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.
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Tam J, Merino D (2015) Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods. J Neurochem 135:643–658
Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544
Sun T, Qiao H, Pan PY, Chen Y, Sheng ZH (2013) Motile axonal mitochondria contribute to the variability of presynaptic strength. Cell Rep 4:413–419
Wong MY, Zhou C, Shakiryanova D, Lloyd TE, Deitcher DL, Levitan ES (2012) Neuropeptide delivery to synapses by long-range vesicle circulation and sporadic capture. Cell 148:1029–1038
Orenbuch A, Shalev L, Marra V, Sinai I, Lavy Y, Kahn J et al (2012) Synapsin selectively controls the mobility of resting pool vesicles at hippocampal terminals. J Neurosci 32:3969–3980
Staras K, Mikulincer D, Gitler D (2013) Monitoring and quantifying dynamic physiological processes in live neurons using fluorescence recovery after photobleaching. J Neurochem 126:213–222
Bancaud A, Huet S, Rabut G, Ellenberg J (2010) Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: Frap, photoactivation, photoconversion, and flip. Cold Spring Harb Protoc 2010:pdb top90
Berkovich R, Wolfenson H, Eisenberg S, Ehrlich M, Weiss M, Klafter J et al (2011) Accurate quantification of diffusion and binding kinetics of non-integral membrane proteins by FRAP. Traffic 12:1648–1657
Kang M, Day CA, Kenworthy AK, DiBenedetto E (2012) Simplified equation to extract diffusion coefficients from confocal FRAP data. Traffic 13:1589–1600
Kang M, Andreani M, Kenworthy AK (2015) Validation of normalizations, scaling, and photofading corrections for FRAP data analysis. PLoS One 10:e0127966
Schmidt H, Brown EB, Schwaller B, Eilers J (2003) Diffusional mobility of parvalbumin in spiny dendrites of cerebellar purkinje neurons quantified by fluorescence recovery after photobleaching. Biophys J 84:2599–2608
Scott DA, Das U, Tang Y, Roy S (2011) Mechanistic logic underlying the axonal transport of cytosolic proteins. Neuron 70:441–454
Tang Y, Scott D, Das U, Gitler D, Ganguly A, Roy S (2013) Fast vesicle transport is required for the slow axonal transport of synapsin. J Neurosci 33:15362–15375
Tsuriel S, Geva R, Zamorano P, Dresbach T, Boeckers T, Gundelfinger ED et al (2006) Local sharing as a predominant determinant of synaptic matrix molecular dynamics. PLoS Biol 4:e271
Staras K, Branco T, Burden JJ, Pozo K, Darcy K, Marra V et al (2010) A vesicle superpool spans multiple presynaptic terminals in hippocampal neurons. Neuron 66:37–44
Schmidt H, Arendt O, Brown EB, Schwaller B, Eilers J (2007) Parvalbumin is freely mobile in axons, somata and nuclei of cerebellar purkinje neurones. J Neurochem 100:727–735
Shulman Y, Stavsky A, Fedorova T, Mikulincer D, Atias M, Radinsky I et al (2015) Atp binding to synaspsin IIa regulates usage and clustering of vesicles in terminals of hippocampal neurons. J Neurosci 35:985–998
Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877
Subach FV, Patterson GH, Manley S, Gillette JM, Lippincott-Schwartz J, Verkhusha VV (2009) Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6:153–159
Chudakov DM, Lukyanov S, Lukyanov KA (2007) Tracking intracellular protein movements using photoswitchable fluorescent proteins ps-cfp2 and dendra2. Nat Protoc 2:2024–2032
Roy S, Yang G, Tang Y, Scott DA (2011) A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution. Nat Protoc 7:62–68
Royo NC, Vandenberghe LH, Ma JY, Hauspurg A, Yu L, Maronski M et al (2008) Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity. Brain Res 1190:15–22
Shevtsova Z, Malik JM, Michel U, Bahr M, Kugler S (2005) Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp Physiol 90:53–59
Groh A, de Kock CP, Wimmer VC, Sakmann B, Kuner T (2008) Driver or coincidence detector: modal switch of a corticothalamic giant synapse controlled by spontaneous activity and short-term depression. J Neurosci 28:9652–9663
Wu Z, Yang H, Colosi P (2010) Effect of genome size on AAV vector packaging. Mol Ther 18:80–86
Hirsch ML, Agbandje-McKenna M, Samulski RJ (2010) Little vector, big gene transduction: fragmented genome reassembly of adeno-associated virus. Mol Ther 18:6–8
Dotti CG, Sullivan CA, Banker GA (1988) The establishment of polarity by hippocampal neurons in culture. J Neurosci 8:1454–1468
Tsibidis GD (2009) Quantitative interpretation of binding reactions of rapidly diffusing species using fluorescence recovery after photobleaching. J Microsc 233:384–390
Brown EB, Wu ES, Zipfel W, Webb WW (1999) Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery. Biophys J 77:2837–2849
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This work was supported by Israel Science Foundation (ISF) grant 1427/12 (DG).
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Tevet, Y., Gitler, D. (2016). Using FRAP or FRAPA to Visualize the Movement of Fluorescently Labeled Proteins or Cellular Organelles in Live Cultured Neurons Transformed with Adeno-Associated Viruses. In: Schwartzbach, S., Skalli, O., Schikorski, T. (eds) High-Resolution Imaging of Cellular Proteins. Methods in Molecular Biology, vol 1474. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6352-2_8
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DOI: https://doi.org/10.1007/978-1-4939-6352-2_8
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