Murphy Lab

 Cytometry Development Workshop

 Flow Cytometry



 Carnegie Mellon University
 Computational Biology Department
 Center for Bioimage Informatics
 Biological Sciences Department
 Biomedical Engineering Department
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Movies and Maps of protein distributions during T cell signalling

This page contains raw data and computationally-derived maps from the manuscript
K. T. Roybal, T. E. Buck, X. Ruan, B. H. Cho, D. J. Clark, R. Ambler, H. M. Tunbridge, J. Zhang, P. Verkade, C. Wülfing, and R. F. Murphy (2016) Automated spatiotemporal analysis identifies WAVE2 and Cofilin as joint regulators of costimulation-mediated T cell actin dynamics. Science Signaling 9:rs3. DOI: 10.1126/scisignal.aad4149


Movies consist of frames of DIC and fluorescence containing fields of T cells expressing an eGFP-tagged protein and antigen-presenting cells expressing the appropriate antigen. Frames were taken at various times, and the time and position of synapse formation has been manually annotated. The movies and annotations files are are grouped into ZIP files for each tagged protein. The ZIP files range in size from 1 GB to 8 GB.
ProteinFull StimulusB7 BlockedB7 Block with active Rac and cofilin

The annotations that accompany these movies, marking the locations and times of synapse formations, are contained in Excel spreadsheets in this zip file:


The ZIP file below contains maps for both the average values and the standard deviations across all cells for each sensor, time point and condition. Maps are contained in separate files for each time point and consist of fluorescence values for each voxel of a 3D template. Within each ZIP file is a folder containing images (scaled to the maximum average value for a given map) and a folder containing comma-separated value (csv) files containing the relative fluorescence intensity values (scaled to a fraction of the total fluorescence). Also included is a csv file containing the average cellular concentration of each probe (except MRLC). A Matlab function that can read the csv files and return 3D images either as relative fluorescence or concentration values is also available. [24 MB]
README file for the ZIP file

Generated Movies

Movie S1.4D Maps of three representative sensors. Each frame of the movie depicts the distribution of three proteins at a given time point, shown as slices perpendicular (top) or parallel (bottom) to the immunological synapse. Those voxels that contain the top 25% of the fluorescence of WAVE2, MRLC, and cofilin are shown in red, green and blue, respectively (with voxels containing more than one protein shown in composite colors). Note that the three proteins have different distributions before immunological synapse formation, that MRLC addition to the synapse is delayed relative to the other two proteins, and that cofilin and WAVE2 leave the synapse in sequence.
Movie S2.4D Maps as in Movie S1 but shown as surface perspective plots. The colors for Cofilin and WAVE2 are switched: Cofilin is shown in red and WAVE2 is shown in blue.


Please contact Bob Murphy for questions or help.

Last Updated: 19 Apr 2016

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