Fluorescence Recovery After Photobleaching (FRAP) with a Focus on F‐actin

Lori R. Hardy1

1 Department of Basic Sciences, Philadelphia College of Osteopathic Medicine, Suwanee, Georgia
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.17
DOI:  10.1002/0471142301.ns0217s61
Online Posting Date:  October, 2012
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Abstract

In this unit on fluorescence recovery after photobleaching (FRAP), an imaging approach to study protein‐protein interactions in situ is described. The protocols presented use confocal microscopy to examine the mobility of a fluorescent protein by measuring the recovery of the protein in a bleached area. The data gained in FRAP studies is qualitative and yields insight into relative binding affinity, binding characteristics, and the effect of treatments or mutations on protein mobility. Curr. Protoc. Neurosci. 61:2.17.1‐2.17.12. © 2012 by John Wiley & Sons, Inc.

Keywords: FRAP; actin; protein mobility; GFP

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: FRAP Imaging of Fluorescently Tagged Actin in Cells
  • Basic Protocol 2: Analysis of the FRAP Time‐Lapse Image Series
  • Basic Protocol 3: FRAP of Immobilized Actin
  • Basic Protocol 4: Determination of Diffusion‐Coupled or Diffusion‐Uncoupled Recovery
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: FRAP Imaging of Fluorescently Tagged Actin in Cells

  Materials
  • Cells of choice (cell lines, primary cell cultures, etc.)
  • Medium of choice
  • Fluorescently tagged actin or protein of interest
  • Culture external base (CEB; see recipe)
  • Glass‐bottom dishes, 35 mm
  • Microscope eguipped with epifluorescence capability
  • Confocal laser‐scanning microscope equipped with photobleaching protocols

Basic Protocol 2: Analysis of the FRAP Time‐Lapse Image Series

  Materials
  • Time‐lapse image series from a photobleaching experiment ( protocol 1)
  • Image analysis software (typically part of the confocal microscope software)
  • Data analysis software (e.g., Microsoft Office Excel or Origin 6.1)

Basic Protocol 3: FRAP of Immobilized Actin

  Materials
  • Cells expressing tagged actin, cultured on 35‐mm glass‐bottom dish ( protocol 1)
  • Culture external base (CEB; see recipe)
  • Actin‐immobilizing agent (cytochalasin D or jasplakinolide)
  • Microscope equipped with epifluorescence capability
  • Confocal laser‐scanning microscope equipped with photobleaching protocols
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Figures

Videos

Literature Cited

Literature Cited
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   Campbell, J.J. and Knight, M.M. 2007. An improved confocal FRAP technique for the measurement of long‐term actin dynamics in individual stress fibers. Microsc. Res. Tech. 70:1034‐1040.
   Jacobson, K. and Wojcieszyn, J. 1984. The translational mobility of substances within the cytoplasmic matrix. Proc. Natl. Acad. Sci. U.S.A. 81:6747‐6751.
   Jönsson, P., Jonsson, M.P., Tegenfeldt, J.O., and Höök, F. 2008. A method improving the accuracy of fluorescence recovery after photobleaching analysis. Biophys. J. 95:5334‐5348.
   Kang, M., Day, C.A., DiBenedetto, E., and Kenworthy, A.K. 2010. A quantitative approach to analyze binding diffusion kinetics by confocal FRAP. Biophys. J. 99:2737‐2747.
   Lin, Y‐C. and Redmond, L. 2008. CaMKIIβ binding to stable F‐actin in vivo regulates F‐actin filament stability. Proc. Natl. Acad. Sci. U.S.A. 105:15791‐15796.
   Lober, R.M., Pereira, M.A., and Lambert, N.A. 2006. Rapid activation of inwardly rectifying potassium channels by immobile G‐protein‐coupled receptors. J. Neurosci. 26:12602‐12608.
   McNeil, P.L. 2003. Direct introduction of molecules into cells. Curr. Protoc. Cell Biol. 18:20.1.1‐20.1.7.
   Peters, R., Brunger, A., and Schulten, K. 1981. Continuous fluorescence microphotolysis: A sensitive method for study of diffusion processes in single cells. Proc. Natl. Acad. Sci. U.S.A. 78:962‐966.
   Reits, E.A.J., Benham, A.M., Plougastel, B., Neefjes, J., and Trowsdale, J. 1997. Dynamics of proteasome distribution in living cells. EMBO J. 16:6087‐6094.
   Schliwa, M. 1982. Action of cytochalasin D on cytoskeletal networks. J. Cell Biol. 92:79‐91.
   Sprague, B.L., Pego, R.L., Stavreva, D.A., and McNally, J.G. 2004. Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys. J. 86:3473‐3495.
   Star, E.N., Kwiatkowski, D.J., and Murthy, V.N. 2002. Rapid turnover of actin in dendritic spines and its regulation by activity. Nat. Neurosci. 5:239‐246.
   Trembecka, D.O., Kuzak, M., and Dobrucki, J.W. 2010. Conditions for using FRAP as a quantitative technique—influence of the bleaching protocol. Cytometry 77:366‐370.
   Zacharias, D.A., Violon, J.D., Newton, A.C., and Tsien, R.Y. 2002. Partitioning of lipid‐modified monomeric GFPs into membrane microdomains of live cells. Science 296:913‐916.
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