Multi-color molecular visualization of signaling proteins reveals how C-terminal Src kinase nanoclusters regulate T cell receptor activation

Sabrina Simoncelli, Juliette Griffié, David J. Williamson, Jack Bibby, Cara Bray, Rose Zamoyska, Andrew P. Cope, Dylan M. Owen

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Elucidating the mechanisms that controlled T cell activation requires visualization of the spatial organization of multiple proteins on the submicron scale. Here, we use stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-PAINT) to image the nanoscale spatial architecture of the primary inhibitor of the T cell signaling pathway, Csk, and two binding partners implicated in its membrane association, PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we find that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and are re-recruited at later time points. Unexpectedly, these clusters do not co-localize with PAG at the membrane but instead provide a ready pool of monomers to downregulate signaling. By generating CRISPR-Cas9 knockout T cells, our data also identify that a major risk factor for autoimmune diseases, the protein tyrosine phosphatase non-receptor type 22 (PTPN22) locus, is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population.
Original languageEnglish
Article number108523
JournalCell Reports
Issue number12
Publication statusPublished - 22 Dec 2020

Bibliographical note

Funding Information:
This work has been supported by BBSRC grant BB/R007365/1 and Versus Arthritis grant 20525 . S.S. acknowledges financial support from the Human Frontier Science Program Organization and the Royal Society through HFSP ( LT001463/2017-C ) and Dorothy Hodgkin ( DHF\R1\191019 ) fellowships, respectively. R.Z. acknowledges funding from Wellcome Trust Investigator Award WT25014/Z/16/Z . We acknowledge the use of the Nikon Imaging Facility (NIC) at King’s College London for data acquisition.


  • Bayesian statistics
  • Csk
  • PTPN22
  • T cells
  • multiplexed
  • nanoclusters
  • quantitative imaging
  • single-molecule localization microscopy
  • super-resolution microscopy

ASJC Scopus subject areas

  • General Biochemistry,Genetics and Molecular Biology


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