Microtubules (MTs) constitute the largest components of the eukaryotic cytoskeleton and facilitate a plethora of diverse functions including intracellular transport, cellular motility, and, cell division. During mitosis, MTs aggregate to form the mitotic spindle, making them a potent drug target for many successful chemotherapeutic agents, including paclitaxel and vinblastine, known as spindle poisons. MT-targeting drugs operate by interfering with dynamic instability (DI): the ability of MTs to rapidly switch from polymerizing to depolymerizing (referred to as catastrophe) and vice-versa. Paclitaxel operates by decreasing catastrophe rate while vinblastine encourages catastrophe and inhibits polymerization. A full understanding of MT catastrophe will greatly aid in the design of spindle poisons with fewer off-target effects, as well as greatly advance general understanding of DI. Each MT is composed of ??-tubulin heterodimers, stacked head-to-tail in protofilaments (PFs) which are aligned laterally to form a hollow tube. Both ?- and ?-tubulin bind guanosine triphosphate (GTP) and hydrolysis of GTP to GDP (guanosine diphosphate) at the ?-tubulin binding site is hypothesized to induce stress on the MT lattice. This stress gradually builds until the subunits at the MT end undergo GTP hydrolysis, at which point PFs begin to peel apart and catastrophe has occurred. Lag between GTP hydrolysis and polymerization creates a construct referred to as the GTP cap: a group of subunits at the MT end that have yet to hydrolyze GTP, release the product inorganic phosphate (Pi), or undergo a structural transition. Recent studies have caused doubt in the field on the nature of this transition and an atomistic understanding of the underlying mechanisms will lead to a full understanding of catastrophe. I propose to computationally resolve three key aspects of catastrophe: the mechanism of GTP hydrolysis, the release of Pi, and the structural coupling between PFs leading to catastrophe. First, I will use enhanced sampling methodology to uncover the enzymatic mechanism of GTP hydrolysis, with emphasis placed on potential catalytic residues belonging to ?-tubulin, which sits atop ?-tubulin upon polymerization to form the active site. Subsequently, I will develop novel computational techniques to determine the pathway of Pi release post-hydrolysis and examine the potential for structural change upon release. Lastly, I will develop a coarse-grained (CG) model of a full MT, using rates determined from the previous studies, able to undergo catastrophe to examine how hydrolysis and Pi release in neighboring subunits affects the potential for these reactions to occur in a particular subunit. This will give an unprecedentedly detailed view of the loss of the GTP cap and the steps leading to catastrophe. Additionally, I will collaborate with two leading experimentalists in the MT community to develop mutants that specifically test my hypotheses and to obtain lattice parameters of MTs doped with spindle poisons. This will allow me to integrate the effects of drugs into the CG model and examine how their effects propagate along an MT. These results and the developed models will greatly advance the understanding of DI and hopefully lead to the development of gentler MT-targeting therapies in the future.
Microtubules (MTs) are dynamic cytoskeletal components with the ability to stochastically convert from a polymerizing to a depolymerizing state (called catastrophe) and vice-versa, a property known as dynamic instability (DI). Due to their role in the formation of the mitotic spindle, MTs are targeted by a number of successful chemotherapeutic drugs that specifically alter catastrophe rate and the normal behavior of DI. In this work, a series of multiscale computational investigations, in conjunction with leading experimentalists, will systematically unveil the molecular mechanisms leading to the initiation of catastrophe, as well as their modulation by MT- targeting drugs, to provide a much clearer understanding of this critical process and pave the road ahead for gentler therapies targeting MTs.
