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

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 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.

• Authors: Sabrina Simoncelli, Juliette Griffié, David J Williamson, Jack Bibby, Cara Bray, Rose Zamoyska, Andrew P Cope, Dylan M Owen
• Journal: Cell reports (Cell Rep) Vol. 33 Issue 12 Pg. 108523 (12 22 2020) ISSN: 2211-1247 [Electronic] United States
• PMID: 33357425 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

Chemical References

• Receptors, Antigen, T-Cell
• src-Family Kinases

Topics

• Humans
• Nanomedicine (methods)
• Receptors, Antigen, T-Cell (metabolism)
• Signal Transduction
• src-Family Kinases (metabolism)