nn6b01294_si_002.mpg (558 kB)
Engineering Circular Gliding of Actin Filaments Along Myosin-Patterned DNA Nanotube Rings To Study Long-Term Actin–Myosin Behaviors
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posted on 2016-08-29, 00:00 authored by Rizal F. Hariadi, Abhinav J. Appukutty, Sivaraj SivaramakrishnanNature has evolved molecular motors
that are critical in cellular
processes occurring over broad time scales, ranging from seconds to
years. Despite the importance of the long-term behavior of molecular
machines, topics such as enzymatic lifetime are underexplored due
to the lack of a suitable approach for monitoring motor activity over
long time periods. Here, we developed an “O”-shaped
Myosin Empowered Gliding Assay (OMEGA) that utilizes engineered micron-scale
DNA nanotube rings with precise arrangements of myosin VI to trap
gliding actin filaments. This circular gliding assay platform allows
the same individual actin filament to glide over the same myosin ensemble
(50–1000 motors per ring) multiple times. First, we systematically
characterized the formation of DNA nanotubes rings with 4, 6, 8, and
10 helix circumferences. Individual actin filaments glide along the
nanotube rings with high processivity for up to 12.8 revolutions or
11 min in run time. We then show actin gliding speed is robust to
variation in motor number and independent of ring curvature within
our sample space (ring diameter of 0.5–4 μm). As a model
application of OMEGA, we then analyze motor-based mechanical influence
on “stop-and-go” gliding behavior of actin filaments,
revealing that the stop-to-go transition probability is dependent
on motor flexibility. Our circular gliding assay may provide a closed-loop
platform for monitoring long-term behavior of broad classes of molecular
motors and enable characterization of motor robustness and long time
scale nanomechanical processes.