Exocytosis and Endocytosis

About this book

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. 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.

Contents

Vesicular Trafficking: Molecular Tools and Targets.- Pharmacological Inhibition of Endocytic Pathways. Is It Specific Enough to be Useful?.- 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 Non-epithelial Cells by OptiPrepTM Density Gradient Ultracentrifugation.- Identification and Characterization of Interacting Partners of Rab GTPases by Yeast Two-Hybrid Analyses.- 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 Madin-Darby 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 Non-Sectioned Cells by Transmission Electron 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.- Application of RNAi Technology and Fluorescent Protein Markers to Study Membrane Traffic in C. elegans.- FM 1-43 Labeling of Synaptic Vesicle Pools at the Drosophila Neuromuscular Junction.- Probing E-cadherin Endocytosis by Morpholino-Mediated Rab5 Knock-Down in Zebrafish.- Quantifying Endocytosis in vivo Using Intravital Two-Photon Microscopy.

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