AbstractMicrotubules are highly dynamic structures with a diverse range of functions including cell division and intracellular transport. This has made them excellent targets for pharmaceuticals and agrochemicals. To understand their dynamic behaviour, microtubules need to be imaged live. However, current imaging probes, such as GFP-tubulin, suffer from high background and both EMTB-3xGFP and SiR-Tubulin are known to stabilise microtubules. Additionally, many of the current microtubule imaging methods only work in one or a few different experimental systems. Therefore, our aim was to produce a new imaging probe that overcame these deficiencies. We present three new live imaging microtubule probes, MnIV, Dynein 2219, and Kif5c that produce microtubule images on a par with or better than existing microtubule imaging probes. All three are protein-based and contain one or more microtubule-binding domains attached to an interchangeable fluorescent tag. Through the use of automated particle tracking and cell scoring we demonstrate that all of the probes analysed, including commonly used probes, modify normal microtubule function to some degree. These effects should be considered when choosing which microtubule-imaging probe to use in any imaging experiment. Increases in microtubule stabilisation were observed, with EMTB-3xGFP showing the largest increase. Additionally, tracking of microtubule plus-tips showed that all the probes we tested, including currently used probes, slowed microtubule dynamics by a small amount. Organelle movement was reduced, to a varying degree, but the overall distribution of lysosomes within the cell was not significantly different from transfected controls. This suggests that the effects on movement are not large enough to cause gross changes to organelle distribution. Of the probes we tested, Kif5c provided the best balance between clear microtubule images and its effects on normal microtubule behaviour. Further testing also showed that Kif5c is a useful tool for imaging microtubules in both Xenopus and Drosophila and is non-toxic to Drosophila development, providing the possibility of one probe that can be used to provide clear microtubule images in a wide range of organisms. This would be a useful tool in the development of new microtubule targeting compounds, as it could be used to directly observe a compounds effect on microtubules in a range of organisms, providing insight into its mode of action and specificity.
|Date of Award||1 Aug 2020|
|Supervisor||Viki Allan (Supervisor) & Sarah Woolner (Supervisor)|
- Cell Biology
- Live Cell Imaging