Horizontal Magnetic Tweezers to Directly Measure the Force–Velocity Relationship for Multiple Kinesin Motors

Abstract

Transport of intracellular cargo is often achieved by the concerted operation of more than a single motor protein. Characterizing the force production during such transport events has therefore been of interest to biophysicists and cell biologists for a long time. However, up to date force generation by multiple motors is poorly understood although the biophysical properties of single motors have intensively been characterized in vitro. [1][2][3][4] Transport of intracellular cargo along cytoskeletal filaments is often achieved by the concerted action of multiple motor molecules. While single-molecule studies have provided profound insight into the mechano-chemical principles and force generation of individual motors, studies on multi-motor systems are less advanced. Here, a horizontal magnetic-tweezers setup is applied, capable of producing up to 150 pN of horizontal force onto 2.8 µm superparamagnetic beads, to motor-propelled cytoskeletal filaments. It is found that kinesin-1 driven microtubules decorated with individual beads display frequent transitions in their gliding velocities which we attribute to dynamic changes in the number of engaged motors. Applying defined temporal force-ramps the force-velocity relationship is directly measured for multi-motor transport. It is found that the stall forces of individual motors are approximately additive and collective backward motion of the transport system under super-stall forces is observed. The magnetic-tweezers apparatus is expected to be readily applicable to a wide range of molecular and cellular motility assays.