A toolkit of customised tension sensors for interrogating mechanical forces in the cell

蛋白质是细胞的主力，但尽管我们对其结构和功能背后的物理和化学原理的理解取得了重大进展，但蛋白质的一个基本性质-蛋白质机制-仍然知之甚少。机械力涉及多种生物过程，如肌肉中的受力蛋白质和细胞分裂过程中染色体分离的张力，细胞感知周围环境机械特性的能力中断是许多疾病的特征，包括肌肉营养不良、动脉硬化、心肌病和癌症。因此，很明显，特定的蛋白质必须能够感知机械信号，并将其转化为生物反应，但确定它们如何做到这一点面临着一些重大挑战：首先，需要对单个分子进行测量，因为力不能作用于蛋白质分子的整体溶液，而是作用于单个蛋白质分子，反应高度依赖于力的作用方式和位置。其次，所涉及的微小(皮牛顿)力尺度需要极其灵敏的仪器，这种仪器很难建造和使用，这就需要在研究中的蛋白质中引入复杂的修饰。第三，为了真正理解力学如何转化为功能，我们需要描述细胞复杂生理环境中的这些力，这又增加了一层难度。我们提议的核心是所谓的“重复蛋白”--一类惊人的蛋白质，看起来就像纳米级的生物弹簧。这些无处不在的蛋白质具有独特的性质。与球形的“典型”蛋白质不同，重复蛋白是由连续多次重复的小结构单元堆积而成的线性马蹄形结构，就像螺旋楼梯上的台阶或一列乐高积木。这种简单、模块化的体系结构使得从头开始设计新的重复蛋白质变得简单，并具有精致的精确度，我们现在才开始意识到这种“设计能力”可以被开发的潜在应用范围。我们将使用一类我们已经发现具有非常特殊的、可开发的物理特征的人工重复蛋白。应用我们的蛋白质工程知识，我们将改变这些蛋白质的弹簧性质。最重要的是，这将使我们能够破译使我们的弹簧以它们的方式工作的代码。一旦我们有了这个代码，我们就可以对弹簧进行编程以采用我们喜欢的任何刚度。我们的主要目标是将我们的发现转化为一种弹簧式张力传感器工具包的开发，该工具包能够解析活细胞中的力，远远超出当前的技术，并可以根据个人研究人员的需求进行定制。传感器将与发光的荧光分子相连，从而使我们能够“看到”活细胞内的作用机制。建造者受到他们工具的质量和多功能性的限制。此外，工具越专业化，使用它们的人就越少。因此，尽管现有的力传感器突出了这类工具的潜力，为我们提供了对细胞内部工作原理的非凡新见解，但除非我们以一种易于定制的方式设计它们，否则它们的价值将是有限的。这是我们的目标。我们将创造一种新型的传感器--一种研究人员可以根据他们所需的任何规格进行编程的传感器。我们相信，我们的设计策略将被证明是任何想要研究生命系统中机械感觉过程的研究人员的通用资源。就像一套螺丝刀一样，我们的工具包将有一系列不同的“比特”，可以根据正在解决的生物问题进行选择。

Proteins are the workhorses of the cell, but despite significant advances in our understanding of the physical and chemical principles underlying their structures and functions, one fundamental property - protein mechanics - remains poorly understood. Mechanical forces are involved in varied biological processes such as force-bearing proteins in the muscle and tension upon chromosomes separation during cell division, and disruption of the cell's ability to sense the mechanical properties of its surroundings represents a hallmark of many diseases, including muscular dystrophy, arteriosclerosis, cardiomyopathies, and cancer. It is clear, therefore, that specific proteins must be able to sense mechanical signals and convert them into biological responses, but determining how they do so presents some major challenges: First, measurements on single molecules are required, as force cannot be applied to a bulk solution of protein molecules but rather to individual protein molecules, the response being highly dependent on how and where the force is applied. Second, the tiny (piconewton) force scales involved require extremely sensitive instrumentation that is difficult to build and use of which necessitates the introduction of complex modifications into the proteins under study. Third, for a true understanding of how mechanics translates into function we will need to characterise these forces within the complex physiological environment of the cell, adding a further layer of difficulty. At the heart of our proposal are so-called "repeat proteins" - a striking class of proteins that appear to behave as nano-sized biological springs. These ubiquitous proteins have unique properties. Unlike "typical" proteins, which are globular in shape, repeat proteins form linear, horseshoe-shaped structures from the stacking of small structural units repeated multiple times in tandem, like steps in a spiral staircase or a column of Lego blocks. This simple, modular architecture makes it straightforward to design novel repeat proteins from scratch and with exquisite precision, and we are only now starting to realise the range of potential applications for which this "design-ability" can be exploited. We will use a class of artificial repeat proteins that we have found to possess very special, exploitable physical characteristics. Applying our knowledge of protein engineering we will alter the spring-like properties of these proteins. Foremost, this will enable us to decipher the code that makes our springs work in the way they do. Once we have this code, we can then program the spring to adopt any stiffness we like. Our primary aim is to translate our findings into the development of a toolkit of spring-like tension sensors able to resolve forces in living cells well beyond the current technologies and which can be customised to the individual researcher's needs. The sensors will be coupled to fluorescent molecules that light up and thereby enable us to "see" the mechanics in action inside living cells.A builder is limited by the quality and versatility of their tools. Moreover, the more specialised the tools, the fewer people can use them. Thus, although existing force sensors highlight the potential for such tools to provide us with remarkable new insights into the inner workings of the cell, they will be of limited value unless we design them in such a way that they can be readily customised. This is our goal. We will create a new type of sensor - one that researchers can program with whatever specifications they require. We believe that our design strategies will prove to be a versatile resource for any researcher wanting to investigate mechanosensory process in living systems. Just like a screwdriver set, our toolkit will have a range of different "bits" that can be selected according to the biological question being tackled.