The keratin-filament cycle of assembly and disassembly

角蛋白中间丝是上皮细胞骨架的主要成分。它们对细胞空间的组织功能对于需要高度角蛋白网络可塑性的多种上皮功能是重要的。我们最近提出，角蛋白细丝的连续组装和拆解周期提供了快速和精确的适应不同的功能要求和情况。第一个项目阶段的目标是建立和应用工具来证明/反驳我们关于角蛋白周期的概念，并检查其拟议的方面。我们的实验观察到了荧光标记的角蛋白多肽，完全支持角蛋白周期的主要特征。此外，它们使我们能够定量测量角蛋白网络特性、角蛋白移动速度和局部周转。我们进一步证明，新的工具能够检测在细胞接种后发生的角蛋白循环的细微差异，以及在生长因子添加、迁移诱导、角蛋白磷酸化和细胞骨架交联物下调以及对底物硬度改变的反应中的细微差异。第二个项目阶段打算从角蛋白周期的描述阶段过渡到对分子的理解。为了实现这一目标，我们将(I)进一步改进监测和操纵角蛋白周期及其调节器的方法和工具，(Ii)专注于识别和表征在角蛋白网络中参与角蛋白运动和张力产生的分子马达，以及(Iii)检查在遗传筛选中确定的角蛋白周期调节器。我们将把新开发的图像记录和分析程序应用于改良的荧光报告细胞系的高分辨率显微图像集上，系统地研究下调所有相关的马达蛋白和角蛋白周期的其他调节器对角蛋白网络组织、动力学和功能的影响。对上皮功能的影响，即细胞迁移、细胞粘弹性和僵硬、增殖和应激反应将与这些发现相关。我们的长期目标是在上皮病理生理学的背景下了解角蛋白网络可塑性的分子机制。

Keratin intermediate filaments are major constituents of the epithelial cytoskeleton. Their organizing function of cellular space is important for diverse epithelial functions requiring a high degree of keratin network plasticity. We have recently proposed that a continuous cycle of keratin filament assembly and disassembly affords rapid and precise adaptation to the different functional requirements and situations. The goal of the first project phase was to establish and apply tools for proving/disproving our concept of the keratin cycle and to examine its proposed aspects. Our experiments observing fluorescently-tagged keratin polypeptides fully supported the main features of the keratin cycle. Furthermore, they allowed us to quantitatively measure keratin network properties, speed of keratin movement and local turnover. We were further able to demonstrate that the new tools are capable detecting slight differences in keratin cycling taking place after seeding of cells and upon growth factor addition, migration induction, keratin phosphorylation, and downregulation of cytoskeletal cross linkers as well as in response to altered substrate stiffness. The second project phase intends to move from a descriptive stage of the keratin cycle to a molecular understanding. To achieve this, we will (i) further refine methods and tools for monitoring and manipulating the keratin cycle and its modulators, (ii) focus on identifying and characterizing the molecular motors involved in keratin movement and tension generation within the keratin network, and (iii) examine modulators of the keratin cycle that are identified in genetic screens. We will apply the newly developed image recording and analysis routines on high resolution microscopic image sets of improved fluorescent reporter cell lines to systematically study the effect of downregulating all of the relevant motor proteins and other modulators of the keratin cycle on keratin network organization, dynamics and function. Consequences on epithelial function, i.e. cell migration, cellular viscoelasticity and stiffness, proliferation and stress response will be correlated to these findings.Our long-term goal is to understand molecular mechanisms of keratin network plasticity in the context of epithelial pathophysiology.