Collaborative Research: Uncovering the Biophysical Mechanisms of Single-cell Wound-healing

创造赋予类似生命属性的系统一直是科学和工程领域的主要努力。近年来，构建合成细胞的研究呈爆炸式增长，不仅是研究生命起源和规律的一种方法，而且是生物化学工程提高分子制造产量的一种新方法。然而，目前的合成细胞研究忽略了生命物质最基本的特性之一——损伤后的自我修复能力。活细胞通常是柔软的，很容易被损坏，但它们中的一些在被机械刺穿、撕裂甚至撕成两半后可以自我修复。如果能在合成细胞中构建这种自我修复能力，就有可能深入了解细胞的一个决定性特征。同时，通过允许合成细胞系统在工业过程的潜在恶劣环境下稳健地运行，它可以打开生化工程的新领域。获得自我修复的合成细胞的一种方法是，从一个已经显示出在单个细胞内愈合大型机械伤口的强大能力的生命系统中调整自我修复机制，并在合成细胞内构建这些机制的类似物。一个这样的系统是Stentor coerleus，一种单细胞的自由生活纤毛虫，它比大多数其他细胞具有更强大的伤口愈合能力。然而，Stentor的自我修复机制在很大程度上是未知的。因此，这项提议的总体目标是了解支架细胞从大型机械伤口中稳健愈合的机制。这项工作的结果将导致对伤口愈合的基本见解，这是生命的定义特征之一。它还将为构建自我修复的合成细胞奠定基础。研究人员之间的合作将为细胞生物学和工程学的培训和劳动力发展提供一个独特的机会。将建立一个关于“超级细胞和细菌”的网站，以提高公众对拥有自我修复和太空生存等“超能力”的非模式生物的兴趣。研究人员将继续努力，通过社交媒体，针对K-12学生的外展活动，以及他们积极参与湾区科学节和创客大会，招募未被充分代表的少数民族进入科学和工程领域。本研究的总体目标是在物理和分子水平上了解蓝突细胞如何从导致质膜开口的大机械伤口中健壮地愈合。研究的关键生物学问题包括：细胞能恢复的最大伤口的极限是什么？是细胞的大尺寸促进了伤口愈合，还是伤口愈合在这个细胞中进化得特别快？关注Stentor的理由是：1)它的伤口愈合能力比大多数其他细胞更强大，能够从严重的伤口中恢复，并在24小时内从小到原细胞大小的1/27的细胞碎片中再生。2)进行高通量基因敲低和损伤实验的能力。研究目标是：1)建立单细胞伤口愈合的极简全细胞数学模型。2)通过测量细胞愈合动力学作为伤口大小和细胞大小的函数来测试模型的预测。3)通过磷酸化蛋白质组学鉴定膜修补、荷包收缩或其他机制对愈合过程的贡献。这项研究的智力价值在于确定了在单个细胞中修复大型机械伤口的原理，以及愈合过程成功或失败的条件。从根本上说，自愈能力是区分生物和非生物的关键特征之一。这项研究将揭示一些生物系统如何比其他生物系统更有效地自愈的问题。实际上，这项工作将为在合成细胞中设计一种新的功能——自我修复——奠定基础，并将使这项技术在实际工业应用的潜在规模上更加强大。该奖项由分子和细胞生物科学部的细胞动力学和功能与系统以及合成生物学集群共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Creating systems that confer life-like properties has been a major endeavor in both science and engineering. Recent years have witnessed an explosion in research on building synthetic cells, not only as a way to study the origins and the rules of life, but also as a new approach to biochemical engineering to increase the yield in making molecules. Nevertheless, current synthetic cell research has neglected one of the most fundamental properties of living matter – the ability to self-repair following damage. Living cells are generally soft and easily damaged, yet a number of them can repair themselves after being mechanically punctured, torn, or even ripped in half. If one could construct such self-repairing capability in synthetic cells, it should be possible to gain insights into one of the defining features of cells. At the same time, it could open new realms of biochemical engineering by allowing the synthetic cell systems to operate robustly under the potentially harsh environment of industrial processes. One approach to attaining self-repairing synthetic cells is to adapt self-repair mechanisms from a living system which already demonstrates robust capacity to heal from large mechanical wounds within a single cell, and build analogs of these mechanisms inside synthetic cells. One such system is Stentor coeruleus, a single-celled free-living ciliate, which possess a more robust wound healing capacity than most other cells. However, the self-repair mechanisms of Stentor are largely unknown. The overall goal of this proposal, therefore, is to understand the mechanisms by which Stentor cells can heal robustly from large mechanical wounds. The results of this work will lead to fundamental insights into wound healing, one of the defining features of life. It will also lay the foundation for constructing self-repairing synthetic cells. The collaboration between the researchers will provide a unique opportunity for training and workforce development at the interface of cell biology and engineering. A website on “superhero cells and bugs” will be created to raise public interest in non-model organisms possessing “superpowers” such as self-healing and survival in space. The researchers will continue their efforts to recruit underrepresented minorities to science and engineering via social media, outreach activities targeted to K-12 students, and their active participation in the Bay Area Science Festival and the Maker Faire.The overall goal of this research is to understand at a physical and molecular level how Stentor coeruleus cells can heal robustly from large mechanical wounds that cause an opening in the plasma membrane. The key biological questions probed include: What sets the limit of the biggest wound the cell can recover from? Does the large size of the cell facilitate its wound healing, or has wound healing evolved to be particularly rapid in this cell? The rationale to focus on Stentor are: 1) its wound healing capacity is more robust than most other cells, capable of recovering from drastic wounds and regenerating from cell fragments as small as 1/27th of original cell size in 24 hours. 2) The ability to perform high-throughput gene knockdown and wounding experiments. The research objectives are to: 1) Develop a minimalistic whole-cell mathematical model of single-cell wound healing. 2) Test predictions of the model by measuring the kinetics of healing in cells as a function of wound size and cell size. 3) Identify contributions to the healing process from membrane patching, purse-string constriction, or other mechanisms as identified by phosphoproteomics. The intellectual merit of this research lies in the identification of the principles for repairing large mechanical wounds in a single cell, and the conditions at which the healing process will succeed or fail. Fundamentally, the ability to heal is one of the key features that distinguish living matter from non-living matter. This study will shed light into the problem of how some biological systems can heal more robustly than others. Practically, the work will lay the foundation for engineering a new function—self-repair—in synthetic cells, and will make the technology more robust for potential scale-up for practical industrial applications.This award was co-funded by the Cellular Dynamics and Function and Systems and Synthetic Biology clusters of the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.