A Piconewton Forcemeter Assembled from Microtubules and Kinesins

Abstract

A forcemeter capable of detecting piconewton forces was assembled from nanoscale building blocks, using a cantilevered microtubule as a beam of known stiffness, which is loaded by a second microtubule transported by kinesin motor proteins adsorbed to the surface and connected to the cantilevered microtubule by the link whose strength is to be determined. Due to the loading rate of less than 1 pN/s, this forcemeter is ideally suited to study the strength of biological receptor/ligand pairs, such as streptavidin/biotin. Here we introduce a new technique to measure the force necessary to rupture receptor/ligand bonds: a molecular forcemeter self-assembled from microtubules and motor proteins. Measurements of the dynamics of receptor-ligand interactions under an applied external force, termed dynamic force spectroscopy, probe the intricate relationship between mechanical force, bond lifetime, and bond (stereo)chemistry. The distribution of rupture forces provides a fingerprint of the energy barriers traversed in the energy landscape along the unbinding pathway. 1 Typical forces to rupture noncovalent bonds range from 1 pN to 1 nN, depending on the rate at which the force is ramped up (the loading rate). A variety of experimental setups have been developed to measure pN forces over a wide range of loading rates down to 0.1 pN/s, which is typical for many biological systems. 2 Our forcemeter utilizes two microtubules - one microtubule, which is clamped on one side and freely swinging on the other, serves as a molecular cantilever, and a second microtubule moving on a kinesin-coated surface 3 applies the force. The perpendicular movement of the second microtubule bends the freely swinging end of the cantilevered microtubule once a link through the receptor/ligand pair of interest is established. The bending is imaged by fluorescence microscopy, and the applied force is calculated from the known flexural rigidity of the microtubules. 4 In our experiment we utilize streptavidin/biotin as the model receptor/ligand pair, because it has been studied repeatedly by force microscopy. Using established protocols, tubulin was biotinylated and polymerized into microtubules that expose biotin on their surface. Streptavidin-coated beads were used as links between the cantilevered microtubule and