The dynamics and underlying mechanisms controlling cell size and canonical Wnt signaling

控制细胞大小和经典 Wnt 信号传导的动力学和潜在机制

• 批准号: 10797294
• 负责人: MARC Wallace KIRSCHNER
• 金额: $ 13.3万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-22 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797294
• 关键词: Address; Biochemistry; Biological Assay; Biology; Cell Cycle; Cell Differentiation process; Cell Size; Cell physiology; Cells; Chemicals; Chimeric Proteins; Complex; Computers; Coupled; Cryoelectron Microscopy; Cultured Cells; Development; Developmental Gene; Disease; Dryness; Enzymes; Event; Failure; Feedback; Future; Growth; Growth Factor; Homeostasis; In Vitro; Kinetics; Lipids; Malignant Neoplasms; Mammalian Cell; Measures; Metabolism; Methods; Microscopy; Mus; Nutrient; Nutritional; Organ; Pathology; Pathway interactions; Phase; Physiological; Physiology; Positioning Attribute; Proliferating; Property; Protein Biosynthesis; Proteins; Raman Spectrum Analysis; Science; Structure; System; Time; Tissues; Transcriptional Regulation; WNT Signaling Pathway; cell growth; fluorescence imaging; mathematical model; pharmacologic; preservation; protein degradation; protein purification; response; scaffold; single molecule; tool

Project Summary/ Abstract Some of the most challenging problems in biology and disease concern dynamical features of the cell. The Wnt pathway is one of the most important developmental and cancer pathways. Control of growth and size is a universal property of all cells, whose dynamics are hard to measure accurately and poorly understood. The Wnt pathway is made up of conserved scaffolds and enzymes that control the stability of catenin, which regulates important developmental genes. Cell growth control, responds to metabolism and differentiation in complex physiological circuits. Components of the Wnt pathway have been long known but how the Wnt signal traverses several kinetic steps before interacting with the catenin is still unclear. We are trying to understand the Wnt pathway from: 1) single molecule imaging of fluorescent chimeric proteins knocked into the endogenous loci, thereby preserving the exact level of expression and transcriptional regulation and 2) the development of an in vitro system that preserves the kinetic response of the downstream events of the pathway. From the in vitro system we can assay purified proteins, and assess their activity. We can quickly isolate complexes and study their posttranslational state, and potentially determine the structure of kinetically important forms by Cryo-electron microscopy. We have in the past and will in the future combine mathematical modeling with biochemistry to identify key features of this system. For cell size control we have used quantitative methods to define the cell’s structural and physiological state. We found that mammalian cell size is controlled, not just at G1/S, but throughout the cell cycle by feedback from cell size onto growth rate. How cells know how large they are and regulate their growth is still a mystery. Further understanding will be facilitated by two tools we developed: computer enhanced Quantitative Phase microscopy (ceQPM) and Normalized Raman Imaging (NoRI). The former is the most accurate method for measuring cell dry mass for attached cells. The latter can also independently measure protein and lipid mass densities and total mass of cells, even deep within tissues. Furthermore, NoRI can measure the rate of protein synthesis and degradation at the single cell level within tissues or in culture in real time. We will use ceQPM and NoRI simultaneously with cultured cells to measure protein synthesis and turnover as a function of cell size and as a function of position in the cell cycle, coupled with pharmacological, growth factor, and nutrient perturbation to identify pathways involved in sensing size and regulating growth. The mechanism of cell size control in differentiated organs under different nutritional states in mouse tissues will also be explored with NoRI.

项目摘要/摘要 生物学和疾病中最挑战的问题涉及的动态特征 细胞。 WNT途径是最重要的发育和癌症途径之一。 对生长和大小的控制是所有细胞的普遍特性，其动力很难 准确且理解不足。 Wnt途径由组成的脚手架组成 和控制stenin的稳定性的酶，可调节重要的发展 基因。细胞生长控制，对复杂生理的新陈代谢和分化反应 电路。 Wnt途径的组成部分已知已知，但是Wnt信号如何 在与Catenin相互作用之前，遍历几个动力学步骤仍不清楚。我们正在尝试 从：1）荧光嵌合的单分子成像中了解Wnt途径 蛋白质被撞入内源基因座，从而保留了确切的表达水平和 转录调节和2）维护体外系统的发展 途径下游事件的动力学响应。从体外系统中，我们可以 测定纯化的蛋白质，并评估其活性。我们可以快速隔离复合物并研究 它们的翻译后状态，并有可能确定动力学重要的结构 通过冷冻电子显微镜形成。我们过去和将来会结合在一起 具有生物化学的数学建模，以识别该系统的关键特征。用于单元格大小 控制我们已经使用定量方法来定义细胞的结构和物理状态。 我们发现哺乳动物细胞的大小不仅在G1/s，而且在整个细胞周期中受到控制 通过从细胞大小到生长速率的反馈。细胞如何知道它们有多大并调节 他们的成长仍然是一个谜。我们将通过两个工具来准备进一步的理解 开发：计算机增强的定量相显微镜（CEQPM）并归一化 拉曼成像（NORI）。前者是测量细胞干质量的最准确的方法 用于附着的细胞。后者还可以独立测量蛋白质和脂质质量密度 和总细胞的总质量，甚至在组织内部。此外，诺里可以测量 蛋白质的合成和降解在组织内或实时培养的单细胞水平上。 我们将简单地与培养细胞一起使用CEQPM和NORI来测量蛋白质合成 并与单元大小的函数以及在单元格周期中的位置的函数，并与 药理，生长因子和养分扰动，以识别涉及的途径 感知大小和调节增长。分化器官中细胞尺寸控制的机制 在小鼠组织中的不同营养状态下，Nori也将探索。