Microtubule organization into tactoids with active and passive crosslinkers

The microtubule cytoskeleton provides significant structural support for cells, much like bones in the human body. This support is especially important for processes where the cell undergoes major structural changes, such as mitosis with the formation of the mitotic spindle. Biophysical mechanisms regulating the characteristic shape of the mitotic spindle involve microtubule associated proteins (MAP's), crosslinkers, and motor proteins, etc. We have been studying microtubule interactions with MAP65, a plant-derived, antiparallel crosslinking protein, from the MAP65/PRC1/Ase1 family. We have observed in vitro formation of microtubule tactoids, whose shape is reminiscent of the mitotic spindle. However, these tactoids are rigid and thin, unlike a mitotic spindle, which is more liquid crystal-like. To create more fluid-like tactoids, we introduce K401, a dimeric kinesin construct with the N-terminal 401 amino acid motor domain, as a dynamic microtubule crosslinker. We can modulate the relative concentrations of crosslinking MAP65 and translocating K401motors to determine how the competition influences the formation and mechanics of microtubule tactoids.