Cellular Integration of Information in the Detection and Response to Epithelial Damage

项目摘要 当上皮组织受伤时，第一个细胞反应是胞质内毒素的急剧增加。 钙水平，在组织受损后立即开始。这种钙的增加不仅在 细胞在伤口边缘，但在伤口周围的细胞的大域。随着遗传学的出现， 编码的钙指标，如GCaMP，这种伤口诱导的钙反应已被观察到在生活中 动物王国中的生物，但相互矛盾的机制已被提出来解释， 诱导钙，这样就没有意义的保守的基本过程。我们的合作团队 生物药理学家和发育遗传学家最近发表的工作确定了一个主要的潜在障碍： 有几种同时存在的机制是创伤诱导的钙反应的基础，我们 已经能够通过我们高度定量的方法和我们的遗传学方法的结合， 操纵由于不了解诱导钙反应的多种机制， 不可能在文献和创伤模型中得出相似之处，也不可能完全 阻断钙反应以分析伤口愈合的下游后果。 我们的分析工具确定了刻板的钙反应，具有不同的振荡模式， 在距离伤口不同的径向距离处有明显的特征。我们假设这些钙离子 这些反应告知细胞其与伤口的距离，并决定其下游细胞的行为。像 钙反应、伤口诱导的细胞行为和转录身份根据以下方式形成模式 远离伤口，迁移细胞和JNK信号传导靠近边缘和增殖细胞， JAK-STAT信号在更远端的环中。在第一个目的中，我们研究了钙离子如何在细胞内形成的机制。 信号传导在这些模式中的每一种模式中都被启动，既与个体途径一起工作， 这些钙模式是如何整合的数学模型。在第二个目标中，我们扰动特定的 钙模式的各个方面，并询问下游细胞的行为和身份是如何改变的。我们能够 实现这些目标，因为我们独特的协作技能，因为我们已经开发了一个 无与伦比的创伤模型，允许基因操作与高时间控制的一边， 只有伤口。因为它是内部控制的，比较两边可以精确量化， 检测钙信号和创伤诱导的细胞行为中甚至很小的变化。 在这个项目完成后，我们希望已经产生了一个高精度的模型，细胞如何 检测组织损伤的距离，以及他们如何解释这些信息，以选择一个空间适当的 修复程序这些基础知识对细胞生物学的许多领域都很重要， 研究，以及伤口愈合程序被不适当激活的癌症等病理。

Project Summary When an epithelial tissue is wounded, the first cellular response is a dramatic increase in cytosolic calcium levels, beginning immediately upon tissue damaged. This calcium increase is observed not just in cells at the wound margin but in large domain of cells surrounding the wound. With the advent of genetically encoded calcium indicators like GCaMP, this wound-induced calcium response has been observed in living organisms across the animal kingdom, yet conflicting mechanisms have been proposed to explain the induction of calcium, such that there is no sense of a conserved fundamental process. Our collaborative team of biophysicists and developmental geneticists recently published work identifying a major underlying obstacle: there are several contemporaneous mechanisms underlying the wound-induced calcium response, which we have been able to tease apart with our combination of highly quantitative approaches and our genetic manipulations. Without understanding the multiple mechanisms inducing calcium responses, it has been impossible to draw parallels across the literature and across wounding models, and it has impossible to fully block calcium responses to analyze the downstream consequences for wound healing. Our analysis tools identified stereotyped calcium responses, with different oscillatory patterns or signatures evident at different radial distances from the wound. We hypothesize that these patterned calcium responses inform the cell about its distance from the wound and determine its downstream cell behaviors. Like the calcium responses, wound-induced cell behaviors and transcriptional identities are patterned according to distance from the wound, with migratory cells and JNK signaling near the margin and proliferative cells and JAK-STAT signaling in a more distal ring. In the first Aim, we investigate the mechanisms of how calcium signaling is initiated in each of these patterns, working both with individual pathways and developing mathematical models for how these calcium patterns are integrated. In the second Aim, we perturb specific aspects of calcium patterns and ask how downstream cell behavior and identity are altered. We are able to achieve these Aims because of our unique collaborative skill-set, and because we have developed an unparalleled wounding model that allows genetic manipulation with high temporal control on one side of the wound only. Because it is internally controlled, comparing the two sides allows precise quantification and detection of even small changes, in both calcium signaling and wound-induced cell behaviors. At the completion of this project, we expect to have generated a high-precision model of how cells detect tissue damage at a distance, and how they interpret this information to select a spatially-appropriate repair program. This fundamental knowledge will be important to many areas of cell biology, to wound-healing studies, and to pathologies like cancer where wound-healing programs are inappropriately activated.