Reconstitution of basic mitotic spindles in cell-like confinement

Bipolar organization of the mitotic spindle is the result of forces generated by dynamic microtubules and associated proteins in interaction with chromosomes and the cell boundary1-4. Biophysical experiments on isolated spindle components have provided important insights into the force-generating properties of different components5-8, but a quantitative understanding of the force balance that results from their concerted action is lacking. Here we present an experimental platform based on water-in-oil emulsion droplets that allows for the bottom-up reconstitution of basic spindles. We find a typical metaphase organization, where two microtubule asters position symmetrically at moderate distance from the mid-zone, is readily obtained even in the absence of chromosomes. Consistent with simulations, we observe an intrinsic repulsive force between two asters that can be counterbalanced alternatively by cortical pulling forces, anti-parallel microtubule crosslinking, or adjustment of microtubule dynamics, emphasizing the robustness of the system. Adding motor proteins that slide anti-parallel microtubules apart drives the asters to maximum separation, as observed in cells during anaphase9,10. Our platform offers a valuable complementary approach to in vivo experiments where essential mitotic components are typically removed, instead of added, one by one.