Cilia最新文献

Cilia appear to be derived, evolutionarily, from structures present in the ancestral (pre-ciliary) eukaryote, such as microtubule-based vesicle trafficking and chromosome segregation systems. Experimental observations suggest that the ciliary gate, the molecular complex that mediates the selective molecular movement between cytoplasmic and ciliary compartments, shares features with nuclear pores. Our hypothesis is that this shared transport machinery is at least partially responsible for the observation that a number of ciliary and ciliogenesis-associated proteins are found within nuclei where they play roles in the regulation of gene expression, DNA repair, and nuclear import and export. Recognizing the potential for such nuclear roles is critical when considering the phenotypic effects that arise from the mutational modification of ciliary proteins.

Background: Skeletal ciliopathies comprise a spectrum of ciliary malfunction disorders that have a profound effect on the skeleton. Most common among these disorders is short rib polydactyly syndrome (SRPS), a recessively inherited perinatal lethal condition characterized by a long narrow chest, markedly shortened long bones, polydactyly and, often, multi-organ system involvement. SRPS shows extensive locus heterogeneity with mutations in genes encoding proteins that participate in cilia formation and/or function.

Results: Herein we describe mutations in IFT43, a satellite member of the retrograde IFT-A complex, that produce a form of SRPS with unusual bending of the ribs and appendicular bones. These newly described IFT43 mutations disrupted cilia formation, produced abnormalities in cartilage growth plate architecture thus contributing to altered endochondral ossification. We further show that the IFT43 SRPS phenotype is similar to SRPS resulting from mutations in the gene encoding IFT121 (WDR35), a direct interactor with IFT43.

Conclusions: This study defines a new IFT43-associated phenotype, identifying an additional locus for SRPS. The data demonstrate that IFT43 is essential for ciliogenesis and that the mutations disrupted the orderly proliferation and differentiation of growth plate chondrocytes, resulting in a severe effect on endochondral ossification and mineralization. Phenotypic similarities with SRPS cases resulting from mutations in the gene encoding the IFT43 direct interacting protein IFT121 suggests that similar mechanisms may be disrupted by defects in these two IFT-A satellite interactors.

Background: The development of a ciliary axoneme requires the correct docking of the basal body at cytoplasmic vesicles or plasma membrane. In the multiciliated cell Paramecium, three conserved proteins, FOR20, Centrin 2, and Centrin 3 participate in this process, FOR20 and Centrin 2 being involved in the assembly of the transition zone. We investigated the function of two other evolutionary conserved proteins, OFD1 and VFL3, likely involved in this process.

Results: In Paramecium tetraurelia, a single gene encodes OFD1, while four genes encode four isoforms of VFL3, grouped into two families, VFL3-A and VFL3-B. Depletion of OFD1 and the sole VFL3-A family impairs basal body docking. Loss of OFD1 yields a defective assembly of the basal body distal part. Like FOR20, OFD1 is recruited early during basal body assembly and localizes at the transition zone between axoneme and membrane at the level of the microtubule doublets. While the recruitment of OFD1 and Centrin 2 proceed independently, the localizations of OFD1 and FOR20 at the basal body are interdependent. In contrast, in VFL3-A depleted cells, the unanchored basal bodies harbor a fully organized distal part but display an abnormal distribution of their associated rootlets which mark their rotational asymmetry. VFL3-A, which is required for the recruitment of Centrin 3, is transiently present near the basal bodies at an early step of their duplication. VFL3-A localizes at the junction between the striated rootlet and the basal body.

Conclusion: Our results demonstrate the conserved role of OFD1 in the anchoring mechanisms of motile cilia and establish its relations with FOR20 and Centrin 2. They support the hypothesis of its association with microtubule doublets. They suggest that the primary defect of VFL3 depletion is a loss of the rotational asymmetry of the basal body which specifies the sites of assembly of the appendages which guide the movement of basal bodies toward the cell surface. The localization of VFL3 outside of the basal body suggests that extrinsic factors could control this asymmetry.

Background: Primary cilia are immotile, microtubule-based organelles present on most cells. Defects in primary cilia presence/function result in a category of developmental diseases referred to as ciliopathies. As the cilia field progresses, there is a need to consider both the ciliary and extraciliary roles of cilia proteins. However, traditional fixation methods are not always suitable for examining the full range of localizations of cilia proteins. Here, we tested a variety of fixation methods with commonly used cilia markers to determine the most appropriate fixation method for different cilia proteins.

Methods: Mouse inner medullary collecting duct and human retinal pigmented epithelial cells were grown to confluence, serum starved, and fixed with one of the following fixation agents: paraformaldehyde-sucrose, paraformaldehyde-PBS, methanol, cytoskeletal buffer followed by methanol, or three variations of cytoskeletal buffer-paraformaldehyde fixation. Each cell type and fixation method combination was probed with the following ciliary markers: acetylated α-tubulin, detyrosinated tubulin, polyglutamylated tubulin, β-tubulin, adenylyl cyclase 3 (AC3), ADP-ribosylation factor-like protein 13b (Arl13b), centrosome and spindle pole associated protein 1 (CSPP1), or intraflagellar transport protein 20 (IFT20). Intraflagellar transport protein 88 (IFT88) and GM130 (Golgi marker) were also used. We assessed actin (via phalloidin) and microtubule integrity, centrioles, cilia, and two extraciliary sites (mitotic figures and Golgi).

Results: For the cilia markers examined, paraformaldehyde fixation preserved cilia immunolabeling of cilia-membrane proteins (AC3 and Arl13b), but failed to reveal cilia immunostaining of axonemal proteins (CSPP1 and IFT20). Methanol revealed cilia labeling for some axonemal proteins, but not others, and this depended on cell type. Generally, any method that first included a wash in cytoskeletal buffer, before fixing, revealed more distinct cilia immunolabeling for axonemal proteins (CSPP1, IFT20, and IFT88), but resulted in the loss of cilia labeling for cilia-membrane proteins (AC3 and Arl13b). All three different post-translational modifications of tubulin antibodies positively immunolabeled cilia in all fixation methods tested. Ultimately, we found that fixing cells in a solution of paraformaldehyde prepared in cytoskeletal buffer allowed for the preservation of cilia immunolabeling for most cilia proteins tested and allowed visualization of two extraciliary sites (mitotic figures and Golgi).

Conclusion: Some general patterns were observed to guide in the choice of a fixation agent. Cilia-membrane proteins generally benefit from quick fixation with no prior permeabilization, whereas axonemal proteins tend to benefit from permeabilization and use of cytoskeletal buffer.

Background: Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical surface facing the outside and accessible for analysis of ciliary function.

Methods: We performed a two-group comparison study of ciliary beat pattern and ciliary beat frequency in spheroids derived from nasal airway epithelium in patients with primary ciliary dyskinesia (PCD) and in healthy controls. Nasal ciliary cells and sheets were removed on day 1 by nasal brush biopsy and analyzed with regard to ciliary beat pattern-and frequency using high-speed video imaging for standard reference values. Three-dimensional explant spheroid formation was initiated in the same individual on the same day by incubation of cells and sheets from a separate brush biopsy. Harvested spheroids were analyzed earliest possible and values of spheroid ciliary beat pattern and frequency were compared to the corresponding reference values from day 1.

Results: Spheroids formed fast in serum-free culture medium. Formation was successful in 15 out of 18 (82%) sampled individuals. Thus, formation was successful in seven healthy controls and eight PCD patients, while unsuccessful in 3 with PCD due to infection. Median (range) number of days in culture before harvesting of spheroids was 4 (1-5) in healthy versus 2 (1-5) in PCD. Spheroid ciliary beat pattern and frequency were unchanged compared to their corresponding day 1 standard reference values. Spheroid ciliary beat frequency discriminated highly significant between healthy controls (9.3 Hz) and PCD patients (2.4 Hz) (P < 0.0001). Survival of spheroids was 16 days in a single healthy person.

Conclusion: Patient-specific three-dimensional explant spheroid formation from a minimal invasive nasal brush biopsy is a feasible, fast and valid ex vivo method to assess ciliary function with potential of aiding the diagnosis of PCD. In addition, it may be a useful model in the investigation of pathophysiological aspects and drug effects in human nasal airway epithelium.

This is a history of cilia research before and after the discovery of intraflagellar transport (IFT) and the link between primary cilia ciliogenesis and polycystic kidney disease (PKD). Before IFT, ca. the beginning of the new millennium, although sensory and primary cilia were well described, research was largely focused on motile cilia, their structure, movement, and biogenesis. After IFT and the link to PKD, although work on motile cilia has continued to progress, research on primary cilia has exploded, leading to new insights into the role of cilia in cell signaling and development. Genomics, proteomics, and new imaging techniques have unified the field and pointed out the critical role of cilia as a restricted cell organellar compartment, functionally integrated with other cell organelles including the autophagosome and the nucleus.

Background: La-related protein 6 (LARP6) is an evolutionally conserved RNA-binding protein. Vertebrate LARP6 binds the 5' stem-loop found in mRNAs encoding type I collagen to regulate their translation, but other target mRNAs and additional functions for LARP6 are unknown. The aim of this study was to elucidate an additional function of LARP6 and to evaluate the importance of its function during development.

Methods: To uncover the role of LARP6 in development, we utilized Morpholino Oligos to deplete LARP6 protein in Xenopus embryos. Then, embryonic phenotypes and ciliary structures of LAPR6 morphants were examined. To identify the molecular mechanism underlying ciliogenesis regulated by LARP6, we tested the expression level of cilia-related genes, which play important roles in ciliogenesis, by RT-PCR or whole mount in situ hybridization (WISH).

Results: We knocked down LARP6 in Xenopus embryos and found neural tube closure defects. LARP6 mutant, which compromises the collagen synthesis, could rescue these defects. Neural tube closure defects are coincident with lack of cilia, antenna-like cellular organelles with motility- or sensory-related functions, in the neural tube. The absence of cilia at the epidermis was also observed in LARP6 morphants, and this defect was due to the absence of basal bodies which are formed from centrioles and required for ciliary assembly. In the process of multi-ciliated cell (MCC) differentiation, mcidas, which activates the transcription of genes required for centriole formation during ciliogenesis, could partially restore MCCs in LARP6 morphants. In addition, LARP6 likely controls the expression of mcidas in a Notch-independent manner.

Conclusions: La-related protein 6 is involved in ciliated cell differentiation during development by controlling the expression of cilia-related genes including mcidas. This LARP6 function involves a mechanism that is distinct from its established role in binding to collagen mRNAs and regulating their translation.

Background: The discovery of disease pathogenesis requires systematic agnostic screening of multiple homeostatic processes that may become deregulated. We illustrate this principle in the evaluation and diagnosis of a 5-year-old boy with Joubert syndrome type 10 (JBTS10). He carried the OFD1 mutation p.Gln886Lysfs*2 (NM_003611.2: c.2656del) and manifested features of Joubert syndrome.

Methods: We integrated exome sequencing, MALDI-TOF mass spectrometry analyses of plasma and cultured dermal fibroblasts glycomes, and full clinical evaluation of the proband. Analyses of cilia formation and lectin staining were performed by immunofluorescence. Measurement of cellular nucleotide sugar levels was performed with high-performance anion-exchange chromatography with pulsed amperometric detection. Statistical analyses utilized the Student's and Fisher's exact t tests.

Results: Glycome analyses of plasma and cultured dermal fibroblasts identified abnormal N- and O-linked glycosylation profiles. These findings replicated in two unrelated males with OFD1 mutations. Cultured fibroblasts from affected individuals had a defect in ciliogenesis. The proband's fibroblasts also had an abnormally elevated nuclear sialylation signature and increased total cellular levels of CMP-sialic acid. Ciliogenesis and each glycosylation anomaly were rescued by expression of wild-type OFD1.

Conclusions: The rescue of ciliogenesis and glycosylation upon reintroduction of WT OFD1 suggests that both contribute to the pathogenesis of JBTS10.

Centriole structure and function in the sea urchin zygote parallel those in mammalian somatic cells. Here, I briefly introduce the properties and attributes of the sea urchin system that make it an attractive platform for the study of centrosome and centriole duplication. These attributes apply to all echinoderms readily available from commercial suppliers: sea urchins, sand dollars, and starfish. I list some of the practical aspects of the system that make it a cost- and time-effective system for experimental work and then list properties that are a "tool kit" that can be used to conduct studies that would not be practical, or in some cases not possible, with mammalian somatic cells. Since centrioles organize and localize the pericentriolar material that nucleates the astral arrays of microtubules (Bobinnec et al. in J Cell Biol 143(6):1575-1589, 1998), the pattern of aster duplication over several cell cycles can be used as a reliable measure for centriole duplication (Sluder and Rieder in J Cell Biol 100(3):887-896, 1985). Descriptions of the methods my laboratory has used to handle and image echinoderm zygotes are reviewed in Sluder et al. (Methods Cell Biol 61:439-472, 1999). Also included is a bibliography of papers that describe additional methods.

Background: Nearly all cells have a primary cilia on their surface, which functions as a cellular antennae. Primary cilia assembly begins intracellularly and eventually emerges extracellularly. However, current ciliogenesis assays, which detect cilia length and number, do not monitor ciliary stages.

Methods: We developed a new assay that detects antibody access to a fluorescently tagged ciliary transmembrane protein, which revealed three ciliary states: classified as 'inside,' 'outside,' or 'partial' cilia.

Results: Strikingly, most cilia in RPE cells only partially emerged and many others were long and intracellular, which would be indistinguishable by conventional assays. Importantly, these states switch with starvation-induced ciliogenesis and the cilia can emerge both on the dorsal and ventral surface of the cell. Our assay further allows new molecular and functional studies of the 'ciliary pocket,' a deep plasma membrane invagination whose function is unclear. Molecularly, we show colocalization of EHD1, Septin 9 and glutamylated tubulin with the ciliary pocket.

Conclusions: Together, the IN/OUT assay is not only a new tool for easy and quantifiable visualization of different ciliary stages, but also allows molecular characterization of intermediate ciliary states.