Large-Scale Analysis of Microtubule Pharmacology via Phosphoproteomics and High-Content Imaging
Pineda, Javier Jesus
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AbstractThe dynamic instability of microtubules is a critical phenomenon that regulates several biological processes. Chemical perturbation of microtubules disrupts these processes and is known to stimulate phosphorylation of downstream substrates. Despite decades of research into microtubule pharmacology, unanswered questions remain regarding the relationship between anti-tubulin drugs and downstream signaling and phenotypes. The following work addresses and answers three outstanding questions regarding the biology of anti-tubulin drugs. It first addresses an important metric for anti-tubulin drug efficacy - site occupancy - and relates classic drug-induced phenotypes to drug occupancy of the taxane site on microtubules in live cells and tissues. The data presented suggest that, when considering site occupancy, the spindle-assembly checkpoint is relatively insensitive to microtubule-stabilizing drugs. Next, after probing the relationship between site occupancy and drug-relevant phenotypes, work is described which utilized mass spectrometry-based phosphoproteomics to elucidate differences in phospho-regulation induced by microtubule-stabilizing and destabilizing drugs. Analysis of this data revealed a large wave of rapid phosphorylation that was common to both classes of drug and highlighted the prominent activity of a protein phosphatase stimulated by microtubule destabilization. Furthermore, the data demonstrated a surprising increase of JNK activity without additional JNK activation in response to microtubule stabilization. Lastly, after comparing drug effects on phospho-signaling in an averaged cell population, work is presented which involved high-content analysis at the single-cell level to elicit information regarding cell heterogeneity in anti-tubulin drug responses. This required development of a novel approach to quantify microtubule dynamics, namely EB3 comet counting in segmented single cells. The data revealed drug-induced single-cell heterogeneity with respect to microtubule dynamics upon treatment with a panel of drugs. Moreover, this occurred in the presence of a pan-efflux pump inhibitor, suggesting that drug-induced heterogeneity can occur independently of drug pump action. This biological finding, along with those mentioned above, is likely important for understanding the therapeutic efficacy of these drugs in cancer chemotherapy.
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