Department of Physics and Biophysics, University of San Diego.
Department of Physics and Biophysics, University of San Diego; W. M. Keck Science Department, Scripps College, Pitzer College, and Claremont McKenna College.
J Vis Exp. 2022 Aug 25(186). doi: 10.3791/64228.
The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.
复合细胞骨架由相互作用的半刚性肌动蛋白丝和刚性微管网络组成，利用肌球蛋白 II 和驱动蛋白等马达蛋白重排并产生力，从而驱动迁移、胞质分裂、黏附和机械感觉等关键过程。尽管肌动蛋白-微管相互作用是细胞骨架多功能性和适应性的关键，但对它们与肌球蛋白和驱动蛋白活性相互作用的理解仍处于起步阶段。这项工作描述了如何工程设计可调节的三维复合网络，其中交织的肌动蛋白丝和微管经历主动重排和弹道运动，由肌球蛋白 II 和驱动蛋白马达驱动，并通过肌动蛋白、微管、马达蛋白和被动交联剂的相对浓度进行调节。还详细介绍了用于荧光标记微管和肌动蛋白丝的方案，以便使用多光谱共聚焦成像最有效地可视化复合重排和运动。最后，提出了可用于定量表征非平衡结构、动力学和力学的数据分析方法的结果。重现和研究这个可调谐的仿生平台提供了有价值的见解，了解如何从有丝分裂到极化到机械感觉等多种细胞过程中，如何通过偶联的马达活性、复合力学和细丝动力学来实现。
