Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Summary

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

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 decades¹. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules^2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude^5-9.

Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay¹⁰. 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 used¹¹. 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.

Introduction

The cytoskeleton, a network of biopolymers found in most eukaryotic cells, plays a role in cellular organization, intracellular transport, and cell mechanics. The mechanical characteristics of the biopolymers of the cytoskeleton (primarily actin and microtubules) play a significant role in determining the mechanical characteristics of the cell as a whole¹². Since whole cell mechanics can characterize healthy and diseased cells^13,14 and is involved in cellular motility¹⁵, the mechanical properties of the underlying cytoskeletal components have been an active area of study for the past two decades¹.

Protocol

1. Microtubule Gliding Assay Stock Solutions

Prepare ahead of gliding assay.

1. Polymerize 0.5 mg microtubules sparsely labeled with bright organic fluorophore ²². The target label concentration is 1 fluorophore per micrometer of microtubule, or a labeling density of approximately 1 fluorophore per 1,500 tubulin dimers. Store at room temperature, light protected with aluminum, foil for up to two weeks.
2. Purify biotin-kinesin²¹ at approximately 1 μM. Store at -80 °C in 5 μl aliquots for use in individual experiments.
3. Prepare the assay buffer (AB) of 50 mM imidiazo....

Results

A snapshot from a gliding assay is shown in Figure 2. A good microtubule density is 1-10 microtubules per field of view; substantially more will result in mistracking as microtubules cross each other. A plot of the 11 microtubule trajectories from the gliding assay in Figure 2 is shown in Figure 3. Typical trajectories are 10 to 30 μm long; some trajectories have gaps where one microtubule crosses another. These trajectories may be discarded from analysis.

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Discussion

Persistence length measurements are a good characterization of the mechanical properties of individual biopolymers. In this article, we have described a method of measuring the persistence length of microtubules. As noted in the introduction, this method is readily extended to examining microtubule mechanical properties in a variety of conditions simply by varying the reagents, temperature, or viscosity in the final step of the gliding assay, 3.9, or by polymerizing microtubules, step 1.1, under different condition.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
imidazole	Sigma-Aldrich	I2399
potassium chloride	Sigma-Aldrich	P9541
magnesium chloride	Sigma-Aldrich	M8266
EGTA	Sigma-Aldrich	E3889
BSA	Calbiochem	126615
biotinylated BSA	Thermo Scientific	29130
α-casein	Sigma-Aldrich	C6780
streptavidin	Thermo Scientific	21125
dithiothreitol	Sigma-Aldrich	D0632
paclitaxel	LC Laboratories	P-9600
glucose oxidase	Sigma-Aldrich	G2133
catalase	Sigma-Aldrich	C100
glucose	Sigma-Aldrich	G8270
ATP	Sigma-Aldrich	A2383
2-mercapt–thanol	Sigma-Aldrich	M3148	Toxic. Buy small amount.
24X60 mm No. 1 1/2 cover glass	VWR	48393-252
22X22 mm No. 1 cover glass	Gold Seal	3306
High Vacuum Grease	Dow-Corning	NA
Equipment
TIRF microscope	many	NA	The TIRF microscope used in this method was home-made.
IDL (software)	Exelis	NA	Could substitute MATLAB, ImageJ, or other image analysis software.