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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

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

Original languageEnglish
Article number108523
Number of pages19
JournalCell Reports
Volume33
Issue number12
Early online date22 Dec 2020
DOIs
Accepted/In press24 Nov 2020
E-pub ahead of print22 Dec 2020
Published22 Dec 2020

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King's Authors

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.

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