Decoding Molecular Mechanisms of Nuclear Organization with Multiplexed Nanoscopy

• Applicant: Dr. Johannes Stein
• Subject Area: Biophysics ; Structural Biology ; Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 561460937

A fundamental challenge in modern biology is understanding the relationship between higher-order genome organization and genome function. While the intricate interplay between 3D chromatin structure and gene regulation is ultimately mediated by molecular complexes, our ability to spatially probe these molecular interactions remains constrained. Current genomics approaches often rely on population averages, obscuring single-cell heterogeneity, and frequently examine few individual targets. Combinatorial fluorescence imaging enables examinations of numerous targets within single cells but is resolution-limited. To address this, my recent work focused on developing tokPAINT, a novel imaging technique that integrates ultrathin Tokuyasu cryosectioning with DNA-PAINT super-resolution microscopy. This approach overcomes limitations of classic DNA-PAINT imaging in crowded cellular compartments such as the nucleus, enabling multiplexed imaging at the single-protein level and counting of target molecules. With this technology, the proposed research aims to provide novel insights into two central processes linked to mammalian genome organization: i) cohesin-mediated chromatin loop extrusion and ii) chromatin organization around nuclear speckles, membraneless bodies believed to assemble through phase separation.The first research aim will use tokPAINT to quantitatively examine the stoichiometry and spatial distribution of cohesin complexes as they form chromatin loops, addressing longstanding questions about cohesin’s functional assembly. By precisely quantifying cohesin and CTCF interactions in 3D and analyzing additional regulatory factors, this work will provide insights into the molecular mechanisms governing loop extrusion. The second aim focuses on nuclear speckle ultrastructure, exploring their anisotropic shape and internal organization through multiplexed imaging of speckle components, such as SRRM2, SON, and RNA polymerase II. By examining how nuclear speckles interact with chromatin and change in response to transcriptional inhibition, this project will illuminate the regulatory roles of nuclear speckles in gene expression.From a technological perspective, the project will enhance tokPAINT’s capabilities by integrating automated fluidics and machine learning for high-throughput imaging and serial volumetric reconstruction. The nucleus serves as an ideal model system for crowded compartments, paving the way for harnessing the full potential of tokPAINT in ultrathin sections of tissues, bacteria, and plants. This research will not only advance our understanding of genome organization but also demonstrate the potential of single-protein imaging to dissect molecular mechanisms in biology. Finally, the insights gained from this work could have broad implications, clarifying the origins of diseases linked to disrupted genome organization, such as cancer and neurodevelopmental disorders, and opening new avenues for therapeutic intervention.

• DFG Programme: Emmy Noether Independent Junior Research Groups
• Major Instrumentation: TIRF super-resolution microscopy setup (self-assembled)
• Instrumentation Group: 5040 Spezielle Mikroskope (außer 500-503)