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Methods Enzymol [journal]
- Methods in Enzymology. Cilia, part B. Preface. [Introductory Journal Article]
- Methods Enzymol 2013.:xvii-xix.
- Centrosome isolation and analysis by mass spectrometry-based proteomics. [Journal Article, Research Support, Non-U.S. Gov't]
- Methods Enzymol 2013.:371-93.
Centrioles are microtubule-based scaffolds that are essential for the formation of centrosomes, cilia, and flagella with important functions throughout the cell cycle, in physiology and during development. The ability to purify centriole-containing organelles on a large scale, combined with advances in protein identification using mass spectrometry-based proteomics, have revealed multiple centriole-associated proteins that are conserved during evolution in eukaryotes. Despite these advances, the molecular basis for the plethora of processes coordinated by cilia and centrosomes is not fully understood. Considering the complexity and dynamics of centriole-related proteomes and the first-pass analyses reported so far, it is likely that further insight might come from more thorough proteome analyses under various cellular and physiological conditions. To this end, we here describe methods to isolate centrosomes from human cells and strategies to selectively identify and study the properties of the associated proteins using quantitative mass spectrometry-based proteomics.
- Chemical screening methods for flagellar phenotypes in Chlamydomonas. [Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't]
- Methods Enzymol 2013.:351-69.
Cilia and flagella are important organelles used for sensing the external cellular environment or for motility. Abnormalities in ciliary structure or function can have devastating pathological consequences ranging from sinusitis and obesity to polycystic kidney disease, retinal degeneration, and mental retardation. Chlamydomonas flagella are excellent models to study the regulation and normal function of cilia. We utilized the 1280 compound Sigma LOPAC annotated library to screen for phenotypes in Chlamydomonas flagellar length, motility, deflagellation, and cellular toxicity. Phenotypes were assessed by quantitation from direct microscopic visualization and custom-designed motility/viability assays. Compounds were clustered based on data across all assays to facilitate the identification of novel pathways regulating flagella in Chlamydomonas. These methods can both aid our understanding of the basic biology of flagellar regulation and provide useful points of therapeutic intervention for cilia-related disorders.
- Finding ciliary genes: a computational approach. [Journal Article, Research Support, Non-U.S. Gov't]
- Methods Enzymol 2013.:327-50.
In the nematode worm Caenorhabditis elegans and several other animal species, many ciliary genes are regulated by RFX (Regulatory Factor binding to the X-box) transcription factors (TFs), which bind to X-box promoter motifs and thereby directly activate ciliary gene expression. This setup (RFX TF/X-box/ciliary gene) makes it possible to search for novel ciliary gene candidates genome-wide by using the X-box promoter motif as a search parameter. We present a computational approach that (i) identifies and extracts from whole genomes genes and the corresponding promoter sequences and annotations; (ii) searches through promoters for regulatory sequence elements (like promoter motifs) by using training sets of known instances of these elements; (iii) scores (evaluates) and sorts all positive hits in a database; and (iv) outputs a list of candidate genes and promoters with a given regulatory sequence element. Evolutionary conservation across species (orthology) of genes, promoters, or regulatory sequence elements is used as an important strengthening feature during the overall search approach. Our computational approach is set up in a modular fashion: not every part needs to be used for a particular search effort. In principle, our approach has broad applications. It applies to any group of genes that share common (conserved) regulation through common (conserved) regulatory sequence elements.
- Isolation of mammalian primary cilia. [Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't]
- Methods Enzymol 2013.:311-25.
Primary cilia are microtubule-based organelles found on most types of cells in the human body. Although primary cilia were long thought to be vestigial remnants of motile cilia, it is now known that primary cilia play important roles in development and physiology, and defects of primary cilia cause a wide range of human disease symptoms, termed ciliopathies. To understand ciliary functions and the molecular mechanisms underlying ciliopathies, it is important to know the components of primary cilia, but primary cilia have proven to be more difficult to isolate than motile cilia. This chapter describes the isolation and imaging of mammalian primary cilia for biochemical and cell biological analyses.
- Analysis of ciliogenesis in primary culture mouse tracheal epithelial cells. [Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't]
- Methods Enzymol 2013.:285-309.
Cell biological and molecular characterization of structural and functional ciliary components and regulators of mammalian motile ciliogenesis is made possible by the development of a robust and biologically faithful mouse tracheal epithelial cell (MTEC) culture system and complementary research techniques. Here, we describe the air-liquid interface culture of mouse airway epithelial progenitor cells that undergo motile ciliogenesis de novo. Multiciliated cells differentiate rapidly, and distinct stages of the ciliogenesis pathway can be identified and characterized with centriolar and ciliary immunofluorescence markers. Immunolabeled structures correlate with morphological features previously identified by electron microscopy, facilitating light microscopy analysis. MTEC cultures can be successfully transduced by lentiviral RNAi or epitope-tagged cDNA constructs to perturb gene expression. Also, motile ciliogenesis can be manipulated by drug treatment. Distinct cell populations can be isolated by cell sorting to facilitate comparison among the multiciliated and other cell types in the in vitro differentiated epithelium. The MTEC system uniquely offers the study of ciliogenesis in cells from genetically modified mouse strains.
- Discovery and functional evaluation of ciliary proteins in Tetrahymena thermophila. [Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't]
- Methods Enzymol 2013.:265-84.
The ciliate Tetrahymena thermophila is an excellent model system for the discovery and functional studies of ciliary proteins. The power of the model is based on the ease with which cilia can be purified in large quantities for fractionation and proteomic identification, and the ability to knock out any gene by homologous DNA recombination. Here, we include methods used by our laboratories for isolation and fractionation of cilia, in vivo tagging and localization of ciliary proteins, and the evaluation of ciliary mutants.
- Analysis of ciliary assembly and function in planaria. [Journal Article]
- Methods Enzymol 2013.:245-64.
Planarians are free-living invertebrates that employ motile cilia for locomotion. Specifically, cilia that populate the ventral epithelium of the planarian body are highly conserved, with a 9+2 axoneme and a full complement of inner and outer arm dynein motors. The abundance of cilia on the planarian body, their unique accessibility, and high degree of conservation make this organism an attractive experimental model system for cilia biology. Moreover, planarians are genetically amenable and defects that compromise the function and structure of the cilia are not detrimental for their overall health, making them an ideal system for cilia gene loss-of-function studies. In this chapter, we provide information for introducing and maintaining planarians for experimental purposes in the laboratory and describe protocols for RNAi-induced gene knockdown studies. Furthermore, we elaborate on different imaging techniques used to analyze cilia physiology and structure, including live video microscopy, immunofluorescence analysis, and electron microscopy. Last, we provide assays for evaluating physical parameters of ciliary motility, including quantification of planarian gliding locomotion and measurement of ciliary beat frequency.
- Zebrafish cilia. [Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't]
- Methods Enzymol 2013.:219-44.
Zebrafish are ideally suited for analysis of genes required for ciliogenesis and cilia function. Combining genetic manipulation with high quality in vivo imaging, zebrafish embryos provide a high-throughput system for annotation of the cilia proteome. The specific advantages of the system are the availability of cilia mutants, the ability to target genes of unknown function using antisense methods, the feasibility of observing cilia in living embryos, and the ability to image fixed cilia in wholemount at high resolution. Techniques are described for analysis of mutants, gene knockdown using antisense morpholino oligos, visualizing cilia and cilia orientation in wholemount zebrafish embryos, live imaging cilia, and electron microscopy of zebrafish cilia.
- Using Xenopus skin to study cilia development and function. [Journal Article, Research Support, N.I.H., Extramural]
- Methods Enzymol 2013.:191-217.
Cilia are prevalent biological structures that are important for cell signaling and for generating fluid flow (or motility). Cilia are found throughout biology from single-celled organisms to vertebrates, and many model systems have been employed for their analysis. Here, we describe the use of Xenopus larval skin as a system for the study of ciliogenesis and ciliary function. In particular, we describe basic molecular and embryological manipulations and imaging techniques that have proven particularly useful for understanding the polarized beating of cilia and the generation of directed fluid flow (Werner & Mitchell, 2012). However, these same tools have the potential to benefit a large number of cilia-related biological questions.