Nanoimaging of DNA nanoagents interacting with cells

Many fundamental processes in biology happen at interfaces. Especially the cellular plasma membrane is of major interest for cell biological and biophysical studies, as it links the inner workings of cells to their surroundings and allows them to sense, interact, and react. Interactions of cell surface receptors with their ligands and other extracellular matrix constituents are crucial for various biological processes such as signaling, carcinogenesis, or adhesion. However, despite their paramount importance, little to no direct evidence of their interaction mechanism and single-molecule distribution on the cell surface is known. Fluorescence microscopy has evolved as an invaluable tool to investigate cell biology. Especially the advent of methods circumventing the classical diffraction limit of light, also known as super-resolution modalities, has already led to a remarkable success in revealing hidden details in subcellular structures.

In this funding period, we plan to develop methodologies using DNA-based super-resolution microscopy (DNA-PAINT) and self-assembled DNA nanostructures (DNA origami nanoagents) to probe and manipulate cell surface receptor molecules on the level of single proteins with thus far unprecedented detail and precision in control.
The first project is aimed to devise and optimize high-efficiency DNA-conjugated binders against the ErbB family of surface receptors for high-performance DNA-PAINT microscopy. One prominent member of the ErbB family is the epidermal growth factor receptor EGFR, which is frequently overexpressed in several types of cancers including glioblastoma, breast, lung, and colorectal carcinomas. The spatial organization of EGFR at the cell membrane is an important factor controlling downstream signaling and related functions. However, little is known about the detailed arrangement of single EGFR at the nanoscale. We will use novel affinity reagents combined with multiplexed, single-protein-sensitive DNA-PAINT to unravel some of the long-standing questions in the field of receptor tyrosine kinases: Are there pre-formed EGFR dimers prior to stimulation? How are oligomers arranged in small signaling clusters? How many EGF ligands are necessary to locally trigger downstream signaling? The latter will be achieved by using novel DNA-origami-based nanoagents (pioneered in the last phase of this CRC), which will carry a specific number of EGF ligands in defined distances to probe receptor-ligand interaction and downstream signaling.
In a second project, we will turn our attention to interactions of cells with their microenvironment through cell-matrix adhesions. Most studies focus on focal adhesions, which are integrin receptor mediated junctions. Ligands for integrin receptors are extracellular matrix proteins, like fibronectin or collagen. Integrins play a key role in force transduction from the microenvironment to cells and vice versa. Integrin-ligand interactions as well as force transduction via integrins were extensively studied over the last years, however little is known about single integrin-ligand bindings and force transduction on the level of single proteins. To fill this gap, we propose to use DNA-PAINT super-resolution microscopy to study single integrin-ligand binding events. A DNA hairpin-based force sensor, enabling investigation of forces and directionality of cells pulling on single integrin ligands by DNA-PAINT, will be developed. Hairpins will be bound to PEG-passivated glass surfaces and contain cyclic Arg-Gly-Asp (cRGD) ligands for integrin engagement and cell attachment. We will assay the distribution and single-molecule arrangement of open hairpin sensors, measure the directionality of force transduction and investigate the location and proximity of activated integrin molecules labeled using genetically encoded tags (Halo, SNAP, GFP, Alfa) in combination with talin, vinculin, and ultimately actin using Exchange-PAINT technology and novel labeling probes.
Taken together, we will shed light on single receptor-ligand interactions and the established tools and protocols will open an entirely new chapter of how researchers investigate cell signaling at the single receptor level.