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A Modular Toolkit for Nanoscale Interrogation of Multiprotein Assemblies Inside Living Cells
Arthur Felker1, Michael Philippi1, Michael Holtmannspötter1
1Department of Biology/Chemistry and Center for Cellular Nanoanalytics, Osnabrück University, Barbarastr. 11, Osnabrück 49076, Germany.
ACS Nano
|March 6, 2026
Summary
We developed a nanopatterning toolbox to create nanodot arrays for studying protein assembly in live cells. This method visualizes the nanoscale organization of the myddosome complex, advancing our understanding of cellular protein networks.
Area of Science:
- Cellular Biology
- Nanotechnology
- Biophysics
Background:
- Quantitative analysis of protein interactions and higher-order assembly in living cells is challenging.
- Existing methods lack the resolution and control for studying nanoscale protein organization.
Purpose of the Study:
- To introduce a versatile nanopatterning toolbox for creating biofunctionalized nanodot arrays (bNDAs).
- To enable spatially controlled enrichment and analysis of cytosolic proteins within live cells.
- To investigate the nanoscale assembly and organization of the myddosome complex.
Main Methods:
- Capillary nanostamping of functionalized polymers to generate bNDAs (<500 nm diameter).
- Orthogonal adaptor designs for robust immobilization of fluorescent protein fusions.
- Super-resolution microscopy for nanoscale imaging of protein assemblies.
Main Results:
- Demonstrated density-dependent recruitment and colocalization of myddosome components (MyD88, IRAK4, IRAK1, TRAF6) within cytosolic nanodot arrays (cNDAs).
- Revealed distinct nanoscale clustering of MyD88 and IRAK4.
- Uncovered the ultrastructural architecture of IRAK4 oligomers in the native cellular context.
Conclusions:
- Cytosolic nanodot arrays (cNDAs) provide a powerful platform for reconstituting and analyzing intricate multiprotein assemblies in live cells.
- The findings offer new opportunities for elucidating the mechanistic principles of complex protein networks at the nanoscale.
- Established a novel approach for high-resolution quantitative analysis of protein organization and function in vivo.
