Multi-color Molecular Visualization of Signaling Proteins Reveals How C-Terminal Src Kinase Nanoclusters Regulate T Cell Receptor Activation

Sabrina Simoncelli, Juliette Griffie, David Williamson, Jack Bibby, Cara Bray, Rose Zamoyska, Andrew Cope, Dylan Owen

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)
79 Downloads (Pure)

Abstract

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 pri- mary inhibitor of the T cell signaling pathway, Csk, and two binding partners implicated in its membrane as- sociation, 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-re- cruited 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 phospha- tase non-receptor type 22 (PTPN22) locus, is essential for Csk nanocluster re-recruitment and for mainte- nance of the synaptic PAG population.
Original languageEnglish
Article number108523
Number of pages19
JournalCell Reports
Volume33
Issue number12
Early online date22 Dec 2020
DOIs
Publication statusPublished - 22 Dec 2020

Keywords

  • Bayesian statistics
  • DNA-PAINT
  • T cells
  • Multiplexed
  • Nanoclusters
  • Single-molecule localization microscopy
  • Super-resolution microscopy
  • Csk

Fingerprint

Dive into the research topics of 'Multi-color Molecular Visualization of Signaling Proteins Reveals How C-Terminal Src Kinase Nanoclusters Regulate T Cell Receptor Activation'. Together they form a unique fingerprint.

Cite this