Journal
SMALL
Volume 17, Issue 18, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/smll.202007388
Keywords
cytoskeletal motors; kinesin; microtubules; molecular motors; single‐ molecule force spectroscopy
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Funding
- Dresden International Graduate School for Biomedicine and Bioengineering (DIGS-BB)
- German Excellence Initiative (TU Dresden Support-the-Best grant)
- Human Frontier Science Program [LT000180/2012-L]
- Max Planck Society
- Projekt DEAL
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This study introduces a highly-parallel, microfluidics-based method for rapid collection of force-dependent motility parameters of cytoskeletal motors, significantly improving throughput. DNA-tethered beads are used to apply tunable hydrodynamic forces to stepping kinesin-1 motors, allowing for simultaneous tracking of various motility parameters of hundreds of individual molecules. The approach, applicable to other molecular systems, represents a new methodology for parallelized single-molecule force studies on cytoskeletal motors.
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 mu m 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.
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