The microtubule (MT) cytoskeleton plays an essential role in mitosis, intracellular transport, cell shape, and cell migration. The assembly and disassembly of MTs, which can occur through the addition or loss of subunits at either the plus- or minus-ends of the polymer, is essential for MTs to carry out their biological functions. In Chapter 1, I describe my work on a recently described family of MT minus-end binding proteins called CAMSAP/Patronin/Nezha that act on MT ends to regulate their dynamics. Patronin, the single member of this family in Drosophila, was previously shown to stabilize MT minus-ends against depolymerization in vitro and in vivo. Here, I show that all three mammalian CAMSAP family members also bind specifically to MT minus-ends and protect them against kinesin-13-induced depolymerization. However, these proteins differ in their abilities to suppress tubulin addition at minus-ends and to dissociate from MTs. CAMSAP1 does not interfere with polymerization and tracks along growing minus-ends. CAMSAP2 and CAMSAP 3 decrease the rate of tubulin incorporation and remain bound, thereby creating stretches of decorated MT minus-ends. Using truncation analysis, I find that somewhat different minimal domains of CAMSAP and Patronin are involved in minus-end localization. However, I find that in both cases, a highly conserved C-terminal domain and a more variable central domain cooperate to suppress minus-end dynamics in vitro and that both regions are required to stabilize minus-ends in Drosophila S2 cells. These results show that members of the CAMSAP/Patronin family all localize to and protect minus-ends but have evolved distinct effects on MT dynamics. In Chapter 2, I describe work characterizing a set of inhibitors of a kinesin-12 family member called Kif15, which has a role in spindle organization during mitosis. Kif15 has been shown in previous work to cooperate with the tetrameric kinesin Eg5 to facilitate bipolar spindle formation; we were interested in the possible application of these molecules to spindle disruption in hopes that the combination of Eg5 and Kif15 inhibition might be clinically efficacious. I show that these small molecules have activity against Kif15 through an uncompetitive mechanism and that Kif15 inhibition with these molecules affects spindle organization and cell division in vivo.

Transport of cargos not only plays a critical role on the meter scale in our daily life when we travel from A to B but also plays an essential role for cellular processes on the nanometer scale without which we would not exist. This cellular transport is carried out by motor proteins which walk on cellular highways and are responsible for almost all directed transport in cells. Moreover, these motor proteins play key roles in other cellular processes including mitosis and cilia motility. One of these motor proteins is the microtubule-based motor dynein. Dynein is a complex, flexible, and large machine that has to coordinate it’s two engines and feet in order to achieve directed and continuous motility. Recent structural and biochemical studies uncovered key molecular mechanisms contributing to dynein motility. However, a comprehensive understanding of how dynein steps along its microtubule track, and how its different domains are coordinated to achieve this movement were lacking in the field.

Therefore, I first set out to determine how dynein’s ATPase activity and mechanics are coupled among the motor domain of dynein and showed that the ~15 nm long coiled-coil linking the catalytic AAA ring and its microtubule-binding domain is indispensable in regulating motor activity. Moreover, I found that the length rather than the sequence of this coiled-coil is remarkably well conserved and that the length conservation is paramount for directional motility. Integrating these observations allowed us to generate an updated model for the internal regulation of dynein.

Our understanding of how the different domains of dynein move relative to each other has been limited by insufficient high spatiotemporal resolution. To overcome this, I first created a method that enables three-color image registration and distance measurements with one nanometer accuracy and second, I developed DNA FluoroCubes that enabled me to track the position of multiple domains of dynein for a prolonged time with nanometer precision. Combining both of these methods enabled me to gain insights into the conformational changes of dynein’s domains while moving along microtubules. I found that the motor domain of dynein is very flexible and that this flexibility is important for dynein motility and enables dynein to adopt a large variety of conformations. Together, these findings revealed a new model for dynein stepping that defines the minimal requirements to facilitate directed and continuous motility.