Reconstitution of asymmetric mitotic spindle positioning in cell-like compartments.

Regulated asymmetric cell division is crucial during development and for maintaining healthy tissues and stem-cell populations. The site of cell division is determined by the position of the mitotic spindle, a microtubule-based structure that physically links the replicated chromosomes to the cell periphery. Formation and positioning of the mitotic spindle is driven by extensive interplay between numerous cooperating and antagonizing force-generating molecules. Due to the complex nature of this process, it will be important to use bottom-up approaches to fundamentally understand mitotic spindle assembly and positioning. I propose to reconstitute essential aspects of asymmetric spindle positioning in cell-like compartments. We recently developed methods to reconstitute microtubule aster assembly within the geometrical confinement of spherical water-in-oil emulsion droplets. By encapsulating microtubule motor proteins and cross-linkers in these cell-like compartments, microtubule asters can be converted into spindle-like structures. Essential lacking components in these reconstitutions are kinetochores, the protein complexes that dynamically link the replicated chromosomes to spindle microtubules. I aim to study these kinetochore-derived forces by introducing isolated kinetochore complexes and purified chromosomes into these reconstituted spindles. Together, this will lead to an integrated picture of the force-balance within the mitotic spindle. Asymmetric positioning of the mitotic spindle often occurs in non-spherical cells and depends on asymmetry in the forces acting on the spindle. Using advanced microfluidics techniques and light-inducible protein localisation, I aim to reconstitute both of these aspects essential to asymmetric spindle positioning. The distinguished know-how on microtubule-reconstitutions in the Dogterom lab, in combination with my expertise in kinetochore-biology and -biochemistry provide a unique starting point for this project. Together, this combination of innovative approaches allows for a comprehensive quantitative understanding of the forces that drive asymmetric spindle positioning.