Self-organization and mechanics of actomyosin networks attached to artificial and cellular plasma membranes

动物细胞的皮质是一个薄的、高度动态的交联型肌动蛋白网络，直接与质膜相连。尽管皮质肌动蛋白在细胞力学和形态发生中发挥着关键作用，但人们对附着在质膜上的皮质的组织、其集体动力学以及它的动力学如何影响最终产生功能的力学性质知之甚少。深刻理解需要研究允许控制网络组成和体系结构的模型系统。这个项目的总体目标是解开网络结构、对质膜的动态附着和收缩是如何影响细胞皮质的粘弹性特性的。自下而上方法得到的人工皮质和自上而下方法得到的自然皮质的力学行为的收敛是人们追求的。该项目的起点是最近建立的最小肌动蛋白皮质(MAC)，它通过Ezrin蛋白将掺入受体脂质PtdIns(4，5)P2的脂膜与F-肌动蛋白动态交联。在以前的工作中，我们量化了F-肌动蛋白网络的组织作为膜上Ezrin钉扎位置的函数，并将F-肌动蛋白/膜复合材料的粘弹性与肌动蛋白组织联系起来。基于已建立的MAC，我们现在能够通过改变F-肌动蛋白结合位点的Ezrin突变体来阐明Ezrin交联剂的动态性质对F-肌动蛋白结构及其粘弹性的影响。我们将进一步解决F-肌动蛋白交联剂如何改变膜上肌动蛋白网络的组织以及这如何影响系统的动力学和流变性的问题。我们将通过添加非肌肉肌球蛋白II马达和ATP来监测网络中的集体行为并量化相关的非热波动，从而主动推动肌动蛋白皮质失去平衡。与MACs相比，我们计划在保留皮质的情况下操纵天然细胞膜碎片的附着位置，以确定不同组件对于动态网络的介观组织的重要性。主动和被动微观流变学的使用将有助于我们研究非热波动对皮质粘弹性特性的影响。网络的非线性行为将通过外部施加高幅噪声的主动微观流变学来探索。这将使我们能够解决人工网络的流变性和活细胞的软玻璃流变学之间的差异。

The cortex of animal cells is a thin, highly dynamically crosslinked actin network directly connected to the plasma membrane. Even though cortical actin plays a pivotal role in cellular mechanics and morphogenesis, only very little is known about the organization of the cortex attached to the plasma membrane, its collective dynamics, and how its dynamics affects mechanical properties that eventually generate function. A profound understanding requires to study model systems that permit control over composition and architecture of the network. The overarching goal of this project is to disentangle how network architecture, dynamic attachment to the plasma membrane, and contractility contribute to the viscoelastic properties of the cellular cortex. Convergence of the mechanical behavior of artificial cortices obtained in bottom-up approaches and natural cortices derived in a top-down approach is sought-after. Starting point of the project is a recently established minimal actin cortex (MAC), which dynamically cross-links a lipid membrane doped with the receptor lipid PtdIns(4,5)P2 with F-actin through the protein ezrin. In prior work, we quantified the organization of the F-actin network as a function of ezrin pinning sites on the membrane and related the viscoelastic properties of the F-actin/membrane composite to actin organization. Based on the established MAC, we are now in the position to elucidate the influence of the ezrin cross-linker’s dynamic nature on the F-actin architecture and its viscoelastic properties using ezrin mutants with an altered F-actin binding site. We will further address the question how F-actin cross-linkers alter the organization of the actin network on the membrane and how this influences the dynamics and rheological properties of the system. We will actively drive the actin cortex out of equilibrium by adding non-muscle myosin II motors and ATP to monitor the collective behavior in the networks and quantify the associated athermal fluctuations. In comparison to the MACs, we plan to manipulate the attachment sites of natural cell membrane fragments with preserved cortices to be able to pin down the importance of the different components for mesoscopic organization of the dynamic network. The use of both active and passive microrheology will help us to investigate the effect of athermal fluctuations on the viscoelastic properties of the cortex. Nonlinear behavior of the networks will be explored by using active microrheology with externally applied high-amplitude noise. This will permit us to address the discrepancy between rheological properties of artificial networks and the soft glassy rheology found for living cell.