Self-Organization and Mechanical Properties of Dynamic Cytoskeletal Networks in vitro
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摘要: 细胞骨架是由微管、肌动蛋白丝和中间纤维三种蛋白丝为主要成分组成的复合动态网络结构，在结合蛋白、辅助调节蛋白和马达蛋白的参与下帮助细胞实现运动、分裂和生长等基本生命过程。研究体外纯化的细胞骨架蛋白和马达蛋白网络，可以深入了解控制自组织亚细胞结构动力学行为的基本原理，为设计类似生命的活性物质和机器提供方向。本文综述了近年来基于纯化蛋白在体外简化环境中实现的细胞骨架蛋白-马达蛋白网络，重点介绍其非平衡本质、活性应力和动态网络的产生，以及这种动态网络对亚细胞结构和宏观尺度活性材料自组织过程的影响。此外，还简要介绍了细胞骨架蛋白-马达蛋白网络在构建体外仿生系统中的应用。Abstract: Cytoskeletal networks usually refer to cellular structures that are comprised of microtubules, actin filaments, intermediate filaments, and their associated accessory proteins and motor proteins, facilitating a range of cellular functions such as cell motion, division and growth. In addition to the narrow definition of “cytoskeleton” that provides frame support to an otherwise fluidic cell, cytoskeletal polymers play an important role in many other cellular functions. For example, bacteria flagella and spindle apparatus are essentially microtubules in different forms. In living cells, there are also hundreds of proteins and biochemical factors regulating the structure and dynamics of such networks, which makes it extremely difficult to elucidate the physical mechanisms behind these processes. In recent years, study of in vitro cytoskeletal polymer-motor protein networks built from purified protein components not only helps to understand the fundamental principles of non-equilibrium self-organization and dynamic behavior of cytoskeletal polymers and motor proteins on the subcellular level, but also sheds light on the design of active matter system and active machines that may operate far from equilibrium with life-like behaviors and functions. One notable success is the artificial active nematics built upon microtubules and kinesin motors, in which active stresses are used to generate macroscopic active flow and guide materials assembly. The active stress and order emergence of materials organization can also be tuned by a set of external parameters, such as external magnetic fields and light-activated proteins, in addition to the concentration of protein building blocks, ATP, and crowding agents. In this review, we focus on in vitro cytoskeleton-motor protein networks based on purified components including tubulin, actin, kinesin, and myosin, emphasizing on the non-equilibrium nature of microtubule and F-actin polymerization, generation of active stress and formation of dynamic networks, as well as the self-organization and dynamic behavior of subcellular structures on a larger scale. We conclude with the application of such networks in the study of active matter and artificial cells.
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Key words:
- cytoskeletal network /
- self-organization /
- microtubule /
- actin filament /
- artificial cell
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图 1 细胞骨架蛋白网络结构：(a) 三种细胞骨架蛋白纤维的组成[7]；(b) 微管蛋白单体的成核与组装[18]；(c) 肌动蛋白纤维的组装[19]；(d) 微管/肌动蛋白(左[20])与马达蛋白(中[7])、交联剂(右[7])之间的相互作用；(e) 细胞骨架纤维与单个/簇状马达之间的相对运动[17]；(f) 微管的生长与解离[19]
Figure 1. Structure of cytoskeletal networks: (a) Compose of three types of cytoskeletal fibers[7]; (b) Nucleation and assembly of tubulin monomers[18]; (c) Assembly of actin fibers[19]; (d) Interactions between microtubules/F-actin (Left)[20] and motor proteins (Middle)[7]、passive crosslinkers (Right)[7]; (e) Relative motion between cytoskeletal fibers and individual/cluster motor proteins[17]; (f) Growth and shrinkage of microtubules[19]
图 2 马达蛋白参与的动态细胞骨架网络构建：(a) 马达蛋白成簇[24]；(b) 极性分选形成星状体[24]；(c) 计算机模拟微管生长速率和马达浓度对马达组织网络形成的影响[24]；(d) 计算机模拟程序形成的向列型网络/星状体[24]；(e) 基于控制变量规则的正常双极纺锤体形成以及选择性马达活性缺失导致纺锤体无法形成[24]；(f) 相邻的星状体合并形成收缩网络[26]
Figure 2. Construction of dynamic cytoskeletal network by motor proteins: (a) Clustering of motor proteins[24]; (b) Aster formation by polarity sorting[24]; (c) Dependence of the organizational states on microtubule growth speed and number of motors per microtubule in computer simulations[24]; (d) Nematic network/aster in computer simulations[24]; (e) Relevance of the control parameter-based rules for normal bipolar spindle assembly in cells and their consequences for the characteristic shapes of defective spindles after motor inactivation[24]; (f) Adjacent asters merge to form contractile network[26]
图 3 细胞骨架网络的有序性和活性应力[16]：(a) 细胞骨架结构的对称性；(b) 活性液晶相中的拓扑缺陷及拓扑电荷；(c) 伸展型与收缩型细胞骨架网络
Figure 3. Symmetries and active stress generation in cytoskeletal networks[16]: (a) Symmetry of cytoskeletal structure; (b) Topological defects and charges in active liquid crystal phase; (c) Extensile and contractile cytoskeletal networks
图 4 边界效应对动态细胞骨架网络的影响：(a) 走向稳定微管端的光二聚电机的示意图[32]；(b) 星状体的形成与消失[32]；(c) 星状体在两个连通的光斑照射下的合并[32]；(d) 通过拉伸/收缩柔性基底实现对微管网络组织能力的调节[33]
Figure 4. Boundary effects on dynamic cytoskeletal networks: (a) Schematic of light-dimerizable motors that walk towards the plus ends of stabilized microtubules[32]; (b) Images of labelled microtubules during aster assembly and decay[32]; (c) Aster merging operation under illumination of a connected pair of light spots[32]; (d) Stretch-and-compression of soft substrates organizes the patterns of an in vitro gliding assay of microtubules[33]
图 5 原型合成细胞中基于微丝的模拟细胞骨架结构和功能[36]：（a） 基于细菌的原型合成细胞中的微丝纤维组装；（b） 人工细胞实现的七种类细胞特征；（c） 共聚焦显微图像的三维重构显示出原型合成细胞由于内部活细菌的存在而呈现出类似阿米巴虫的形貌变化
Figure 5. Structure and function of the proto-cytoskeletal network in an artificial cell[36]： (a) F-actin assembly in bacteriogenic protocells; (b) Seven cell-mimic functions of artificial cells; (c) 3D construction of confocal microscopic images showing progressive transformation of a protocell into amoeba-like morphology due to on-site E. coli activity
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