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

Research output: Contribution to journalArticlepeer-review

Authors

  • Sabrina Simoncelli
  • Juliette Griffié
  • David J. Williamson
  • Jack Bibby
  • Cara Bray
  • Rose Zamoyska
  • Andrew P. Cope

Colleges, School and Institutes

Abstract

Summary
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.

Bibliographic 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.

Details

Original languageEnglish
Article number108523
JournalCell Reports
Volume33
Issue number12
Publication statusPublished - 22 Dec 2020

Keywords

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