Collaborative Research: Mechanics of Reconstituted Self-Organized Contractile Actomyosin Systems

许多细胞功能依赖于细胞骨架，这是一组动态的生物聚合物网络，为细胞提供了机械性能和改变形状的能力。这些网络具有不同的体系结构，通常在单元中共存。尽管对整个细胞的研究已经能够表征这些网络，但它们如何自组织以产生观察到的结构和动力学仍然是一个悬而未决的问题。通过从纯化的组件中重构网络结构和动态，该项目试图定义自组织的基本元素，并使良好控制的测量和建模能够探测其机制。迄今为止，此类重构研究主要集中在散装溶液或固体载体中的蛋白质组装上。然而，细胞被脂质双层膜包围，这被认为是许多细胞功能和它们背后的机制所必需的。膜不仅限制分子种类；它还提供了一个边界，可以锚定细胞骨架结构，但在细胞中的典型力下变形。该项目将以最新的技术进步为基础，研究自组织合成脂质囊泡的蛋白质网络。这项工作的广泛影响包括研究本身的内在价值，因为所有细胞都含有某种形式的细胞骨架。其他活动包括培训高中学生和他们的老师，以及本科生和博士后研究人员。pi还将为伍兹霍尔海洋生物实验室正在开发的合成细胞艺术装置做出贡献。通过系统地表征囊泡内不同细胞骨架蛋白组成的结构和动力学，该项目将促进对肌动蛋白网络自组织和力产生的理解，以及膜约束和偶联在这些过程中所起的作用。该项目将特别侧重于重建细胞分裂的特征，特别是由丝状肌动蛋白和运动蛋白肌球蛋白II组成的收缩环的形成和收缩，以及额外的结构（例如，锚定和捆绑）蛋白质。我们的实验策略的基础是一个强大的平台，在巨大的单层囊泡中重建细胞骨架网络。这将与细胞骨架网络的粗粒度模拟配对。该项目将通过描述肌动蛋白、肌动蛋白结合蛋白（α -肌动蛋白、束状蛋白和/或纤蛋白）和运动蛋白（肌凝蛋白和/或其截断形式）混合物的自组织，在没有特定膜相互作用的情况下，确定网络/环形成的基本要素。然后，将测试在膜上组装肌动蛋白环与肌动蛋白皮质共存的策略，以研究膜结合如何改变肌动蛋白网络的结构。最后，模式运动激活将用于驱动膜相关网络/环收缩和囊泡收缩，并将研究由此产生的力。为了达到最大的影响，研究、教育和推广目标将紧密结合在一起。该项目由分子和细胞生物科学部的系统和合成生物学以及细胞动力学和功能项目共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Many cellular functions rely on the cytoskeleton, a collection of dynamic networks of biopolymers that provide cells with their mechanical properties and abilities to change shape. These networks have diverse architectures, which often coexist in cells. Although studies of whole cells have enabled characterization of these networks, how they self-organize to give rise to their observed structures and dynamics remains an outstanding question. By reconstituting network structures and dynamics from purified components, this project seeks to define the essential elements for self-organization and enable well-controlled measurements and modeling to probe its mechanisms. To date, such reconstitution studies have largely focused on protein assemblies in bulk solution or on solid supports. However, cells are enclosed by a lipid bilayer membrane, which is thought to be essential for many cellular functions and for the mechanics underlying them. The membrane does not just confine molecular species; it also provides a boundary that can anchor cytoskeletal structures yet deform under typical forces in cells. The project will build on recent technological advances to study protein networks that self-organize into synthetic lipid vesicles. The Broader Impacts of the work include the intrinsic merit of research itself as all cells contain some form of cytoskeleton. Additional activities include training of high school students and their teachers, along with undergraduates and post-doctoral research fellows. The PIs will also contribute to an art installation on synthetic cells that is being developed at the Marine Biological Laboratory at Woods Hole. By systematically characterizing the structures and dynamics accessible to different compositions of cytoskeletal proteins within vesicles, the project will advance understanding of self-organization and force generation by actin networks and the roles membrane confinement and coupling play in these processes. The project will specifically focus on reconstituting features of cell division, in particular the formation and constriction of a contractile ring composed of filamentous actin and the motor protein myosin II, along with additional structural (e.g., anchoring and bundling) proteins. The foundation of our experimental strategy is a powerful platform for reconstituting cytoskeletal networks in giant unilamellar vesicles. This will be paired with coarse-grained simulations of cytoskeletal networks. The project will determine the essential elements for network/ring formation by characterizing the self-organization of mixtures of actin, actin-binding proteins (alpha-actinin, fascin, and/or fimbrin), and motor proteins (myosin and/or a truncated form of it) in the absence of specific membrane interactions. Then, strategies to assemble an actin ring in coexistence with an actin cortex at the membrane will be tested to investigate how membrane binding alters the architecture of actin networks. Finally, patterned motor activation will be used to drive membrane-associated network/ring contraction and vesicle constriction, and the resulting forces will be investigated. To achieve the greatest impact, research, education, and outreach objectives will be closely integrated. This project was co-funded by the Systems and Synthetic Biology, and the Cellular Dynamics and Functions programs, both in the Molecular and Cellular Biosciences Division.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.