Biology Reference
In-Depth Information
CHAPTER
a Microtubule Gliding Assay
2
Douglas S. Martin
Lawrence University, Appleton, Wisconsin, USA
Measuring Microtubule
Persistence Length Using
CHAPTER OUTLINE
Introduction .............................................................................................................. 14
2.1 Theory............................................................................................................... 15
2.2 Methods ............................................................................................................ 17
2.2.1 Polymerization of Fluorescently Labeled Microtubules ........................ 18
2.2.2 Microtubule Gliding Assay................................................................ 19
2.2.3 Image Analysis and Persistence Length Calculation............................ 20
2.3 Discussion......................................................................................................... 22
Summary .................................................................................................................. 23
Acknowledgments ..................................................................................................... 23
References ............................................................................................................... 23
Abstract
The mechanical properties of microtubules have been an area of active research for the
past two decades, in part because understanding the mechanics of individual microtu-
bules contributes to modeling whole-cell rigidity and structure and hence to better un-
derstanding the processes underlying motility and transport. Moreover, the role of
microtubule structure andmicrotubule-associated proteins (MAPs) in microtubule stiff-
ness remains unclear. In this chapter, we present a kinesin-driven microtubule gliding
assay analysis of persistence length that is amenable to simultaneous variation of micro-
tubule parameters such as length, structure, or MAP coverage and determination of per-
sistence length. By combining sparse fluorescent labeling of individual microtubules
with single particle tracking of individual fluorophores, microtubule gliding trajectories
are tracked with nanometer-level precision. The fluctuations in these trajectories, due to
thermal fluctuations in the microtubules themselves, are analyzed to extract the micro-
tubule persistence length. In the following, we describe this gliding assay and analysis
and discuss two example microtubule variables, length and diameter, in anticipation that
the method may be of wide use for
in vitro
study of microtubule mechanical properties.
