Expression Studies of Other Unconventional Myosins

与牛津大学的Philipp Kukura合作，我们使用了一种基于光学显微镜的干涉散射技术来研究肌球蛋白5 HMM在肌动蛋白上的前进运动。使用这项技术，我们能够成像单个的，未标记的肌球蛋白分子5 HMM，以几纳米的精度沿着肌动蛋白移动。该分子的步长为36 nm，移动速度与先前报道的荧光标记肌球蛋白5的移动速度相同。通过将20 nm的金粒子连接到氨基末端，我们能够在高达1000赫兹的采样率下测量运动，并跟踪未连接的标记肌球蛋白头部的运动。有趣的是，即使附着了20纳米的金粒子，肌球蛋白的运动速度也与未标记的分子相同。未标记的头部并不像先前提出的那样自由扩散，而是将大部分时间花在一个固定的、脱离肌动蛋白的位置上，它偶尔会从这个位置探索前向肌动蛋白结合部位。对两个头部的同时跟踪显示，在指南针般的旋转运动中，连续的步伐沿着相同的路径到达肌动蛋白的同一侧，展示了对称的行走模式。在与Tom Friedman的实验室合作中，我们发现肌球蛋白-15需要伴侣蛋白的共同表达才能在Sf9/杆状病毒系统中成功表达，并且它结合了一个调节的和必要的轻链。

In collaboration with Philipp Kukura of Oxford University, we have used a light microscopy based interferometric scattering technique to examine the processive movement of myosin 5 HMM on actin. Using this technique we were able to image single, unlabeled molecules of myosin 5 HMM move along actin with a precision of a few nanometers. The molecule took 36 nm steps and moved at the same speed as previously reported for fluorescently-labeled myosin 5. By attaching a 20 nm gold particle to the amino-terminus we are able to measure the movement at sampling rates up to 1000 Hz and follow the movement of the unattached labeled myosin head. Interesting, even with a 20 nm gold particle attached the myosin moves at the same velocity as the unlabeled molecule. The unlabeled head does not freely diffuse as previously proposed, but rather spends most of its time in a fixed, off actin, position from which it occasionaly explores the forward actin binding sites. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. In collaboration with the lab of Tom Friedman we showed that myosin-15 requires the co-expression of chaperones for successful expression in the Sf9/baculovirus system and that it binds a regulatory and an essential light chain.