Measuring the force of adhesion between multiple kinesins and a microtubule using the fluid force produced by microfluidic flow

Abstract

We developed a method to measure the adhesion force between the motor protein, kinesin, and a microtubule. Compared with conventional methods that use optical tweezers, our method employs the fluid force that acts on the interaction between a kinesin-coated microbead and a microtubule in a microfluidic channel. When the fluid force just exceeds the kinesin-microtubule adhesion force, the beads are released from the microtubules. Having modeled the kinesins that are bound to the microtubules and the beads as mechanical springs, adhesion forces were measured as 31.3 or 362.9 pN for fluid containing 1 mM ATP or 0 M ATP, respectively. These forces are much larger than those measured when optical tweezers were used to measure the adhesion force between a single kinesin and a microtubule. For our multi-kinesin system we elucidated the relationship between the binding force of a single kinesin molecule and that of all kinesin molecules in a contact area by varying one of two parameters: either the contact area length or the kinesin density on a bead. This study provides insight into the behavior of a bead that is supported by several kinesins in a microfluidic system, which is essential knowledge if a motor protein is to be used as a nanoactuator for in vitro molecular transport.

Automated Dielectrophoretic Tweezers-Based Force Spectroscopy System in a Microfluidic Device

“…Fifteen-micrometer carboxyl-terminated functionalized polystyrene particles (Kisker Biotech GmbH & Co. KG, Steinfurt, Germany) suspended in a DI water (~4 × 10 4 particles/mL) solution were introduced into the reservoir, and a glass slide was used to cover the top of it. For the formation of electrostatic repulsion and hydrogen bonding between the oxide surface and particles, the pH of the suspended solution was adjusted with hydrochloric acid (HCl) at 7 and 3.8 in the experiment, respectively [ 13 , 14 , 16 ]. Despite the electrostatic repulsion was generated between the oxide surface and beads at pH 7, a Van der Waals interaction was formed when the beads closely approached the oxide surface [ 14 ].…”

“…For the formation of electrostatic repulsion and hydrogen bonding between the oxide surface and particles, the pH of the suspended solution was adjusted with hydrochloric acid (HCl) at 7 and 3.8 in the experiment, respectively [ 13 , 14 , 16 ]. Despite the electrostatic repulsion was generated between the oxide surface and beads at pH 7, a Van der Waals interaction was formed when the beads closely approached the oxide surface [ 14 ]. Therefore, in the experiment using the automatic DFS system, Van der Waals interactions and hydrogen bonding were investigated.…”

“…The dielectrophoretic force corresponding to the applied voltage was calculated in the following way. The DEP force is described by where n is the force order, Φ refers to the electrostatic potential of the external electric field, and K n is the n th -order Clausius–Mossotti factor [ 14 ]. This equation was used to convert the voltage applied to the electrode into the DEP force.…”

“…After that, the fluid is applied into the binding system as varying the velocity of it, resulting in the movement of the particles. Associating the movement of the particles with the velocity of the fluid, the biological binding force can be measured [ 13 , 14 ]. This fluidic technique can measure a substantial amount of the binding force at the same time under the same environment so that it can achieve simple, efficient, and high-throughput measurement, compared with the AFM and optical tweezers described above.…”

Highly-parallel microfluidics-based force spectroscopy on single cytoskeletal motors

“…Hence, the number of constant-force experiments performed at a time is in principle limited only by the size of the imaged area and the surface density of the bead-coupled mechanosystems. Low-throughput experiments using hydrodynamic flow have previously been performed to study single-molecule forces in protein unfolding 17 , to measure rupture forces of streptavidin-biotin bonds 18 , to investigate the confining potential felt by individual membrane-embedded receptors 19 , and, in the context of cytoskeletal motors, to measure the adhesion forces of beads covered with multiple kinesin-1 motors to microtubules 20 . Moreover, high-throughput experiments using hydrodynamic flow have been performed to study DNA mechanics and DNA-protein interactions.…”