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.

项目总结/摘要 生物学和疾病中一些最具挑战性的问题涉及微生物系统的动力学特征。 cell. Wnt通路是最重要的发育和癌症通路之一。 对生长和大小的控制是所有细胞的普遍属性，其动力学很难被理解。 准确测量，不太了解。Wnt通路由保守的支架组成 和控制β-catenin稳定性的酶，β-catenin调节重要的发育 基因.细胞生长控制，对复杂生理过程中的代谢和分化作出反应， 电路. Wnt信号通路的组成部分早已为人所知，但Wnt信号是如何通过 在与β-catenin相互作用之前经过几个动力学步骤仍然不清楚。我们正在努力 为了从以下方面理解Wnt途径：1）荧光嵌合体的单分子成像， 敲入内源基因座的蛋白质，从而保持精确的表达水平， 转录调控和2）在体外系统的发展，保留 该途径的下游事件的动力学响应。从体外系统中我们可以 分析纯化的蛋白质，并评估其活性。我们可以快速分离复合物并进行研究 它们的翻译后状态，并可能决定结构的动力学重要的 冷冻电子显微镜下的形态。我们过去和将来都将联合收割机 数学建模与生物化学，以确定该系统的关键特征。对于像元大小 控制我们已经使用定量方法来定义细胞的结构和生理状态。 我们发现，哺乳动物细胞的大小不仅在G1/S期，而且在整个细胞周期中都受到控制 通过细胞大小对生长速率的反馈。细胞如何知道它们有多大并调节 它们的生长仍然是个谜。我们将通过两个工具来促进进一步的理解， 开发：计算机增强定量相位显微镜（ceQPM）和归一化 拉曼成像（NoRI）。前者是测量细胞干质量最准确的方法 for attached附着cells细胞.后者还可以独立测量蛋白质和脂质质量密度 以及细胞的总质量，甚至是组织深处的细胞。此外，NoRI可以测量 在组织内或培养物中真实的实时地在单细胞水平上的蛋白质合成和降解。 我们将同时使用ceQPM和NoRI与培养的细胞来测量蛋白质合成 和周转作为细胞大小的函数和作为细胞周期中位置的函数， 药理学，生长因子和营养扰动，以确定参与 感知大小和调节生长。分化器官中细胞大小控制的机制 在不同的营养状态下，小鼠组织中的NoRI也将被探索。