Multi-colour DNA-qPAINT reveals how Csk nano-clusters regulate T-cell receptor signalling

Abstract

Phosphorylation of the negative regulatory element of the tyrosine kinase Lck by Csk downmodulates 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 co-clustering analysis framework, we provide a powerful resource for dissecting signalling pathways regulated by spatio-temporal 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 co-localise 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 rerecruitment and for maintenance of the synaptic PAG population.

Quantitative single molecule analysis of podoplanin clustering in fibroblastic reticular cells uncovers CD44 function

“…The high level of nanometre accuracy attained by DNA-PAINT (i.e. it can routinely obtain sub-10 nm localization precision) has proven capable of imaging individual molecular targets on synthetic samples that simulates biomolecular nanoclusters [18], as well as protein clusters in fixed cells [19][20][21]. Furthermore, due to the predictable binding kinetics between imager and docking strands, it is possible to correlate the frequency of single-molecule events with the underlying number of labelled molecular targets [22] overcoming 'overcounting' artefacts observed with other SMLM techniques.…”