Thin film microrheology of the living actomyosin cortex in the C. elegans embryo

细胞皮层是细胞膜下由交联的肌动蛋白丝和肌球蛋白运动蛋白组成的薄层。除了为细胞提供机械稳定性外，这个动态网络还驱动大规模的皮质流动。皮层流对细胞极化和细胞分裂等关键细胞过程至关重要。尽管我们对单个皮层成分有详细的了解，但它们在皮层内的相互作用仍然不清楚，因为很难从可观察到的皮层的大规模行为中推断出皮层特性的分子贡献。需要一种中间层次的描述，详细描述大脑皮层涌现的物理特性。在这个项目中，我们建议使用一种新的活性薄膜微流变学（ATHUR）方法直接研究皮层的力学特性。我们最近建立了一种在单细胞秀丽隐杆线虫胚胎中对微米大小的磁性颗粒施加校准力的方法。通过将磁性颗粒显微注射到雌雄同体成虫的性腺中，将胚胎置于磁场梯度中，对磁性颗粒施加外力。为了研究皮质力学，我们将把植入的磁珠拉到皮层，首先，研究它们作为与皮层相关的被动示踪剂的运动，然后，通过在皮质平面上对磁珠施加校准的力，研究皮层对这些主动探针的反应。虽然被动和主动微流变学实验已被用于描述体外重建肌动球蛋白凝胶的体粘弹性，但提出的实验将使我们首次能够获得活细胞薄的准二维皮质网络的力学特性。然后，通过RNAi将机械特性与肌动蛋白结合蛋白的遗传扰动结合起来，以确定这些蛋白质在皮层中调节的机械参数。使用流体力学的描述皮层作为一个活跃的复杂流体薄膜，我们将把皮层的物质特性的变化与皮层流动表型联系起来。总之，这一建议旨在提供从分子到皮质行为机制的联系，这将对理解生命系统中的皮质流动至关重要。

The cell cortex is a thin layer of cross-linked actin filaments and myosin motor proteins beneath the cell membrane. In addition to providing mechanical stability to the cell, this dynamic network also drives large-scale cortical flows. Cortical flows are crucial to key cellular processes such as cell polarization and cell division. Despite our detailed understanding of individual cortical components, their interplay within the cortex remains obscure because of the difficulty to infer molecular contributions to cortical properties from the observable large-scale behavior of the cortex alone. An intermediate level of description is required, detailing the emergent physical properties of the cortex.In this project, we propose to study the mechanical properties of the cortex directly, using a novel Active THin-film microRheology (ATHUR) approach. We have recently established a method to apply calibrated forces on micrometer-sized magnetic particles in the one-cell C. elegans embryo. The magnetic particles are introduced into embryos by microinjection into the gonad of adult hermaphrodite worms, and external forces are exerted on the incorporated particles by placing the embryo in a magnetic field gradient. To investigate cortical mechanics, we will pull the incorporated magnetic beads to the cortex and study, first, their motion as passive tracers associated with the cortex, and then, by applying a calibrated force on the beads in the cortical plane, the response of the cortex to these active probes.While passive and active microrheology experiments have been used to describe the bulk viscoelasticity of reconstituted actomyosin gels in vitro, the proposed experiments will allow us to access, for the first time, the mechanical properties of the thin, quasi-2D cortical network of a living cell. The mechanical characterization will then be combined with genetic perturbations of actin-binding proteins by RNAi to identify the mechanical parameters that these proteins tune in the cortex. Using a hydrodynamic description of the cortex as a thin film of active complex fluid, we will then relate the changes in the material properties of the cortex to the cortical flow phenotypes.In summary, this proposal aims to provide a link from molecules to the mechanics of cortical behavior, which will be crucial to the understanding of cortical flows in living systems.