Department of Chemical Engineering, Ben Gurion University of the Negev.
Department of Chemical Engineering, Ben Gurion University of the Negev; Department of Chemical Engineering, Ilse Kats Institute for Nanoscale Science and Technology, Ben Gurion University of the Negev;
J Vis Exp. 2023 Mar 10(193). doi: 10.3791/64377.
Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This feature is attributed to the mechanical and dynamic properties of the cell cytoskeleton, notably, the actomyosin cytoskeleton, which is an active gel of polar actin filaments, myosin motors, and accessory proteins that exhibit intrinsic contraction properties. The usually accepted view is that the cytoskeleton behaves as a viscoelastic material. However, this model cannot always explain the experimental results, which are more consistent with a picture describing the cytoskeleton as a poroelastic active material-an elastic network embedded with cytosol. Contractility gradients generated by the myosin motors drive the flow of the cytosol across the gel pores, which infers that the mechanics of the cytoskeleton and the cytosol are tightly coupled. One main feature of poroelasticity is the diffusive relaxation of stresses in the network, characterized by an effective diffusion constant that depends on the gel elastic modulus, porosity, and cytosol (solvent) viscosity. As cells have many ways to regulate their structure and material properties, our current understanding of how cytoskeleton mechanics and cytosol flow dynamics are coupled remains poorly understood. Here, an in vitro reconstitution approach is employed to characterize the material properties of poroelastic actomyosin gels as a model system for the cell cytoskeleton. Gel contraction is driven by myosin motor contractility, which leads to the emergence of a flow of the penetrating solvent. The paper describes how to prepare these gels and run experiments. We also discuss how to measure and analyze the solvent flow and gel contraction both at the local and global scales. The various scaling relations used for data quantification are given. Finally, the experimental challenges and common pitfalls are discussed, including their relevance to cell cytoskeleton mechanics.
细胞可以主动改变形状并变得具有运动性，这种特性取决于它们主动重组内部结构的能力。这种特性归因于细胞细胞骨架的机械和动态特性，特别是肌动球蛋白细胞骨架，它是一种具有内在收缩特性的极性肌动蛋白丝、肌球蛋白马达和辅助蛋白的活性凝胶。通常认为细胞骨架表现为粘弹性材料。然而，这种模型并不能总是解释实验结果，这些结果更符合描述细胞骨架为多孔弹性活性材料的图像，即嵌入细胞溶质的弹性网络。肌球蛋白马达产生的收缩性梯度驱动细胞溶质流过凝胶孔，这推断出细胞骨架和细胞溶质的力学紧密耦合。多孔弹性的一个主要特征是网络中应力的扩散松弛，其特征在于有效扩散常数取决于凝胶弹性模量、孔隙率和细胞溶质（溶剂）粘度。由于细胞有许多调节其结构和材料特性的方法，我们对细胞骨架力学和细胞溶质流动动力学如何耦合的理解仍然知之甚少。在这里，采用体外重组方法来表征多孔弹性肌动球蛋白凝胶的材料特性，作为细胞细胞骨架的模型系统。凝胶收缩是由肌球蛋白马达的收缩性驱动的，这导致渗透溶剂的流动。本文描述了如何制备这些凝胶并进行实验。我们还讨论了如何在局部和全局尺度上测量和分析溶剂流动和凝胶收缩。给出了用于数据量化的各种比例关系。最后，讨论了实验挑战和常见陷阱，包括它们与细胞细胞骨架力学的相关性。
