In vitro reconstitution of spindle bipolarity. (360G-Wellcome-100145_Z_12_Z)

The proposed study aims to answer the key question of what constitutes the mechanochemical core of the mitotic spindle using an in vitro reconstitution approach. Supported by microfluidics, mathematical modelling and experiments in Xenopus egg extracts, I will develop a novel experimental setup, consisting entirely of purified proteins, to test the central hypothesis of how a combination of spatially restricted microtubule nucleation and antagonistic motor activities generates symmetric bipolar spindles of appropriate size. To mimic the role of chromatin and locally promote microtubule formation, I will engineer different molecular gradients inducing microtubule polymerisation around functionalised glass beads, based on the Aurora B and the Ran pathways. Analysing the biophysical properties of these gradients will explain how high microtubule densities form in the proximity of chromosomes in vivo. Second, I will evaluate the role of the main microtubule crosslinking motors, the plus-en d-directed kinesin-5 and the minus-end-directed kinesin-14, in generating bipolar topology. By combining here, for the first time, the anti-parallel microtubule sorting potential of kinesin-5, the pole focussing ability of kinesin-14 and the continous turnover of locally generated microtubules, I will elucidate the relative contribution of each factor in ensuring a bipolar spindle-like state instead of mono- or multi-polar microtubule assemblies.