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

项目摘要/摘要 生物学和疾病中的一些最具挑战性的问题涉及到 手机。Wnt通路是最重要的发育和癌症通路之一。 控制生长和大小是所有细胞的普遍属性，其动力学很难 测量准确，但人们对此理解不足。WNT路径由保守的脚手架组成 和控制连环蛋白稳定性的酶，它调节重要的发育 基因。细胞生长调控，在复杂的生理条件下对新陈代谢和分化作出反应 电路。Wnt途径的组成部分早已为人所知，但Wnt如何发出信号 在与连接素相互作用之前，经历了几个动力学步骤仍不清楚。我们正在努力 从1)荧光嵌合体的单分子成像来理解Wnt途径 蛋白质被敲入内源基因，从而保持准确的表达水平和 转录调控和2)体外系统的发展，以保存 该通路下游事件的动力学响应。从体外系统我们可以 检测纯化的蛋白质，并评估其活性。我们可以快速分离络合物并进行研究 它们的翻译后状态，并潜在地决定了运动学上重要的结构 通过低温电子显微镜观察。我们过去有，将来也会结合起来 用生物化学进行数学建模，以确定这一系统的关键特征。对于单元格大小 我们已经使用定量方法来定义细胞的结构和生理状态。 我们发现哺乳动物的细胞大小受到控制，不仅在G1/S，而且在整个细胞周期中 通过从细胞大小到生长速度的反馈。细胞如何知道它们有多大并调节 它们的成长仍是一个谜。我们将通过两个工具来促进进一步的理解 已开发：计算机增强定量位相显微镜(CeQPM)和归一化 拉曼成像(NORI)。前者是测量电池干质量最准确的方法 用于附加的单元格。后者还可以独立测量蛋白质和脂肪的质量密度 细胞的总质量，甚至在组织中的深处。此外，Nori还可以测量 在单细胞水平的蛋白质合成和降解，在组织内或在培养中实时进行。 我们将在培养细胞中同时使用ceqpm和nori来测量蛋白质的合成。 以及作为细胞大小和细胞周期中位置的函数的周转率，结合 药理、生长因子和营养扰动以确定参与的途径 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，小鼠的组织中也将与Nori一起探索。

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.