Effects of Therapeutic Concentrations of Tubulin-binding Drugs on Microtubule Dynamics.

Microtubules are dynamic structures that are formed by the polymerization of alpha-beta tubulin heterodimers into long, hollow tubes. They mediate a number of critical processes, including mitosis, intracellular transport, and cell migration. During mitosis, microtubules form into a complex structure known as the spindle apparatus, which orchestrates the segregation of chromosomes into daughter cells. This process depends on an intrinsic feature of microtubules called dynamic instability, where microtubules abruptly switch between phases of sustained growth and rapid shortening. This allows microtubules to quickly probe the intracellular volume to locate and couple to chromosomes before synchronous division. Any aberrant function of this delicate process, if not detected and resolved by a mitotic spindle checkpoint, can lead to aneuploidy and tumorigenesis. Microtubule-targeting drugs are commonly used as chemotherapeutic agents, given their ability to disrupt native microtubule dynamics, which prevents cell division. These drugs have been used to successfully treat a range of cancers; however, their use is associated with a number of debilitating side effects, and in some cases, acquired drug resistance. The detailed mechanisms of action of microtubule-binding drugs are not well understood, and therefore, the development of efficacious drugs with lower collateral toxicity is slow. In this work I investigated the effects of four microtubule-binding drugs, which each target distinct sites on the tubulin subunit. First, I adapted previously established methods to determine how the kinetic rates of tubulin exchange from microtubules are affected by microtubule-binding drugs. I found that the drugs colchicine and vinblastine potently suppress the kinetic rates, while paclitaxel and peloruside A have little effect, even though they are able to completely suppress dynamic instability. Futhermore, I related these effects to changes in the microtubule tip structure and explored methods for measuring changes in microtubule rigidity. Finally, experimental data and modeling show that the fluorescent paclitaxel-site probe Flutax-2 undergoes surface interactions, which can lead to non-monotonic self-quenching effects. Moreover, Flutax-2 aggregates in solution, leading to a substantial reduction in the monomeric, microtubule-binding form.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133211/1/sethmc_1.pd