A Fluorescent GTP Analogue as a Single Molecule Fluorescence Label of Microtubules

Abstract

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Dynamic studies of microtubule function make use of fluorescent labels via antibodies, paclitaxel, and direct attachment to the tubulin protein. However, these labels suffer from drawbacks such as transient labeling, occlusion of functional sites on the microtubule surface, or structural non-specificity. Here we report a new, complementary fluorescent labeling technique that avoids these drawbacks. A fluorescently modified GTP analogue is used to polymerize microtubules from tubulin dimers. This GTP analogue binds selectively to the exchangeable GTP-binding site (E-site) on the tubulin dimer, which is available only during polymerization. The E-site affinity of this GTP analogue is about 100 fold weaker than that of GTP. Because this labeling technique places a bright fluorophore at a defined location within the microtubule lattice, it may facilitate observations of microtubule dynamics with increased precision.