By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of Förster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.
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Method Article
In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy
In This Article
Summary
Abstract
The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. Förster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 α-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.
Protocol
1. Plasmid design
The proteins of interest are fused to one of two different fluorescent labels, as described next. The fluorescent labels not only provide information about the location of the proteins within the cell, but also quantitative information regarding the homo-oligomerization of the protein1. The technique of Fluorescence Resonance Energy Transfer (FRET) 2-4 is used to accumulate the information about the protein interactions 5-10. FRET utilizes the principle that a transfer of energy can occur from an optically excited molecule (typically referred to as the donor, D) to an unexcited molecule ....
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Discussion
In this presentation, we illustrated how to determine the size and structural information about a protein oligomer complex in vivo. While the data presented was obtained from a specific membrane receptor (i.e. Ste2p) expressed in yeast cells, the method is all encompassing in that it can be applied to any type of protein expressed in any type of cell, the only stipulation being that the proteins are tagged with the appropriate fluorescent markers. Future modifications to this protocol will involve enhancing the.......
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Disclosures
No conflicts of interest declared.
Acknowledgements
This work was supported by the UW-Milwaukee Research Growth Initiative, the Wisconsin Institute for Biomedical and Health Technologies, and the Bradley Foundation.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Peptone | Fisher Scientific | BP1420 | |
| Yeast Extract | Fisher Scientific | BP1422 | |
| Polyethlyene Glycol 4000 | Hampton Research | HR2-605 | |
| Yeast Nitrogen Base w/o (NH4)2SO4 and amino acids | Fisher Scientific | DF0335-15-9 | |
| Yeast Synthetic Drop-out Medium Supplements | Sigma-Aldrich | Y2001 | |
| D-Glucose | Fisher Scientific | D16-1 | |
| Agar | Fisher Scientific | S70210 | |
| Ammonium Sulfate | Fisher Scientific | A702-500 | |
| Potassium Chloride | Acros Organics | 424090010 | |
| Leucine | Fisher Scientific | BP385 | |
| Histidine | Fisher Scientific | BP382 | |
| Plan Achromat Infinity Corrected 100x Oil Immersion Objective NA=1.43 | Nikon Instruments | ||
| Spectrally resolved two photon microscope |
References
- Raicu, V. Nanoscopy and Multidimensional Optical Fluorescence Microscopy. Diaspro, A. , CRC Press. Boca Raton. (2010).
- Lakowicz, J. R. Principles of Fluorescence Spectroscopy. , Springer. New York. (2006).
- Raicu, V., Popescu, A.
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