title: Photobleaching methods to study Golgi complex dynamics in living cells.
abstract: The Golgi complex (GC) is a highly dynamic organelle that constantly receives and exports proteins and lipids from both the endoplasmic reticulum and the plasma membrane. While protein trafficking can be monitored with traditional biochemical methods, these approaches average the behaviors of millions of cells, provide modest temporal information and no spatial information. Photobleaching methods enable investigators to monitor protein trafficking in single cells or even single GC stacks with subsecond precision. Furthermore, photobleaching can be exploited to monitor the behaviors of resident GC proteins to provide insight into mechanisms of retention and trafficking. In this chapter, general photobleaching approaches with laser scanning confocal microscopes are described. Importantly, the problems associated with many fluorescent proteins (FPs) and their uses in the secretory pathway are discussed and appropriate choices are suggested. For example, Enhanced Green Fluorescent Protein (EGFP) and most red FPs are extremely problematic. Finally, options for data analyses are described.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, USA.
authors.forename: Erik Lee
authors.initials: EL
authors.lastname: Snapp
mesh_terms: D000818:Animals; D004058:Diffusion; D004721:Endoplasmic Reticulum / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D038761:Photobleaching; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Diffusion; FLIP; FRAP; Membrane; Superfolder GFP