蒂宾根大学：锗纳米球可用于测量细胞马达蛋白力之大小

诸平

Ultraresolution kinesin traces with optically trapped germanium nanospheres.

Kinesins are molecular machines that transport vesicles along microtubules inside cells. Membrane-coated germanium nanospheres (TEM micrograph, left) improved the spatiotemporal resolution of optical tweezers and allowed the measurement of substeps during the normal kinesin stepping cycle. Under load, kinesins did not detach but slipped along the microtubule, which led to the discovery of rescues for vesicle transport.

据德国蒂宾根大学（Universität Tübingen）2021年2月12日提供的消息，该校科学家通过锗纳米球对马达蛋白（Motor proteins）的运动和力进行分辨率更高、更加精确地测量和跟踪。

马达蛋白为我们细胞内的机械过程产生动力。它们以纳米级为单位，驱动我们的肌肉或在细胞内运输物质。德国蒂宾根大学细胞纳米科学教授埃里克·谢弗（Erik Schäffer）可以用肉眼看到这样的运动：通过用专门开发的显微镜“光学镊子”跟踪分子机器的工作。他在植物分子生物学中心的团队现在进一步完善了这项技术。通过锗纳米球可以使分辨率更高、测量更加精确，可以对马达蛋白的运动和力进行跟踪测量。相关研究结果于2021年2月12日已经在《科学》（Science）杂志网站发表——Swathi Sudhakar, Mohammad Kazem Abdosamadi, Tobias Jörg Jachowski, Michael Bugiel, Anita Jannasch, Erik Schäffer. Germanium nanospheres for ultraresolution picotensiometry of kinesin motors. Science， 12 Feb 2021: Vol. 371, Issue 6530, eabd9944. DOI: 10.1126/science.abd9944。

所研究的马达蛋白只有60 nm大小，确实很小，但是对于细胞过程却是必不可少的。除其他外，它们通过将染色体机械地拉开来帮助细胞分裂，或者在细胞内运输“包裹”。如果这些马达蛋白不起作用，可能会导致神经系统疾病，例如神经细胞中的阿尔茨海默氏病（Alzheimer's）。
为了追踪这些分子机器的机理，生物物理学家埃里克·谢弗（Erik Schäffer）开发了超精密光学镊子。它们基于天文学家约翰内斯·开普勒（Johannes Kepler）在1609年发现的原理，物理学家亚瑟·阿什金（Arthur Ashkin）为此获得了2018年诺贝尔奖，他所发明的光镊及光镊在生物系统领域被广泛应用。通过这些发明，如今我们可以用一种崭新的方法来观察极端微小的物体和超高速过程，先进的精确设备正在开发全新领域，具有广阔的工业和医学应用前景。

光的辐射压力用于用激光束将小球体固定在适当的位置并测量最小的力。借助这种工具，埃里克·谢弗在最近几年已经能够证明马达蛋白驱动蛋白（motor protein kinesin）像舞蹈一样四处移动：通过两个“脚”，每次需要8 nm的步幅并转动半圈–类似于维也纳华尔兹(Viennese waltz)舞姿。

埃里克·谢弗的博士生斯瓦蒂·苏达卡尔（Swathi Sudhakar）现在进一步发展了光镊技术。使用所谓的锗纳米球，是更小、更高分辨率的探针，其大小仅仅相当于一块巧克力重量的五万亿分之一；而分辨率可以达到使人几乎无法达到的生物马达5皮牛顿（5 piconewtons, 5 pN）之力。这意味着即使最小和快速的移动也可以被测量。到目前为止，由于典型的小颗粒快速运动，无法精确地观察到这些现象。

可以对驱动蛋白（kinesin）进行实时观察，斯瓦蒂·苏达卡尔能够在他的运动中展示出另一个“中间步骤”，这使华尔兹几近完美。埃里克·谢弗说：“是否有中间步骤已经在科学家之间争论了20年。” “我们首次能够用光镊（optical tweezers）对其直接测量。” 此外，纳米球还揭示了一种以前未知的马达蛋白“滑移机理”（“slip mechanism” of the motor protein）：埃里克·谢弗说：“这是一种安全线，既是负载过高，它也可以将马达保持在轨道上。”该机制解释了细胞中物质运输的高效率。“如果您要知道驱动蛋白马达的详细工作原理，那么您需要更好地了解驱动它们的重要细胞过程以及可能导致疾病的故障。”

科学家将这种新技术与深入研究进行了比较，可以说是分子机器的“幕后（under the hood）”。这样，不仅可以精确地观察马达蛋白的各个运动，而且可以更好地理解例如蛋白质如何获得其结构。“作为半导体，纳米球具有其他令人兴奋的光学和电学性质，还可以用于纳米和材料科学的其他领域，例如用于更好的锂离子电池等。”更多信息请注意浏览原文或者相关报道。

Kinesin takes substeps

Simultaneously measuring the nanoscale motion and forces that molecular machines generate provides insights into how they work mechanically to fulfill their cellular function. To study these machines, Sudhakar et al. developed germanium semiconductor nanospheres as probes for so-called optical tweezers. With these high–refractive index nanospheres, they improved the resolution of optical tweezers and discovered that the motor kinesin takes 4-nanometer substeps. Further, instead of detaching from their microtubule track under load, motors slid back on it, enabling rapid reengagement in transport. The new technology will allow investigation of a range of other proteins and their behaviors at nanometer scales.

Science, this issue p. eabd9944

Structured Abstract

INTRODUCTION

Cytoskeletal motors drive many essential mechanical processes inside cells. For example, kinesin motors are key for cell division or vesicle transport. Kinesin-1 transports cargo along microtubules by coupling adenosine 5′-triphosphate (ATP) hydrolysis to perform mechanical work against piconewton loads. This force generation and overall transport distance are limited by motor detachment. However, how kinesins walk and detach is still unclear.

To simultaneously measure the nanoscale motion and forces of molecular machines, optical tweezers are often used. In the tight, mostly infrared laser focus of optical tweezers, small dielectric particles can be trapped and used as handles for sensitive position and force measurements. Because optical forces scale with the particle volume, piconewton force measurements with nanoparticles require a high laser power. This high power leads to excessive heating and precludes biological measurements. Therefore, for biological single-molecule measurements, micrometer-sized probes are used. However, such probes have a large hydrodynamic drag and therefore lack the spatiotemporal resolution to unravel important fast or small details in the mechanochemistry of molecular machines. These details remain hidden in the storm of Brownian motion.

RATIONALE

To overcome this practical resolution limit of optical tweezers and resolve so-far hidden conformational changes of proteins, we sought to compensate the volume scaling of optical trapping forces by the use of probe materials with a very high refractive index and low light absorption. This compensation should allow the use of nanometer-sized probes and the generation of piconewton optical forces without detrimental heating but with improved temporal response and spatial precision. Promising materials include silicon and germanium that become transparent in the near-infrared, with very high refractive indices exceeding 4. However, efficient methods to fabricate such semiconductor nanospheres suitable for optical trapping do not exist.

RESULTS

We developed a solution-based method to synthesize germanium nanospheres. With a diameter of roughly 70 nm, they are about an order of magnitude smaller as compared with commonly used microspheres and still allowed piconewton force measurements. To find out how kinesin works mechanically, we developed an in vitro reconstituted assay. To this end, we coated the nanospheres with a lipid bilayer [the white rim in the transmission electron microscopy (TEM) image] to mimic vesicles and to be roughly their size inside cells. When we bound kinesin-1 to these “vesicles” and measured the interaction of single motors with microtubules under piconewton tension, we discovered that each hydrolysis cycle is broken up into two 4-nm center-of-mass substeps. The durations of these substeps alternated in their force and ATP dependence, with the duration of one of the substeps being nearly independent of both parameters. Furthermore, when subjected to hindering loads, motors never detached from the microtubule. Instead, motors slipped along the microtubule in 8-nm steps on microsecond time scales. These slip steps are consistent with a bond-rupture model that involves protein friction between the motor and its track. Unexpectedly, motors usually did not detach after a slip event but reengaged in motility that rescued cargo transport.

CONCLUSION

Germanium nanospheres are promising for bioimaging, sensing, optoelectronics, nanophotonics, and energy storage. For optical trapping, the nanospheres open a new temporal window by which to uncover hidden dynamics in molecular machines. The direct observation of load-bearing kinesin substeps resolves a long-standing controversy. Slipping and rescues should allow load distribution and synchronization when motors operate in teams. Understanding their mechanochemistry is important for a better understanding of cellular transport and other essential molecular functions of kinesins, with implications, for example, for neurodegenerative diseases and cancer.