Expression Studies of Other Unconventional Myosins

与牛津大学的Philipp Kukura合作，我们使用了基于光学显微镜的干涉散射技术来研究肌球蛋白5 HMM在肌动蛋白上的进行性运动。 使用这种技术，我们能够成像单个的，未标记的肌球蛋白5 HMM分子沿沿着肌动蛋白移动，精度为几纳米。 该分子以36 nm的步长移动，并以与先前报道的荧光标记肌球蛋白5相同的速度移动。 通过将20 nm的金颗粒连接到氨基末端，我们能够以高达1000 Hz的采样率测量运动，并跟踪未连接的标记肌球蛋白头部的运动。 有趣的是，即使有一个20纳米的金颗粒连接的肌球蛋白以相同的速度移动的未标记的分子。 未标记的头部并不像以前提出的那样自由扩散，而是将大部分时间花在固定的、远离肌动蛋白的位置上，从该位置开始探索前向肌动蛋白结合位点。 同时跟踪的两个头显示，连续的步骤遵循相同的路径，在同一侧的肌动蛋白在一个指南针般的旋转运动，展示了对称的步行模式。 在与Tom Friedman实验室的合作中，我们发现肌球蛋白-15需要伴侣蛋白的共表达才能在Sf 9/杆状病毒系统中成功表达，并且它结合了一条调控和一条必需的轻链。

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.