RESUMEN
Loss of the cilium is important for cell cycle progression and certain developmental transitions. Chytrid fungi are a group of basal fungi that have retained centrioles and cilia, and they can disassemble their cilia via axoneme internalization as part of the transition from free-swimming spores to sessile sporangia. While this type of cilium disassembly has been observed in many single-celled eukaryotes, it has not been well characterized because it is not observed in common model organisms. To better characterize cilium disassembly via axoneme internalization, we focused on chytrids Rhizoclosmatium globosum and Spizellomyces punctatus to represent two lineages of chytrids with different motility characteristics. Our results show that each chytrid species can reel in its axoneme into the cell body along its cortex on the order of minutes, while S. punctatus has additional faster ciliary compartment loss and lash-around mechanisms. S. punctatus retraction can also occur away from the cell cortex and is partially actin dependent. Post-internalization, the tubulin of the axoneme is degraded in both chytrids over the course of about 2 hr. Axoneme disassembly and axonemal tubulin degradation are both partially proteasome dependent. Overall, chytrid cilium disassembly is a fast process that separates axoneme internalization and degradation.
Asunto(s)
Axonema/metabolismo , Cilios/metabolismo , Hongos/patogenicidadRESUMEN
The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.
Asunto(s)
Cilios/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Tetrahymena/citología , Regulación de la Expresión Génica , Locomoción , Proteínas Protozoarias/metabolismo , Tetrahymena/metabolismo , Tetrahymena/fisiologíaRESUMEN
Cilia, essential motile and sensory organelles, have several compartments: the basal body, transition zone, and the middle and distal axoneme segments. The distal segment accommodates key functions, including cilium assembly and sensory activities. While the middle segment contains doublet microtubules (incomplete B-tubules fused to complete A-tubules), the distal segment contains only A-tubule extensions, and its existence requires coordination of microtubule length at the nanometer scale. We show that three conserved proteins, two of which are mutated in the ciliopathy Joubert syndrome, determine the geometry of the distal segment, by controlling the positions of specific microtubule ends. FAP256/CEP104 promotes A-tubule elongation. CHE-12/Crescerin and ARMC9 act as positive and negative regulators of B-tubule length, respectively. We show that defects in the distal segment dimensions are associated with motile and sensory deficiencies of cilia. Our observations suggest that abnormalities in distal segment organization cause a subset of Joubert syndrome cases.
Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cilios/metabolismo , Microtúbulos/metabolismo , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Proteínas del Dominio Armadillo/genética , Proteínas de Ciclo Celular/genética , Cerebelo/anomalías , Cerebelo/metabolismo , Cilios/genética , Anomalías del Ojo/genética , Anomalías del Ojo/metabolismo , Humanos , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/metabolismo , Microtúbulos/genética , Proteínas Protozoarias/genética , Retina/anomalías , Retina/metabolismo , Tetrahymena thermophila/genéticaRESUMEN
Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin-14 is a conserved minus-end directed microtubule motor that cross-links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin-14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP-tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin-14 is important for nuclear divisions that involve a bipolar spindle.
Asunto(s)
Segregación Cromosómica , Cilióforos/genética , Cinesinas/genética , Cinesinas/fisiología , Meiosis , Mitosis , Tetrahymena thermophila/genética , Animales , Núcleo Celular , Cilióforos/citología , Técnicas de Inactivación de Genes , Cinesinas/clasificación , Cinesinas/ultraestructura , Macronúcleo , Profase Meiótica I , Metafase , Microtúbulos , Mutación , Filogenia , Proteínas Recombinantes , Huso Acromático , Polos del Huso , Tetrahymena/genética , Tetrahymena thermophila/citología , Tetrahymena thermophila/metabolismoRESUMEN
Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo-electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.
Asunto(s)
Axonema/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Proteínas Protozoarias/fisiología , Tetrahymena/metabolismo , Cilios/metabolismo , Cilios/fisiología , Tomografía con Microscopio Electrónico , Técnicas de Inactivación de Genes , Microtúbulos/ultraestructura , Modelos Moleculares , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Tetrahymena/genética , Tetrahymena/ultraestructuraRESUMEN
Kinesin-13, an end depolymerizer of cytoplasmic and spindle microtubules, also affects the length of cilia. However, in different models, depletion of kinesin-13 either lengthens or shortens cilia, and therefore the exact function of kinesin-13 in cilia remains unclear. We generated null mutations of all kinesin-13 paralogues in the ciliate Tetrahymena. One of the paralogues, Kin13Ap, localizes to the nuclei and is essential for nuclear divisions. The remaining two paralogues, Kin13Bp and Kin13Cp, localize to the cell body and inside assembling cilia. Loss of both Kin13Bp and Kin13Cp resulted in slow cell multiplication and motility, overgrowth of cell body microtubules, shortening of cilia, and synthetic lethality with either paclitaxel or a deletion of MEC-17/ATAT1, the α-tubulin acetyltransferase. The mutant cilia assembled slowly and contained abnormal tubulin, characterized by altered posttranslational modifications and hypersensitivity to paclitaxel. The mutant cilia beat slowly and axonemes showed reduced velocity of microtubule sliding. Thus kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and likely indirectly, through its effects on the axonemal microtubules, affects the ciliary dynein-dependent motility.
Asunto(s)
Axonema/fisiología , Cilios/fisiología , Cinesinas/genética , Tubulina (Proteína)/metabolismo , Acetiltransferasas/genética , Axonema/genética , Cilios/genética , Técnicas de Inactivación de Genes , Morfogénesis/efectos de los fármacos , Morfogénesis/genética , Mutación , Paclitaxel/farmacología , Procesamiento Proteico-Postraduccional , Tetrahymena thermophila/genética , Tetrahymena thermophila/fisiologíaRESUMEN
ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.
Asunto(s)
Actinas/metabolismo , Cofilina 1/genética , Proteínas de Microfilamentos/genética , Fagocitosis/genética , Tetrahymena thermophila/crecimiento & desarrollo , Tetrahymena thermophila/metabolismo , Citoesqueleto de Actina/metabolismo , Infecciones por Cilióforos/genética , Infecciones por Cilióforos/microbiología , Cofilina 1/metabolismo , Citocinesis/genética , Técnicas de Inactivación de Genes , Homología de Secuencia de Aminoácido , Tetrahymena thermophila/patogenicidad , Vacuolas/metabolismoRESUMEN
The ciliate Tetrahymena thermophila is an excellent model system for the discovery and functional studies of ciliary proteins. The power of the model is based on the ease with which cilia can be purified in large quantities for fractionation and proteomic identification, and the ability to knock out any gene by homologous DNA recombination. Here, we include methods used by our laboratories for isolation and fractionation of cilia, in vivo tagging and localization of ciliary proteins, and the evaluation of ciliary mutants.