Biophotonics

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Linked publications & trials

Abstract

Fluorescent protein development 1) We are studying photoswitching behaviors of photoswitchable fluorescent proteins and their use in Forster Resonance Energy Transfer (FRET) experiments. This includes an ongoing project to develop photoswitchable yellow fluorescent proteins, better photoswitchable red fluorescent proteins, and far-red fluorescent proteins for use in conventional diffraction-limited microscopy as well as super-resolution molecular localization microscopy. Current emphases are on developing multi-label FRET experiments(3-4 interacting proteins) using combinations of psFRET and acceptor photoswitching methods and well as developing mutants with disparate photoswitching kinetics. 2) We continue our collaboration with Joy Zhao and Peter Schuck on surveying numerous fluorescent proteins to better define their oligomerization characteristics. These characteristics have not been rigorously determined and unforeseen oligomerization leads to aberrant fluorescent protein behavior. Our efforts are to develop variants with little to no self-association behaviors. 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 and Scarlet variants with much decreased self-association, increased brightness, and faster maturation. We have upgraded our instrumentation and data analyses to better screen mutant forms expressed in bacteria colonies growing on agar plates. Cell biology projects 1) We have completed a project to image the localization of Golgi apparatus enzymes using Stochastic Optical Reconstruction Microscopy (STORM). The multi-color super-resolution experiments have required development an ImageJ plugin for single molecule co-localization analyses. 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. We are currently developing electron microscopy methods in collaboration with Maria Aronova and Richard Leapman in our efforts to more precisely determine the relative localizations of these molecules. 2) We collaborate with Jim Kochenderfer (NCI) to study the self-association properties of chimeric antigen receptor (CAR) molecules using our psAFRET technique (see below). 3) We are utilizing our psFRET technique in conjunction with photoswitchable fluorescent protein tagged histone proteins to determine the binding sites of anti-cancer drugs, such as doxorubicin, within the chromatin of live cells. In related studies, we are also monitoring the proximity of PS-FP tagged histone proteins during chromatin compaction using psAFRET. 4) We collaborate with the Koret Hirschberg (Tel Aviv University) to image endoplasmic reticulum (ER) exit site machinery using super-resolution fluorescence techniques. In addition, we are utilizing our psFRET imaging technique to monitoring protein-protein interactions within the secretory pathway. 5) We collaborate with the Rafael Carazo Salas lab to develop new FUCCI sensors to help expand the multiplexing imaging capabilities of cells as they proceed through the stages of the cell cycle. Instrumentation and imaging development 1) Forster Resonance Energy Transfer (FRET) is a powerful approach to study the interactions of fluorescent molecules. We are continuing to develop 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 photoswitching kinetics of a photoswitchable fluorescent protein in the presence and absence of an acceptor. We are exploring new psFRET approaches which combine photoswitching kinetics and fluorescence lifetime imaging to maximize the informational content of the experiments. 2) Another FRET based project using psFPs derived directly from the psFRET studies involves imaging homo-FRET by monitoring the anisotropy of the PS-FPs during the photoswitching process as a read-out of protein-protein interactions. This technique, which we called photoswitching anisotropy FRET (psAFRET), differs from measurements of hetero-FRET (energy transfer between two different color proteins) since it monitors energy transfer between copies of the same probe. This project has been expanded to monitoring hetero-FRET using anisotropy changes in the acceptor molecule as an approach to improve the sensitivity of FRET measurements. 3) We have developed and continuing to upgrade image analysis macros that can be run within the ImageJ application to analyze psFRET and psAFRET data. We have developed and are continuing to upgrade ImageJ plugins that are more versatile and can be used on almost any dataset. In addition, plugins available on the ImageJ update site (https://imagej.net/User:Pattersg) to analyze the exponential decay in fluorescence at each pixel as the photoswitchable fluorescent protein switches off. We collaborate with the Hari Shroff lab on the development of improved forms of TIRF microscopy. These have resulted in improvements to lateral super-resolution levels while maintaining the acquisition speeds necessary for live cell imaging. Our collaboration is currently focused on improving axial resolution through the use of photoswitchable fluorescent proteins. --- Zhao H, Wu D, Nguyen A, Li Y, Ado RC, Valkov E, Patterson GH, Piszczek G, Schuck P. Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids. 2021. iScience. May 7:102523. doi: 10.1016/j.isci.2021.102523. Online ahead of print. PMID: 33997662