This technology is an approach to simultaneously track donor-acceptor-tagged proteins or pairs of proteins and observe their FRET signal during diffusion at the plasma membrane, thereby linking protein structural dynamics and protein-protein interactions to their movement at the cell membrane.
Single-molecule fluorescence resonance energy transfer (smFRET) imaging has emerged as one of the leading methods for studying the structural dynamics of proteins but is still used almost exclusively to study purified, surface-immobilized proteins. Since the function of proteins can change depending on their environmental conditions, characterizing the structural dynamics of proteins or protein-protein interactions in a native cellular environment is of central interest to molecular biophysics and critical to understanding protein function. Moreover, the motion state of a protein can provide information about microdomain localization that could influence the protein’s structure and function. As such, there is a need for a technology that links the structural dynamics of proteins with their dynamics of motion in a native cell membrane.
The in-cell study of structural protein dynamics and protein-protein interactions requires high resolution tracking of donor-acceptor labeled proteins and the measurement of the smFRET signal during protein diffusion. In contrast to existing confocal smFRET techniques (i. e. ALEX1), the smFRET method presented here is based on a wide-field imaging technique (TIRF) and offers the advantage of simultaneously detecting the time-dependent fluorescence emission of multiple diffusing proteins. Most importantly, the method allows for long observation times with single-molecule tracks on the order of seconds with ~10 millisecond time resolution. This time resolution can be sufficient for detecting large-scale protein domain motions as well as protein-protein interactions. The key invention is a computational approach necessary for integrating spatial trajectory data (data set 1) and two-color smFRET fluorescence intensity data (data set 2)2. With this approach, individual tracked molecules or pairs of molecules can be analyzed for their diffusion state and simultaneously for their calculated FRET signal. Both results are linked so that a) conformational changes in the protein and/or b) interactions of proteins can be correlated to their spatiotemporal motion profiles.
The software smCellFRET streamlines the data analysis of spatiotemporal smFRET data. Before employing the pipeline, smFRET imaging data are pre-analyzed using the open source particle tracking software u-track 3. Tracking results generated with u-track then serve as the main input for the analysis pipeline. Furthermore, the smCellFRET analysis pipeline is based on a co-tracking procedure, which is primarily controlled by a locally weighted mean transformation function. The transformation function is specific to the imaging system in use and must be measured by tracking a set of control points in both fluorescence emission channels of the microscope as described4. In subsequent steps, the program will then co-track the donor and sensitized acceptor data, calculate the background-corrected donor and acceptor intensities, filter out unwanted traces and finally calculate and correct the FRET values. Additionally, a DC-MSS (Divide-and-Conquer Moment Scaling Spectrum) motion analysis5 of acceptor tracks is performed to detect diffusion segments in individual tracks and to classify them as free, confined, directional and immobile diffusion. All results of the tracking, diffusion and FRET signal analysis are linked and saved in a generic structure variable that can be evaluated via a graphical user interface.
With the graphical user interface (GUI) cellFretViewtraces (see Figure) the user can manually browse through the output data of the smCellFRET pipeline. The GUI allows the user to play back the originally recorded smCellFRET movie and to observe the performance of the tracker for each individual single-molecule, frame by frame. Each detected particle in a smCellFRET image has a specific identification number that allows the user to examine the associated data (intensity, signal-to-noise ratio, track length, etc.). Data such as the donor and acceptor fluorescence intensity, total intensity, FRET-efficiency, diffusion mode and the donor-acceptor single molecule tracks are displayed directly in the GUI. This level of control allows the user to adjust specific settings in the pipeline and to repeat the data analysis until an optimized result is obtained.
Jonathan Javitch, M.D., Ph.D.
Currently, there is no software package that allows the processing of wide-field single-molecule FRET and single-molecule tracking data.
Jonathan A. Javitch, Peter Geggier, Wesley B. Asher, Signe Mathiasen, Scott C. Blanchard, Daniel S. Terry, Khuloud Jaqaman
live cell imaging, fluorescence resonance energy transfer, FRET, single molecule, tracking, software, Matlab, structural protein dynamics, diffusion.
To request a license to download and use the smCellFRET software, please click on "Express Licensing", above.