Analyzing microtubule dynamics in living cells using fluorescence photoactivation

Microtubules play an important role in different aspects of cell physiology, including intracellular transport, mitosis and dynamics of the cytoskeleton. In neuronal cells, the formation and maintenance of axons and dendrites requires a coordinated and dynamic reorganization of microtubules. Modification of microtubule dynamics can have a major influence on structure and function of neuronal cells, e.g. in Alzheimer’s Disease, where the hyperphosphorylation and aggregation of tau leads to a break down of the microtubule array. We established a method to determine microtubule dynamics in living neural cells by transfection of PC12 cells with photoactivatable GFP (PAGFP)-tagged α-tubulin followed by live cell imaging after focal fluorescence activation. By measuring the intensity decay of activated PAGFP-tubulin we quantified microtubule dynamics by determining the ratio of mobile to immobile tubulin over time. We validated the approach by demonstrating that the microtubule stabilizing drug taxol decreases microtubule dynamics, and cholchicin, which is known to depolymerize microtubules, increases microtubule dynamics in our system. The data indicate that our method provides a novel approach to determine the effect of various parameters on the dynamics of microtubules in living neurons.