丁香实验_LOGO
登录
提问
我要登录
|免费注册
点赞
收藏
wx-share
分享

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

互联网

1558
  • Abstract
  • Table of Contents
  • Materials
  • Figures
  • Literature Cited

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   Figure Figure 2.17.1 Schematic illustrating FRAP. A cell expressing fluorescently tagged protein undergoes high‐power laser photobleaching in a defined region of interest (ROI) in the cell. Recovery from the laser bleach is monitored over time by low‐power laser imaging. Due to imaging there is a reduction in whole‐cell fluorescence concurrent with a recovery of fluorescence in the bleached ROI.
    View Image
  •   Figure Figure 2.17.2 Fluorescence recovery curve. The data have been normalized for background reduction in whole‐cell fluorescence and normalized to prebleach fluorescence intensity [ F (−1)]. Immobile and mobile fractions are indicated. F (−1) = prebleach background‐normalized fluorescence in the ROI at t = −1. F (∞) = final recovered fluorescence value in the ROI that is a constant value or plateau (recovery is considered complete at F (∞)). F (0) = fluorescence value at time zero after the bleach. t 0 = the first time point after the bleach. t 1/2 = half‐time of fluorescence recovery, i.e., the time required to reach 50% of final recovered fluorescence.
    View Image
  •   Figure Figure 2.17.3 FRAP of protein after immobilization of actin. The fluorescence recovery of an actin‐interacting protein in untreated control (black line), after 1 µM jasplakinolide treatment (JS, dark gray line), and after 10 µg/ml cytochalasin D treatment (CD, light gray line) is shown. Reprinted with permission from Lin and Redmond; © 2008 National Academy of Sciences, U.S.A
    View Image

Videos

Literature Cited

Literature Cited
   Bubb, M.R., Senderowicz, A.M., Sausville, E.A., Duncan, K.L., and Korn, E.D. 1994. Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F‐actin. J. Biol. Chem. 269:14869‐14871.
   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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
 
提问
扫一扫
丁香实验小程序二维码
实验小助手
丁香实验公众号二维码
扫码领资料
反馈
TOP
打开小程序