CytoskeletonCoupling · Active actin-microtubule crosstalk in reconstituted systems

Living cells maintain a high degree of functional and structural organization which largely depends on a highly dynamic scaffold, the cytoskeleton, with microtubules (MTs) and actin filaments as the central elements. While both components are comparably well characterized individually, their interplay and mutual regulation remains poorly understood. Precisely their cooperative functioning, however, is increasingly regarded as a central element for many cellular key processes including cell division, cell migration, and adhesion.The central aim of this proposal is to achieve a more profound and quantitative understanding of actin-MT crosstalk using a simple yet realistic reconstituted model system. Centrosome nucleated microtubule asters will be embedded into passive and active actin networks. Coupling between the two cytoskeleton components will be introduced in form of transient binding of growing MT plus ends to actin filaments mediated by tip-tracking EB3 and MACF, a highly relevant actin-MT linker.I will study the mutual influence between a surrounding actin matrix and the dynamic MT asters. Addition of myosin motors will induce actin network contractions that should strongly affect MT growth dynamics. Applying computer-based image analysis routines will allow me to quantify centrosome motion and microtubule tip dynamics. Further, I will investigate the additional effects of cell size confinement on the spatial self-organization of actin and MT asters. Finally, I aim at mimicking the actin-MT crosstalk at the leading edge of migrating cells by establishing an actively treadmilling actin network growing against the aster-based microtubules of opposite polarity.The proposed work will be carried out at the AMOLF institute, simultaneously in the groups of Prof. M. Dogterom and Prof. G. Koenderink which allows me to integrate their combined MT and actin expertise.