Cilia. Part B [electronic resource] / edited by Wallace F. Marshall.

Print version record.

Includes bibliographical references and indexes.

Non-Motile Primary Cilia as Fluid Shear Stress Mechanosensors -- Biomechanical Measurement of Kinocilium -- Analysis of Primary Cilia in Directional Cell Migration in Fibroblasts -- Sperm Patch-Clamp -- Laser Trap Measurements of Flagellar Membrane Motility -- Probing Ciliogenesis Using Micropatterned Substrates -- Fluorescent Imaging of Drosophila melanogaster Sperm in the Reproductive Tract: A New Model of Flagellar Motility -- Visualization of Mouse Nodal Cilia and Nodal Flow -- Dissecting the Functional Interplay Between the TOR Pathway and the Cilium in Zebrafish -- Using Xenopus Skin to Study Cilia Development and Function -- Zebrafish Cilia -- Analysis of Ciliary Assembly and Function in Planaria -- Discovery and Functional Evaluation of Ciliary Proteins in Tetrahymena thermophila -- Analysis of Ciliogenesis in Primary Culture Mouse Tracheal Epithelial Cells -- Isolation of Mammalian Primary Cilia -- Finding Ciliary Genes: A Computational Approach -- Chemical Screening Methods for Flagellar Phenotypes in Chlamydomonas -- Centrosome Isolation and Analysis by Mass Spectrometry-Based Proteomics.

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers cilia and includes chapters on such topics as methods for studying ciliary polarity in Xenopus, analysis of signaling pathways in mammalian spermatozoa, and biochemical and physiological analysis of axonemal dyneins. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers cilia Contains chapters on such topics as methods for studying ciliary pol.

Front Cover; Cilia, Part B; Copyright; Contents; Contributors; Preface; Methods in Enzymology; Chapter One: Non-Motile Primary Cilia as Fluid Shear Stress Mechanosensors; 1. Introduction; 2. Identification of Primary Cilium; 2.1. Cellular characterizations; 2.2. Ciliary polycystins; 3. Mechanosensory Cilia; 3.1. Experimental settings; 3.2. Calcium readout; 3.3. Nitric oxide readout; 3.4. Protein readout; 4. Summary; Acknowledgments; References; Chapter Two: Biomechanical Measurement of Kinocilium; 1. Introduction; 1.1. Vestibular hair cells.

1.2. Theoretical background: Beam models for kinocilia1.3. Accuracy of small-deflection beam theory; 1.4. Rotational stiffness; 1.5. Previous measurements of tubule and kinocilium mechanical properties; 1.6. Current approach to kinocilia mechanics; 1.7. Kinocilium motion in fluid as a measure of its mechanical properties; 2. Sample Preparation and Measurement; 2.1. Experimental preparation; 2.2. Force-displacement technique; 2.3. Error analysis of force-deflection technique; 3. Determining Mechanical Properties from Experimental Measurements; 3.1. Properties of the kinocilium shaft.

3.2. Rotational stiffness of the insertion4. Method Discussion; 4.1. Euler-Bernoulli versus Timoshenko beam analysis; 4.2. Comparison with literature; Acknowledgments; References; Chapter Three: Analysis of Primary Cilia in Directional Cell Migration in Fibroblasts; 1. Introduction; 2. Localization of Primary Cilia by Immunofluorescence Microscopy; 2.1. Fibroblast cultures and immunofluorescence microscopy analysis; 2.1.1. Cell cultures; 2.1.2. Immunofluorescence microscopy; 3. Cell Migration and Orientation of Primary Cilia in Scratch Assays; 3.1. Setting up the scratch assay.

3.2. Live-cell imaging and analysis3.3. Orientation of the primary cilium during cell migration; 4. Cell Migration and Ciliary Signaling in Micropipette Assays; 4.1. Setting up the micropipette assay; 4.2. Live-cell imaging and computational analysis; 5. Summary; Acknowledgments; References; Chapter Four: Sperm Patch-Clamp; 1. Introduction; 2. Equipment; 3. Isolation of Spermatozoa for Patch-Clamp Experiments; 3.1. Selection of a suitable sperm developmental stage; 3.2. Isolation of epididymal mouse spermatozoa; 3.3. Collection and isolation of ejaculated human spermatozoa.

4. Patch-Clamp Recording4.1. Difference in sperm patch-clamp recording from traditional recordings; 4.2. Recording from the whole sperm plasma membrane (both human and mouse); 4.3. Recording from spermatozoan cell fragments; 4.4. Compositions of recording solutions; 5. Potential Pitfalls Associated with Sperm Patch Clamp; 6. Conclusions; Acknowledgments; References; Chapter Five: Laser Trap Measurements of Flagellar Membrane Motility; 1. Introduction; 2. Laser Trap Overview; 3. Measuring Flagellar Membrane Transport; 3.1. Cells; 3.2. Laser trap measurements of flagellar membrane transport.