MECASPREAD

Collective cell migration is involved in morphogenesis, tissue regeneration and cancer. Collectively migrating cells must coordinate through the directional propagation of signals. In epithelial collective migration, actomyosin cytoskeleton conveys mechanical information to cell-cell adherens junctions (AJ) and cell-matrix focal adhesions (FA). Our recent studies revealed how mechanosensitive adhesions encode the mechanical information transmitted by actomyosin to form stable complexes between a-catenin, vinculin and VASP in AJs, and between talin and vinculin in FAs, that polymerize actin to feed the actomyosin cytoskeleton. We hypothesize that the coordination of FA and AJ mechanosensitive and actin assembly activities control the propagation of mechanochemical signals to coordinate cells during their collective migration. The global objectives of this project are to determine 1) the molecular mechanisms by which the activities of the FA and AJ mechanosensors coordinate during collective migration 2) the role of FA and AJ mechanosensors in the propagation of signals throughout a migrating monolayer, and 3) the importance of these mechanisms for the parameters of collective migration such as speed, collectiveness, directionality and persistence. To this aim, our team will develop an interdisciplinary strategy combining biochemistry, biophysics and cell biology. First, we will develop a minimal system made of micropatterned purified proteins, including FA and AJ mechanosensors, whose spatial organization mimics the 2D architecture of the basal face of a migrating cell cluster. Here, the mechanosensitive protein a-catenin will be immobilized on the edges of micropatterned hexagons forming a honeycomb structure, mimicking its distribution in AJs, while the mechanosensitive protein talin will be immobilized inside the hexagons, mimicking its localization on the basal face of the cells. The binding of fluorescent actin, vinculin, and VASP, to talin and a-catenin areas will be stimulated by the actomyosin force and observed in TIRF microscopy. The development of innovative FRET sensors will enable to measure the actomyosin force applied to talin and a-catenin areas. Finally, we will determine the mechanisms governing the actomyosin-dependent spatiotemporal propagation of vinculin binding to talin and a-catenin areas, and the accompanying traction force, following the local stimulation of actomyosin by optogenetic tools. The information obtained from this in vitro assay, such as the general concepts associated with the spatiotemporal coordination of FA/AJ mechanosensor activities and the corresponding molecular tools, such as mutants with defects in mechanosensitive and actin polymerisation activities, will be transposed to collectively migrating MDCK II cells. These mutants will be expressed in MDCK II cells to determine their role in 1) the coordination of AJs and FAs during collective migration, 2) the propagation of intrinsic and optogenetically-induced mechanochemical waves, 4) the key parameters of collective migration (speed, persistence, directionality and collectiveness).