UCSF

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.