Bi-directional microtubule-focal contact regulation

The turnover of adhesion sites is necessary for the reorganisation of the actin cytoskeleton and essential for cell motility. Also, the dynamic properties of microtubules are important for cell migration, organelle movement and a number of other essential functions in a motile cell. The dynamic rearrangements of these two cytoskeletal systems are interdependent and involve a number of regulatory factors. Functioning of those factors is established on the whole cell level. The spatial picture of such a regulation is required to explain morphological asymmetry and existence of differential cytoskeletal pattern in different cell domains. Using video microscopy of living cells, we have recently discovered that the growing ends of microtubules specifically target the focal adhesions. In frame of this phenomenon, microtubules facilitates adhesion turnover, and adhesions, in turn, are able to alter dynamic properties of microtubules. This spatial interaction between the elements of two cytoskeletal systems is direct and precise, allowing for differential regulation of even closely spaced adhesion foci or microtubules. Now, we aim to concentrate on a targeting event itself, in order to identify which molecular players are engaged in the bi-directional control. The project, therefore, is divided in two parts. First part includes studies how microtubule dynamics influence the regulation of adhesions by such regulators as GEF H1, mDia, Rho G. Second part is devoted to candidate factors for microtubule stabilisation at adhesion sites, such as paxillin and microtubule tip protein complex. For this purpose, we are going to 1. establish precise drug application system to change microtubule dynamic properties in a small cell region; 2. establish a cell-free system that allows to simulate intra-structural interactions; 3. use mutant versions of regulatory molecules of interest; 4. combine these approaches with high-resolution video microscopy. This project is of general significance, since it will shed light on the mysterious phenomenon of spatial regulation of polarized cell motility.