Highly‐Parallel Microfluidics‐Based Force Spectroscopy on Single Cytoskeletal Motors

Abstract

Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, a highly‐parallel, microfluidics‐based method that allows for rapid collection of force‐dependent motility parameters of cytoskeletal motors with two orders of magnitude improvement in throughput compared to currently available methods is introduced. Tunable hydrodynamic forces to stepping kinesin‐1 motors via DNA‐tethered beads and utilize a large field of view to simultaneously track the velocities, run lengths, and interaction times of hundreds of individual kinesin‐1 molecules under varying resisting and assisting loads are applied. Importantly, the 16 µm long DNA tethers between the motors and the beads significantly reduces the vertical component of the applied force pulling the motors away from the microtubule. The approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single‐molecule force studies on cytoskeletal motors.

The force required to remove tubulin from the microtubule lattice by pulling on its α-tubulin C-terminal tail

“…In addition, we sought a high-throughput assay, unlike the tweezer assay in which we can only pull on one tubulin at a time. We therefore developed a motor-pulling assay in which many force events can be measured simultaneously 45 . To this end, we first prepared GDP-microtubules capped with GMP-CPP tubulin (GMP-CPP is a slowly hydrolysable GTP analogue) to prevent their spontaneous depolymerization.…”

Key residue on cytoplasmic dynein for asymmetric unbinding and unidirectional movement along microtubule

“…The effects of the mutation could be explained by the loss of the asymmetric unbinding property of dynein MTBD, as expected from the MD simulations. To evaluate this, we applied hydrodynamic forces to dynein-coated polystyrene beads on microtubules by shear flow caused by a microfluidic system (28). Consistent with this notion, the shear flow needed to unbind R3423D-coated beads from microtubules did not show directional dependence, while the wild-type coated beads required twice faster flow when pulled towards the plus-end of the microtubule compared to when pulled to the minus-end (Fig.…”

High-throughput force measurement of individual kinesin-1 motors during multi-motor transport

“…On the other hand, several single-molecule force sensing techniques have been developed with DNA-based designs for increasing the throughput of force measurement. 16–24…”

“…On the other hand, several single-molecule force sensing techniques have been developed with DNA-based designs for increasing the throughput of force measurement. [16][17][18][19][20][21][22][23][24] Here, we develop a Force Sensing by Individual Motors (FSIM) assay to directly measure forces of individual kinesins as they collectively move a common cargo while measuring the displacement and velocity of the common cargo, and we do so in high-throughput measurements of forces. Specifically, we have used a mammalian kinesin-1 motor for our study (referred to as kinesin; see methods section "Kinesin Purification").…”