Proximity Labeling Expansion Microscopy (PL-ExM) resolves structure of the interactome

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Park, S.; Wang, X.; Li, X.; Huang, X.; Fong, K. C.; Yu, C.; Tran, A. A.; Scipioni, L.; Dai, Z.; Huang, L.; Shi, X.


Elucidating the spatial relationships within the protein interactome is pivotal to understanding the organization and regulation of protein-protein interactions. However, capturing the 3D architecture of the interactome presents a dual challenge: precise interactome labeling and super-resolution imaging. To bridge this gap, we present the Proximity Labeling Expansion Microscopy (PL-ExM). This innovation combines proximity labeling (PL) to spatially biotinylate interacting proteins with expansion microscopy (ExM) to increase imaging resolution by physically enlarging cells. PL-ExM unveils intricate details of the 3D interactome\'s spatial layout in cells using standard microscopes, including confocal and Airyscan. Multiplexing PL-ExM imaging was achieved by pairing the PL with immunofluorescence staining. These multicolor images directly visualize how interactome structures position specific proteins in the protein-protein interaction network. Furthermore, PL-ExM stands out as an assessment method to gauge the labeling radius and efficiency of different PL techniques. The accuracy of PL-ExM is validated by our proteomic results from PL mass spectrometry. Thus, PL-ExM is an accessible solution for 3D mapping of the interactome structure and an accurate tool to access PL quality.

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