Establishing a High-content Analysis Method for Tubulin Polymerization to Evaluate Both the Stabilizing and Destabilizing Activities of Compounds

Abstract

Microtubules are important components of the cellular cytoskeleton that play roles in various cellular processes such as vesicular transport and spindle formation during mitosis. They are formed by an ordered organization of α-tubulin and β-tubulin hetero-polymers. Altering microtubule polymerization has been known to be the mechanism of action for a number of therapeutically important drugs including taxanes and epothilones. Traditional cell-based assays for tubulin-interacting compounds rely on their indirect effects on cell cycle and/or cell proliferation. Direct monitoring of compound effects on microtubules is required to dissect detailed mechanisms of action in a cellular setting. Here we report a high-content assay platform to monitor tubulin polymerization status by directly measuring the acute effects of drug candidates on the cellular tubulin network with the capability to dissect the mechanisms of action. This high-content analysis distinguishes in a quantitative manner between compounds that act as tubulin stabilizers versus those that are tubulin destabilizers. In addition, using a multiplex approach, we expanded this analysis to simultaneously monitor physiological cellular responses and associated cellular phenotypes.

Honokiol, a Polyphenol Natural Compound, Attenuates Cisplatin-Induced Acute Cytotoxicity in Renal Epithelial Cells Through Cellular Oxidative Stress and Cytoskeleton Modulations

“…Quantification of polymerized α-tubulin was followed by method described by Sum et al (2014) . In brief, MDCK cells were seeded on the coverslips and were grown for 2–3 days to achieve an 80% confluence.…”

Toward Discovery of Novel Microtubule Targeting Agents: A SNAP-tag–Based High-Content Screening Assay for the Analysis of Microtubule Dynamics and Cell Cycle Progression

“…However, a small number of more sophisticated and elaborate HCS assays that address microtubule dynamics have also been described. [12][13][14] We have developed a new cell-based HCS assay that allows microtubule dynamics to be investigated in living cells, with multiparametric readouts captured by automated confocal microscopy and image analysis. We used SNAPtag technology, which is based on a modified human O 6 alkylguanine-DNA alkyltransferase.…”

Pipeline for illumination correction of images for high‐throughput microscopy

“…Although the BBBC021 experiment was not specifically designed as an assay for Taxol's effects, we examined images and noted that the intensity of tubulin staining distinctly increases when cells are treated with Taxol, compared to DMSO; there is prior evidence for this effect ( Sum et al , 2014 ). We thus chose this simple intensity metric as the readout for our illumination correction tests (vs. more complex readouts that are more heavily influenced by other factors such as segmentation quality).…”