Fluorescent protein development 1) We are studying photoswitching behaviors of photoswitchable fluorescent proteins and their use in Frster Resonance Energy Transfer (FRET) experiments. These studies have led to the development of techniques and reagents to test new approaches to performing FRET measurements as well as developing new photoswitchable fluorescent proteins with the necessary characteristics to properly perform these experiments. This includes an ongoing project to develop a photoswitchable yellow fluorescent proteins for use in conventional diffraction-limited microscopy as well as super-resolution molecular localization microscopy. 2) We collaborate with Joy Zhao and Peter Schuck on their development of new fluorescence ultracentrifugation techniques. In convert, we are surveying numerous fluorescent proteins to better define their oligomerization characteristics. Literature surveys have suggested these characteristics have not been rigorously determined and that unforeseen oligomerization leads to aberrant fluorescent protein behavior. 3) We have an ongoing project to develop improved red fluorescent proteins. Current variants display low fluorescence, slow maturation, and/or oligomerization. Biochemical analyses of wild type proteins coupled with site-directed mutagenesis has led to our discoveries of mRuby variants with much decreased self-association, increased brightness, and faster maturation. 4) We have continued efforts to improve two-step fluorescent probes. In addition to mutagenesis experiments to improve the folding and photoswitching behaviors of Padron and Kohinoor, the current standards, we are attempting to engineering positive photoswitching behaviors in other photoswitchable fluorescent proteins. Cell biology projects 1) We collaborated with Anamaris Colberg-Poley on super-resolution imaging of human cytomegalovirus infected cells. Our interest was in gaining insight into the transfer of pUL37x1 protein from mitochondria associated membranes to the outer mitochondria membrane. Recent work has been performed using 2-color imaging to compare the localizations of multiple proteins of interest has been reported 2) With Gaetan Herbomel, a post-doctoral fellow in the lab of Lawrence Tabak, we have a project to image the localization of Golgi apparatus enzymes using Stimulated Emission Depletion (STED) microscopy, Multifocal Structured Illumination Microscopy (MSIM), Photoactivated Localization Microscopy (PALM), and Stochastic Optical Reconstruction Microscopy (STORM). The multi-color super-resolution experiments have required development new data analyses which we have now implemented. These studies are intended to help in our understanding of where the enzymes are located within the Golgi and what role these locations may play in the enzymatic activity. Instrumentation and imaging development 1) Frster Resonance Energy Transfer (FRET) is a powerful approach to study the interactions of fluorescent molecules. We are developing new approaches which can be performed on a conventional widefield or confocal microscope to image FRET between a donor photoswitchable fluorescent protein, Dronpa, and an acceptor based on a conventional fluorescent protein. The technique which we call photoswitching FRET (psFRET) is based on Dronpas photoswitching properties in the presence and absence of an acceptor. This technique is currently be used to monitor interactions between histone proteins within living cells.
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