Programmable Synthetic Organelles Built from Disordered Proteins for Cellular Engineering

摘要 细胞决策的工程学--如进入细胞周期或干细胞分化--是 对我们再生功能细胞和组织的能力至关重要。这些技术的发展是有限的。 取决于一个人可以在多大程度上操纵内生的细胞机械。无膜细胞器是 天然存在的固有无序蛋白质(IDPs)的集合，形成富含蛋白质的流体状相 在牢房里。通过有洞察力的工程，这些细胞器可以提供一种通过 调节分子的区隔和释放。最近，我们开发了一个新的平台，用于 无膜细胞器的组装，赋予了无数新的性质和控制基序。我们的 这些方法是基于工程上内在无序的富含精氨酸-甘氨酸的RGG结构域，这些结构域足够 以驱动相分离。我们应用的前提是开发大量最先进的IdP 用于组装新的、设计的无膜细胞器的材料。我们已经开发出方法来动态 调节RGG序列和引入的蛋白质相互作用基序的多价性以实现封存 指定货物的数量。我们建议制定控制快速释放和封存的策略 货物蛋白对光和热刺激的反应。此外，我们还提出了开发光学和酶制剂的建议。 细胞器装配和拆卸的控制器。重要的是，我们已经演示了表达式 的序列足以在酵母和哺乳动物细胞系中产生无膜细胞器。在这里我们 在合成生物学中提出一种新的、创新的范式，通过 无膜细胞器的操纵。这些细胞器将被设计成隔离控制 细胞周期承诺或细胞命运决定的线索，对光或生化刺激的反应。终极的 拟议工作的目标是通过细胞周期和细胞命运的决定来控制动态进展- 制造用于再生医学的造血干细胞。在目标1中，我们将扩展SELF的工具箱 通过在无序的蛋白质材料中设计光学和热开关来组装IDPs以实现动态 细胞器组装、合成和释放的门控。在目标2中，我们将使用无膜细胞器作为细胞 决策，快速隔离和释放目标蛋白，动态控制细胞增殖和 干细胞的命运。利用隔间的力量，我们设计的细胞器承诺可调 生化生态位和一种可推广的策略，用于精确设计能够响应 具有可预测结果的特定刺激。

Abstract The engineering of cellular decision-making – such as entry into the cell cycle or stem cell differentiation – is critical to our ability to regenerate functional cells and tissues. The development of these technologies is limited by the extent to which one can manipulate the endogenous cellular machinery. Membraneless organelles are naturally-occurring assemblies of intrinsically disordered proteins (IDPs) that form protein-rich, fluid-like phases in cells. With insightful engineering, these organelles can provide a means for directing cell physiology through the compartmentalization and release of regulatory molecules. Recently, we developed a novel platform for the assembly of membraneless organelles endowed with a myriad of novel properties and control motifs. Our methods are based on engineering intrinsically-disordered arginine-glycine rich RGG domains that are sufficient to drives phase separation. The premise of our application is the development of numerous, state-of-the-art IDP materials for assembly of new, designer membraneless organelles. We have developed methods to dynamically modulate the multivalency of RGG sequences and introduced protein-interaction motifs to allow sequestration of designated cargo. We propose to develop strategies for controlling the rapid release and sequestration of cargo proteins in response to optical and thermal stimuli. Further, we propose the develop optical and enzymatic controllers for organelle assembly and disassembly. Importantly, we have already demonstrated the expression of the sequences is sufficient to generate membraneless organelle in yeast and mammalian cell lines. Here we propose a novel, innovative paradigm in synthetic biology for controlling cellular responses through the manipulation of membraneless organelles. These organelles will be designed to sequester factors that control cell cycle commitment or cell fate decisions on cue, in response to light or biochemical stimulus. The ultimate goal of the proposed work is to control the dynamic progression through the cell cycle and cell fate decision- making in hematopoietic stem cells for regenerative medicine. In aim 1, we will expand the toolbox of self- assembling IDPs by engineering optical and thermal switches within disordered protein materials for dynamic gating of organelle assembly, composition and release. In aim 2, we will use membranless organelles for cell decision-making, rapidly sequestering and releasing target proteins to dynamically control cell proliferation and stem cell fate. Harnessing the power of compartmentalization, our designer organelles promise a tunable biochemical niche and a generalizable strategy for precisely engineering cell systems capable of responding to specific stimuli with predictable outcomes.