Exocytosis and Endocytosis

Exocytosis and endocytosis remain among the most popular subjects in biology and biomedical sciences. Tremendous progress in understanding these complex intracellular processes has been achieved by employing a wide array of research tools ranging from classical biochemical methods to modern imaging techniques. In this book, skilled experts provide the most up-to-date, step-by-step laboratory protocols for examining molecular machinery and biological functions of exocytosis and endocytosis in vitro and in vivo. The volume presents detailed lists of tested reagents that really work, and notes on troubleshooting from leading laboratories in the field. Insightful to both newcomers and seasoned professionals, the book offers a unique and highly practical guide to versatile laboratory tools developed to study various aspects of intracellular vesicle trafficking in simple model systems and living organisms.

Provides classic, state of art, and cutting edge methods in a simple, easily reproducible format Contains a comprehensive set of experimental models including cell-free systems, different specialized cells, and model organisms Presents detailed lists of tested reagents that really work Notes on troubleshooting from leading laboratories in the field

Klappentext

Due to their vital involvement in a wide variety of housekeeping and specialized cellular functions, exocytosis and endocytosis remain among the most popular subjects in biology and biomedical sciences. Tremendous progress in understanding these complex intracellular processes has been achieved by employing a wide array of research tools ranging from classical biochemical methods to modern imaging techniques. In Exocytosis and Endocytosis, skilled experts provide the most up-to-date,step-by-step laboratory protocols for examining molecular machinery and biological functions of exocytosis and endocytosis in vitro and in vivo. Following the highly successful Methods in Molecular Biology™ series format, the chapters present an introduction outlining the principle behind each technique, a list of the necessary materials, an easy to follow, readily reproducible protocol, and a Notes section offering tips on troubleshooting and avoiding known pitfalls.

Insightful to both newcomers and seasoned professionals, Exocytosis and Endocytosis offers a unique and highly practical guide to versatile laboratory tools developed to study various aspects of intracellular vesicle trafficking in simple model systems and living organisms.

Inhalt
An Overview of Methods for Analyzing Vesicular Trafficking.- Vesicular Trafficking: Molecular Tools and Targets.- Pharmacological Inhibition of Endocytic Pathways: Is It Specific Enough to Be Useful?.- Cell-Free and Biochemical Assays of Exocytosis and Endocytosis.- In Vitro Assays to Measure SNARE-Mediated Vesicle Fusion.- Imaging Ca2+-Triggered Exocytosis of Single Secretory Granules on Plasma Membrane Lawns from Neuroendocrine Cells.- FRAP Analysis of Secretory Granule Lipids and Proteins in the Sea Urchin Egg.- A Cell-Free Assay for Endocytosis of E-Cadherin.- Cell-Surface Biotinylation to Study Endocytosis and Recycling of Occludin.- Fractionation of Subcellular Membrane Vesicles of Epithelial and Nonepithelial Cells by OptiPrep™ Density Gradient Ultracentrifugation.- Identification and Characterization of Interacting Partners of Rab GTPases by Yeast Two-Hybrid Analyses.- Analysis of Endocytosis and Exocytosis in Different Cell Systems.- High-Throughput Analysis of the Dynamics of Recycling Cell Surface Proteins.- Studying Phagocytosis by Live-Cell Scintillation Proximity Assay.- Transcytosis of Polymeric Immunoglobulin A in Polarized MadinDarby Canine Kidney Cells.- The Use of Syntaxin Chimeras to Study Polarized Protein Trafficking in Epithelial Cells.- Targeting the Epithelial SNARE Machinery by Bacterial Neurotoxins.- Exocytosis of Endothelial Cells Is Regulated by N-Ethylmaleimide-Sensitive Factor.- Molecular Dissection of HCl Secretion in Gastric Parietal Cells Using Streptolysin O Permeabilization.- Addressing Membrane Protein Topology Using the Fluorescence Protease Protection (FPP) Assay.- Visualizing Clathrin-Mediated IgE Receptor Internalization by Electron and Atomic Force Microscopy.- Analyzing Endosomes in Nonsectioned Cells by TransmissionElectron Microscopy.- Imaging Exocytosis of Single Insulin Secretory Granules With TIRF Microscopy.- Reconstitution of Depolarization and Ca2+-Evoked Secretion in Xenopus Oocytes Monitored by Membrane Capacitance.- Low-Noise Recording of Single-Vesicle Capacitance Steps in Cell-Attached Patches.- Good Practices in Single-Cell Amperometry.- Analysis of Single-Vesicle Exocytotic Events Recorded by Amperometry.- Analysis of Exocytosis and Endocytosis in Model Organisms.- Application of RNAi Technology and Fluorescent Protein Markers to Study Membrane Traffic in Caenorhabditis elegans.- FM 1-43 Labeling of Synaptic Vesicle Pools at the Drosophila Neuromuscular Junction.- Probing E-Cadherin Endocytosis by Morpholino-Mediated Rab5 Knockdown in Zebrafish.- Quantifying Endocytosis In Vivo Using Intravital Two-Photon Microscopy.