Fluorescence Correlation Spectroscopy: Detecting and Interpreting the Mobility of Transmembrane Proteins In Vivo

Nina Malchus1

1 German Cancer Research Center, Heidelberg, Germany
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.19
DOI:  10.1002/0471140856.tx0219s48
Online Posting Date:  May, 2011
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Mobility of proteins is crucial for their functionality. Fluorescence correlation spectroscopy (FCS) is a sensitive tool for assessing dynamics in vivo. It can reveal properties of diffusing proteins, as well as of the surrounding medium. Hence, subtle changes in the dynamics after treatment with toxic substances can be visualized. On biological membranes, the high concentration of transmembrane and peripheral membrane proteins leads to molecular crowding, and thus to a change in the diffusion behavior, i.e., to anomalous diffusion of membrane proteins. Presented here is a protocol for conducting and evaluating FCS measurements of membrane proteins before and after treatment. Curr. Protoc. Toxicol. 48:2.19.1‐2.19.16. © 2011 by John Wiley & Sons, Inc.

Keywords: fluorescence correlation spectroscopy; anomalous diffusion; membrane proteins

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: FCS Sample Preparation
  • Basic Protocol 2: FCS Data Acquisition
  • Basic Protocol 3: FCS Data Analysis
  • Basic Protocol 4: Data Analysis Using XMGrace
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: FCS Sample Preparation

  • Cells of interest in culture
  • Transfection reagent (e.g., Fugene 6, Roche Diagnostics)
  • Plasmid with GFP‐fusion protein of interest
  • Serum‐free cell culture medium appropriate to the cells being studied
  • Live‐imaging medium: appropriate standard cell culture medium without phenol red (which gives a high fluorescent background), optionally with antibiotics and serum (for longer imaging times), and buffered with HEPES, if necessary
  • 2‐well Lab‐Tek chambers (Nunc)
  • Additional reagents and equipment for growing mammalian cells in culture (Phelan, ), preparing cell culture medium (Sato and Kan, ), and preparing and transfecting plasmids (see appendix 3A)

Basic Protocol 2: FCS Data Acquisition

  • Distilled water
  • Sample to be tested in Lab‐Tek chambers ( protocol 1)
  • Leica SP2‐TCS confocal laser scanning microscope with
    • Argon laser (488‐nm excitation)
    • Software
    • 63× water objective (HCX PL APO 63× 1.2W CORR)
  • Climate box (Life Imaging Services)
  • Mercury arc lamp
  • ISS FCS unit and software (ISS Vista) (Leica Microsystems)
  • External avalanche photodiode (APD) for single‐photon detection with a 500 to 530 nm bandpass filter (Leica Microsystems)

Basic Protocol 3: FCS Data Analysis

  • XMGrace (http://plasma‐gate.weizmann.ac.il/Grace)
  • PC with operating system capable of running XMGrace (see http://plasma‐gate.weizmann.ac.il/Grace)
  • Autocorrelation data from the FCS measurements, saved as *.csv files
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Literature Cited

Literature Cited
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   Griffiths, G., Warren, G., Quinn, P., Mathieu‐Costello, O., and Hoppeler, H. 1984. Density of newly synthesized plasma membrane proteins in intracellular membranes. i. Stereological studies. J. Cell. Biol. 98:2133‐2141.
   Guigas, G., Kalla, C., and Weiss, M. 2007. The degree of macromolecular crowding in the cytoplasm and nucleoplasm of mammalian cells is conserved. FEBS Lett. 581:5094‐5098.
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