Biology Reference
In-Depth Information
stabilized by small molecules such as paclitaxel (as in this experiment) or slowly hy-
drolyzing GTP analogs (GMP-CPP or GTP-
g
S), or stabilized by high temperatures
on a microscope designed to operate at 37
C. In addition, this experiment requires
that the microtubules support kinesin motility; hence, the uses of conditions which
disrupt motility (such as certain MAPs or temperatures) are not appropriate for this
experiment. Finally, while equipment required for single-fluorophore imaging is in
wide use (in the form of TIRF and confocal microscopes), the up-front costs of such
equipment compared to a standard fluorescence microscope can be substantial.
Ultimately, however, we hope that this combination of rapid experiments and
analysis, which are reproducible from day to day, will make this technique useful
to a range of workers studying biophysical properties of microtubules. Indeed, the
analysis technique described here is not limited to microtubules driven by kinesin;
actin polymers in myosin-driven gliding assays are amenable to exactly the same
technique.
SUMMARY
We have described a technique to measure microtubule mechanical properties using
kinesin-driven gliding assays. This technique is complementary to existing tech-
niques relying on active forces or passive, nondriven, microtubule fluctuations.
The key advantages it offers are a simple, well-characterized, biochemical system
(kinesin motility); high precision through the tracking of single fluorophores; and
the ability to combine mechanical and structural measurements on microtubules.
The software necessary to perform this analysis is relatively straightforward to
use and available from the author upon request.
Acknowledgments
We would like to thank Melissa Klocke for help preparing
Fig. 2.2
, and Anna Ratliff for sub-
stantially simplifying the gliding assay described in
Section 2.2
. This work was supported by
the Research Corporation for Science Advancement.
References
