Experimental Characterization and Theoretical Modeling of Circular Dorsal Ruffles

圆形背侧褶边的实验表征和理论建模

• 批准号: 237405144
• 负责人: Professor Dr. Hans-Günther Döbereiner, Ph.D.
• 金额: --
• 依托单位: Institut für Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/237405144?language=en
• 关键词: Experimental; Characterization; Theoretical; Modeling; Circular

Circular Dorsal Ruffles (CDRs) are dynamic actin structures propagating on the dorsal cell side. Our goal is to understand the mechanism of CDR propagation. They play an important role in the uptake of growth factors via endocytosis and the reorganization of the cytoskeleton. We are concerned with experimental characterization and theoretical modeling of actin spatiotemporal dynamics with respect to free, branched, and polymerized actin, as well as CDR ultrastructure. We are interested to characterize and model inhibition of actin polymerization. In order to probe the system, we interfere with i) upstream signaling, ii) direct actin regulators, and iii) actin itself by biochemical and optogenetic means. Phenotypic statics and dynamics are altered as a function of growth factor and biochemcial inhibitor concentrations as well as expression levels of selected proteins. CDRs are prepared and observed as lateral waves in the closed circular space between the nucleus and the cellular periphery. They are forced to propagate on ring-like trajectories via disk-shaped micro-contact printed adhesion patches for cells of appropriate size. CDR propagation is characterized by mean velocity, lifetime and repeat frequency. We aim to verify the existing model describing CDRs as bistable states and to investigate the general role of fluctuations. There are two conceptually different effects to be analyzed. First, protein activity and density exhibit temporal fluctuations at overall constant number of molecules. Second, variations in gene expression alter the number of molecular copies. The first effect is mainly relevant on short time scales of a few minutes. On longer timescales the second effect becomes important. We expect random gene expression and drift to affect trajectories in state space, i.e., total (regulating) protein concentrations cannot be considered constant. This means, one is not just observing trajectories in a given fixed phase space, but the phase space itself is changing over time. Experimental tools used include, but are not limited to, optical microscopy (fluorescent, PH, DIC, RICM), microfluidics and microcontact printing, as well as optogenetics. Preparation of defined cell morphologies via adhesion onto disk-like domains ensuring defined boundary conditions turned out to be absolutely critical for reproducible data. Optogenetics allows to manipulate protein expression in vitro and observe in vivo variations in phenotype with genotype. Theoretical tools are image correlation analysis, fitting of numerical solutions to experimental data via parameter matching, and AI-based cluster analysis. Especially important is the identification of type and location of bifurcations of solutions in comparison to experiment. Detailed analysis allows to construct phase diagrams.

圆形背褶（CDR）是在背侧细胞侧传播的动态肌动蛋白结构。我们的目标是了解CDR传播的机制。它们在通过内吞作用和细胞骨架重组吸收生长因子方面发挥重要作用。我们关注的是肌动蛋白时空动力学的实验表征和理论建模方面的自由，分支和聚合的肌动蛋白，以及CDR超微结构。我们有兴趣描述和模型抑制肌动蛋白聚合。为了探测该系统，我们通过生物化学和光遗传学手段干扰i）上游信号传导，ii）直接肌动蛋白调节剂，和iii）肌动蛋白本身。表型静态和动态改变作为生长因子和生化抑制剂浓度以及所选蛋白质的表达水平的函数。制备CDR，并在细胞核和细胞外周之间的封闭圆形空间中观察到侧波。它们被迫通过用于适当大小的细胞的盘形微接触印刷粘附贴片在环状轨迹上传播。CDR传播的特征在于平均速度，寿命和重复频率。我们的目标是验证现有的模型描述CDR的双态，并调查波动的一般作用。有两种概念上不同的效应需要分析。首先，蛋白质活性和密度在总体恒定分子数下表现出时间波动。第二，基因表达的变化改变了分子拷贝的数量。第一种效应主要与几分钟的短时间尺度有关。在更长的时间尺度上，第二种效应变得重要。我们期望随机基因表达和漂移影响状态空间中的轨迹，即，总（调节）蛋白质浓度不能被认为是恒定的。这意味着，人们不仅仅是在给定的固定相空间中观察轨迹，而且相空间本身也会随着时间的推移而变化。使用的实验工具包括但不限于光学显微镜（荧光、PH、DIC、RICM）、微流体和微接触印刷以及光遗传学。通过粘附到盘状结构域上来制备确定的细胞形态，确保确定的边界条件对于可重复的数据是绝对关键的。光遗传学允许在体外操纵蛋白质表达并观察表型与基因型的体内变化。理论工具是图像相关性分析，通过参数匹配和基于AI的聚类分析来拟合实验数据的数值解。特别重要的是识别的类型和位置的分叉的解决方案相比，实验。详细的分析允许构建相图。