Experimental model for studying the primary cilia in tissue culture cells.
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In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.Keywords:
Centriole
Interphase
Immunofluorescence
Ciliogenesis
Primary cell
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In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.
Centriole
Interphase
Immunofluorescence
Ciliogenesis
Primary cell
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Cilia, either motile or immotile, exist on most cells in the human body. There are several different mechanisms of ciliogenesis, which enable the production of many kinds of cilia and flagella: motile and immotile, transient and long-lived. These can be linked to the cell cycle or associated with differentiation. A primary cilium is extended from a basal body analogous to the mitotic centrioles, whereas the several hundred centrioles needed to form the cilia of a multi-ciliated cell can be generated by centriolar or acentriolar pathways. Little is known about the molecular control of these pathways and most of our knowledge comes from ultrastructural studies. The increasing number of genetic diseases linked to dysfunctional cilia and basal bodies has renewed interest in this area, and recent proteomic and cell biological studies in model organisms have helped to shed light on the molecular components of these enigmatic organelles.
Ciliogenesis
Basal body
Centriole
Motile cilium
Organelle
Intraflagellar Transport
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A fluorescent-labelled antibody against detyrosinated alpha-tubulin, ID5 (Wehland and Weber, 1987), allows primary cilia and centrioles to be detected rapidly and accurately for analysis in cell cultures. Many features of primary cilia have been re-examined, including frequency of expression in relation to levels of confluency, different sera, different cell cycle stages, and following trypsinization and centrifugation procedures. The detection of multiple ciliation per cell, and the precise positioning of cilia in cells in monolayers are readily recorded. The findings are discussed in relation to cilium expression in cell growth and cycling, to previous findings based on ultrastructural analysis, and to their potential role in sensing the local environment.
Centriole
Trypsinization
Immunofluorescence
Ciliogenesis
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Microtubule organizing center
Immunofluorescence
Interphase
Organelle
Microtubule nucleation
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Citations (134)
Abstract Centrioles and cilia are conserved, microtubule-based structures critical for cell function and development. Their structural and functional defects cause cancer and developmental disorders. How microtubules are organized into ordered structures by microtubule-associated proteins (MAPs) and tubulin modifications is best understood during mitosis but is largely unexplored for the centrioles and the ciliary axoneme, which are composed of remarkably stable microtubules that maintain their length at steady state. In particular, we know little about the identity of the centriolar and ciliary MAPs and how they work together during the assembly and maintenance of the cilium and centriole. Here, we identified Enkurin domain containing 1 (ENKD1) as a component of the centriole wall and the axoneme in mammalian cells, and showed that it has extensive proximity interactions with these compartments and MAPs. Using in vitro and cellular assays, we found that ENKD1 is a new MAP that promotes microtubule polymerization and regulates microtubule organization and stability. Consistently, overexpression of ENKD1 increased tubulin polymerization and acetylation and disrupted microtubule organization. Cells depleted for ENKD1 were defective in ciliary length and content regulation and failed to respond to Hedgehog pathway activation. Together, our results establish ENKD1 as a new centriolar and ciliary MAP that regulate primary cilium structure and function, and advances our understanding of the functional and regulatory relationship between MAPs and the primary cilium.
Centriole
Axoneme
Basal body
Microtubule nucleation
Ciliogenesis
Microtubule polymerization
Intraflagellar Transport
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We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-α-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells.
Basal body
Centriole
Interphase
Intraflagellar Transport
Ciliogenesis
Chlamydomonas
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Citations (220)
The primary cilium is a microtubule-based structure protruded from the basal body analogous to the centriole. CPAP (centrosomal P4.1-associated protein) has previously been reported to be a cell cycle-regulated protein that controls centriole length. Mutations in CPAP cause primary microcephaly (MCPH) in humans. Here, using a cell-based system that we established to monitor cilia formation in neuronal CAD (Cath.a-differentiated) cells and hippocampal neurons, we found that CPAP is required for cilia biogenesis. Overexpression of wild-type CPAP promoted cilia formation and induced longer cilia. In contrast, an exogenously expressed CPAP-377EE mutant that lacks tubulin-dimer binding significantly inhibited cilia formation and caused cilia shortening. Furthermore, depletion of CPAP inhibited ciliogenesis and such effect was effectively rescued by expression of wild-type CPAP, but not by the CPAP-377EE mutant. Taken together, our results suggest that CPAP is a positive regulator of ciliogenesis whose intrinsic tubulin-dimer binding activity is required for cilia formation in neuronal cells.
Ciliogenesis
Basal body
Centriole
Motile cilium
Intraflagellar Transport
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As mammalian cells enter mitosis, the Golgi complex is disorganized and the remnants are dispersed throughout the cytoplasm in the form of a few short cisternae and small clusters of vesicles. Once the separation of the chromosomes is completed and nuclei reform, stacks of flattened cisternae reappear and a united Golgi complex of interphase type starts to be reorganized. This process is believed to ensure an approximately equal partitioning of the Golgi complex on the daughter cells. Here, the configuration of the Golgi complex and its relation to the cytoplasmic microtubule system was studied at the end of cytokinesis using synchronized cultures of L929 mouse fibroblasts and rat dermal fibroblasts. One hour after the release of the mitotic block, the Golgi complex (visualized immunocytochemically with antibodies against mannosidase II) was most frequently located on the proximal side of the nucleus as related to the intercellular bridge (visualized immunocytochemically with antibodies against tyrosinated alpha-tubulin). One hour later, it was preferentially found on the distal side of the nucleus as related to the intercellular bridge. Immunocytochemical demonstration of the radiating pattern of microtubules, and direct demonstration of the centrioles using antibodies against detyrosinated or acetylated alpha-tubulin, showed that the microtubule organizing center (MTOC) shifted position in a similar manner as the Golgi complex. Moreover, double staining with antibodies against mannosidase II and tyrosinated alpha-tubulin revealed that the Golgi complex and the MTOC codistributed at both times after the release of the mitotic block. Electron microscopic analysis confirmed that the reforming Golgi stacks first gathered close to the centrosome (a pair of centrioles with associated structures, constituting the main MTOC in the cell) on the proximal side of the nucleus and that the Golgi stacks and the centrosome were subsequently both relocated to the distal side of the nucleus as related to the intercellular bridge. Taken together, the findings indicate that the Golgi complex goes through a characteristic translocation in the terminal phase of cytokinesis and confirm the idea that the cytoplasmic microtubule system plays an important role in the organization of this organelle system. A possible function of the shift in location of the Golgi complex at the end of cytokinesis could be to direct membrane traffic first to the elongating intercellular bridge and thereafter to the leading edge as the cells are about to separate and move away from each other.
Microtubule organizing center
Interphase
Centriole
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Summary Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs) such as polyglycylation, polyglutamylation or acetylation, are determinants of microtubule (MT) functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme.
Axoneme
Ciliopathies
Ciliogenesis
Motile cilium
Centriole
Basal body
Intraflagellar Transport
Organelle
Bardet–Biedl Syndrome
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Citations (47)
Microtubule organizing center is composed of a pair of centrioles and active pericentriolar materials. Its nucleating activity enhances at mitosis to organize a mitotic spindle. We raised a monoclonal antibody against microtubule associated protein 1. Indirect immunofluorescence by this antibody was located in the microtubule organizing center and in the nucleus. The intranuclear antigen was bound to the nuclear skeleton during interphase, and released to cytoplasm concentrating to the mitotic spindle and its poles. The nuclear immunofluorescence reversibly disappeared in growth-inhibited normal cells, but was always apparent in transformed cells.
Microtubule organizing center
Centriole
Interphase
Microtubule nucleation
Spindle pole body
Immunofluorescence
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