RESUMO
Cilia are ubiquitous and highly conserved extensions that endow the cell with motility and sensory functions. They were present in the first eukaryotes and conserved throughout evolution (Carvalho-Santos et al., 2011). Paramecium has around 4,000 motile cilia on its surface arranged in longitudinal rows, beating in waves to ensure movement and feeding. As with cilia in other model organisms, direction and speed of Paramecium ciliary beating is under bioelectric control of ciliary ion channels. In multiciliated cells of metazoans as well as paramecia, the cilia become physically entrained to beat in metachronal waves. This ciliated organism, Paramecium, is an attractive model for multidisciplinary approaches to dissect the location, structure and function of ciliary ion channels and other proteins involved in ciliary beating. Swimming behavior also can be a read-out of the role of cilia in sensory signal transduction. A cilium emanates from a BB, structurally equivalent to the centriole anchored at the cell surface, and elongates an axoneme composed of microtubule doublets enclosed in a ciliary membrane contiguous with the plasma membrane. The connection between the BB and the axoneme constitutes the transition zone, which serves as a diffusion barrier between the intracellular space and the cilium, defining the ciliary compartment. Human pathologies affecting cilia structure or function, are called ciliopathies, which are caused by gene mutations. For that reason, the molecular mechanisms and structural aspects of cilia assembly and function are actively studied using a variety of model systems, ranging from unicellular organisms to metazoa. In this review, we will highlight the use of Paramecium as a model to decipher ciliary beating mechanisms as well as high resolution insights into BB structure and anchoring. We will show that study of cilia in Paramecium promotes our understanding of cilia formation and function. In addition, we demonstrate that Paramecium could be a useful tool to validate candidate genes for ciliopathies.
RESUMO
Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.
Assuntos
Movimento Celular/fisiologia , Centrossomo/metabolismo , Cílios/metabolismo , Paramecium/citologia , Paramecium/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Centrossomo/ultraestrutura , Cílios/ultraestrutura , Flagelos/metabolismo , Teste de Complementação Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Paramecium/genética , Proteínas de Plantas/genética , Proteínas de Protozoários/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
Human cytomegalovirus (CMV) causes severe disease in immunosuppressed patients and notably infects the gastrointestinal tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used the intestinal epithelial cell line Caco-2 and demonstrated that CMV enters predominantly through the basolateral surface of polarized Caco-2 cells. As shown by expression of all three classes of CMV proteins and by visualization of nucleocapsids by transmission electron microscopy, both poorly and fully differentiated Caco-2 cells were permissive to CMV replication. However, infection failed to produce infectious particles in Caco-2 cells, irrespective of the state of differentiation.
Assuntos
Diferenciação Celular , Citomegalovirus/patogenicidade , Mucosa Intestinal/virologia , Antígenos Virais/imunologia , Membrana Basal/virologia , Células CACO-2 , Citomegalovirus/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/ultraestrutura , Microscopia Eletrônica , Nucleocapsídeo/biossínteseRESUMO
Ciliates assemble basal bodies in great number at many stages of the life-cycle. In order to understand their assembly mechanisms, we screened a library of monoclonal antibodies directed against pericentriolar material. One of these antibodies, CTR210, was used previously to follow steps of this assembly process: in Paraurostyla, new basal bodies appear along a scaffold of linear structures recognized by this antibody. The very unusual behavior of this antigen deserved confirmation in other species. In the present study, we show by immunofluorescence that, in another phylogenetically very distant species, Euplotes, basal bodies are assembled in the same pathway during division. In addition, this antibody recognizes a filamentous ring located at the division furrow and linking many basal body assemblages. By cell fractionation and cytoskeletal extraction, we obtained fractions enriched in basal bodies and associated material. Such fractions still display a high complexity in protein composition. These fractions were used to characterize the main target of the antibody as a doublet of 45 kDa. These results confirm previous results in terms of functionality of the protein recognized by the antibody, but raise new questions in terms of the assignment of the recognized protein to the HSP70 family as hypothesized previously.
Assuntos
Antígenos de Protozoários/metabolismo , Centrossomo/imunologia , Euplotes/crescimento & desenvolvimento , Euplotes/imunologia , Animais , Anticorpos Monoclonais , Divisão Celular , Centrossomo/ultraestrutura , Cilióforos/crescimento & desenvolvimento , Cilióforos/imunologia , Cilióforos/ultraestrutura , Euplotes/ultraestrutura , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/metabolismo , Imunoquímica , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Peso Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismoRESUMO
The secretion of proteins and fluids from the exorbital lacrymal gland of rat is mainly controlled by muscarinic receptors. In a recent pharmacological study. Mauduit et al (Am J Physiol (1993) 264, C1550-C1560) identified a homogeneous population of M3 muscarinic receptors in preparations of acini from these tissues. In order to define the cellular composition of these acini and localize the muscarinic receptors, we have performed an immunofluorescent labelling study combined with confocal scanning microscopy. Antibodies raised against components of the different cytoskeletal networks (alpha-smooth muscle actin, cytokeratin peptide 14 and alpha-tubulin) revealed the presence of two different cell types. Cells with a stellate form are identified as myoepithelial cells, whereas rounded cells are secretory acinar cells. Both cell types are reactive with an antibody specifically directed against the muscarinic receptor. However, myoepithelial cells appear more intensely labelled than acinar cells. The roles of myoepithelial cells and secretory cells in the physiological function of the gland are discussed in terms of the distribution of muscarinic receptors.
Assuntos
Aparelho Lacrimal/química , Receptores Muscarínicos/análise , Actinas/análise , Animais , Citoesqueleto/química , Epitélio/química , Imunofluorescência , Imuno-Histoquímica , Aparelho Lacrimal/citologia , Masculino , Microscopia Confocal , Ratos , Ratos Sprague-DawleyRESUMO
In rat parotid gland, 3H-protein secretion is stimulated by beta-adrenergic receptor activation (via cAMP) and also by cholinergic receptor activation (via IP3, calcium and diacylglycerol). The disorganization of microfilament system by cytochalasin D induced an inhibition of beta-adrenergic induced 3H-protein secretion whereas it did not modify the cholinergic muscarinic one. Cytochalasin D induced the formation of vacuoles in the parotid cell. In this work we show that the activation of muscarinic receptors (with carbachol) partially abolished the inhibitory effect of cytochalasin D on beta-adrenergic induced secretion. Since carbachol induced both intracellular calcium increase and protein kinase C activation, we decided to test separately the effect of calcium (using the calcium ionophore A23187) and protein kinase C activation (using phorbol ester) on the inhibitory effect of cytochalasin D on beta-adrenergic induced secretion. A23187, in the presence of calcium in the external medium was able to partially abolish cytochalasin D effect (ie re-establishing protein secretion) whereas activation of protein kinase C by phorbol 12-13 di-butyrate had no effect. These results suggest that protein kinase C is not involved in re-establishing a 'normal' secretion phenomenon whereas calcium does interfere. Furthermore, our fluorescence study shows that, when cytochalasin D is present in the incubation medium, the actin network is disturbed even in the presence of carbachol. This indicates that a calcium entry in the cell is not sufficient to restore a 'normal' actin network.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cálcio/farmacologia , Glândula Parótida/fisiologia , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Animais , Calcimicina/farmacologia , Carbacol/farmacologia , Citocalasina D/farmacologia , Isoproterenol/farmacologia , Masculino , Ésteres de Forbol/farmacologia , Ratos , Ratos Endogâmicos , Estimulação QuímicaRESUMO
In ciliated cells of metazoa, striated rootlets associated with basal bodies anchor the ciliary apparatus to the cytoskeleton. We have used here a monoclonal antibody against a 175 kDa protein associated with the striated rootlets of quail ciliated cells, to study ciliated cells of different species. In mussel gill epithelium the antibody recognized a protein of 92 kDa which shows a periodic distribution along the striated rootlets. In frog ciliated palate epithelium, two different rootlets are associated with basal bodies, both are decorated and only one protein of 48 kDa is recognized on immunoblot. The antigen is arranged in a helix around the striated rootlets. In rabbit ciliated oviduct epithelium, we detected the presence of very small and thin rootlets which are weakly labeled. We have shown that an epitope associated with the striated rootlets is preserved through evolution although the molecular weight of the peptide varies. We have also observed the appearance of this epitope on protein associated with junctional complexes in rabbit and cytoskeleton component in quail oviduct.
Assuntos
Evolução Biológica , Cílios/imunologia , Epitélio/ultraestrutura , Epitopos , Animais , Western Blotting , Epitélio/imunologia , Imunofluorescência , Microscopia EletrônicaRESUMO
In quail oviduct, a 175K (K = 10(3) Mr) protein associated with striated rootlets was previously identified by Klotz and co-workers using monoclonal antibody CC310. As this monoclonal antibody recognizes several proteins on immunoblots of ciliated cells, we prepared a polyclonal antibody monospecific to the 175K protein by intrasplenic immunization of mice. Immunofluorescence study confirmed the distribution of the 175K protein at the apical part of the ciliated cell and its absence in other epithelial cells. Immunogold staining showed that this protein was strongly associated with the fibrillar axis of striated rootlets. The absence of labeling on striation suggested that rootlets were composed of several proteins, with one group forming the fibrillar axis and the second forming the striation. The formation of striated rootlets during ciliogenesis was studied using this polyclonal antibody. The 175K protein appeared at the beginning of centriologenesis in fibrillar material located around dense granules, and then around the generative complex. The formation of rootlets began at the basal pole of migrating basal bodies. The elongation of the rootlet axes took place when basal bodies were anchored to the plasma membrane.
Assuntos
Cílios/fisiologia , Oviductos/fisiologia , Codorniz/fisiologia , Animais , Cílios/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Feminino , Immunoblotting , Imuno-Histoquímica , Microscopia de FluorescênciaRESUMO
The different steps of ciliogenesis occurring in quail oviduct were compared to the ciliogenesis pattern described in other metazoan species. Centrioles are generated according to pathways that are found within the same cell: the centriolar and the acentriolar pathways. In the acentriolar pathway, centrioles are generated in the Golgi area, without contact with the preexisting centrioles of the centrosomes, and they migrate toward the apical membrane. The control of this polarized migration was studied by means of several drugs (colchicine, nocodazol, taxol, cytochalasin D, benzodiazepines) and immunocytochemistry. It was suggested that an actin-myosin system was involved in the migration of centrioles, whereas labile microtubules were not necessary. Basal bodies must dock with plasma membrane or cytoplasmic vesicles for the initiation of axonemal microtubule polymerization. This signal is necessary even in the presence of taxol.
Assuntos
Cílios/fisiologia , Citoesqueleto/fisiologia , Alcaloides/farmacologia , Animais , Benzimidazóis/farmacologia , Centríolos/efeitos dos fármacos , Centríolos/ultraestrutura , Cílios/ultraestrutura , Colchicina/farmacologia , Citocalasina D , Citocalasinas/farmacologia , Citoesqueleto/ultraestrutura , Diazepam/farmacologia , Epitélio , Morfogênese , Nocodazol , Paclitaxel , CodornizRESUMO
Ciliated cells are characterized by a highly organized cytoskeleton which is connected with the ciliary apparatus. The organization of microtubules, microfilaments, and cytokeratin filaments is described and the relationships of each network with the ciliary apparatus are emphasized. Possible functions of such a complex cytoskeleton are discussed.
Assuntos
Cílios/ultraestrutura , Citoesqueleto/ultraestrutura , Citoesqueleto de Actina/ultraestrutura , Actinas/fisiologia , Animais , Membrana Celular/ultraestrutura , Cílios/fisiologia , Citoesqueleto/fisiologia , Filamentos Intermediários/ultraestrutura , Microtúbulos/ultraestrutura , Movimento , Tubulina (Proteína)/fisiologiaRESUMO
Myosin has been localized during ciliogenesis of quail oviduct by immunocytochemistry (immunofluorescence, immunoperoxidase, immunogold labeling) using a previously characterized monoclonal antibody. In ovariectomized quail oviduct many undifferentiated epithelial cells present a primary cilium arising from one of the diplosome centrioles. Myosin is associated with material located between the two centrioles. In contrast, in estrogen-stimulated quail oviduct, the material preceding the procentioles is never labeled. Basal bodies become labeled just before their migration toward the apical plasma membrane. During the anchoring phase, the labeling is mainly associated with the basal feet. In mature ciliated cells, myosin appears associated with an apical network embedding the basal bodies. This network is connected to a myosin-rich belt associated with the apical junctional complex which differentiates at the beginning of centriologenesis. The association of myosin with migrating basal bodies suggests that myosin could be involved in basal body movements.
Assuntos
Cílios/ultraestrutura , Miosinas/análise , Oviductos/ultraestrutura , Animais , Coturnix , Epitélio/ultraestrutura , Feminino , Imunofluorescência , Microscopia Eletrônica , Oviductos/citologiaRESUMO
In the quail oviduct, the mature ciliated cells contain a well developed and polarized cytokeratin network which is bound to desmosomes and in close contact with the striated rootlets associated with basal bodies. In ovariectomized quail, the immature epithelial cells of oviduct present a rudimentary cytokeratin network associated with the centrioles of the diplosome (one of them forming a primary cilium) and with the short striated rootlets. The development of the cytokeratin network which occurs simultaneously with the ciliogenesis was observed by electron microscopy and immunocytochemistry (immunofluorescence and immunogold staining) using a prekeratin antiserum. During estrogen-induced ciliogenesis, cytokeratin intermediate filaments are always found associated with the different ciliogenic structures i.e. [dense granules, deuterosomes, procentrioles and centrioles]. In ciliogenic cells, the procentrioles and centrioles seem to be associated with the intermediate filaments by their pericentriolar material. These direct contacts decrease once the centrioles/basal bodies are anchored to the plasma membrane. Simultaneously the striated rootlets develop and associate with cytokeratin. The ciliogenic cells appear as a suitable system for studying in vivo, the possible association between centrioles and intermediate filaments and its functional meaning.