NANO LETTERS

A Piconewton Forcemeter Assembled from Microtubules and Kinesins

Henry Hess,*, Jonathon Howard, and Viola Vogel
Department of Bioengineering, UniVersity of Washington, Seattle, Washington, and Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Received July 30, 2002; Revised Manuscript Received September 2, 2002

2002 Vol. 2, No. 10 1113-1115

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 surface3 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
* Corresponding author: Henry Hess, Dept. of Bioengineering, Box 351721, University of Washington, Seattle, WA 98195. Phone: (206) 6164194. Fax: (206) 685-4434. E-mail: hhess@u.washington.edu University of Washington. Max-Planck-Institute of Molecular Cell Biology and Genetics. 10.1021/nl025724i CCC: $22.00 Published on Web 09/25/2002 2002 American Chemical Society

Figure 1. The cantilevered microtubule (MT) is attached on one end to a clump of beads, while the other end is suspended above the surface. Swiveling due to Brownian motion causes the smearedout appearance of the free end. The moving microtubule is propelled by kinesin motor proteins adsorbed to the surface and carries a clump of beads. The snapshots show how the moving microtubule approaches (0 s), makes contact (10 s), bends (20 s, 30 s), and releases (40 s) the cantilevered microtubule.

the moving microtubule (Figure 1). We relied on chance to attach one end of a microtubule to a clump of beads adsorbed to the surface, while the other end of the microtubule was

Figure 2. (left top) The moving microtubule (bottom) is transported on a kinesin-coated surface, while connected through biotin linkers and a streptavidin-coated bead to the cantilevered microtubule. (right top) An average force on the bonds can be found by calculating the bending energy Eb stored in the cantilevered microtubule, measuring the distance s covered by the moving microtubule, and the angle R between the moving microtubule and the cantilevered microtubule. (bottom) The shape of the cantilevered microtubule (fitted by third-order polynomials) and the path of the moving microtubule (as defined by the leading tip) is shown for all time points.

suspended above the surface and moved freely, as indicated by its Brownian motion (Figure 1, 0 s). The moving microtubule, propelled by the kinesins on the surface, picked up a small clump of beads and approached the other microtubule, which acts as the cantilever (Figure 1, 0 s). Upon contact of the beads with the cantilevered microtubule, a biotin/streptavidin link was established (Figure 1, 10 s). After contact, the moving microtubule proceeded along its path while starting to bend the cantilevered microtubule (Figure 1, 20 s). The cantilevered microtubule was drastically bent (Figure 1, 30 s). In the next image (not shown), acquired 5 s later, the bonds between the moving microtubule and beads had ruptured. The cantilevered microtubule snapped back into its original position together with the transferred beads, while the moving microtubule continued on its path (Figure 1, 40 s). Taking into account the bending of the cantilevered microtubule, the distance covered by the moving microtubule, and the angle between the moving microtubule and the cantilevered microtubule, the force on the link can be calculated for every acquired frame (Figure 2). The flexural rigidity of microtubules has been measured4 but can also be directly determined from the shape fluctuations of the cantilevered microtubule due to thermal forces (Figure 1, 0 s). As seen in Figure 1, a force of 5 pN is sustained by the streptavidin/biotin bond before unbinding occurs. This
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compares very well with the measurement by Merkel et al.5 of the rupture force between biotin and streptavidin of 5 pN at a loading rate of 0.2 pN/s using the biomembrane force probe. This agreement indicates that it is indeed possible to self-assemble a pN forcemeter from molecular components. A number of issues have to be addressed for the proposed approach, among them the possibility of simultaneous binding of multiple ligands, nonspecific adhesion between the two microtubules, and a limited lifetime of the device. Binding between multiple streptavidin/biotin pairs is currently possible because the interaction region between a biotinylated microtubule and the streptavidin-coated bead stretches over the length of 60 tubulin subunits (assuming a bead radius of 1 m, and a biotin linker length of 30 nm). However, shrinking the diameter of the bead reduces the size of the interaction region, so that ultimately a single biotinylated tubulin subunit in the cantilevered microtubule can connect only via the streptavidin-coated bead to a single biotinylated tubulin subunit in the perpendicularly oriented moving microtubule. Nonspecific adhesion is a difficult problem for all force measurements on the molecular scale. In this particular case, the negative charge on both streptavidin6 and microtubules7 will reduce nonspecific adhesion, while longrange steering effects facilitate biotin-streptavidin association.8 Due to the limited lifetime of the receptor proteins, the forcemeter does not have to be kept in a functional state for much longer, and we routinely observe kinesin-mediated motility of microtubules over many hours. In addition, assemblies of microtubules may be kept in a frozen state until use, as suggested by Bohm et al.9 Balancing these challenges, the suggested forcemeter design also incorporates several unique advantages. The loading of the forcemeter by motor proteins inherently leads to loading rates comparable to in-vivo conditions, because the rates are determined by the speed at which the microtubule is propelled by the kinesins (which can be adjusted by changing the ATP concentration). Thus, the measured rupture forces are representative for rupture events actually occurring in biology. A second advantage of this molecular forcemeter, selfassembled from microtubules, kinesins, and coated beads, is its relative simplicity, requiring only commercially available proteins and a fluorescence microscope as compared to current technologies where force is applied to receptorligand bonds via optical tweezers,10 AFM cantilevers,11 glass fibers,12 or liposomes attached to capillaries.13 Detailed information about the energy landscape of protein-ligand interactions requires a statistical analysis of many rupture events. Currently, this is done sequentially by probing one interaction at a time. In contrast, the compactness of our forcemeter allows the fabrication of an array of forcemeters, so that many rupture events can be observed in parallel within the field of view of the microscope. Because the self-assembly of the presented forcemeter is difficult to reproduce reliably, we do not strive to produce further copies. Instead, our research focuses on creating a structured surface to guide the assembly process. This surface will have elevated regions selectively adsorbing and supNano Lett., Vol. 2, No. 10, 2002

porting one end of the cantilevered microtubule, and recessed tracks of kinesin motors, which move the second microtubule under a defined angle to the cantilevered microtubule. Building onto the concept of a molecular shuttle,14,15 a nanoscale transport system based on motor proteins, which has been used previously to image surfaces,16 we have assembled a functional instrument from nanoscale building blocks with the sensitivity required for the measurement of molecular events. Materials and Methods. We performed a kinesin gliding motility assay according to ref 17. A glass flow cell was perfused with a casein solution to precoat the surfaces, then a kinesin solution to adsorb the kinesin motors to the surfaces, and finally a motility solution containing biotinylated microtubules labeled with rhodamine,18 streptavidincoated beads (Magnabind, Pierce), 1 mM ATP, and an antifade solution, as described in ref 19. One surface of the flow cell was imaged every 5 s using an epi-fluorescence microscope with differential interference contrast (DIC) capability (Leica DMIRBE, 100 oil objective) and a CCD camera (Orca II, Hamamatsu, 500 ms exposure time). Fluorescence imaging of the microtubules and DIC imaging of the beads was conducted simultaneously by carefully adjusting the DIC illumination intensity. Data Analysis. The shape of the cantilevered microtubule was tracked manually by recording 12 points along the microtubule in every frame. A fit of a third-order polynomial gave a good representation of the microtubule shape. The average of the shape before contact and after rupture was used to define the orientation of the microtubule at zero load. The difference between the shape under load and the orientation at zero load was used to calculate the bending energy stored in the microtubule. The path of the moving microtubule was tracked and the intersections of this path with the shapes of the cantilevered microtubule at different time points were recorded. The force exerted by the moving microtubule was calculated by dividing the change in bending energy between two frames by the distance between the respective intersection points. The force exerted by the moving microtubule divided by the sine of the angle between the moving microtubule and the cantilevered microtubule at

the intersection point gives the force on the biotin/streptavidin bond. We measured deformations of the cantilevered microtubule corresponding to forces on the bond of 0 pN (0 s), 0.2 pN (5 s), 0.3 pN (10 s), 0.2 pN (15 s), 1.8 pN (20 s), 3.7 pN (25 s), 5.4 pN (30 s), and 0.1 pN (35 s). Supporting Information Available: A movie in AVI format of the described forcemeter is available free of charge via the Internet at http://pubs.acs.org. References
(1) Evans, E. Annu. ReV. Biophys. Biomol. Struc.t 2001, 30, 105-128. (2) Clausen-Schaumann, H.; Seitz, M.; Krautbauer, R.; Gaub, H. E. Curr. Opin. Chem. Biol. 2000, 4, 524-530. (3) Howard, J. Mechanics of Motor Proteins and the Cytoskeleton; Sindauer: Sunderland, MA, 2001. (4) Gittes, F.; Mickey, B.; Nettleton, J.; Howard, J. J. Cell Biol. 1993, 120, 923-934. (5) Merkel, R.; Nassoy, P.; Leung, A.; Ritchie, K.; Evans, E. Nature 1999, 397, 50-53. (6) Sivasankar, S.; Subramaniam, S.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12961-12966. (7) Stracke, R.; Bohm, K. J.; Wollweber, L.; Tuszynski, J. A.; Unger, E. Biochem. Biophys. Res. Commu. 2002, 293, 602-609. (8) Leckband, D. E.; Israelachvili, J. N.; Schmitt, F.-J.; Knoll, W. Science 1992, 255, 1419-1421. (9) Bohm, K. J.; Stracke, R.; Muhlig, P.; Unger, E., 2001; Vol. 12, no.3, pp 238-244. (10) Visscher, K.; Schnitzer, M. J.; Block, S. M. Nature 1999, 400, 184189. (11) Oberhauser, A. F.; Marszalek, P. E.; Erickson, H.; Fernandez, J. Nature 1998, 393, 181-185. (12) Meyhofer, E.; Howard, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 574-578. (13) Evans, E.; Ritchie, K.; Merkel, R. Biophys J. 1995, 68, 2580-2587. (14) Hess, H.; Vogel, V. ReV. Mol. Biotechnol. 2001, 82, 67-85. (15) Hess, H.; Clemmens, J.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Appl. Phys. A 2002, 75, 309-313. (16) Hess, H.; Clemmens, J.; Howard, J.; Vogel, V. Nano Lett. 2002, 2, 113-116. (17) Howard, J.; Hunt, A. J.; Baek, S. Methods Cell Biol. 1993, 39, 137147. (18) Hyman, A. A.; Drechsel, D. N.; Kellog, D.; Salser, S.; Sawin, K.; Steffen, P.; Wordeman, L.; Mitchison, T. J. Methods Enzymol. 1991, 196, 478-485. (19) Hess, H.; Clemmens, J.; Qin, D.; Howard, J.; Vogel, V. Nano Lett. 2001, 1, 235-239.

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