A fluorescent GTP analog as a specific, high-precision label of microtubules

Abstract

Fluorescent imaging of cytoskeletal structures permits studies of both organization within the cell and dynamic reorganization of the cytoskeleton itself. Traditional fluorescent labels of microtubules, part of the cytoskeleton, have been used to study microtubule localization, structure, and dynamics, both in vivo and in vitro. However, shortcomings of existing labels make imaging of microtubules with high precision light microscopy difficult. In this paper, we report a new fluorescent labeling technique for microtubules, which involves a GTP analog modified with a bright, organic fluorophore (TAMRA, Cy3, or Cy5). This fluorescent GTP binds to a specific site, the exchangeable site, on tubulin in solution with a dissociation constant of 1.0±0.4 µM. Furthermore, the label becomes permanently incorporated into the microtubule lattice once tubulin polymerizes. We show that this label is usable as a single molecule fluorescence probe with nanometer precision and expect it to be useful for modern subdiffraction optical microscopy of microtubules and the cytoskeleton.

Keywords

• Microtubules
• fluorescence
• TIRF
• FIONA
• cytoskeleton