Photoactivatable green fluorescent protein-tubulin

Abstract

Direct observations of live cells expressing fluorescently tagged tubulin have led to important advances in our understanding of mitosis. A limitation of this approach is that all of the cells' microtubules are fluorescent and thus observation of the behavior of specific subsets of microtubules is precluded. To address this problem, we have tagged tubulin with a photoactivatable variant of green fluorescent protein (PA-GFP), thereby allowing one to follow the behavior of a subset of tagged molecules in the cell. Here, we describe methods to tag and express proteins with PA-GFP, locally photoactivate the recombinant protein and record the dynamic behavior of the photoactivated molecules in live cells. Use of photoactivatable proteins is a powerful approach to examine dynamic processes, including spindle formation, in diverse cells.