Structural studies of the microtubule and its regulation

Microtubules (MTs) are highly conserved, cytoskeletal polymers that are involved in a large number of cellular processes. These include cell motility, in which they form the central scaffold of the beating machinery in cilia or flagella, to intracellular transport, in which they function as the cellular highways for the shipment of organelles and other cargos by motor proteins of the kinesin and dynein families, or cell division, in which they form the mitotic spindle to carefully segregate genetic material. These polymers undergo dynamic instability, the stochastic switching between growth and shrinkage, which give them the capacity to rapidly rearrange to allow the cell to grow, move, or divide1. Dynamic instability misregulation can cause cancer, Alzheimer’s disease, and other life-threatening diseases. Therefore, it is crucial to understand how MTs, and the chemicals or proteins that regulate them, work. For example, the molecular mechanism of α-tubulin acetylation (αK40) has remained elusive, although this chemical modification has been shown to correlate with stability of MTs and serves as a prognostic marker for breast cancer2,3. In addition, how MAPs transform large MT substructures is an emerging frontier waiting to be explored./nWhile it is known that αK40 acetylation is associated with more stable MTs, it is not clear whether the relationship between this chemical modification and stability is causative. To tackle this controversy, I applied a reductionist approach to tease out the direct effects of this modification on MT structure by using cryo-electron microscopy (cryo-EM) to visualize acetylated and deacetylated MTs. I showed that acetylation changes the conformational ensemble of the intraluminal αK40 loop in α-tubulin and may serve as an evolutionarily conserved ‘electrostatic switch’ to regulate MT stability4. Due to the high flexibility of the loop, unlocking the effects of this modification required an exciting hybrid EM-MD approach, designed in collaboration with Dr. James Fraser at UCSF and Dr. Massiliano Bonomi at the University of Cambridge./nAs a result of my studies, I have become very interested in conformational heterogeneity and plasticity in the MT lattice. For example, the structure and mechanics of the lattice are not only dependent on the modification state of each tubulin, but also on tubulin isotypes, interacting drugs, or MT-binding partners, all of which can cause changes in tubulin structure and subunit packing within the MT and affect local mechanical strain and other physical properties of the lattice5. I started to appreciate the MT as an allosteric macromolecular machine that interprets multifaceted inputs and reacts by transforming its conformation, stiffness and dynamics5. This appreciation launched an exploratory project into the complex world of therapeutic agents, specifically on lankacidins, a unique class of antibiotics. Similar to PTMs and MAPs, therapeutic agents can affect MT structure and stability. Taxol, a major breast cancer chemotherapy agent, can block the cell cycle in its G1 or M phases by stabilizing MTs and limiting MT critical dynamics6,7. Surprisingly, lankacidins (LCs) were shown to have both in vivo antitumor activity in multiple cancer cell lines and antimicrobial activity against Gram-positive pathogens. We observed that LC did not polymerize MTs in a taxol-like manner and now believe that the proposal that its in vivo antitumor activity occurs via MTs could be due to previous lack of proper experimental controls. Thus, we shifted our focus to the effects of LC on bacterial translation and its role as an antibiotic. We present a 2.8 Å structure of the LC-ribosome complex, which improves upon the existing model and shows that LC ring closure is imperative for its mechanism of action. This mechanism may be conserved across related metabolites in the lankacidin class for their antibiotic activity.

Advisor: Nogales, Eva