Phosphorylation of the negative regulatory element of the tyrosine kinase Lck by Csk down modulates T cell receptor induced signalling. Being constitutively active, Csk spatial organization is responsible for regulating this signalling interaction. Here, we used stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA qPAINT) to image the nanoscale spatial architecture of Csk and two binding partners implicated in its membrane association PAG and TRAF3. Combined with a newly developed coclustering analysis framework, we provide a powerful resource for dissecting signalling pathways regulated by spatiotemporal organisation. We found that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post stimulation and rerecruited at later time points. Unexpectedly, these clusters do not directly colocalise with PAG at the membrane, but instead provide a ready pool of monomers to down-regulate signalling. By generating CRISPR/Cas9 knock-out T cells, our data also identify that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population.
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