Title

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Authors

Douglas S. Martin, Lawrence University
Lu Yu, Lawrence University
Brian L. Van Hoozen Jr., Lawrence UniversityFollow

Document Type

Article

Publication Date

11-9-2012

Publication Title

Journal of Visualized Experiments

Keywords

Biophysics, Issue 69, Bioengineering, Physics, Molecular biology, Cellular biology, Microtubule, Persistence length, Flexural rigidity, Gliding assay, Mechanics, Cytoskeleton, Actin

ISSN

1940-087X

Volume

69

First Page

50117

Abstract

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules, and even long microtubules have measured persistence lengths which vary by an order of magnitude.

Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.

This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

Comments

©2012 Journal of Visualized Experiments

NSUWorks Citation

Martin, D. S., Yu, L., & Van Hoozen, B. L. (2012). Flexural Rigidity Measurements of Biopolymers Using Gliding Assays. Journal of Visualized Experiments, 69, 50117 - . https://doi.org/10.3791/50117. Retrieved from https://nsuworks.nova.edu/cnso_chemphys_facarticles/201

ORCID ID

0000-0002-0343-3181

DOI

10.3791/50117