Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics.
Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems.
School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK. Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Kole T
KT
Roybal
Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Taráz E
TE
Buck
Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Xiongtao
X
Ruan
Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Baek Hwan
BH
Cho
School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK.
Danielle J
DJ
Clark
School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK.
Rachel
R
Ambler
School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK.
Helen M
HM
Tunbridge
Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Jianwei
J
Zhang
School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
Paul
P
Verkade
School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK. Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. christoph.wuelfing@bristol.ac.uk murphy@cmu.edu.
Christoph
C
Wülfing
Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA. Departments of Biological Sciences, Biomedical Engineering, and Machine Learning, Carnegie Mellon University, Pittsburgh, PA 15213, USA. Freiburg Institute for Advanced Studies and Faculty of Biology, Albert Ludwig University of Freiburg, Freiburg im Breisgau 79104, Baden-Württemberg, Germany. christoph.wuelfing@bristol.ac.uk murphy@cmu.edu.
Robert F
RF
Murphy
D008841:Actin Cytoskeleton / Q000378:metabolism*; D051339:Actin Depolymerizing Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D018106:CD28 Antigens / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D019295:Computational Biology / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054992:Immunological Synapses / Q000378:metabolism; D007700:Kinetics; D008213:Lymphocyte Activation; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D059008:Time-Lapse Imaging / Q000379:methods; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000235:genetics / Q000378:metabolism*
D051339:Actin Depolymerizing Factors; D018106:CD28 Antigens; D011948:Receptors, Antigen, T-Cell; D051300:Wiskott-Aldrich Syndrome Protein Family; D049452:Green Fluorescent Proteins