Engineering Circular Gliding of Actin Filaments Along Myosin-Patterned DNA Nanotube Rings To Study Long-Term Actin-Myosin Behaviors.
Engineering Circular Gliding of Actin Filaments Along Myosin-Patterned DNA Nanotube Rings To Study Long-Term Actin-Myosin Behaviors.
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DOI：
10.1021/acsnano.6b01294
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发表时间：
2016-09-27
期刊：
影响因子：
17.1
通讯作者：
Sivaramakrishnan S
中科院分区：
文献类型：
--
作者：
Hariadi RF;Appukutty AJ;Sivaramakrishnan S
大自然已经进化出分子马达，这些马达在细胞过程中至关重要，发生在从几秒到几年的广泛时间尺度上。尽管分子机器的长期行为的重要性，但由于缺乏长时间监测运动活动的合适方法，酶寿命等主题尚未得到充分研究。在这里，我们开发了一种“O”形肌球蛋白增强滑动试验（OMEGA），该试验利用工程化的微米级DNA纳米管环，具有肌球蛋白VI的精确排列来捕获滑动的肌动蛋白丝。这种环形滑动测定平台允许相同的单个肌动蛋白丝在相同的肌球蛋白整体（每个环50-1000个马达）上滑动多次。首先，我们系统地表征了具有4、6、8和10个螺旋周长的DNA纳米管环的形成。单个肌动蛋白丝沿着纳米管环以高的持续性滑动，在运行时间内可达12.8转或11分钟。然后，我们表明肌动蛋白的滑动速度是强大的马达数量的变化和独立的环曲率在我们的样本空间（环直径为0.5-4 μm）。作为OMEGA的一个模型应用，我们分析了基于电机的机械影响肌动蛋白丝的“停-走”滑行行为，揭示了停-走转换概率取决于电机的灵活性。我们的循环滑动试验可以提供一个闭环平台，用于监测广泛类别的分子马达的长期行为，并能够表征马达的鲁棒性和长时间尺度的纳米机械过程。
Nature has evolved molecular motors that are critical in cellular processes occurring over broad timescales, 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 minutes 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 timescale nanomechanical processes.
DOI：
10.1083/jcb.81.1.115
发表时间：
1979-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Young RB;Bergen WG;Blauwiekel PB
通讯作者：
Blauwiekel PB