Molecular Insights into the Formation of the Activated Transport Complex of Dynein

Abstract

The ability of cells to dynamically reorganize their interior is critical for cellular function. The molecular motors dynein and kinesins enable the dynamic organization of cells by transporting cargo, like organelles, along microtubules. Dynein is the primary retrograde motor in most eukaryotes and transports a wide range of cellular cargo. Dynein activity is subject to regulation by several essential binding partners. To achieve activation and walk along microtubules, dynein must bind to the multi-subunit complex, dynactin, and to one of several cargo adaptors, forming an activated transport complex capable of moving processively along microtubules. Different adaptors tether dynein to different cargo types, thus enabling dynein trafficking of hundreds of cargoes. The regulatory proteins, Lis1 and the orthologs Nde1 and Ndel1 also play critical roles in dynein function. Lis1 promotes the formation of the activated transport complex, while Nde1/Ndel1 inhibits the formation of the activated transport complex. Despite extensive research on the proteins involved in dynein activation and regulation, the precise mechanism of how the activated transport complex assembles remains unknown. This dissertation investigates the molecular mechanism underlying dynein activation. The research employs single-molecule fluorescence microscopy to visualize the real-time association and dynamics of purified dynein, dynactin, and adaptor. Chapter 1 introduces molecular motors, with a focus on microtubules, dynein, dynactin, adaptors, and dynein-associated regulatory proteins Lis1 and Ndel1. Chapter 2 describes the experimental setup, including TIRF microscopy and kinetic association assays to study single-molecule interactions. Chapter 3 compares the order of association between dynactin-dynein and adaptor-dynein, highlighting the stability of the activated transport complex. Chapter 4 explores how different adaptors influence the association kinetics and stability of the dynein-dynactin-adaptor complex. Chapter 5 investigates the influence of microtubules on the assembly of the activated transport complex. Chapter 6 outlines the roles of the dynein regulators, Lis1 and Ndel,1 in modulating the assembly of the tripartite complex. Finally, Chapter 7 discusses the implications of these findings, future research directions, and potential therapeutic applications. This study enhances our understanding of dynein activation and regulation by elucidating the interactions between dynein, dynactin, adaptors, and regulatory proteins. These insights pave the way for new therapeutic strategies for diseases linked to dynein dysfunction and provide a framework for studying other molecular motors in cellular transport.