Regulation of Microtubule Plus-End Dynamics by Molecular Motors

The microtubule cytoskeleton is the structural base of key cellular processes such as axonal growth, intracellular transport and cell division. These carefully orchestrated processes rely on the precise control of microtubule function and architecture. Microtubules, however, are dynamic polymers that stochastically transition between periods of growth and shrinkage. Therefore, the cell must regulate microtubule dynamics to achieve control over the length and stability of the microtubule cytoskeleton to accurately perform its cellular functions. Kinesins are best known for their ability to move processively on microtubules and carry cargo. Nevertheless, some kinesin families can also regulate microtubule dynamics and organize microtubule structures. Kinesin-8s play a central role in regulating the microtubule cytoskeleton by controlling microtubule length. Although extensive studies have been conducted, the mechanism by which kinesin-8 disassemble microtubules has remained unclear. My thesis work identified the central features of this mechanism for the yeast kinesin-8/Kip3. Like other kinesins, kinesin-8/Kip3, uses ATP hydrolysis for stepping on the microtubule lattice. Upon binding to curved tubulin at the plus-end, Kip3 undergoes a switch in activity resulting in strong tubulin binding, thereby promoting microtubule depolymerization. Our model is supported by the identification of the structural elements in the motor domain and in tubulin required for plus-end recognition, long-range processivity and microtubule depolymerization by Kip3. To obtain a comprehensive understanding of the Kip3-tubulin interaction we obtained high-resolution structures using cryo-electron microscopy of Kip3 bound to stabilized microtubules. This approach identified intra and intermolecular interactions that underlie Kip3’s ability to switch from a motile kinesin to a depolymerase at the microtubule end. The coexistence of the two activities in kinesin-8/Kip3, gives rise to its emergent property of length-dependent microtubule disassembly. Another challenge in the regulation of microtubule structures is in the control of the nucleation and architecture of the microtubule polymer. To overcome these challenges we developed a programmable DNA-origami microtubule seed, which can template defined microtubule structures. This tool will enable future studies on the regulation of microtubule architecture, such as the microtubule doublet structures found in cilia. Together, these findings elucidate regulatory mechanisms whereby nanometer-size molecules can measure and regulate micron-sized cellular microtubule structures.