Using fluorescent myosin to directly visualize cooperative activation of thin filaments.
Using fluorescent myosin to directly visualize cooperative activation of thin filaments.
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DOI：
10.1074/jbc.m114.609743
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发表时间：
2015-01-23
期刊：
影响因子：
--
通讯作者：
Kad NM
中科院分区：
文献类型：
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作者：
Desai R;Geeves MA;Kad NM
背景：钙如何调节细丝活化尚不确定。结果：利用单分子成像技术报道了细丝在不同溶液条件下的活化情况。结论：激活细丝需要两个肌凝蛋白头，以三种状态发生，肌凝蛋白结合为后续结合物开放了11个位点。意义：这是第一次直接可视化合作行为，揭示了复杂系统的功能。横纹肌的收缩受肌细胞内钙的释放和隔离的严格调节。在分子水平上，钙调节肌凝蛋白进入细丝的通道。一旦结合，肌凝蛋白被认为可以增强进一步肌凝蛋白的结合。在这里，我们直接成像肌球蛋白的单分子结合和激活细丝。利用这种方法，肌凝蛋白沿细丝的协同结合已被量化。我们发现需要两个肌凝蛋白头来横向激活细丝的调节单元。发现调节单元能够容纳11个额外的肌凝蛋白。具有不同肌球蛋白结合能力的三种细丝激活状态也可见。为了描述这个系统，我们制定了一个简单的协同活化化学动力学模型，适用于广泛的溶液条件。在次优激活条件下获得的数据强烈强调了激活的随机性，在次优激活条件下，可以观察到激活波的产生和它们的灾难性崩溃。这表明细灯丝具有以二进制方式完全打开或关闭的潜力。
Background: How calcium regulates thin filament activation is uncertain. Results: Single molecule imaging is used to report on thin filament activation across relevant solution conditions. Conclusion: Two myosin heads are required to activate a thin filament, which occurs as three states, and myosin binding opens up 11 sites for subsequent binders. Significance: This first direct visualization of cooperativity in action exposes how complex systems function. Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin's access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here, we directly image single molecules of myosin binding to and activating thin filaments. Using this approach, the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 additional myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system, we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off in a binary fashion.