Protein-PAINT: Superresolution microscopy with signaling proteins

Area of Science:

• Cellular biology
• Microscopy
• Molecular signaling

Background:

• Superresolution microscopy advances cellular understanding but often requires bulky labels.
• Existing methods can lead to underlabeling and artifacts.
• Native protein interaction domains offer an alternative labeling strategy.

Purpose of the Study:

• To develop a novel single-molecule localization microscopy (SMLM) technique for visualizing nanoscale protein binding.
• To investigate T cell signaling by targeting Src homology 2 (SH2) domains.
• To overcome limitations of bulky fluorophore attachments in superresolution imaging.

Main Methods:

• Developed and applied protein point accumulation in nanoscale topography (pPAINT), a new SMLM technique.
• Utilized transient binding of SH2 domains to phosphorylated tyrosine residues for stochastic blinking.
• Employed total internal reflection microscopy for live-cell imaging and quantification.

Main Results:

• Visualized nanoscale binding sites of multiple T cell receptor-proximal signaling molecules (Zap70, PI3K, Grb2, Syk, Eat2, SHP2).
• Demonstrated multiplexing capability of pPAINT probes.
• Correlated binding half-life with binding site cluster size for PI3K and Eat2 SH2 domains.

Conclusions:

• pPAINT enables visualization of signaling protein binding at the nanoscale using native domains.
• The technique provides insights into phosphotyrosine-mediated signaling dynamics at the plasma membrane.
• pPAINT offers a powerful tool for studying cellular signaling with improved specificity and reduced artifacts.