in vitro micromanipulation of microtubules buck wilcox advisor: dr. dahong zhang zoology

In Vitro Micromanipulation

of Microtubules

Buck Wilcox

Advisor: Dr. Dahong Zhang

Zoology

Eukaryotic Cytoskeleton

•Structural Stability•Motility•Intracellular Transport

•Microtubules•Actin Filaments•Intermediate Filaments

Nature 408, 423 (2000)Alexey Khodjakov

MicrotubulesMajor structural component of the mitotic spindle.

caAA

FFEE

CCBB

DD

Bar = 10 m

Microtubule Polymerization

•Microtubules are in equilibrium with a pool of tubulin subunits.

•Requires GTP hydrolysis

•Polarized Plus end Minus end

•Dynamic

25 nm25 nm

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

In Vitro vs. In Vivo

Piezoelectric Micromanipulator

Glass microneedleTip diameter ~ 0.1 m

Slide with ~1.0 cm diameter hole

Coverslip

Microtubules

In vitroMicrotubule Polymerization

Fluorescently-labeled tubulin protein

Buffer

EGTA – chelator Ca++ inhibits microtubule polymerization

MgCl2 – Mg++ promotes polymerization

Glycerol – promotes polymerization

GTP – energy for protein

Taxol (paclitaxel) – stabilizes microtubules

Individual Taxol-Stabilized Microtubules

Bar = 10 m

Results of Polymerization

Bar = 10 m

Microtubule Asters

•Naturally occuring

•Artificially induced

By what mechanism does Taxol induce microtubule asters to form?

Several theories, but none conclusive.

Bar = 10 m

So, how does Taxol cause microtubules to bundle and form

asters?

It’s a sticky crystal.

Taxol crystalsBar = 10 m

+ =

Taxol crystal

Microtubule asters

Mechanism for Taxol-Induced Microtubule Asters

Not drawn to scale

MicrotubulesAND/OR

tubulin dimers

OR

•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

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.

Acknowledgements

Howard Hughes Medical Institute

National Science Foundation

Brad Alsop

Dr. Kevin Ahern

Dr. Dahong Zhang