loading . . . A Modular Toolkit for Nanoscale Interrogation of Multiprotein Assemblies Inside Living Cells Quantitative analysis of protein interactions and the formation of higher-order assemblies in living cells remain major challenges. 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 orthogonal adaptor designs, we achieve robust immobilization of diverse fluorescent protein fusions, enabling simultaneous and selective spatially controlled enrichment of cytosolic proteins into high-density cytosolic nanodot arrays (cNDAs). Focusing on the assembly of the multimeric myddosome complex, we demonstrate density-dependent recruitment and colocalization of the core components MyD88, IRAK4, IRAK1, and TRAF6 within cNDAs. Super-resolution microscopy revealed the distinct nanoscale clustering of MyD88 and IRAK4 and uncovered the ultrastructural architecture of IRAK4 oligomers. These analyses highlight the spatial organization and hierarchical assembly 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 exciting opportunities for elucidating the mechanistic principles underlying complex protein networks. https://doi.org/10.1021/acsnano.5c14657
