Programmable Synthetic Organelles Built from Disordered Proteins for Cellular Engineering

摘要 细胞决策的工程--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 取决于人们能在多大程度上操纵内源性细胞机制。无膜细胞器是 天然存在的内在无序蛋白质（IDP）组装体，形成富含蛋白质的流体状相 在细胞中。通过有见地的工程，这些细胞器可以提供一种指导细胞生理学的手段， 调节分子的区室化和释放。最近，我们开发了一个新颖的平台， 无膜细胞器的组装，赋予了无数的新特性和控制基序。我们 这些方法是基于工程化的内在无序的富含精氨酸-甘氨酸的RGG结构域， 以驱动相分离。我们的应用程序的前提是众多的，国家的最先进的IDP的发展 组装新型无膜细胞器的材料。我们已经开发出了动态地 调节RGG序列的多价性并引入蛋白质相互作用基序以允许隔离 指定的货物。我们建议制定战略，控制快速释放和封存的 货物蛋白响应光和热刺激。此外，我们还提出了开发光学和酶的方法。 用于细胞器组装和拆卸的控制器。重要的是，我们已经证明了表达式 这些序列中的每一个都足以在酵母和哺乳动物细胞系中产生无膜细胞器。这里我们 提出了一种新的，创新的合成生物学范式，通过控制细胞反应， 操纵无膜细胞器。这些细胞器将被设计为隔离控制因子， 细胞周期的承诺或细胞命运的决定线索，响应光或生化刺激。最终 所提出的工作的目标是通过细胞周期和细胞命运决定来控制动态进展- 为再生医学制造造血干细胞。在目标1中，我们将扩大自我的工具箱， 通过在无序蛋白质材料内设计光学和热开关来组装IDP， 门控细胞器组装、组成和释放。在目标2中，我们将使用无膜细胞器进行细胞培养， 决策，快速螯合和释放靶蛋白，以动态控制细胞增殖， 干细胞命运利用区室化的力量，我们的设计师细胞器承诺一个可调的 生物化学生态位和一个可推广的战略，精确工程细胞系统能够响应 特定的刺激与可预测的结果。

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.