Kinesin superfamily motors have a well-characterized ability to move along microtubules and transport cargo. However, some members of the kinesin superfamily can also remodel microtubule networks by controlling tubulin polymerization dynamics and by organizing microtubule structures. The kinesin-8 family of motors play a central role in cellular microtubule length control and in the regulation of spindle size. These motors move in a highly processive manner along the microtubule lattice towards plus ends. Once at the microtubule plus end, these motors have complex effects on polymerization dynamics: kinesin-8s can either destabilize or stabilize microtubules, depending upon the context. My thesis work identified a tethering mechanism that facilitates the processivity and plus end-binding activity of Kip3 (kinesin-8 in budding yeast), which is essential for the destabilizing activity of kinesin-8 in cells. A concentration-dependent model was proposed to explain the divergent effects of Kip3 on microtubule dynamics. Moreover, a novel activity of Kip3 in organizing microtubules was discovered: Kip3 can slide anti-parallel microtubules apart. The sliding activity of Kip3 counteracts the depolymerizing activity of Kip3 in controlling spindle length and stability. A lack of sliding activity causes fragile spindles during the process of chromosome segregation in anaphase. The tail domain of Kip3, which binds both microtubules and tubulin dimers, plays a critical role in all these activities. Together, my work defined multiple mechanisms by which Kip3 remodels the microtubule cytoskeleton. The physiological importance of these regulatory mechanisms will be discussed.

During embryogenesis, a single cell gives rise to a multi-cellular embryo through successive rounds of cell division. As cells become smaller, cellular organelles adapt their sizes accordingly. The size of the mitotic spindle—the microtubule-based structure controlling these divisions—is particularly important as it determines the distance over which chromosomes are segregated. To perform its function properly, spindle size scales with cell size. However, we still lack a mechanistic understanding of the underlying microtubule-based processes that regulate spindle scaling. In this thesis, I combined quantitative microscopy and laser ablation in zebrafish embryos and Xenopus laevis egg extract encapsulated in oil droplets. My measurements revealed the influence of microtubule length dynamics, transport, and nucleation on cell size-dependent spindle scaling. Strikingly, I discovered a hierarchical regulation of spindle size. In large cells, microtubule nucleation exclusively scales spindle size relative to cell size by changing the number of microtubules within the spindle. In small cells, microtubule dynamics fine-tune spindle size by modulating microtubule length. To understand the mechanism of spindle scaling, I proposed a theoretical model based on a limiting number of microtubule nucleators and microtubule-associated proteins that regulate microtubule length. The transition from nucleation- to dynamics-based scaling requires that microtubule number and the number of microtubule-associated proteins that promote microtubule growth scale differently with cell size. This can be achieved by sequestering an inhibitor of microtubule nucleation to the cell membrane, which is consistent with my measurements of microtubule nucleation. The differential regimes of spindle scaling modulated by microtubule nucleation and dynamics imply a gradual change in spindle architecture, which may ensure faithful chromosome segregation by spindles of all sizes.