Control mechanisms of microtubule overlap regions

Microtubule organization in cells is an important process. An example of careful microtubule organization is the mitotic spindle. The spindle is a bipolar structure with microtubules emanating from the poles at both sides. These microtubules form antiparallel overlaps in the centre of the spindle where they are bundled by bundling proteins. The overlaps are centred in the spindle and their constant length is regulated. The overlaps are important for the stability of the microtubule network, without bundling proteins the overlaps are lost and the spindle collapses. The antiparallel overlaps are also the site where microtubules slide apart to induce spindle elongation. Sliding is induced by tetrameric motor proteins that can bind to two bundled microtubules. Spindle elongation also requires microtubule growth at the overlaps. All these different functions at the overlap, sliding, growth/shrinkage and bundling, have to cooperate to maintain overlap length. While sliding reduces overlap length, growth will increase overlap length. These activities have to be coordinated for the maintenance of a constant overlap length. We propose that a feedback mechanism is present where growth of the microtubules is limiting the sliding in the overlap. This would prevent sliding when the overlap decreases and helps to maintain the overlap. We designed in vitro experiments to make antiparallel overlaps in vitro. In these experiments we use purified proteins from S. pombe. We combine ase1 and klp9 in a relative sliding assay to mimic the sliding in the midzone. In our experiments we combine relative sliding with dynamic microtubules for the first time. This allows us to test how these activities are coordinated. In other experiments we combine ase1 and cls1 with dynamic microtubules to see if the rescue activity of cls1 can be confined to the overlaps. Furthermore, interactions between motor proteins and diffusive proteins are investigated on single microtubules.