Preparation of Dual-Color Polarity-Marked Fluorescent Microtubule Seeds
Assaying microtubule dynamics in vitro requires stabilized nucleation centers, a method to immobilize individual microtubules onto a surface, and a specialized microscope to image the microtubule. Microtubules are polar structures with different dynamic properties at the plus and minus ends. However, the dynamics of the two ends can be modified by the addition of other proteins, such as microtubule plus-end-tracking proteins (+TIPs), so that it becomes impossible to distinguish the microtubule polarity by measuring the differences in the dynamic properties of the ends alone. In this chapter, we describe a method for labeling tubulin protein with N-hydroxysuccinimide ester fluorescent dyes, enabling the formation of dual-color polarity-marked stable microtubule seeds that can be immobilized onto a microscopic cover glass for imaging by fluorescence microscopy. These seeds create functional nucleation centers for the growth of dynamic microtubules.
Key wordsN-hydroxysuccinimide ester Succinimidyl ester Tubulin Microtubule In vitro microtubule dynamics assay Polarity-marked microtubules Guanylyl-(alpha, beta)-methylene-diphosphonate
We thank Dr Douglas Drummond for his comments on the manuscript. We gratefully acknowledge the Association for International Cancer Research (09-0221), Cancer Research UK (C19638/A6211), and Marie Curie Cancer Care for supporting this work.
- 1.Walker, R. A., O’Brien, E. T., Pryer, N. K., Soboeiro, M. F., Voter, W. A., Erickson, H. P., and Salmon, E. D. (1988) Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies, J. Cell Biol. 107, 1437–1448.PubMedCrossRefGoogle Scholar