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Sci Signal. 2016 Apr 19;9(424):rs3. doi: 10.1126/scisignal.aad4149.

Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics.

Author information

1
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.
2
Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
3
School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK.
4
School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
5
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.
6
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.

Abstract

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.

PMID:
27095595
PMCID:
PMC4871116
DOI:
10.1126/scisignal.aad4149
[Indexed for MEDLINE]
Free PMC Article

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