Microtubules are large cellular structures that grow and shrink on the order of seconds to minutes. Chemical disruption of microtubule dynamics typically leads to death in both cycling and non-cycling cells, though the mechanisms responsible for this are not well characterized. Interestingly, microtubule drugs stimulate kinase- mediated stress signaling prior to cell death, which suggests that cells can detect and respond to microtubule disruption. I propose to address the questions surrounding this intriguing stress phenomenon. First, I would like to understand the general impact that microtubule perturbations have on cellular signaling. Given the precedent of phosphorylation stress signals in response to microtubule disruption, I propose to generate highly quantitative mass spectrometry measurements of protein phosphorylation that occurs immediately following microtubule drug treatment. Second, I want to determine whether different microtubule perturbations (e.g. stabilization and destabilization) have different effects on cellular signaling. To do this, I willuse different drugs to perturb microtubules and compare effects on protein phosphorylation. Finally, there is currently very little knowledge regarding how microtubules might directly regulate signaling, especially in response to chemical insults. I hope to elucidate mechanisms by which signaling proteins (e.g. kinases, phosphatases) are regulated by microtubule dynamics and activated in response to microtubule disruption. To this end, I will use immunofluorescence imaging, conventional transfection techniques, and statistical inference methods.
Many cell processes depend on microtubule dynamics to function properly, yet most work on microtubules has focused on their role in mitotic spindle formation and DNA segregation. Recent studies have demonstrated that disrupting microtubule dynamics triggers cell stress in cycling and non-cycling cells, although by unknown mechanisms. A systematic analysis of microtubule disruption may elucidate the mechanisms by which stress signals are stimulated, some of which may be the same signals that precede tumor shrinkage in cancer chemotherapy.
| Pineda, Javier J; Miller, Miles A; Song, Yuyu et al. (2018) Site occupancy calibration of taxane pharmacology in live cells and tissues. Proc Natl Acad Sci U S A 115:E11406-E11414 |
