Dynein-Mediated Positioning of Microtubule Asters in 3D Confinement

Abstract

Important functions of eukaryotic cells such motility and division depend sensitively on cytoskeletal mechanics and organization. In particular, microtubules are stiff dynamic polymers that can generate pushing and pulling forces. To fulfill biological functions, microtubules adopt specific spatial patterns, like the mitotic spindle during cell division. How the shape and size of cells, as well as the presence of pushing and/or pulling forces influence this organization is in many cases still unclear. To assess the influence of confinement on microtubule self-organization, we reconstitute a dynamic microtubule cytoskeleton inside 3D water-in-oil emulsion droplets, using lipids that can optionally be functionalized with active dynein molecular motors. We study the positioning of centrosomes, from which microtubules are nucleated that exert pushing and/or dynein-mediated pulling forces when reaching the boundary. We show that the central position of one centrosome confined in a spherical droplet is drastically destabilized by pushing forces, while pulling forces tend to center the centrosome. Interestingly, when two centrosomes are present, pushing forces cause the centrosomes to find a stable position at opposite sides of the droplet. When both pushing and pulling forces are present, two centrosomes adopt an equilibrium position balancing the centering effect of the dynein-mediated pulling forces with the repulsion effect of the two centrosomes, thereby reproducing a ‘mitotic spindle’ like organization. These experiments set the stage for a better understanding of the role of additional mitotic spindle components such as mitotic motors and crosslinkers that we plan to add to our system.