Imaging
A primary goal of our imaging studies is to confirm protein complexes identified by the Center for Molecular and Cellular Systems and to provide biological and physical context to these interactions. Live cell-based assays are ideal complements to analysis approaches that require the complexes to be removed from the cell. Measurements in situ, within the cell, obviate the need for approximating physical and chemical conditions and eliminate the need for protein isolation.
Figure 1. Diagram of protein interaction detected by FRET
Fluorescence microscopy
Fluorescence microscopy is the premiere technique for localizing and identifying molecular species within live cells. It has become the standard for live cell imaging as a variety of labels and instruments are readily available. Enabling these approaches are recombinantly introduced fluorescent protein tags. Green fluorescent protein (GFP), and its spectral derivatives YFP (yellow fluorescent protein) or CFP (cyan fluorescent protein), are relatively small, well characterized monomeric proteins (~27-40 kDa) that do not require exogenously added substrate or co-factors for fluorescence. This makes them ideal for live cell imaging studies. Additionally, specialized techniques such as fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP), as well as new reagents are aiding in localizing and characterizing dynamic events and biomolecular interactions.
The technique of FRET is commonly employed for assessing protein interactions in live cells. FRET efficiency is proportional to the inverse sixth power of the distance between two fluorescent molecules (“donor” and “acceptor” pairs), thus leading to relatively large changes in FRET efficiency with subtle changes in the distance between the two dyes (Å resolution). This property makes FRET a highly sensitive and reliable reporter of molecular interactions that occur within less than 100 Å (Figure 1).
Another imaging-based approach to characterizing protein interactions in the cell is based on the co-localization of protein fluorescence. Typically, two proteins of interest are tagged with different fluorophores and imaged within the cell. If the fluorescence signals are co-localized, a molecular interaction is suggested, with FRET measurements providing confirmation of a short-range, molecularly dependent interaction.
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Figure 2.: Co-localization based protein interaction assay. (A) Diagram of assay. The DivIVA-POI1 fusion localizes to the cell’s poles following induction with arabinose. If POI2 interacts directly with POI1, the GFP-POI2 fusion protein is recruited to the cell poles. If there is no interaction between POI1 and POI2, the GFP-POI2 fusion protein remains diffuse. (B) Example of positive interaction between R. palustris GroEL2 and GroES2. |
Figure 3. Sample results: interactions among R. palustris GroEL-GroES subunits. (A)Table of all 16 pairwise combinations tested. (+) represents a positive interaction; (-) represents a negative interaction. (B) Images of cells showing GFP-GroEL2 localization before (left) and after (right) DivIVA-GroES1 expression. Recruitment of GFP-GroEL2 to the cell poles is indicative of a positive interaction between GroES1 and GroEL2. |
A modified co-localization assay can be used to confirm a specific association between two proteins of interest. Ding et al. (2002) have described an imaging based assay that depends on the localization properties of a cell division protein. This assay involves co-expression of two fusion proteins in E. coli (Figure 2). The first protein of interest (POI1) is fused to DivIVA, a cell division protein from Bacillus subtilis [Edwards, 1997], which directs localization of the fusion protein to the cell poles. For this assay, expression of the DivIVA fusion protein is tightly regulated by an arabinose inducible promoter. The second protein of interest (POI2) is fused to GFP and expressed in E. coli cells from a T7 promoter. Upon induction of the DivIVA fusion protein, the second protein of interest will be recruited to the cell poles if a protein interaction occurs (Figure 2). The assay is conveniently implemented in E. coli and could be adapted to other Gram-negative bacteria. Detection of interactions between R. palustris GroEL and GroES proteins are shown in Figure 3.
Scanning probe microscopy of membrane bound protein complexes
Figure 4. Diagram of atomic force microscope. Adapted from Hansma 2001. Ann. Rev. Phys. Chem 52:71-92.
The scanning probe microscope (SPM) is ideally suited for characterizations at or near the cell surface (Figure 4). Membrane bound proteins can be studied in viable bacterial spheroplasts, isolated membranes, and in lipid bilayers (Figure 5). The nanometer scale resolution of atomic force microscopy (AFM) can be used to image membrane bound proteins in a liquid environment and in many cases sub-molecular structure of individual proteins can be resolved. In addition to imaging, sensitivity of the AFM cantilever can be exploited to measure forces required to rupture bonds between biomolecules, within single protein molecules, and by tethering specific probe molecules to the cantilever to identify single molecule interactions with specific target molecules on surfaces.
Figure 5. (A) Diagram showing spheroplasts mounted in layer of gelatin. (B-D) Amplitude images of spheroplasts immobilized in gelatin. (B) representative 75µm scan taken at 512 pixels per line at a speed of 2 Hz; (C,D) To demonstrate the stability of the preparation technique, two spheroplasts were imaged (C) after 4 scans at 1.3Hz and compared with the image of the same spheroplasts imaged (D) after 20 scans at 1.3Hz over a 3 hour period.
Measurement of molecular interactions
One attribute of scanning probe microscopy is its ability to measure forces. These forces can be between the surface and the scanning probe tip or between molecules that are specifically tethered to these surfaces. These force measurements can be collected while scanning or from specific locations.
Related Publications
2009
- Abstract: Jennifer L. Morrell-Falvey, et al., An Imaging-Based Assay with High Sensitivity for Confirming and Characterizing Protein Interactions, 2009 Genomics:GTL Awardee Workshop VII, Bethesda, Maryland.
2007
- Article: C.J. Sullivan, S. Venkataraman, S.T. Retterer, D.P. Allison, and M. J. Doktycz, "Comparison of the Indentation and Elasticity of E. coli and its Spheroplasts by AFM," Ultramicroscopy, 107, 934-942, 2007. [PDF]
2006
- Article: S. Venkataraman, D.P. Allison, H. Qi, J.L. Morrell-Falvey, N.J. Kallewaard, J.E. Crowe Jr., and M.J. Doktycz, "Automated image analysis of atomic force microscopy images of Rotavirus particles," Ultramicroscopy 106, 829-37, 2006
- Article: S. Venkataraman, M.J. Doktycz, H. Qi, and J.L. Morrell-Falvey, "Automated analysis of fluorescence microscopy images to identify protein-protein interactions," International Journal of Biomedical Imaging, Article ID 69851, 2006
2005
- Article: C. J. Sullivan, J. L. Morrell, D. P. Allison, M. J. Doktycz, "Mounting of Escherichia coli spheroplasts for AFM imaging," Ultramicroscopy, 2005, Vol. 105, 96-102. [PDF]
2004
- Article: M. Micic, D. Hu, G. Newton, M. Romine, and H. P. Lu, "Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells." Surface and Colloid B. 34:205-12, 2004.
2003
- Article: M. J. Doktycz, C. J. Sullivan, P. R. Hoyt, D. A. Pelletier, S. Wu, and D. P. Allison, "AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces," Ultramicroscopy, Volume 97, Issues 1-4, October-November 2003, Pages 209-216. [Abstract, Full Text, PDF open in new windows]
