Focused on one of the most important and fascinating fields of modern biology, Exocytosis and Endocytosis, Second Edition presents a comprehensive collection of protocols for cutting edge, recently developed methods of studying exocytosis and endocytosis in vitro and in vivo. Divided into four easy-to-follow sections, chapters focus on diverse research topics including cell-free and biochemical assays of exocytosis and endocytosis, probing protein, membrane and organelle trafficking in cultured cells and powerful in vivo imaging approaches to investigate different physiological processes involving exocytosis and endocytosis. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

Highly practical and clearly written, Exocytosis and Endocytosis, Second Edition will serve both seasoned researchers as well as newcomers to the field and will provide a unique resource and expert guidance to modern laboratory techniques developed for examining protein and membrane trafficking in simple cellular systems and model organisms.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Zusammenfassung
Focused on one of the most important and fascinating fields of modern biology, Exocytosis and Endocytosis, Second Edition presents a comprehensive collection of protocols for cutting edge, recently developed methods of studying exocytosis and endocytosis in vitro and in vivo.

Inhalt

Pharmacological Inhibitors of Exocytosis and Endocytosis: Novel Bullets for Old Targets.- Systematic Analysis of Endocytosis by Cellular Perturbations.- Real Time Detection of SNARE Complexes with FRET Using the Tetracysteine System.- Profiling Lysine Ubiquitination by Selective Enrichment of Ubiquitin Remnant-Containing Peptides.- Secretion of Circular Proteins Using Sortase.- Fractionation of Subcellular Membrane Vesicles of Epithelial and Non-Epithelial Cells by OptiPrepTM Density Gradient Ultracentrifugation.- Combining Pulsed SILAC Labeling and Click-Chemistry for Quantitative Secretome Analysis.- Probabilistic Density Maps to Study the Spatial Organization of Endocytosis.- Use of Kaede and Kikume Green-Red Fusions for Live Cell Imaging of G Protein-Coupled Receptors.- Halo Tag as a Tool to Investigate Peroxisome Dynamics in Cultured Mammalian Cells.- SNAP-Tag to Monitor Trafficking of Membrane Proteins in Polarized Epithelial Cells.- FLAsH-PALM: Super-Resolution Pointillist Imaging with FLAsH-tetracysteine Labeling.- Analysis of Protein Dynamics with Tandem Fluorescent Protein Timers.- Synchronization of Secretory Cargo Trafficking in Populations of Cells.- Use of Transmembrane FRET to Investigate the Internalization of Glycosylated Proteins.- A Method to Rapidly Induce Organelle-specific Molecular Activities and Membrane Tethering.- A Novel Pair of Split Venus Fragments to Detect Protein-Protein Interactions by In Vitro and In Vivo Biomolecular Fluorescence Complementation Assays.- Real Time Investigation of Plasma Membrane Deformation and Fusion Pore Expansion Using Polarized Total Internal Reflection Fluorescence Microscopy.- Nanocones to Study Initial Steps of Endocytosis.- A Novel Permeabilization Protocol to Obtain Intracellular 3D Immunolabeling for Electron Tomography.- VIS2FIX: Rapid Chemical Fixation of Vitreous Sections for Immuno-Electron Microscopy.- Chemical Genomics: Characterizing Target Pathways for Bioactive Compounds Using theEndomembrane Trafficking Network.- Application of RNAi Technology and Fluorescent Protein Markers to Study Membrane Traffic in C. elegans.- Visualization of Clathrin-Mediated Endocytosis in Live Drosophila Egg Chambers.- Novel Extraction Protocol to Probe the Role of Cholesterol in Synaptic Vesicle Recycling.- Microfluidic Devices for Imaging Trafficking Events In Vivo Using Genetic Model Organisms.- The In Situ Proximity Ligation Assay to Probe Protein-Protein Interactions in Intact Tissues.- Probing the Role of the Actin Cytoskeleton During Regulated Exocytosis by Intravital Microscopy.- Measurement of Dynamic F-actin Changes During Exocytosis.