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

TY - JOUR

T1 - Multi-color Molecular Visualization of Signaling Proteins Reveals How C-Terminal Src Kinase Nanoclusters Regulate T Cell Receptor Activation

AU - Simoncelli, Sabrina

AU - Griffie, Juliette

AU - Williamson, David

AU - Bibby, Jack

AU - Bray, Cara

AU - Zamoyska, Rose

AU - Cope, Andrew

AU - Owen, Dylan

PY - 2020/12/22

Y1 - 2020/12/22

N2 - 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.

AB - 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.

KW - Bayesian statistics

KW - DNA-PAINT

KW - T cells

KW - Multiplexed

KW - Nanoclusters

KW - Single-molecule localization microscopy

KW - Super-resolution microscopy

KW - Csk

UR - http://www.scopus.com/inward/record.url?scp=85098055087&partnerID=8YFLogxK

U2 - 10.1016/j.celrep.2020.108523

DO - 10.1016/j.celrep.2020.108523

M3 - Article

SN - 2211-1247

VL - 33

JO - Cell Reports

JF - Cell Reports

IS - 12

M1 - 108523

ER -