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

光控Ca2驱动超分子发动机的生物物理机制与合成工程

• 批准号: 10653947
• 负责人: SAAD BHAMLA
• 金额: $ 39.55万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10653947
• 关键词: Actins; Address; Biochemistry; Biophysical Process; Biophysics; Biosensor; Calcium; Calcium ion; Cells; Chemicals; Contracts; Cytoskeleton; Devices; Drug Delivery Systems; Elasticity; Elements; Energy-Generating Resources; Engineering; Generations; Guanosine Triphosphate; In Vitro; Kinesin; Knowledge; Length; Light; Lipids; Mechanics; Microfluidic Microchips; Microscopy; Microtubules; Modeling; Molecular Motors; Motion; Movement; Myosin ATPase; Optics; Output; Physics; Polymers; Proteins; Research; Stroke; Structure; System; Therapeutic; Trimethoprim-Sulfamethoxazole; Vertebral column; Vesicle; Work; biophysical techniques; design; experimental study; in vivo; mathematical theory; millimeter; millisecond; nanomachine; nanoscale; novel; response; self assembly; simulation; synthetic biology

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.

项目摘要 肌力体是钙驱动的超分子蛋白质“弹簧”，形成产生力的细胞骨架 一些原生纤毛虫的结构，例如Spirostomum ambiguum。在 Spirostomum 中，myonemes 极高的功率输出（相当于二冲程柴油发动机），使螺口能够收缩 在不到 5 毫秒的时间内缩小到其身体长度的 1/4（单细胞水平上最快的运动之一）。就条款而言 每单位质量的功率，肌动体产生的力比传统 ATP 驱动的力高六个数量级 分子马达，例如肌球蛋白或驱动蛋白。肌丝不包含传统的细胞骨架元件，例如 如肌动蛋白、微管或肌球蛋白。相反，肌丝体由两种成分的自组装组成：中心蛋白 钙响应蛋白和弹性骨架蛋白 Sfi1。因此，myonemes 提供了有吸引力的 诸如非 ATP 依赖性驱动、超快和高功率传输以及简单的两组件等功能 系统，可以实现潜在的变革性合成生物学应用，例如人工设计 合成细胞或生物杂交细胞的细胞骨架，使它们能够分裂、移动或运输与其自身相似的货物 活着的同行。然而，我们对生物物理控制机制的认识存在重大差距 这些弹簧中产生力的过程，钙离子如何充当化学锁来控制和同步力 提供超过毫米的长度尺度，以及如何合成设计这些超分子组件 以及体外自组装，以利用它们实现所需的功能。 为了解决这些理解上的差距，未来 5 年拟议的研究将采取双管齐下的方式 方法：i) 结合生物物理实验、实时显微镜和基于软物质物理的模型来揭示 活细胞体内肌丝产生力的生物物理机制，以及 ii) 工程师、自我 在体外微流体装置和脂质中组装和整合光控制合成肌细胞 (synMyo) 囊泡。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 Ca2+ 的光驱动材料 产生合成力的细胞骨架组件，在细胞内驱动和传感中的应用， 合成细胞中的治疗药物输送装置和人工细胞骨架。对于合成细胞来说，这些 超分子弹簧可以实现新的机械功能，例如比任何材料更快的收缩 基于微管或肌动蛋白的系统可以提供；不受聚合物轨道影响的局部力产生；可控性 这是从细胞特异性生物化学中正交化的；以及一种新型的、非 ATP 或 GTP 能源 细胞内的能量运动。