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.
Written for:
Cell biologists, physiologists, developmental biologists, immunologists, and neurobiologists
Keywords:
* Cellular physiology
* Eukaryotic cells
* Intracellular processes
* Membrane-coated vesicles
* Plasma membrane
Table of contents
Contents
Preface
Contributors
Part I An Overview of Methods for Analyzing Vesicular Trafficking
1 Vesicular Trafficking: Molecular Tools and Targets
Elena Vassilieva and Asma Nusrat
2 Pharmacological Inhibition of Endocytic Pathways. Is It Specific Enough to be Useful?
Andrei Ivanov
Part II Cell-free and Biochemical Assays of Exocytosis and Endocytosis
3 In Vitro Assays to Measure SNARE-Mediated Vesicle Fusion
Susanne Kreye, Jörg Malsam, and Thomas H. Söllner
4 Imaging Ca2+-Triggered Exocytosis of Single Secretory Granules on Plasma Membrane Lawns from Neuroendocrine Cells.
Thorsten Lang
5 FRAP Analysis of Secretory Granule Lipids and Proteins in the Sea Urchin Egg
Julian L. Wong and Gary M. Wessel
6 A Cell-Free Assay for Endocytosis of E-cadherin
Toshiaki Sakisaka and Yoshimi Takai
7 Cell-Surface Biotinylation to Study Endocytosis and Recycling of Occludin
Noriyuki Nishimura and Takuya Sasaki
8 Fractionation of Subcellular Membrane Vesicles of Epithelial and Non-epithelial Cells by OptiPrepTM Density Gradient Ultracentrifugation
Xuhang Li and Mark Donowitz
9 Identification and Characterization of Interacting Partners of Rab GTPases by Yeast Two-Hybrid Analyses
Mark Kail and Angelika Barnekow
Part III Analysis of Endocytosis and Exocytosis in Different Cell Systems
10 High-Throughput Analysis of the Dynamics of Recycling Cell Surface Proteins
Roland Govers, David E. James, and Adelle C. F. Coster
11 Studying Phagocytosis by Live-Cell Scintillation Proximity Assay
Walter Stockinger and Axel Nohturfft
12 Transcytosis of Polymeric Immunoglobulin A in Polarized Madin-Darby Canine Kidney Cells
Asli Oztan, Christine Rondanino, and Gerard Apodaca
13 The Use of Syntaxin Chimeras to Study Polarized Protein Trafficking in Epithelial Cells
Martin B.A. ter Beest
14 Targeting the Epithelial SNARE Machinery by Bacterial Neurotoxins
Veronique Proux-Gillardeaux and Thierry Galli
15 Exocytosis of Endothelial Cells is Regulated by N-ethylmaleimide Sensitive Factor Munekazu Yamakuchi, Marcella Ferlito, Craig N. Morrell, Kenji Matsushita, Craig A. Fletcher, Wangsen Cao, and Charles J. Lowenstein
16 Molecular Dissection of HCl Secretion in Gastric Parietal Cells Using Streptolysin O Permeabilization
Xia Ding, Fang Wu, Zhen Guo, and Xuebiao Yao
17 Addressing Membrane Protein Topology Using the Fluorescence Protease Protection (FPP) Assay
Holger Lorenz, Dale W. Hailey, and Jennifer Lippincott-Schwartz
18 Visualizing Clathrin-Mediated IgE Receptor Internalization by Electron and Atomic Force Microscopy
Alan R. Burns, Janet M. Oliver, Janet R. Pfeiffer, and Bridget S. Wilson
19 Analyzing Endosomes in Non-Sectioned Cells by Transmission Electron Microscopy Willem Stoorvogel