Imaging
Understanding how organisms grow and respond to their environment requires the ability to interact with and manipulate biochemical pathways in live cells in real time. A comprehensive characterization of live cells involves knowing where proteins localize and how and when they interact with other proteins to form functional pathways. Imaging techniques are especially powerful because they can provide spatial and temporal localization data, as well as generate information about molecular interactions, chemical environment, and physical properties. Our current research interests are focused on using genomics and live cell imaging tools to understand the function of proteins and networks in living cells.
Initial studies have focused on microbial pathways, including the glucose phosphotransferase system in E. coli. In order to visualize and track specific proteins in live cells, the proteins of interest must be labeled. One approach to protein labeling is through the use of recombinantly introduced peptide tags, such as green fluorescent protein (GFP) and its spectral derivatives. By genetically engineering protein fusions, perfect stoichiometry can be achieved, physiological responses can be measured in live cells, and additional labeling steps that might risk altering protein function are avoided.
Atomic force microscopy (AFM) is another imaging tool employed to provide a comprehensive analysis of protein networks. AFM allows molecular scale resolution of biological materials in physiologically relevant conditions and is capable of assessing the large proportion of the proteome that interacts at the cell membrane. In addition to imaging, the sensitivity of the AFM cantilever can be exploited to measure forces required to rupture bonds between biomolecules and within single protein molecules, and to identify single molecule interactions with specific target molecules on surfaces by tethering specific probe molecules to the cantilever.
