cDHC1b activity. The mammalian DHC2 and D2LIC also colocalize in the apical cytoplasm and axonemes of ciliated epithelia in the lung, brain, and efferent duct. These studies, together with the identification of an LIC mutation, xbx-1(ok279), which disrupts retrograde IFT in Caenorhabditis elegans, indicate that the novel LIC is a component of the cDHC1b/DHC2 retrograde IFT motor in a variety of organisms.