Felker, A.; Philippi, M.; Holtmannspötter, M.; Drees, C.; Schäfer, E.; Steinhart, M.; Kurre, R.; You, C.; Piehler, J.

Quantitative analysis of protein interactions and the formation of higher-order assemblies in living cells remains a major challenge. Here, we introduce a versatile nanopatterning toolbox that employs capillary nanostamping of functionalized polymers to generate high contrast biofunctionalized nanodot arrays (bNDAs) with diameters below 500 nm. By leveraging or-thogonal adaptor designs, we achieve robust immobilization of diverse fluorescent protein fusions, enabling simultaneous and selective recruitment of cytosolic and membrane-associated proteins into discrete nanodomains. This approach of forming cytosolic nanodot arrays (cNDAs) provides striking capabilities for dissecting cytosolic multiprotein complexes with molecular precision. Focusing on the assembly of the multimeric myddosome complex, we demonstrate density-dependent recruitment and co-localization of the core components MyD88, IRAK4, IRAK1, and TRAF6 within cNDAs. Super-resolution microscopy reveals dis-tinct nanoscale clustering of MyD88 and IRAK4 and uncovers the ultrastructural architecture of IRAK4 oligomers. These analyses highlight the spatial organization and hierarchical as-sembly of the myddosome at the nanoscale in the native cellular context. Collectively, our findings establish cNDAs as a powerful platform for reconstituting and analyzing intricate multiprotein assemblies in live cells, offering new opportunities for elucidating the principles of complex protein networks.