Hannabuss, JC; (2018) In vitro reconstitution of the anaphase central spindle. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The anaphase central spindle is the major regulatory scaffold for coordinating cytokinesis; the final stage in cell division. It is constructed from two populations of microtubules, which emanate from opposite halves of the cell, until they meet at the centre, form antiparallel overlaps, and are cross-linked by microtubule bundling proteins. This cross-linked overlapping region marks the division plane, and recruits proteins that promote the membrane cleavage furrow, and the final abscission of the two daughter cells. For robust cortical signalling, and localisation of the abscission apparatus, the cell must regulate the length and alignment of the overlapping region to create a clear narrow band. What are the molecular mechanisms underlying this regulation? To address this question, I use a novel assay, which allows me to use TIRF microscopy to study free-floating, dynamic, microtubule bundles, nucleated de novo in solution, and organised by purified recombinant proteins. By reverse engineering the anaphase central spindle in this way, I have discovered that only two human central spindle proteins, PRC1 and KIF4a, are necessary to organise dynamic microtubules into bundled structures with narrow, aligned, antiparallel overlaps. These results also suggest a novel functional role for human KIF4a, as an antiparallel sliding motor, capable of aligning microtubule overlaps in a bundle.

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