Measuring microtubule persistence length using a microtubule gliding assay.

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 microtubules contributes to modeling whole-cell rigidity and structure and hence to better understanding the processes underlying motility and transport. Moreover, the role of microtubule structure and microtubule-associated proteins (MAPs) in microtubule stiffness remains unclear. In this chapter, we present a kinesin-driven microtubule gliding assay analysis of persistence length that is amenable to simultaneous variation of microtubule parameters such as length, structure, or MAP coverage and determination of persistence 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 microtubule 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.