Evanescent wave microscopy to probe cell guidance

The migration of cells is essential to life, as a primary feature of developmental and repair processes. It also contributes to disease states, such as in the dissemination of malignant cells during metastasis. Cell migration is driven by the dynamic formation and reorganisation of actin filaments that form the "actin cytoskeleton". However directional locomotion can only proceed with an advancing front and a retracting rear and this "polarisation" requires the involvement of a dynamic network of microtubules. In the foregoing project, we unveiled the route via which microtubules most likely exert their polarising effect. We showed that microtubules target substrate adhesion foci; sites at which transient transmembrane linkages occur between the actin cytoskeleton and the extracellular matrix. Our collected findings indicate that microtubules promote the turnover of adhesion foci and that by regulating their turnover in a spatially defined way, they influence the organisation of the actin cytoskeleton and, thereby, cell polarity. The present project aims at characterising: 1, the involvement of microtubule- adhesion cross-talk in the polarisation of different cell types; 2, the mechanism of guidance of microtubules into adhesion foci; and 3, the molecular complexes involved in delivering the signal that modulates adhesion complex turnover. It is proposed in this quest to exploit the technique of evanescent wave microscopy which we have recently shown is ideal for analysing the fidelity of microtubule-adhesion interactions. These studies should reveal deeper insights into the fundamental mechanisms underlying directional cell migration.

The development and repair of organs and tissues as well as the targeting of cells to sites of inflammation all rely on the ability of cells to migrate. In other scenarios, cell migration can have adverse consequences, as is the case for cancer cells that disseminate from the primary tumour during metastasis. If we are to influence migration processes, we must understand the cues that tell cells when and where to move. The aim of the present project was to shed light on how cells are guided during their migration. A cell moves in phases of protrusion, adhesion and retraction and all of these processes depend on the turnover of the actin cytoskeleton. However, without the second major cytoskeleton system of polymers, the microtubules, cells are either unable to migrate or move randomly. Collaboration between the actin and microtubule systems is thus required for directed migration. In the foregoing project, studies on tissue fibroblasts showed that this collaboration involves targeted interactions of microtubule ends with the points of adhesion that cells make with the underlying substrate. It was accordingly proposed that microtubules influence the direction of cell movement through a spatial regulation of adhesion turnover. The present project aimed to establish whether or not microtubule-adhesion interactions are a general feature of migrating cells and to elucidate the molecular nature of these interactions. A technical requirement for these studies was a total-internal-reflection-fluorescence (TIRF) microscope that allows selective analysis of the base of migrating cells, where microtubule-adhesion interactions take place. We were first able to demonstrate that rapidly migrating, neutrophil-like cells, exhibit the same microtubule- adhesion targeting interactions as seen with fibroblasts and other cultured cell types, suggesting that these interactions play a general role in tuning cell steering. But how do microtubules find their target sites? Based on studies from yeast, we tested for the involvement of a myosin motor (myosin V) as the link between microtubule tips and actin filament tracks. We engineered into cells defective motor molecules or suppressed their expression with RNAi; however, we could not yet disrupt microtubule-targeting activities. Nevertheless, interesting changes in cell motility were observed that point to a role of myosin V in cell polarisation that is currently being pursued. To undertake a more systematic analysis of molecules involved in microtubule-adhesion cross-talk we developed conditions for a genome wide microscopy-based RNAi screen. In a collaborative effort with EMBL in Heidelberg, this screen is now underway and promises to deliver candidate molecules and pathways involved in adhesion turnover.