RESUMO
Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a "transition zone" (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium-BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.
Assuntos
Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Axonema/fisiologia , Corpos Basais/fisiologia , Células Epiteliais/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Movimento/fisiologia , Traqueia/fisiologiaRESUMO
In multi-ciliated cells, directed and synchronous ciliary beating in the apical membrane occurs through appropriate configuration of basal bodies (BBs, roots of cilia). Although it has been experimentally shown that the position and orientation of BBs are coordinated by apical cytoskeletons (CSKs), such as microtubules (MTs), and planar cell polarity (PCP), the underlying mechanism for achieving the patterning of BBs is not yet understood. In this study, we propose that polarity in bundles of apical MTs play a crucial role in the patterning of BBs. First, the necessity of the polarity was discussed by theoretical consideration on the symmetry of the system. The existence of the polarity was investigated by measuring relative angles between the MTs and BBs using published experimental data. Next, a mathematical model for BB patterning was derived by combining the polarity and self-organizational ability of CSKs. In the model, BBs were treated as finite-size particles in the medium of CSKs and excluded volume effects between BBs and CSKs were taken into account. The model reproduces the various experimental observations, including normal and drug-treated phenotypes. Our model with polarity provides a coherent and testable mechanism for apical BB pattern formation. We have also discussed the implication of our study on cell chirality.
Assuntos
Corpos Basais/fisiologia , Cílios/fisiologia , Citoesqueleto/fisiologia , Animais , Membrana Celular , Polaridade Celular , Simulação por Computador , Elasticidade , Células Epiteliais/citologia , Camundongos , Microtúbulos/fisiologia , Modelos Teóricos , Nocodazol/farmacologia , Fenótipo , Traqueia/fisiologiaRESUMO
Eukaryotic cilia/flagella exhibit two characteristic ultrastructures reflecting two main functions; a 9+2 axoneme for motility and a 9+0 axoneme for sensation and signalling. Whether, and if so how, they interconvert is unclear. Here we analyse flagellum length, structure and molecular composition changes in the unicellular eukaryotic parasite Leishmania during the transformation of a life cycle stage with a 9+2 axoneme (the promastigote) to one with a 9+0 axoneme (the amastigote). We show 9+0 axonemes can be generated by two pathways: by de novo formation and by restructuring of existing 9+2 axonemes associated with decreased intraflagellar transport. Furthermore, pro-basal bodies formed under conditions conducive for 9+2 axoneme formation can form a 9+0 axoneme de novo. We conclude that pro-centrioles/pro-basal bodies are multipotent and not committed to form either a 9+2 or 9+0 axoneme. In an alternative pathway structures can also be removed from existing 9+2 axonemes to convert them to 9+0.
Assuntos
Axonema/metabolismo , Corpos Basais/fisiologia , Flagelos/fisiologia , Leishmania mexicana/fisiologia , Animais , Axonema/ultraestrutura , Divisão Celular , Células Cultivadas , Chlamydomonas reinhardtii , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde , Leishmania mexicana/ultraestrutura , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Organismos Geneticamente Modificados , Proteínas RecombinantesRESUMO
In wild type (WT) tracheal epithelial cells, ciliary basal bodies are oriented such that all cilia on the cell surface beat in the same upward direction. This precise alignment of basal bodies and, as a result, the ciliary axoneme, is termed rotational planar cell polarity (PCP). Rotational PCP in the multi-ciliated epithelial cells of the trachea is perturbed in rats lacking myosin Id (Myo1d). Myo1d is localized in the F-actin and basal body rich subapical cortex of the ciliated tracheal epithelial cell. Scanning and transmission electron microscopy of Myo1d knock out (KO) trachea revealed that the unidirectional bending pattern is disrupted. Instead, cilia splay out in a disordered, often radial pattern. Measurement of the alignment axis of the central pair axonemal microtubules was much more variable in the KO, another indicator that rotational PCP is perturbed. The asymmetric localization of the PCP core protein Vangl1 is lost. Both the velocity and linearity of cilia-driven movement of beads above the tracheal mucosal surface was impaired in the Myo1d KO. Multi-ciliated brain ependymal epithelial cells exhibit a second form of PCP termed translational PCP in which basal bodies and attached cilia are clustered at the anterior side of the cell. The precise asymmetric clustering of cilia is disrupted in the ependymal cells of the Myo1d KO rat. While basal body clustering is maintained, left-right positioning of the clusters is lost.
Assuntos
Epêndima/fisiologia , Células Epiteliais/citologia , Miosina Tipo I/fisiologia , Miosinas/fisiologia , Actinas/fisiologia , Animais , Animais Geneticamente Modificados , Axonema/fisiologia , Corpos Basais/fisiologia , Encéfalo/fisiologia , Polaridade Celular , Cílios/fisiologia , Marcação In Situ das Extremidades Cortadas , Intestinos/fisiologia , Masculino , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Miosina Tipo I/genética , Miosinas/genética , Neuroglia/fisiologia , Fenótipo , Ratos , Ratos Endogâmicos F344 , Rotação , Traqueia/fisiologiaRESUMO
Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.
Assuntos
Proteínas de Transporte/metabolismo , Cílios/metabolismo , Células Epiteliais/citologia , Proteínas dos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Mucosa Respiratória/citologia , Motivos de Aminoácidos/genética , Animais , Corpos Basais/fisiologia , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Centríolos/fisiologia , Cílios/genética , Quinases do Centro Germinativo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Depuração Mucociliar/genética , Naftalenos , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
CYLD is a tumour suppressor gene mutated in familial cylindromatosis, a genetic disorder leading to the development of skin appendage tumours. It encodes a deubiquitinating enzyme that removes Lys63- or linear-linked ubiquitin chains. CYLD was shown to regulate cell proliferation, cell survival and inflammatory responses, through various signalling pathways. Here we show that CYLD localizes at centrosomes and basal bodies via interaction with the centrosomal protein CAP350 and demonstrate that CYLD must be both at the centrosome and catalytically active to promote ciliogenesis independently of NF-κB. In transgenic mice engineered to mimic the smallest truncation found in cylindromatosis patients, CYLD interaction with CAP350 is lost disrupting CYLD centrosome localization, which results in cilia formation defects due to impairment of basal body migration and docking. These results point to an undiscovered regulation of ciliogenesis by Lys63 ubiquitination and provide new perspectives regarding CYLD function that should be considered in the context of cylindromatosis.
Assuntos
Corpos Basais/fisiologia , Comunicação Celular/fisiologia , Centrossomo/fisiologia , Cílios/fisiologia , Cisteína Endopeptidases/fisiologia , Células Epiteliais/fisiologia , Animais , Células Cultivadas , Cisteína Endopeptidases/genética , Proteínas do Citoesqueleto/fisiologia , Enzima Desubiquitinante CYLD , Células Epiteliais/citologia , Feminino , Humanos , Rim/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microtúbulos/fisiologia , NF-kappa B/fisiologia , Proteínas Nucleares/fisiologia , Retina/citologia , Transdução de Sinais/fisiologiaRESUMO
Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified flgC as a potential binding mutant of SE. Therefore, we hypothesize FlgC which plays a significant role in the binding ability of SE to the intestinal mucosa of poultry. To test our hypothesis, we created a mutant of SE in which flgC was deleted. We then tested the in vitro and in vivo binding ability of ∆flgC when compared to the wild-type SE strain. Our data showed a significant decrease in the binding ability of ∆flgC to intestinal epithelial cells as well as in the small intestine and cecum of poultry. Furthermore, the decrease in binding correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Overall, these studies indicate FlgC is a major factor in the binding ability of Salmonella to the intestinal mucosa of poultry.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Corpos Basais/fisiologia , Células Epiteliais/microbiologia , Flagelos/fisiologia , Salmonella enteritidis/fisiologia , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Ceco/microbiologia , Galinhas , Flagelos/genética , Deleção de Genes , Humanos , Intestino Delgado/microbiologia , Fígado/microbiologia , Salmonella enteritidis/genética , Baço/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Basal bodies and centrioles are conserved microtubule-based organelles the improper assembly of which leads to a number of diseases, including ciliopathies and cancer. Tubulin family members are conserved components of these structures that are integral to their proper formation and function. We have identified the ε-tubulin gene in Tetrahymena thermophila and detected the protein, through fluorescence of a tagged allele, to basal bodies. Immunoelectron microscopy has shown that ε-tubulin localizes primarily to the core microtubule scaffold. A complete genomic knockout of ε-tubulin has revealed that it is an essential gene required for the assembly and maintenance of the triplet microtubule blades of basal bodies. We have conducted site-directed mutagenesis of the ε-tubulin gene and shown that residues within the nucleotide-binding domain, longitudinal interacting domains, and C-terminal tail are required for proper function. A single amino acid change of Thr150, a conserved residue in the nucleotide-binding domain, to Val is a conditional mutation that results in defects in the spatial and temporal assembly of basal bodies as well as their stability. We have genetically separated functions for the domains of ε-tubulin and identified a novel role for the nucleotide-binding domain in the regulation of basal body assembly and stability.