Protein-PAINT: Superresolution microscopy with signaling proteins

• DOI: 10.1126/scisignal.abg9782
• Publication Date: 2022-02-01T18:58:08Z
• Authors: Megan V. Farrell; Andrea C. Nunez; Zhengmin Yang; Pablo Pérez-Ferreros; Katharina Gaus; Jesse Goyette
• Keywords: Cell Signaling; Cell membrane

Superresolution techniques have advanced our understanding of complex cellular structures and processes but require the attachment fluorophores to targets through tags or antibodies, which can be bulky result in underlabeling. To overcome these limitations, we developed a technique visualize nanoscale binding locations signaling proteins by taking advantage their native interaction domains. Here, demonstrated that pPAINT (protein point accumulation topography) is new, single-molecule localization microscopy (SMLM) used it investigate T cell visualizing Src homology 2 (SH2) domain, common molecules. When SH2 domain–containing relocate plasma membrane, domains selectively, transiently, reversibly bind preferred phosphorylated tyrosine residues on receptors. This transient yields stochastic blinking events necessary for SMLM when observed with total internal reflection enables quantification coefficients intact cells. We reveal sites several receptor–proximal molecules, including Zap70, PI3K, Grb2, Syk, Eat2, SHP2, showed probes could multiplexed. half-life tandem domain PI3K correlated site cluster size at immunological synapses cells, longer lifetimes were associated smaller clusters monovalent Eat2. These results demonstrate potential investigating phosphotyrosine-mediated membrane.