1. In Vitro Micromanipulation of Microtubules Buck Wilcox Advisor: Dr. Dahong Zhang Zoology

2. Eukaryotic Cytoskeleton Structural Stability Motility Intracellular Transport Microtubules Actin Filaments Intermediate Filaments Nature 408, 423 (2000) Alexey Khodjakov

3. Microtubules Major structural component of the mitotic spindle. c a A A F F E E C C B B D D Bar = 10  m

4. Microtubule Polymerization Microtubules are in equilibrium with a pool of tubulin subunits. Requires GTP hydrolysis Polarized Plus end Minus end Dynamic   25 nm

5. Micromanipulation Using a glass microneedle with tip diameter ~ 0.1 µm we can: Detach and remove chromosomes from cells Cut cells Dissect and reconstruct the mitotic spindle apparatus Make force measurements

6. In Vitro vs. In Vivo Piezoelectric Micromanipulator Glass microneedle Tip diameter ~ 0.1  m Slide with ~1.0 cm diameter hole Coverslip Microtubules

7. In vitro Microtubule Polymerization Fluorescently-labeled tubulin protein Buffer EGTA – chelator Ca ++ inhibits microtubule polymerization MgCl 2 – Mg ++ promotes polymerization Glycerol – promotes polymerization GTP – energy for protein Taxol (paclitaxel) – stabilizes microtubules

8. Individual Taxol-Stabilized Microtubules Bar = 10  m

9. Results of Polymerization Bar = 10  m

10. Microtubule Asters Naturally occuring Artificially induced By what mechanism does Taxol induce microtubule asters to form? Several theories, but none conclusive. Bar = 10  m

11. So, how does Taxol cause microtubules to bundle and form asters? It’s a sticky crystal. Taxol crystals Bar = 10  m

12. + = Taxol crystal Microtubule asters Mechanism for Taxol-Induced Microtubule Asters Not drawn to scale Microtubules AND/OR tubulin dimers OR

13. A pure, simple in vitro system is a valuable tool for studying microtubule dynamics and interactions. It has allowed for the clear identification of how Taxol causes the reorganization of microtubules. It is expected that further use of this system will lead to a greater understanding of the interactions between microtubules and actin filaments. Conclusions

14. Future Directions Test interactions between microtubules and actin filaments using a LED-illuminated microneedle. Test the ability of microtubules to bend and withstand various forces. Identify if other drugs that induce microtubule asters to form act by a mechanism similar to that of Taxol.

15. Acknowledgements Howard Hughes Medical Institute National Science Foundation Brad Alsop Dr. Kevin Ahern Dr. Dahong Zhang