Quantifying Cellular Dynamics by Fluorescence Resonance Energy Transfer (FRET) Microscopy

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The cell is a spatially organized system whose function emerges from the complex interaction of molecular components. Such local interaction of nanometer?sized molecules generates patterns that span throughout the cell. Those patterns, in turn, regulate the molecular interactions. Understanding such simultaneous bidirectional causation requires quantifying the spatio?temporal progression of biochemical reactions in the context of a living cell. Due to its ability to resolve micrometer?sized structures, biological microscopy has been instrumental to the discovery and understanding of living systems. Functional fluorescence microscopy allows a cellular dynamic topographic map of proteins to be overlaid with topological information on the causality that determines protein state. Here we describe how Förster/fluorescence resonance energy transfer (FRET) can be used to measure the phosphorylation state of proteins in the context of the cell. Curr. Protoc. Neurosci. 63:5.22.1?5.22.14. © 2013 by John Wiley & Sons, Inc.

Keywords: quantification of cellular processes; fluorescence microscopy; Förster resonance energy transfer (FRET); acceptor photobleaching; fluorescence lifetime imaging microscopy (FLIM)

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Introduction
Basic Protocol 1: FRET Microscopy on Fixed Cells
Basic Protocol 2: Protein Labeling with Cy3.5
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: FRET Microscopy on Fixed Cells

Materials

Cells of interest (e.g., MCF7, Cos7, A431)
Low‐background‐fluorescence CO₂ ‐independent medium (Life Technologies or see recipe )
Plasmid for mCitrine‐tagged protein (e.g., EGFR‐mCitrine, SRC‐mCitrine)
Transfection reagent (e.g., FuGENE 6 from Roche)
Serum‐free medium or 0.5% serum medium (for cells that undergo apoptosis upon serum deprivation)
Test substance, e.g., growth factor or hormone for stimulation, drug, inhibitor
Phosphate‐buffered saline (PBS; see recipe ), pH 7.4
4% (w/v) formaldehyde fixative solution (see recipe )
Quench solution: 50 mM Tris⋅Cl (pH 8.0)/100 mM NaCl or 0.1 M hydroxylamine or 0.1 M glycine
0.1% (v/v) Triton X‐100 in PBS
0.1% saponin in PBS or −20°C methanol (optional)
Antibody (e.g., PY72 monoclonal anti‐phosphotyrosine antibody) labeled with Cy3.5 (see protocol 2 )
1% (w/v) bovine serum albumin (BSA, fraction V) in PBS
Mowiol mounting medium (see recipe )
Molten agarose or rubber cement (optional)

6‐ and 12‐well tissue culture plates
Coverslips
Jeweler's forceps
Microscope slides
Confocal laser scanning microscope (e.g., Zeiss LSM 710, Leica SP5, Olympus FV1000), equipped with an argon laser (488 nm or 514 nm line) and a diode laser (561 nm); appropriate emission filters for mCitrine and Cy3.5 (such as Chroma ET535/30 and Chroma HQ620/60m)
Imaging software package (e.g., ImageJ, freely available from http://rsbweb.nih.gov/ij/)

NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.NOTE: All incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO₂ to maintain pH 7.4.

Basic Protocol 2: Protein Labeling with Cy3.5

Materials

Antibody (PY72 monoclonal anti‐phosphotyrosine antibody)
Phosphate‐buffered saline (PBS; see recipe ), pH 7.4
100 mM and 10 mM bicine, pH 8.0 (adjusted with NaOH)
1 M bicine, pH 9.0 (adjusted with NaOH)
100 mM citric acid, pH 2.8 (adjusted with NaOH)
1 M NaCl

Labeling buffer: 100 mM bicine (pH 8.0)/100 mM NaCl
Cy3.5 monofunctional sulfoindocyanine succinimide ester (Cy3.5; GE Healthcare PA23501)
Dimethylformamide (DMF) dried by addition of 10 to 20 mesh 3 Å pore diameter molecular sieve dehydrate (Fluka)
1 M Tris adjusted with HCl to pH 8.0
1‐ml protein G or protein A HiTrap columns (Amersham Pharmacia Biotech)

Centricon YM30 concentrators (Amicon)
Biogel P6DG Econopac prepacked size‐exclusion columns (5.5 × 1.5 cm, ∼10 ml; Bio‐Rad)
1 ml and 10 ml syringes with HPLC Luer‐Lok fitted tubing

Additional reagents and equipment for spectrophotometric protein determination ( appendix 1K ) and SDS‐PAGE (Gallagher, )

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Figures

Figure 5.22.1 Measuring the phosphorylation of the epidermal growth factor receptor (EGFR) using acceptor photobleaching with laser scanning confocal microscopy. Left column: Images of the donor (EGFR‐mCitrine) before (upper row) and after (bottom row) photobleaching. Middle column: Corresponding images of the acceptor (PY72‐Cy3.5). Notice how the intensity of the donor increases where the acceptor has been bleached. Right column: Apparent fluorescence resonance energy transfer efficiency (AFE). As schematized below, a high AFE (as seen in the membrane) means that there is a larger fraction of donor in‐complex with the acceptor (phosphorylated receptor). Scale bar: 20 µm.

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Figure 5.22.2 Pharmacological intervention with the Ras acylation cycle affects PDEδ activity. Time‐lapse FLIM analyses of mCit‐NrasG12D‐ and mCh‐PDEδ‐transfected Madin‐Darby canine kidney (MDCK) cells show the loss of PDEδ‐NrasG12D interaction after thioesterase inhibition by palmostatin‐B treatment. Upper two rows: fluorescence intensity distribution of the indicated fluorescent fusion proteins. Lower two rows: average fluorescence lifetime (τ_av ) in nanoseconds and molar fraction (α) of mCit‐Nras^G12D interacting with mCh‐PDEδ. Scale bar: 10 µm. Reproduced from Chandra et al. ()

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Videos

Literature Cited

Literature Cited
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Quantifying Cellular Dynamics by Fluorescence Resonance Energy Transfer (FRET) Microscopy

Abstract

Table of Contents

Materials

Basic Protocol 1: FRET Microscopy on Fixed Cells

Basic Protocol 2: Protein Labeling with Cy3.5

Figures

Videos

Literature Cited