Quantitative super-resolution microscopy unravels nanoscale patterns of membrane receptor networks

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Poster Session 2
Marina S. Dietz (1), Yunqing Li (1), Claudia Catapano (1), Mark S. Schröder (1), Johanna V. Rahm (1), Tim N. Baldering (1), Marie-Lena I.E. Harwardt (1), Christos Karathanasis (1), Mike Heilemann (1)
1. Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, 60438 Frankfurt, Germany

single-molecule localization microscopy, super resolution microscopy, DNA-PAINT, single-particle tracking, membrane receptors, receptor tyrosine kinases

Abstract text

Macromolecular protein complexes that form in the cell membrane are the “communication hubs” of a cell to its environment. Triggered by ligands, cell-cell contact, or pathogens, membrane receptors form complexes of specified composition and function, thereby encoding external stimuli into information that is processed by the cell. This often involves the formation of protein assemblies that constitute through weak interactions of multivalent membrane-associated proteins. The complexity and heterogeneity of these protein assemblies demand for imaging methods that can achieve molecular resolution as well as read out the heterogeneity of such assemblies in the plasma membrane and that can operate in a native and unperturbed state of a cell. 

We determine the composition of membrane protein assemblies directly in cells by developing and applying tools for multi-protein super-resolution microscopy and quantitative image analysis including DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), photoactivated localization microscopy (PALM), and single-particle tracking.[1] With this toolbox, we visualize the organization of membrane receptor assemblies in the plasma membrane of cells and follow the dynamics of their assembly.[2-5] We study protein assemblies of receptor tyrosine kinases including members of the MET, the EGFR, and the FGFR family, their modulation through ligands, and their interaction with other membrane-associated proteins. The imaging and analysis technology is transferable to other multi-protein networks in cells and will help to understand the principles of molecular organization in the plasma membrane.  


[1] Dietz, M.S. and Heilemann, M. (2019) Optical super-resolution microscopy unravels the molecular composition of functional protein complexes. Nanoscale.

[2] Harwardt, M.-L.I.E. et al. (2020) Single-molecule super-resolution microscopy reveals heteromeric complexes of MET and EGFR upon ligand activation. IJMS 21, 2803.

[3] Schröder, M.S., Harwardt, M.-L.I.E., Rahm, J.V., Li, Y., Freund, P., Dietz, M.S. and Heilemann, M. (2020) Imaging the fibroblast growth factor receptor network on the plasma membrane with DNA-assisted single-molecule super-resolution microscopy. Methods. DOI: 10.1016/j.ymeth.2020.05.004.

[4] Baldering, T.N. et al. (2021) CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics. iScience 24, 101895. 

[5] Karathanasis, C. et al. (2020) Single-molecule imaging reveals the oligomeric state of functional TNFa-induced plasma membrane TNFR1 clusters in cells. Science signaling 13, eaax5647.