Catch and slip bond dynamics of cytoskeletal cross-linkers in live cells – a new approach to understand principles of cytoskeletal material engineering

近年来，一些生物物理学研究表明，生物材料，如动物细胞，积极地设计其材料特性。理解这种现象是我们理解细胞力响应、细胞形状动力学和组织结构的重要前提。具体来说，肌动蛋白细胞骨架能够通过肌球蛋白马达引入的主动机械应力来调整其力学。然而，不同的研究发现，通过主动应力的这种机械调谐的定性不同的模式;已经观察到肌动球蛋白网的硬化和软化。在此，笔者建议将联合收割机的实验与理论相结合来阐明主动应力对细胞力学性能的影响。我将集中在我的研究在肌动蛋白细胞骨架中的肌动蛋白交联剂的力感应的影响。以前的实验表明，存在两种性质不同的肌动蛋白交联剂类型-捕获和滑动键交联剂-其键在拉伸应力的存在下被加强或减弱。因此，预期捕获或滑动键交联剂在增加的主动应力下分别引起肌动蛋白细胞骨架的硬化或软化。虽然最先进的测量方法是在体外完成的，但我将向前迈出重要的一步，并描述活细胞皮质中交联剂键的力依赖性。为此，我将利用我最近开发的一种新技术来测量主动皮质应力和细胞骨架刚度。在那里，使用原子力显微镜通过平行板压缩测定法机械探测细胞。该技术将与细胞肌动蛋白皮质处荧光标记的交联剂的光漂白相结合。交联剂荧光的恢复将揭示其解结合动力学。我将使用这种检测来表征的寿命的交联剂在不同水平的积极皮质应力，因此，表征他们作为捕获或滑动债券cross-linkers.Outlook：在第二个资金周期，我计划研究如何结合不同的捕获和滑动债券交联剂的肌动蛋白细胞骨架可以用来定制的材料性能的细胞。为此，我打算开发一个数学模型，其特征在于捕获和滑动键交联剂对肌动蛋白细胞骨架的紧急材料特性的预期影响。该模型将预测交联剂浓度制度，其中主动应力导致硬化或软化，固化或流化的网络。在实验部分，我计划探索这些制度，通过有针对性地敲低细胞中的交联蛋白，从而提供了一个原则的证明，力传感的交联影响肌动蛋白细胞骨架的非线性特性。

In recent years, several biophysical studies have shown that biological material, such as an animal cell, actively engineers its material properties. Understanding this phenomenon is an important prerequisite for our comprehension of cellular force response, cell shape dynamics and tissue organization. Specifically, the actin cytoskeleton is able to tune its mechanics through active mechanical stress introduced by myosin motors. However, different studies find qualitatively different modes of this mechanical tuning through active stress; both stiffening and softening of actomyosin meshworks have been observed. Here, I suggest to combine experiment and theory to elucidate the influence of active stress on cell mechanical properties. I will focus in my study on the influence of the force-sensing of actin cross-linkers in the actin cytoskeleton. Previous experiments have indicated that two qualitatively different actin cross-linker types exist – catch and slip bond cross-linkers – whose bonds are either enforced or weakened in the presence of tensile stress. Therefore, catch or slip bond cross-linkers are expected to give rise to a stiffening or softening of the actin cytoskeleton at increased active stress, respectively. While the state-of-the-art measurements on the load-dependence of bond lifetimes are done in vitro, I will go an important step ahead and characterize the force-dependence of cross-linker bonds in the cortex of live cells. To this end, I will utilize a new technique for the measurement of active cortical stress and cytoskeletal stiffness that I have recently developed. There, cells are mechanically probed by a parallel plate compression assay using an atomic force microscope. This technique will be combined with photo-bleaching of fluorescently labeled cross-linkers at the actin cortex of cells. The recovery of cross-linker fluorescence will reveal their unbinding dynamics. I will use this assay to characterize the lifetime of cross-linkers at varying levels of active cortical stress and, thus, characterize them as catch or slip bond cross-linkers.Outlook: In a second funding period, I plan to examine how a combination of different catch and slip bond cross-linkers in the actin cytoskeleton can be used to custom-tailor the material properties of the cell. To that end, I intend to develop a mathematical model that characterises the expected influence of catch and slip bond cross-linkers on emergent material properties of the actin cytoskeleton. This model will predict cross-linker concentration regimes in which active stress causes stiffening or softening, solidification or fluidization of a meshwork. In an experimental part, I plan to explore these regimes through targeted knock-down of cross-linker proteins in the cell, thereby, delivering a proof of principle that force-sensing of cross-linkers affects the nonlinear properties of the actin cytoskeleton.