Experimental Characterization and Theoretical Modeling of Circular Dorsal Ruffles

圆形背皱纹(CDRs)是一种生长在背侧细胞上的动态肌动蛋白结构。我们的目标是了解CDR的传播机制。它们通过内吞作用和细胞骨架的重组，在摄取生长因子方面起着重要作用。我们关注肌动蛋白自由、分支和聚合的时空动力学的实验表征和理论建模，以及CDR超微结构。我们感兴趣的是肌动蛋白聚合抑制的特征和模型。为了探索该系统，我们干扰i)上游信号，ii)直接肌动蛋白调节，以及iii)通过生化和光遗传手段肌动蛋白本身。随着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变化，表型的静力学和动力学会发生变化。CDR的制备和观察是在细胞核和细胞外围之间的封闭圆形空间中以侧波的形式进行的。它们被迫通过圆盘状的微接触印刷贴片在环形轨迹上传播，以获得合适大小的细胞。CDR的传播由平均速度、寿命和重复频率来表征。我们的目标是验证现有的将CDR描述为双稳状态的模型，并研究涨落的一般作用。有两个概念上不同的影响需要分析。首先，在分子总数不变的情况下，蛋白质的活性和密度呈现时间波动。第二，基因表达的变化改变了分子拷贝的数量。第一个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，第二个效应变得重要。我们预计随机的基因表达和漂移会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，人们不仅要观察给定固定相空间中的轨迹，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)、微流体和微接触打印以及光遗传学。通过粘贴到盘状结构域上来制备定义的细胞形态，确保定义的边界条件被证明是可重现数据的绝对关键。光遗传学允许在体外操纵蛋白质的表达，并在体内观察表型随基因的变化。理论工具是图像相关性分析，通过参数匹配对实验数据进行数值解拟合，以及基于人工智能的聚类分析。与实验相比，尤其重要的是确定解的分叉类型和位置。详细的分析可以构建相图。

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.