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Full Description
This detailed volume presents a series of methods exploring membrane trafficking research, ranging from genetics and high-resolution imaging to in vitro biochemical and biophysical assays. Covering virtually all the major trafficking branches, the book delves into the exocytic pathway, which focuses on cargo transport from the ER to the Golgi, through the Golgi cisternae, and to the plasma membrane and the extracellular space; the endocytic pathway, which includes cargo endocytosis, endosomal recycling, and lysosomal degradation; as well as emerging topics beyond the conventional exocytic and endocytic pathways. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Membrane Trafficking: Methods and Protocols provides techniques with broad applications as an ideal guide for junior researchers new to membrane trafficking as well as established membrane biologists seeking to expand their research programs.
Contents
Characterizing Membrane Traffic in the Early Secretory Pathway Using the RUSH Retention System.- Monitoring Intra-Golgi Transport with Acute Spatiotemporal Control of a Synthetic Cargo.- Imaging Secretory Granule Budding from the Trans-Golgi Network Using Retention Using Selective Hook (RUSH).- Spatial and Temporal Control of Protein Secretion with Light.- Generation of Endogenously Tagged Membrane Trafficking Regulators Using CRISPR Genome Editing.- Determine the Function of the Exocyst in Vesicle Tethering by Ectopic Targeting.- Imaging Insulin Granule Dynamics in Human Pancreatic β-Cells Using Total Internal Reflection Fluorescence (TIRF) Microscopy.- Monitoring Synaptic Exocytosis Using a Whole-Cell Patch Clamp Technique.- Live-Cell Super-Resolution Imaging of Retrograde Axonal Trafficking Using Pulse-Chase Labeling in Cultured Hippocampal Neurons.- Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapse.- In Vitro Reconstitution Studies of SNAREs and Their Regulators Mediating GLUT4 Vesicle Fusion.- Imaging Single Vesicle Exocytosis with Total Internal Reflection Fluorescence Microscopy (TIRFM).- Localizing Proteins on Single Trafficking Organelles in 3D with Semisynthetic Gold Labeling and Platinum Replica Electron Microscopy.- Endocytosis Assays Using Cleavable Fluorescent Dyes.- Reconstituting and Purifying Assembly Intermediates of Clathrin Adaptors AP1 and AP2.- Monitoring Endosomal Cargo Retrieval to the Trans-Golgi Network by Microscopic and Biochemical Approaches.- Measuring Plasma Membrane Recycling Using Microscopic and Biochemical Approaches.- Light Scattering Techniques to Assess Self-Assembly and Hydrodynamics of Membrane Trafficking Proteins.- Measurement of Lysosome Positioning by Shell Analysis and Line Scan.- Optogenetic Control of Membrane Trafficking Using Light-Activated Reversible Inhibition by Assembly Trap of Intracellular Membranes (IM-LARIAT).- Generating Custom Pooled CRISPR Libraries for Genetic Dissectionof Biological Pathways.- Studying Unconventional Secretion of Misfolded Proteins in Cultured Cells and Primary Neurons.- Single-Molecule Optical Tweezers Study of Protein-Membrane Interactions.- Generation of Hybrid Extracellular Vesicles by Fusion with Functionalized Liposomes.- Quantitative Measurement of Extracellular Vesicle Content Delivery within Acceptor Cells.
