UC Berkeley

Microtubule-based motors play essential roles in intracellular organization and cell division by transporting cargos and generating force along microtubules. Kinesin and dynein are microtubule motors that move toward the plus and minus ends of microtubules, respectively. These motors are highly coordinated to enable bidirectional cargo transport and precise spatial organization within cells. However, the molecular mechanisms that regulate kinesin and dynein activity and coordination remain elusive.In my doctoral work, I addressed this question using biochemical reconstitution and single-molecule imaging. First, I studied the activation and regulation of kinesin and dynein by the mitochondrial adaptor protein TRAK. I showed that TRAK activates dynein and enhances kinesin activation in vitro. In addition, I found that TRAK adaptors can recruit kinesin and dynein simultaneously, and these complexes, in which both motors are associated, move exclusively to the plus end at kinesin speed, demonstrating that dynein is carried by kinesin as an inactive passenger. Furthermore, I demonstrated that recruitment of syntaphilin to mitochondria acts as a static anchor that opposes motor-driven motility, leading to mitochondrial pausing.Additionally, I investigated the activation and regulation of dynein by its mitotic cargo adaptor NuMA. Using a biochemical reconstitution approach, I discovered that the N-terminal fragment of NuMA activates dynein. At the same time, the C-terminal region of NuMA binds and suppresses microtubule minus-end dynamics in vitro. The C-terminal region also recognizes microtubule minus ends through its MTBD1 region. Full-length NuMA is autoinhibited during interphase, and its ability to interact with dynein is significantly enhanced by phosphomimetic mutations at CDK1, Aurora A, and Plk1 phosphorylation sites at its C terminus. Together with dynein/dynactin, activated NuMA sorted and focused the minus-ends of microtubules into aster-like structures, resembling spindle pole focusing during prometaphase. This work provides mechanistic insight into how the adaptor protein NuMA regulates dynein activation during mitosis.