NANOTRACKING THE MOLECULE'S FATE IN LIVING CELLS SPLIT SP 75% SH 25% NB 75OHD=FACILITIES DO NOT SPLIT ALL SP

In this joined project between the two Departments, Physiscs and Pharmacy & Pharmacology, we successfully developed a novel methodological approach for selective labelling and mapping of individual protein complexes on the surface of biological cells using magnetic nanobeads. The location of the labelled proteins was detected with an Atomic Force Microscope (AFM) and then confirmed in the Magnetic Force Microscopy (MFM) mode. This approach allowed to specifically detect an individual protein on the native cell surface which has a complex relief and potentially allow the investigation of magnetically labelled protein complexes both on the surface and beneath the cell membrane at significantly higher resolution than modern light imaging techniques.

Specifically, the experiments were performed on single smooth smooth cells isolated from the rat aorta. Endothelin (ET) receptors activated by endothelins, short peptides which upon tight binding to the receptor cause contraction of smooth muscle cells and are important in the regulation of blood pressure in humans) have been chosen as the target protein complex on the surface of the cell. ET-1, an endothelin receptor agonist, was initially biotinylated using a commercial kit and forming biotinylated ET-1 (bET-1). The ability of bET-1 to interact with ET receptors was confirmed in functional contraction studies. bET-1was then allowed to interact with the receptors on the surface of the cell. As the binding affinity of ET-1 to the receptor and therefore the dissociation of the agonist from the receptor is slow, the complex bET-1-receptor was sufficiently stable even after wash out of agonist. Cells were then treated with commercially available anti-biotin coated paramagnetic nanobeads of ~50 nm in diameter (Miltenyi Biotec, UK). This resulted in labelling of bET-1-receptor complexes with magnetic nanoparticles. The complexes were detected with AFM and then with MFM. Our findings demonstrated for the first time that ET-1 receptors distributed both individually and in the form of receptor complexes on the surface of vascular smooth muscle cells. Although detection of membrane proteins with AFM was reported previously, most of the work has been done in cells overexpressing the target protein and using antibodies (which, depending on their selectivity, may also bound to other proteins) against a target protein. In this project, use of a highly specific endogenous receptor agonist as a target allowed us to detect individual receptor molecules on the surface of native cells. The usage of both AMF and MFM ascertain that specific highlighted peaks are indeed the receptors of interest.

The main scientific outcome of the grant is the development of a novel method for selective labelling of proteins using magnetic nanoparticles. This method was demonstrated to have much better resolution (potentially two orders of magnitude) than other labelling techniques. It allows to determine location of individual proteins not only on the cell membrane but also under the membrane on the depth up to 150 nm. In our opinion, this is a significant breakthrough in cell biology.