Global analysis of the biophysical properties of ErbB4

The cell membrane contains receptors whose activation by growth factors induces cells to undergo differentiation or cell proliferation. Understanding of these processes is of utmost importance since both normal ontogeny and organogenesis as well as cancer development are determined by the balance or lack of balance of these signals. The epidermal growth factor (EGF) receptor, also known as ErbB1, has been well characterized at the molecular level in terms of the activation mechanism. It has been established that EGF-induced dimerization of ErbB1 functions as an activation switch, and the same principle has been identified to function for another member of the receptor family, ErbB4. Since detailed molecular characterization of ErbB4 has not been carried out, we propose to investigate the formation of dimers and higher-order clusters in resting and growth factor stimulated cells and to link these events to transmembrane signaling. We plan to join the competences of the Hungarian and Austrian collaboration partners to build upon recent advances in microscopic approaches, which permit the interrogation of single molecules allowing unprecedented insight into these kinds of molecular events. Such a combination of approaches can provide a detailed quantitative model for the activation of the ErbB4 receptor. The receptor planned to be studied in our project, ErbB4, has been shown to play an important role in the development of one of the most malignant tumors, melanoma. Given the importance of receptor interactions in the activation of cells our results can pinpoint diagnostic and therapeutical targets against this devastating disease.

Cells in our body constantly communicate with each other. They do so using tiny "antennae" on their surface, called receptors, which sense signals from the environment and translate them into cellular responses. One such receptor, ErbB4, is known to play important roles in development, especially in the nervous system and heart, and has also been linked to cancer. Despite its importance, we still know surprisingly little about how this receptor is organized and behaves in living cells. In this project, we set out to observe ErbB4 at the most fundamental level possible: one molecule at a time. To do this, we used advanced microscopy methods that allow us to track individual proteins on the surface of living cells in real time. This required not only cutting-edge instrumentation, but also the development of special molecular tools. A key step was the creation of small binding proteins, called DARPins, which specifically attach to ErbB4 without disturbing its function. By labeling these probes with fluorescent dyes, we were able to "light up" individual receptors and follow their movement on the cell membrane. What we found was unexpected. Instead of being controlled mainly by activation signals, the behavior of ErbB4 turned out to depend strongly on its local environment - in particular, the physical properties of the cell membrane. Changes in membrane composition altered how fast the receptors moved and how they were organized, while activation by external signals had only a modest effect. These findings suggest that cells regulate receptor function not only through biochemical signals, but also through the physical state of their membrane. This adds an important new layer to our understanding of how cells process information. Beyond these biological insights, the project also led to technical advances. We improved microscopy hardware and developed robust experimental workflows that make it possible to study single molecules in living cells more reliably. Overall, this work provides new tools and perspectives for studying cell communication at the nanoscale. Such knowledge is essential for understanding complex biological systems and may ultimately contribute to better insights into diseases where receptor signaling is altered, including cancer.