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

合作研究：重建自组织收缩肌动球蛋白系统的力学

• 批准号: 2201235
• 负责人: Aaron Dinner
• 金额: $ 53.32万
• 依托单位: University of Chicago
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2201235&HistoricalAwards=false
• 关键词: Collaborative; Research; Mechanics; Reconstituted; Self

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.

许多细胞功能依赖于细胞骨架，细胞骨架是生物聚合物的动态网络的集合，为细胞提供机械特性和改变形状的能力。这些网络具有不同的架构，通常在小区中共存。虽然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它们所观察到的结构和动力学仍然是一个悬而未决的问题。通过从纯化的组件重构网络结构和动态，该项目旨在定义自组织的基本要素，并使良好控制的测量和建模能够探索其机制。迄今为止，这样的重构研究主要集中在本体溶液或固体支持物中的蛋白质组装。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力学所必需的。膜不仅限制了分子种类，它还提供了一个边界，可以锚细胞骨架结构，但在细胞中的典型力下变形。该项目将建立在最近的技术进步，研究蛋白质网络，自组织成合成脂质囊泡。这项工作的更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及其教师，沿着还有本科生和博士后研究员。 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细胞骨架蛋白的不同组成的结构和动力学，该项目将促进对肌动蛋白网络的自组织和力生成以及膜限制和耦合在这些过程中所起作用的理解。该项目将特别关注细胞分裂的重建特征，特别是由丝状肌动蛋白和马达蛋白肌球蛋白II组成的收缩环的形成和收缩，沿着额外的结构（例如，锚定和捆绑）蛋白质。我们实验策略的基础是在巨大的单层囊泡中重建细胞骨架网络的强大平台。这将与细胞骨架网络的粗粒度模拟配对。该项目将确定网络/环形成的基本要素，通过表征肌动蛋白，肌动蛋白结合蛋白（α-辅肌动蛋白，肌成束蛋白，和/或fimarin）的混合物的自组织，和电机蛋白（肌球蛋白和/或它的截短形式）在特定的膜相互作用的情况下。然后，将测试组装肌动蛋白环与肌动蛋白皮质在膜上共存的策略，以研究膜结合如何改变肌动蛋白网络的结构。最后，模式化的电机激活将用于驱动膜相关的网络/环收缩和囊泡收缩，并将研究所产生的力。为了实现最大的影响，研究、教育和推广目标将紧密结合。 该项目由分子和细胞生物科学部的系统与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NSF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。