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

无论是在科学上还是在工程上，创造出赋予生命般的特性的系统都是一项重大的努力。近年来，建造合成细胞的研究出现了爆炸性的增长，不仅是作为研究生命起源和规律的一种方式，而且作为一种新的生化工程方法，以增加制造分子的产量。然而，目前的合成细胞研究忽略了生物体最基本的属性之一--损伤后的自我修复能力。活细胞通常很软，很容易损坏，但其中一些细胞在被机械刺穿、撕裂甚至撕裂成两半后可以自我修复。如果一个人能够在合成细胞中构建这种自我修复能力，就应该有可能深入了解细胞的定义特征之一。与此同时，它可以通过允许合成细胞系统在潜在恶劣的工业过程环境下稳健地运行，从而开辟生化工程的新领域。获得自我修复合成细胞的一种方法是调整来自已经显示出从单个细胞内的大型机械创伤中愈合的强大能力的生命系统的自我修复机制，并在合成细胞中建立这些机制的类似物。一个这样的系统是Stentor Blue uleus，一种单细胞自由生活的纤毛虫，它比大多数其他细胞拥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还不清楚。因此，这项提议的总体目标是了解支架细胞从大型机械创伤中强劲愈合的机制。这项工作的结果将导致对伤口愈合的基本见解，伤口愈合是生命的定义特征之一。这也将为构建自修复合成细胞奠定基础。研究人员之间的合作将为细胞生物学和工程学领域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“超级英雄细胞和虫子”的网站，以提高公众对非模型生物的兴趣，这些生物具有自我修复和在太空中生存的“超能力”。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旧金山湾区科学节和Maker Faire，招募未被充分代表的少数族裔进入科学和工程专业。这项研究的总体目标是在物理和分子水平上了解蓝柱肌细胞如何从导致质膜开口的大型机械创伤中强劲愈合。探讨的关键生物学问题包括：细胞可以恢复的最大创伤的限制是什么？是细胞的大小促进了伤口的愈合，还是伤口的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，能够从严重的伤口恢复，并在24小时内从小至原始细胞大小的1/27的细胞碎片再生。2)进行高通量基因敲除和损伤实验的能力。研究目的：1)建立单细胞创伤愈合的最小全细胞数学模型。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)确定膜补片、钱袋收缩或磷蛋白质组学所确定的其他机制对愈合过程的贡献。这项研究的学术价值在于确定了在单个细胞中修复大型机械创伤的原则，以及愈合过程成功或失败的条件。从根本上说，治愈的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系统更有力地愈合的问题。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中的潜在放大能力更加强大。该奖项由分子和细胞生物科学部细胞动力学和功能与系统和合成生物学集群共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.