title: Using FRAP or FRAPA to Visualize the Movement of Fluorescently Labeled Proteins or Cellular Organelles in Live Cultured Neurons Transformed with Adeno-Associated Viruses.
abstract: Fluorescence recovery after photobleaching (FRAP) and fluorescence redistribution after photoactivation (FRAPA) are complementary methods used to gauge the movement of proteins or sub-resolution organelles within cells. Using these methods we can determine the nature of the movement of labeled particles, whether it is random, constrained, or active, the coefficient of diffusion if applicable, binding and unbinding constants, and the direction of active transport. These two techniques have been extensively utilized to probe the cell biology of neurons. A practical outline of FRAP and FRAPA in cultured neurons is presented, including the preparation of the neurons and their infection with adeno-associated viral vectors. Considerations in planning such experiments are provided.
authors: Department of Physiology and Cell Biology, Faculty of Health Sciences and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel.
authors: Yaara
authors: Y
authors: Tevet
authors: Department of Physiology and Cell Biology, Faculty of Health Sciences and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel. gitler@bgu.ac.il.
authors: Daniel
authors: D
authors: Gitler
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006624:Hippocampus; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling; D016708:Synaptophysin / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: C583169:Dendra2 protein, Dendronephthya; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D016708:Synaptophysin; C569015:Syp protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Adeno-associated virus; FRAP; Fluorescence microscopy; GFP; Neuronal culture; Photoactivation