Advancing DNA-PAINT super-resolution microscopy to uncover the structural basis of membrane receptor biology

Pachmayr, Isabelle (2025): Advancing DNA-PAINT super-resolution microscopy to uncover the structural basis of membrane receptor biology. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Super-resolution microscopy methods have revolutionized fluorescence imaging by surpassing the diffraction limit of light, a breakthrough recognized with the Nobel Prize in Chemistry in 2014. One class of these techniques is single-molecule localization microscopy (SMLM), which routinely achieves resolutions of ~20 nm by temporally separating fluorophores through stochastic on- and off-switching (“blinking”) and localizing subsets of single molecules with high precision. DNA-PAINT (DNA Points Accumulation for Imaging in Nanoscale Topography) has emerged as a powerful SMLM technique that relies on the transient binding of dye-labeled imager strands to complementary DNA docking strands. DNA-PAINT offers several advantages: it is resistant to photobleaching due to the continuous supply of imager strands in solution, follows predictable DNA hybridization kinetics, and enables high multiplexing. However, in the cellular context, its resolution is typically limited to ~10 nm, primarily due to the limited number of photons that can be collected per localization. Accessing sub-10 nm resolution is critical for detecting and resolving cell membrane receptors. These receptors are key regulators of cellular fate, processing external signals through the cell membrane, and are highly specific drug targets. The nanoscale organization of membrane receptors, and its modulation by ligand or drug binding, governs the activation of downstream cellular processes. To understand molecular arrangements within cells, the first part of my thesis advances DNA-PAINT imaging to the molecular scale in a cellular context by introducing Resolution Enhancement by Sequential Imaging (RESI) (Publication 1). Through stochastic labeling and sequential imaging of sparse subsets, RESI achieves Ångström-resolution in DNA origami structures. Applying RESI at ~1 nm resolution in cells enabled the detection of antibody-induced rearrangements of the membrane receptor CD20 at the molecular scale. In the second part of my thesis, I extended RESI to two-target imaging in 3D to visualize CD20 in complex with therapeutic antibodies (Publication 2). Anti-CD20 antibodies are classified into Type I and Type II based on their functional properties. To investigate how these differences relate to receptor organization, I quantitatively analyzed RESI data before and after antibody treatment, uncovering the distinct structural arrangements of Type I and Type II antibodies. Combining RESI with functional assays revealed a distinct pattern of CD20 oligomerization that drives the shift from Type II to Type I antibody function. These findings support a minimal model describing a continuum between oligomerization state and antibody function for anti-CD20 antibodies. The third part of my thesis presents a novel strategy for DNA-PAINT and RESI imaging of small extracellular ligands, using Epidermal Growth Factor (EGF) as a model system (Publication 3). Understanding the molecular arrangement of EGF during its interaction with the Epidermal Growth Factor Receptor (EGFR) is essential for dissecting receptor signaling pathways that govern cell survival and proliferation. To enable functional ligand labeling for super-resolution imaging, two tagged EGF constructs – ALFA-EGF and DNA-EGF – were compared. The ALFA-tagged variant more effectively preserved EGFR binding and dimerization, showing that ALFA-tagging maintains EGF function while enabling stoichiometric labeling. This establishes ALFA-tagging as a broadly applicable strategy for ligand labeling in DNA-PAINT and RESI microscopy. Together, these advances establish RESI as a powerful method for achieving molecular-scale resolution in cells and expand the capabilities of DNA-PAINT for studying receptor-ligand interactions.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 21. Oktober 2025
• 1. Bericht­erstatter:in: Klein, Christian