RESUMEN
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.
Asunto(s)
Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Axonema/fisiología , Cuerpos Basales/fisiología , Células Epiteliales/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Movimiento/fisiología , Tráquea/fisiologíaRESUMEN
The basal body of the bacterial flagellum is a rotary motor that consists of several rings (C, MS and LP) and a rod. The LP ring acts as a bushing supporting the distal rod for its rapid and stable rotation without much friction. Here, we use electron cryomicroscopy to describe the LP ring structure around the rod, at 3.5 Å resolution, from Salmonella Typhimurium. The structure shows 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners, which explains the structural stability. The inner surface is charged both positively and negatively. Positive charges on the P ring (the part of the LP ring that is embedded within the peptidoglycan layer) presumably play important roles in its initial assembly around the rod with a negatively charged surface.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Flagelos/química , Flagelos/ultraestructura , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/ultraestructura , Proteínas Bacterianas/fisiología , Cuerpos Basales/química , Cuerpos Basales/fisiología , Cuerpos Basales/ultraestructura , Microscopía por Crioelectrón , Flagelos/fisiología , Modelos Moleculares , Proteínas Motoras Moleculares/fisiología , Movimiento/fisiología , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Salmonella typhimurium/química , Salmonella typhimurium/fisiología , Salmonella typhimurium/ultraestructura , Electricidad EstáticaRESUMEN
The bacterial flagellum consists of a long extracellular filament that is rotated by a motor embedded in the cell envelope. While flagellar assembly has been extensively studied,1 the disassembly process remains less well understood. In addition to the programmed flagellar ejection that occurs during the life cycle of Caulobacter crescentus, we and others have recently shown that many bacterial species lose their flagella under starvation conditions, leaving relic structures in the outer membrane.2-7 However, it remains unknown whether the programmed flagellar ejection of C. crescentus leaves similar relics or not. Here, we imaged the various stages of the C. crescentus life cycle using electron cryo-tomography (cryo-ET) and found that flagellar relic subcomplexes, akin to those produced in the starvation-induced process, remain as a result of flagellar ejection during cell development. This similarity suggests that the programmed flagellar ejection of C. crescentus might share a common evolutionary path with the more general, and likely more ancient,3 starvation-related flagellar loss.
Asunto(s)
Caulobacter crescentus/fisiología , Pared Celular/metabolismo , Flagelos/fisiología , Lipopolisacáridos/metabolismo , Peptidoglicano/metabolismo , Cuerpos Basales/fisiología , Cuerpos Basales/ultraestructura , Caulobacter crescentus/metabolismo , Caulobacter crescentus/ultraestructura , Pared Celular/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Fimbrias Bacterianas/fisiología , Fimbrias Bacterianas/ultraestructura , Flagelos/ultraestructuraRESUMEN
The single mitochondrial nucleoid (kinetoplast) of Trypanosoma brucei is found proximal to a basal body (mature (mBB)/probasal body (pBB) pair). Kinetoplast inheritance requires synthesis of, and scission of kinetoplast DNA (kDNA) generating two kinetoplasts that segregate with basal bodies into daughter cells. Molecular details of kinetoplast scission and the extent to which basal body separation influences the process are unavailable. To address this topic, we followed basal body movements in bloodstream trypanosomes following depletion of protein kinase TbCK1.2 which promotes kinetoplast division. In control cells we found that pBBs are positioned 0.4 um from mBBs in G1, and they mature after separating from mBBs by at least 0.8 um: mBB separation reaches ~2.2 um. These data indicate that current models of basal body biogenesis in which pBBs mature in close proximity to mBBs may need to be revisited. Knockdown of TbCK1.2 produced trypanosomes containing one kinetoplast and two nuclei (1K2N), increased the percentage of cells with uncleaved kDNA 400%, decreased mBB spacing by 15%, and inhibited cytokinesis 300%. We conclude that (a) separation of mBBs beyond a threshold of 1.8 um correlates with division of kDNA, and (b) TbCK1.2 regulates kDNA scission. We propose a Kinetoplast Division Factor hypothesis that integrates these data into a pathway for biogenesis of two daughter mitochondrial nucleoids.
Asunto(s)
Cuerpos Basales/fisiología , Quinasa de la Caseína I/metabolismo , ADN de Cinetoplasto/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Quinasa de la Caseína I/antagonistas & inhibidores , Quinasa de la Caseína I/genética , Citocinesis/fisiología , Citoplasma/metabolismo , División del ADN , Replicación del ADN , Mitocondrias/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
The Lyme disease bacterium Borrelia burgdorferi has 7-11 periplasmic flagella (PF) that arise from the cell poles and extend toward the midcell as a flat-ribbon, which is distinct from other bacteria. FlhF, a signal recognition particle (SRP)-like GTPase, has been found to regulate the flagellar number and polarity; however, its role in B. burgdorferi remains unknown. B. burgdorferi has an FlhF homolog (BB0270). Structural and biochemical analyses show that BB0270 has a similar structure and enzymatic activity as its counterparts from other bacteria. Genetics and cryo-electron tomography studies reveal that deletion of BB0270 leads to mutant cells that have less PF (4 ± 2 PF per cell tip) and fail to form a flat-ribbon, indicative of a role of BB0270 in the control of PF number and configuration. Mechanistically, we demonstrate that BB0270 localizes at the cell poles and controls the number and position of PF via regulating the flagellar protein stability and the polar localization of the MS-ring protein FliF. Our study not only provides the detailed characterizations of BB0270 and its profound impacts on flagellar assembly, morphology and motility in B. burgdorferi, but also unveils mechanistic insights into how spirochetes control their unique flagellar patterns.
Asunto(s)
Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/metabolismo , Flagelos/metabolismo , Flagelos/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Bacterianas/genética , Cuerpos Basales/fisiología , Borrelia burgdorferi/genética , Tomografía con Microscopio Electrónico , Flagelos/genética , Eliminación de Gen , Locomoción/genética , Proteínas de Unión al GTP Monoméricas/genéticaRESUMEN
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.
Asunto(s)
Cuerpos Basales/fisiología , Cilios/fisiología , Citoesqueleto/fisiología , Animales , Membrana Celular , Polaridad Celular , Simulación por Computador , Elasticidad , Células Epiteliales/citología , Ratones , Microtúbulos/fisiología , Modelos Teóricos , Nocodazol/farmacología , Fenotipo , Tráquea/fisiologíaRESUMEN
Multi-ciliary arrays promote fluid flow and cellular motility using the polarized and coordinated beating of hundreds of motile cilia. Tetrahymena basal bodies (BBs) nucleate and position cilia, whereby BB-associated striated fibers (SFs) promote BB anchorage and orientation into ciliary rows. Mutants that shorten SFs cause disoriented BBs. In contrast to the cytotaxis model, we show that disoriented BBs with short SFs can regain normal orientation if SF length is restored. In addition, SFs adopt unique lengths by their shrinkage and growth to establish and maintain BB connections and cortical interactions in a ciliary force-dependent mechanism. Tetrahymena SFs comprise at least eight uniquely localizing proteins belonging to the SF-assemblin family. Loss of different proteins that localize to the SF base disrupts either SF steady-state length or ciliary force-induced SF elongation. Thus, the dynamic regulation of SFs promotes BB connections and cortical interactions to organize ciliary arrays.
Asunto(s)
Cuerpos Basales/fisiología , Cilios/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/crecimiento & desarrollo , Tetrahymena thermophila/metabolismo , Fenómenos Mecánicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Protozoarias/genética , Tetrahymena thermophila/genéticaRESUMEN
Motile cilia on multiciliated cells (MCCs) function in fluid clearance over epithelia. Studies with Xenopus embryos and individuals with the congenital respiratory disorder reduced generation of multiple motile cilia (RGMC), have implicated the nuclear protein MCIDAS (MCI), in the transcriptional regulation of MCC specification and differentiation. Recently, a paralogous protein, geminin coiled-coil domain containing (GMNC), was also shown to be required for MCC formation. Surprisingly, in contrast to the presently held view, we find that Mci mutant mice can specify MCC precursors. However, these precursors cannot produce multiple basal bodies, and mature into single ciliated cells. We identify an essential role for MCI in inducing deuterosome pathway components for the production of multiple basal bodies. Moreover, GMNC and MCI associate differentially with the cell-cycle regulators E2F4 and E2F5, which enables them to activate distinct sets of target genes (ciliary transcription factor genes versus basal body amplification genes). Our data establish a previously unrecognized two-step model for MCC development: GMNC functions in the initial step for MCC precursor specification. GMNC induces Mci expression that drives the second step of basal body production for multiciliation.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cilios/fisiología , Ratones Mutantes , Proteínas Nucleares/fisiología , Animales , Cuerpos Basales/fisiología , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Núcleo Celular/fisiología , Ciliopatías , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Dominios Proteicos , Pez CebraRESUMEN
Photoreceptors are polarized neurons, with very specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor contains an outer segment, the site of photon capture that initiates vision, an inner segment that houses the biosynthetic machinery and a synaptic terminal for signal transmission to downstream neurons. Outer segments and inner segments are connected by a connecting cilium (CC), the equivalent of a transition zone (TZ) of primary cilia. The connecting cilium is part of the basal body/axoneme backbone that stabilizes the outer segment. This report will update the reader on late developments in photoreceptor ciliogenesis and transition zone formation, specifically in mouse photoreceptors, focusing on early events in photoreceptor ciliogenesis. The connecting cilium, an elongated and narrow structure through which all outer segment proteins and membrane components must traffic, functions as a gate that controls access to the outer segment. Here we will review genes and their protein products essential for basal body maturation and for CC/TZ genesis, sorted by phenotype. Emphasis is given to naturally occurring mouse mutants and gene knockouts that interfere with CC/TZ formation and ciliogenesis.
Asunto(s)
Cilios/fisiología , Células Fotorreceptoras/fisiología , Animales , Cuerpos Basales/fisiología , Proteínas de la Membrana/metabolismo , Modelos Animales , Transporte de Proteínas/fisiología , Transducción de Señal/fisiologíaRESUMEN
The actin cytoskeleton is critical to shape cells and pattern intracellular organelles, which collectively drives tissue morphogenesis. In multiciliated cells (MCCs), apical actin drives expansion of the cell surface necessary to host hundreds of cilia. The apical actin also forms a lattice to uniformly distribute basal bodies. This apical actin network is dynamically remodeled, but the molecules that regulate its architecture remain poorly understood. We identify the chromatin modifier, WDR5, as a regulator of apical F-actin in MCCs. Unexpectedly in MCCs, WDR5 has a function independent of chromatin modification. We discover a scaffolding role for WDR5 between the basal body and F-actin. Specifically, WDR5 binds to basal bodies and migrates apically, where F-actin organizes around WDR5. Using a monomer trap for G-actin, we show that WDR5 stabilizes F-actin to maintain lattice architecture. In summary, we identify a non-chromatin role for WDR5 in stabilizing F-actin in MCCs.
Asunto(s)
Citoesqueleto de Actina/fisiología , Cuerpos Basales/fisiología , Membrana Celular/metabolismo , Cilios/fisiología , Embrión no Mamífero/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Animales , Embrión no Mamífero/citología , Femenino , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Morfogénesis , XenopusRESUMEN
Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane-although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. We show that PLP helps assemble "pericentriolar clouds" of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells.
Asunto(s)
Centriolos/metabolismo , Centrosoma/metabolismo , Proteínas de Drosophila/fisiología , Microtúbulos/metabolismo , Animales , Animales Modificados Genéticamente , Cuerpos Basales/metabolismo , Cuerpos Basales/fisiología , Proteínas de Unión a Calmodulina , Centriolos/genética , Cilios/genética , Cilios/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Interfase/fisiología , Complejos Multiproteicos/metabolismo , Mutación/fisiología , Multimerización de Proteína/fisiología , Estabilidad Proteica , Células Receptoras Sensoriales/química , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/ultraestructuraRESUMEN
Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance.
Asunto(s)
Cuerpos Basales/fisiología , Caenorhabditis elegans/fisiología , Centriolos/fisiología , Neurogénesis , Células Receptoras Sensoriales/fisiología , Animales , Animales Modificados Genéticamente , Axonema/fisiología , Cuerpos Basales/metabolismo , Cuerpos Basales/ultraestructura , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/metabolismo , Centriolos/metabolismo , Centriolos/ultraestructura , Cilios/fisiología , Tomografía con Microscopio Electrónico , Microscopía Fluorescente , Microscopía por Video , Proteolisis , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/ultraestructura , Factores de Tiempo , Imagen de Lapso de TiempoRESUMEN
Multiciliated cell (MCC) differentiation involves extensive organelle biogenesis required to extend hundreds of motile cilia. Key transcriptional regulators known to drive the gene expression required for this organelle biogenesis are activated by the related coiled-coil proteins Multicilin and Gemc1. Here we identify foxn4 as a new downstream target of Multicilin required for MCC differentiation in Xenopus skin. When Foxn4 activity is inhibited in Xenopus embryos, MCCs show transient ciliogenesis defects similar to those seen in mutants of Foxj1, a known key regulator of genes required for motile ciliation. RNAseq analysis indicates that Foxn4 co-activates some Foxj1 target genes strongly and many Foxj1 targets weakly. ChIPseq suggests that whereas Foxn4 and Foxj1 frequently bind to different targets at distal enhancers, they largely bind together at MCC gene promoters. Consistent with this co-regulation, cilia extension by MCCs is more severely compromised in foxn4 and foxj1 double mutants than in single mutants. In contrast to Foxj1, Foxn4 is not required to extend a single motile cilium by cells involved in left-right patterning. These results indicate that Foxn4 complements Foxj1 transcriptionally during MCC differentiation, thereby shaping the levels of gene expression required for the timely and complete biogenesis of multiple motile cilia.
Asunto(s)
Cilios/metabolismo , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Piel/embriología , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Animales , Cuerpos Basales/fisiología , Sistemas CRISPR-Cas/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/metabolismo , Morfolinos/genética , Proteínas del Tejido Nervioso/genética , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/metabolismoRESUMEN
Centrioles are the foundation of two organelles, centrosomes and cilia. Centriole numbers and functions are tightly controlled, and mutations in centriole proteins are linked to a variety of diseases, including microcephaly. Loss of the centriole protein Asterless (Asl), the Drosophila melanogaster orthologue of Cep152, prevents centriole duplication, which has limited the study of its nonduplication functions. Here, we identify populations of cells with Asl-free centrioles in developing Drosophila tissues, allowing us to assess its duplication-independent function. We show a role for Asl in controlling centriole length in germline and somatic tissue, functioning via the centriole protein Cep97. We also find that Asl is not essential for pericentriolar material recruitment or centrosome function in organizing mitotic spindles. Lastly, we show that Asl is required for proper basal body function and spermatid axoneme formation. Insights into the role of Asl/Cep152 beyond centriole duplication could help shed light on how Cep152 mutations lead to the development of microcephaly.
Asunto(s)
Centriolos/metabolismo , Centriolos/fisiología , Proteínas de Drosophila/metabolismo , Espermatozoides/crecimiento & desarrollo , Espermatozoides/metabolismo , Animales , Axonema/metabolismo , Axonema/fisiología , Cuerpos Basales/metabolismo , Cuerpos Basales/fisiología , Proteínas de Ciclo Celular/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiología , Masculino , Mitosis/fisiología , Espermatozoides/fisiologíaRESUMEN
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.
Asunto(s)
Axonema/metabolismo , Cuerpos Basales/fisiología , Flagelos/fisiología , Leishmania mexicana/fisiología , Animales , Axonema/ultraestructura , División Celular , Células Cultivadas , Chlamydomonas reinhardtii , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes , Leishmania mexicana/ultraestructura , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Organismos Modificados Genéticamente , Proteínas RecombinantesRESUMEN
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.
Asunto(s)
Epéndimo/fisiología , Células Epiteliales/citología , Miosina Tipo I/fisiología , Miosinas/fisiología , Actinas/fisiología , Animales , Animales Modificados Genéticamente , Axonema/fisiología , Cuerpos Basales/fisiología , Encéfalo/fisiología , Polaridad Celular , Cilios/fisiología , Etiquetado Corte-Fin in Situ , Intestinos/fisiología , Masculino , Ratones Noqueados , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Miosina Tipo I/genética , Miosinas/genética , Neuroglía/fisiología , Fenotipo , Ratas , Ratas Endogámicas F344 , Rotación , Tráquea/fisiologíaRESUMEN
Cilia are key organelles in development and homeostasis. The ever-expanding complement of cilia associated proteins necessitates rapid and tractable models for in vivo functional investigation. Xenopus laevis provides an attractive model for such studies, having multiple ciliated populations, including primary and multiciliated tissues. The rapid external development of Xenopus and the large cells make it an especially excellent platform for imaging studies. Here we present embryological and cell biological methods for the investigation of cilia structure and function in X. laevis, with a focus on quantitative live and fixed imaging.
Asunto(s)
Cilios/fisiología , Embrión no Mamífero/citología , Animales , Axonema/fisiología , Cuerpos Basales/fisiología , Técnica del Anticuerpo Fluorescente/métodos , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Morfolinos/genética , Oligonucleótidos Antisentido/genética , Coloración y Etiquetado/métodos , Fijación del Tejido/métodos , Xenopus laevisRESUMEN
Paramecium is a single cell able to divide in its morphologically differentiated stage that has many cilia anchored at its cell surface. Many thousands of cilia are thus assembled in a short period of time during division to duplicate the cell pattern while the cell continues swimming. Most, but not all, of these sensory cilia are motile and involved in two main functions: prey capture and cell locomotion. These cilia display heterogeneity, both in their length and their biochemical properties. Thanks to these properties, as well as to the availability of many postgenomic tools and the possibility to follow the regrowth of cilia after deciliation, Paramecium offers a nice opportunity to study the assembly of the cilia, as well as the genesis of their diversity within a single cell. In this paper, after a brief survey of Paramecium morphology and cilia properties, we describe the tools and the protocols currently used for immunofluorescence, transmission electron microscopy, and ultrastructural immunocytochemistry to analyze cilia, with special recommendations to overcome the problem raised by cilium diversity.