Biophysical mechanism and synthetic engineering of optically-controlled Ca2+-powered supramolecular engines

项目摘要 肌丝是钙驱动的超分子蛋白质“弹簧”，形成了产生力量的细胞骨架。 一些原生动物纤毛虫的结构，如疑似螺旋口。在螺旋口属中，肌丝 超高功率输出(相当于二冲程柴油发动机)，使Sprostomum能够收缩 在不到5毫秒的时间内达到其身体长度的四分之一(在单细胞水平上最快的运动之一)。在以下方面 在单位质量的能量中，肌丝产生的力量比传统的ATP动力多6个数量级 分子马达，如肌球蛋白或肌动蛋白。Myonemes不包含传统的细胞骨架元件，如 如肌动蛋白、微管或肌球蛋白。相反，肌丝是由两个组成部分组成的自组装：中心 对钙有反应的蛋白质和Sfi1，一种弹性的骨架蛋白质。因此，肌位提供了有吸引力的 不依赖于ATP的驱动、超快和高功率传输以及简单的两组分 系统，该系统可以实现潜在的变革性合成生物学应用，例如人工智能的设计 用于合成或生物杂交细胞的细胞骨架，使它们能够分裂、移动或运输类似于其 活着的对应者。然而，我们对控制生物物理机制的认识存在着关键的空白。 这些弹簧中的力的产生，钙离子如何作为化学闩锁来控制和同步力 提供超过毫米长度的规模，以及如何合成这些超分子组件 以及体外自组装，以利用它们实现所需的功能。 为了解决这些认识上的差距，拟议中的未来5年的研究将采取双管齐下的方式 方法：一)结合生物物理实验、活显微镜和基于软物质物理的模型来揭示 活体细胞中肌丝内力产生的生物物理机制，以及II)工程师，自我 在微流控装置和脂类中组装和整合体外合成肌丝(SynMyo)中的光控制 水泡。最后，这项工作还将利用数学理论和数值模拟来支持我们的发现。 从长远来看，这项研究将开辟一类全新的纳米级、基于钙离子和光驱动的材料 合成力产生细胞骨架组件，在细胞内驱动和传感中的应用， 治疗性药物输送装置和人造细胞骨架。对于合成细胞，这些 超分子弹簧可以实现新的机械功能，例如比任何 基于微管或肌动蛋白的系统可以提供；不受聚合物轨道的局部力产生；可控性 这是从细胞特定的生物化学正交化的；以及一种新的、不基于ATP或GTP的能源，以 细胞内的能量运动。

PROJECT ABSTRACT Myonemes are calcium-powered supramolecular protein `springs’ that form the force-generating cytoskeletal structure in some protozoan ciliates such as Spirostomum ambiguum. In Spirostomum, myonemes extraordinarily high-power outputs (equivalent to a 2-stroke diesel engine) that enable Spirostomum to contract to 1/4th of its body length in less than 5 milliseconds (one of the fastest motions at the single cell level). In terms of power per unit mass, myonemes generate six orders of magnitude more force than conventional ATP-powered molecular motors such as myosin or kinesin. Myonemes do not contain conventional cytoskeletal elements such as actin, microtubules or myosin. Rather, myonemes comprise of self-assemblies of two-components: centrin proteins that are calcium-responsive and Sfi1, an elastic backbone protein. Thus, myonemes offer attractive features such as non-ATP dependent actuation, ultrafast and high-power delivery and a simple two-component system, that could enable potentially transformational synthetic biology applications, such as design of artificial cytoskeletons for synthetic or biohybrid cells to enable them to divide, move or transport cargo similar to their living counterparts. However, there exists key gaps in our knowledge on the governing biophysical mechanism of force generation in these springs, how calcium ions act as chemical latches to control and synchronize force deliver over millimeter length scales, and how these supramolecular assemblies can be synthetically engineered and self-assembled in-vitro for harnessing them for desired functionalities. To address these gaps in understanding, the proposed research over the next 5 years will take a two-pronged approach: i) combine biophysical experiments, live microscopy and soft matter physics-based models to uncover the biophysical mechanism of force-generation in myonemes in-vivo in living cells, and ii) engineer, self- assemble and incorporate light-control in synthetic myonemes (synMyo) in-vitro in microfluidic devices and lipid vesicles. Finally, this work will also utilize mathematical theory and numerical simulations to support our findings. Long-term, this research will open up a fundamentally new class of nanoscale, Ca2+-based, and light-actuatable synthetic force generating cytoskeletal assemblies, with applications in intracellular actuation and sensing, therapeutic drug-delivery devices and artificial cytoskeletons in synthetic cells. For synthetic cells, these supramolecular springs can enable new mechanical functionalities, such as faster contraction than any microtubule or actin based system could offer; localized force generation free from polymer tracks; controllability that is orthogonalized from cell-specific biochemistry; and a novel, non-ATP- or GTP-based energy source to power movement inside cells.