Mechanism by Which the Bicaudal D2-Nuclear Pore Protein 358 Interaction Activates Microtubule-based Cargo Transport

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Project Summary/Abstract There are many cargo such as secretory vesicles, autophagosomes, and liquid droplets that are bi-directionally transported along microtubules by the opposing motors cytoplasmic dynein and kinesin. However, it is still unclear how these two biological motors collaborate to position the cargo and achieve cellular functions. Impaired bidirectional transport leads to a variety of neurodegenerative diseases, including Alzheimer's disease. Microtubule-associated protein MAP7 and dynein cofactor Lis1 are two important proteins that play critical roles in cargo transport. While MAP7 enhances the processive motion of kinesin, Lis1 relieves the auto- inhibition of dynein. In this proposal, we aim to reconstitute the DDBE-K (dynein, dynactin, BicD, Egl, kinesin) complex and observe its motion on microtubule tracks in the presence of both MAP7 and Lis1 using total internal reflection fluorescence (TIRF) microscopy. Given that MAP7 has the ability to prolong the attachment of kinesin to microtubules and Lis1 prefers binding two dyneins, we predict that MAP7 and Lis1 will play key roles in determining the direction of motion of the complexes on microtubules. Using single-molecule assays, we will investigate whether the adapter protein BicD not only functions as a linker between cargo and motor but also plays an essential role in sensing cargo and motor binding. These proposed studies will provide novel insights into the roles of dynein and cargo adapter proteins and contribute to our understanding of how cytoplasmic dynein and kinesin work together to transport cargo bidirectionally on microtubules.