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In Vitro Micromanipulation
of Microtubules
Buck Wilcox
Advisor: Dr. Dahong Zhang
Zoology
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Eukaryotic Cytoskeleton
•Structural Stability•Motility•Intracellular Transport
•Microtubules•Actin Filaments•Intermediate Filaments
Nature 408, 423 (2000)Alexey Khodjakov
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MicrotubulesMajor structural component of the mitotic spindle.
caAA
FFEE
CCBB
DD
Bar = 10 m
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Microtubule Polymerization
•Microtubules are in equilibrium with a pool of tubulin subunits.
•Requires GTP hydrolysis
•Polarized Plus end Minus end
•Dynamic
25 nm25 nm
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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
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In Vitro vs. In Vivo
Piezoelectric Micromanipulator
Glass microneedleTip diameter ~ 0.1 m
Slide with ~1.0 cm diameter hole
Coverslip
Microtubules
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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
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Individual Taxol-Stabilized Microtubules
Bar = 10 m
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Results of Polymerization
Bar = 10 m
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Microtubule Asters
•Naturally occuring
•Artificially induced
By what mechanism does Taxol induce microtubule asters to form?
Several theories, but none conclusive.
Bar = 10 m
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So, how does Taxol cause microtubules to bundle and form
asters?
It’s a sticky crystal.
Taxol crystalsBar = 10 m
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+ =
Taxol crystal
Microtubule asters
Mechanism for Taxol-Induced Microtubule Asters
Not drawn to scale
MicrotubulesAND/OR
tubulin dimers
OR
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•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
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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.
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Acknowledgements
Howard Hughes Medical Institute
National Science Foundation
Brad Alsop
Dr. Kevin Ahern
Dr. Dahong Zhang