TubulinTracker

TubulinTracker is a novel fluorescent reporter cell assay to investigate the microtubule polymerisation dynamics, progression of cells through mitosis and aneugenicity. The assay has been developed as followup for the ToxTracker (ACE) assay, to further assess the mode-of-action of genotoxic compounds that were classified as aneugen. However, TubulinTracker can also be used as stand-alone Tubulin polymerisation assay.

Key Features

        • GFP-reporter assay for microtubule disruption detection
        • Detection of both stabilising and destabilising Tubulin poisons
        • Insight into the aneugenic mode-of-action of genotoxic compounds

TubulinTracker

TubulinTracker is a novel in vitro assay using GFP-tagged Tubulin, that allows the direct visualisation of the microtubules using either live cell microscopy to follow the dynamics during the cell cycle or flow cytometry. The assay can be applied to study the effects of compounds on microtubule stability as well as assess the effect on the cell cycle progression and induction of aneuploidy. TubulinTracker can discriminate between Tubulin stabilising and Tubulin destabilising substances and provides more insight into the cause of aneuploidy upon exposure.

The advantage of the TubulinTracker assay is that it can efficiently detect both stabilising and destabilising Tubulin poisons and can be used to detect an aneugenic mode-of-action of genotoxic compounds. The assay can be used to study Tubulin dynamics in the context of a cell throughout the different phases of the cell cycle Insight into the MOA provided is important for hazard identification and as part of AOP and weight of evidence approaches.

Background:
Aneuploidy, the presence of an abnormal number of chromosomes, can be caused by any process that interfere with chromosome segregation during mitosis, including microtubule disruption or inhibition of cell cycle kinases, like Aurora A/B/C. To detect aneugenicity, as result of chemical exposure, typically the micronucleus assay is used. However, both broken DNA fragments caused by clastogenic agents and mis-segregated chromosomes caused by aneugenic agents can lead to the micronuclei formation. To confirm an aneugenic mode of action, a laborious centromere or FISH staining is required.

TubulinTracker assay principle

The assay is build around the reporter technology that was developed for the ToxTracker, TubulinTracker uses a Tubulin-GFP fusion report that allows for direct visualisation of microtubuli, using high content imaging or flow cytometry. In the TubulinTracker assay, the GFP-Tubulin reporter cells are exposed to various concentrations of a test compound. After treatment, cells are permeabilised to extract the free GFP-Tubulin. After fixation, the remaining polymerised GFP-Tubulin in the cells is quantified using flow cytometry.

 

Results

To measure the effect of compounds on Tubulin polymerisation, the amount of GFP-Tubulin signal in microtubules is quantified using flow cytometry. A DNA staining is included during the analysis to simultaneously assess the effect of the agent on the cell cycle progression. Exposure of cell to Tubulin poisons typically results in an accumulation of cells in mitosis. Exposure to compounds that affect cell cycle progression but not microtubule stability, such as DNA reactive compounds or Aurora kinase inhibitors, did not affect polymerised GFP-Tubulin signals.

 

Tubulin stabilising compounds

Exposure to Tubulin stabilisers (i.e. taxol) arrests cells in G2/M and increases the GFP-Tubulin signal.

Left panel shows detected GFP by using live cell microscopy. Middle panel shows the quantification of GFP-tubulin signal in G2/M cells only, showing a significant increase in intracellular polymerised GFP-Tubulin upon exposure to tubulin stabilising compounds.Right panel shows the percentage of cells at each cell cycle phase, after 4h of treatment with taxol. The stabilising compound taxol arrest cells in G2/M.

 

Tubulin destabilising compounds

Exposure to Tubulin destabilisers (i.e. vinblastine) arrests cells in G2/M and decreases the GFP-Tubulin signal.

Similarly to a stabilising compound, mES GFP-Tubulin treated with a destabilising compound (vinblastine) show an increase in G2/M phase after 4h exposure (right stacked columns). However, exposure to the tubulin destabiliser vinblastine decreases the GFP-Tubulin signal (middle panel).

Validation

TubulinTracker has been validated using a broad selection of various Tubulin poisons and other genotoxic compounds with an aneugenic or clastogenic mode-of-action.  Also a number of non-genotoxic compounds were included in the validation.

 

For the validation of TubulinTracker, the GFP-Tubulin cells were exposed to  for 4h to 10 concentrations of the test substances. The fraction of polymerised Tubulin was determined by flow cytometry and quantified by calculating the area under the curve (AUC). Compounds were classified as microtubule stabiliser in case the relative AUC was >2, or as microtubule destabiliser when the relative AUC was <0.5. The tested genotoxic and non-genotoxic compounds that did not affect microtubule stability all resulted in a relative AUC around 1 (dashed line).

 

Service Information

Toxys offers TubulinTracker as a service from our state-of-the-art laboratory in Leiden, the Netherlands. You can send your compounds and receive a full report, in most cases within 2-3 weeks.

Project Specifications:

  • Amount of test material required: < 10 mg
  • Types of solvents which can be used: DMSO/PBS/Water
  • Turn around time: 2-3 weeks

These are some of the typical application of TubulinTracker:

  • Follow-up of ToxTracker (ACE) for compounds with an aneugenic mode-of-action
  • Follow-up of a positive in vitro micronucleus test
  • AOP approach for tubulin poisons
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