Insight into microtubule dynamics: from purified protein to cell

Microtubule-targeting agents have received considerable interest as potential tumour-selective anti-angiogenic and vascular-disrupting agents. To improve targeting of these agents, a detailed characterization of their interaction with tubulin/microtubule system is required and includes the analysis of stabilisation and depolymerisation of microtubules as well as imbalance in microtubule dynamics. We performed in vitro assays using purified tubulin to investigate the effects of microtubule-targeting agents on microtubule assembly in terms of polymer mass, polymerization kinetics, microtubule structure.and dynamics1. Moving from purified protein to cultured cells, we investigated the effects of microtubule-targeting agents on microtubule network by immunofluorescence and confocal analyses. Finally, microtubule dynamics in live cell was investigated by time lapse imaging of cells expressing the microtubule-associated protein EB3-GFP, a protein that binds specifically to the plus end of growing microtubules.