Vinculin and associated signalling networks in the regulation of cell motility

The process whereby cells in the body move from place to place is called cell migration. Cell migration is fundamental for the development of organisms, wound healing, and the immune respose in the body. All these physiological processes are regulated by the interaction of the cell with its environment. The major group of molecules controlling these interactions are cell surface proteins called integrins. Integrins span the cell membrane and are able to attach with their extracellular part to proteins located in the outside surrounding of the cell (extracellular matrix). With their intracellular part, integrins associate with proteins that link them to the cells' highly flexible and dynamic skeleton (actin cytoskeleton). The interaction of integrins with the cytoskeleton is indirect. One of the key molecules, which control the link between integrins and the actin cytoskeleton, is named 'vinculin'. Mice lacking this protein die early during development, and cells deficient in vinculin are highly metastatic. Vinculin is able to interact with 11 other proteins, which have the potential to exert important signals regulating the cell communication with its environment. My aim is to understand how vinculin coordinates the numerous signals and thus modulates the communications between the cell and its outside surroundings. We will explore the function of vinculin by visualizing and monitoring its dynamic behaviour and interactions with other proteins in live cells using advanced imaging techniques. We will genetically modify vinculin and its ability to interact with its potential binding partners and thus test how vinculin controls a network of signals that is important for coordinated cell migration. For coordinated cell movements, the cell needs to exert pulling forces on the extracellular environment. Our recent discoveries suggest that vinculin interactions with the contractile skeleton in the cell are essential for the transmission of forces to the integrins that bind with its extracellular part to the extracellular matrix. Using highly developed nanotechnological devices, we will determine how vinculin signals contribute to such force transmission from the interior to the outside of the cell. Using devices that will mimic forces in the cells outer environment (such as pressure, stretching and shear forces in the body), we will be able to test how vinculin transfers signals from the outside of the cell to the inside cytoskeleton. The outcome of this study will provide us with important information for the better understanding of how cell motility is regulated in health and disease.

The primary sites for cell adhesion to the substrate are focal adhesions (FA). At FAs, over 100 molecules regulate the link of integrins to the actomyosin force machinery. FAs are therefore molecular entities that regulate cell motility as well as the transmission of forces to the substrate, and vice versa. Here I seek support to define and integrate the contribution of vinculin, a central player for focal adhesion regulation. Vinculin in its active state has the potential to bind 11 other FA components, which in turn have multiple binding partners. With its prominent binding sites for talin and actin, vinculin is located at the interface between adhesion receptors (integrins) and the actin cytoskeleton and thus in an ideal position to coordinate a network of signals regulating cell motility. I aim to understand how vinculin and its multiple interaction partners (a) control cell adhesion and motility and (b) how they are involved in the transmission of forces. To date, protein interaction studies in FAs have relied mostly on biochemical analyses, which are unable to probe the enormously complex and dynamic events that occur in cells. We will capitalise on my knowledge on advanced fluorescence microscopy, including fluorescence recovery after photobleaching (FRAP), quantitative image correlation analysis (ICA), and fluorescence resonance energy transfer (FRET), as an essential tool for overcoming some of these barriers. We will complement these techniques with biochemisty and (nano-) technological devices that allow application and measurement of forces to elucidate: 1) How does vinculin control the large network of signals in focal adhesions? 2) How do interaction partners of vinculin vary in time and space? How is vinculin involved in sensing of extracellular forces, the generation of intracellular force, and the regulation of adhesion and migration?