Imaging of thermal activation of actomyosin motors
Imaging of thermal activation of actomyosin motors
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DOI：
10.1073/pnas.96.17.9602
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发表时间：
1999-08-17
影响因子：
11.1
通讯作者：
Ishiwata, S
中科院分区：
文献类型：
--
作者：
Kato, H;Nishizaka, T;Ishiwata, S
我们已经开发了温度脉冲显微术，其中显微样品的温度以方波方式可逆地升高，上升和下降时间为几毫秒，并且局部地在直径约10 μ m的区域中具有高达2 ℃/μ m的温度梯度，通过对附着在（单个）上的罗丹明鬼笔环肽的荧光强度进行图像处理，在短脉冲下，肌动球蛋白马达可以在高于生理温度（峰值高于60摄氏度）时被激活，然后热诱导的蛋白质损伤开始发生。当滑动肌动蛋白丝加热到40-45 ℃时，滑动速度在25 mM KCl时达到30 μ m/s，在50 mM KCl时达到50 μ m/s，这是在通常的体外测定系统中骨骼肌球蛋白的最高速度。当从18 ℃加热到40-45 ℃时，滑动速度和力都增加了一个数量级。温度脉冲显微镜预计将是有用的生物分子和细胞的研究需要时间和/或空间的热调制。
We have developed temperature-pulse microscopy in which the temperature of a microscopic sample is raised reversibly in a square-wave fashion with rise and fall times of several ms, and locally in a region of approximately 10 mu m in diameter with a temperature gradient up to 2 degrees C/mu m, Temperature distribution was imaged pixel by pixel by image processing of the fluorescence intensity of rhodamine phalloidin attached to (single) actin filaments, With short pulses, actomyosin motors could be activated above physiological temperatures (higher than 60 degrees C at the peak) before thermally induced protein damage began to occur. When a sliding actin filament was heated to 40-45 degrees C, the sliding velocity reached 30 mu m/s at 25 mM KCl and 50 mu m/s at 50 mM KCl, the highest velocities reported for skeletal myosin in usual in vitro assay systems. Both the sliding velocity and force increased by an order of magnitude when heated from 18 degrees C to 40-45 degrees C. Temperature-pulse microscopy is expected to be useful for studies of biomolecules and cells requiring temporal and/or spatial thermal modulation.