The role of dynamics in defining the limits of normal developmental signaling

摘要 在这份补充材料中，我们申请资金购买一台活细胞荧光显微镜，以取代故障 基本设备，这将显著提升我们收集细胞信号生物传感器数据的能力 高空间和时间分辨率。本补充材料的父项目以使用活细胞为中心 生物传感器实验，以收集关于激酶活性和基因表达的动力学数据。这些数据集具有唯一性 能够对信号转导途径和基因表达之间的关系进行计算建模。 由于这些长期延时成像实验的时间密集性，我们需要一个专门的 可连续运行多天实验的活细胞显微镜系统。我们目前的系统有 使生长因子信号在概念和方法上取得重大进展，但面临两大 问题：1)由于老化的设备故障导致的频繁停机；2)过时的技术限制了我们的 能够从活细胞中收集高质量的数据。拟议的文书将大大减少 因组件故障而损失的人员时间。此外，拟议的文书将利用 一台大视场的cmos相机，可以在每次实验中从近两倍的细胞中收集数据； 这些额外的数据将通过捕获更多独特的细胞来直接改进我们的计算建模工作 行为。最后，新仪器将包括自动化，以支持使用40倍和60倍的水浸泡 目的研究用于长期时间推移实验的透镜。相对于我们目前依赖于 20X和40X非浸没镜头，这些镜头将提高空间分辨率和灵敏度，使 我们可以将信号转导和基因表达与关键基因的形态变化联系起来的实验 细胞器，如自噬小体和线粒体，我们的工作涉及到调节 动态激酶活性正在研究中。

Summary In this supplement, we request funding to purchase a live-cell fluorescence microscope that will replace failing essential equipment, and which will significantly upgrade our ability to collect cell signaling biosensor data with high spatial and temporal resolution. The parent project for this supplement is centered on using live-cell biosensor experiments to collect kinetic data on kinase activity and gene expression. These datasets uniquely enable computational modeling of the relationship between signal transduction pathways and gene expression. Because of the time-intensive nature of these long-term time-lapse imaging experiments, we need a dedicated live-cell microscopy system that can run continuously for multi-day experiments. Our current systems have enabled significant conceptual and methodological advances in growth factor signaling but face two major problems: 1) frequent down-time due to failures of aging equipment and 2) outdated technology that limits our ability to collect high-quality data from live cells. The proposed instrument would dramatically decrease the amount of personnel time lost to component failures. Furthermore, the proposed instrument will take advantage of a large field of view sCMOS camera that can collect data from nearly twice as many cells in each experiment; these additional data will directly improve our computational modeling efforts by capturing more unique cellular behaviors. Finally, the new instrument will include automation to enable the use of 40X and 60X water immersion objective lenses for long-term time time-lapse experiments. Relative to our current imaging systems that rely on 20X and 40X non-immersion lenses, these lenses will increase spatial resolution and sensitivity, enabling experiments in which we can link signal transduction and gene expression to morphological changes in key organelles such as autophagosomes and mitochondria, which our work has implicated in regulation of the dynamic kinase activity being studied.