Dissecting the nuclear pore-like permeability barrier function of phase separated liquid FG nucleoporin condensates

剖析相分离液体 FG 核孔蛋白凝聚物的核孔样渗透屏障功能

• 批准号: 419070619
• 负责人: Professor Dr. Edward A. Lemke
• 金额: --
• 依托单位: Department of Biomedical Engineering
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/419070619?language=en
• 关键词: Dissecting; nuclear; pore; like; permeability

The nuclear pore complex (NPC) is a ~120 MDa structure built from multiple copies of ~30 different nucleoporins (Nups). The NPC traverses the nuclear envelope and functions as the “gatekeeper” for nucleocytoplasmic transport. The NPC’s permeability barrier consists of ~ ten different “FG-Nups”, primarily disordered Nups containing domains rich in phenylalanine (F) and glycine (G). These FG-domains non-covalently interact to form a supramolecular matrix in the NPC’s central region for which the structure has remained elusive. A promising approach to study the permeability barrier is to develop in vitro reconstitution models of FG-Nups that capture key features of the functional NPC. This includes formation of a natural barrier for cargoes larger than ~ 4 nm, unless accompanied by nuclear transport receptors (NTRs). We are developing a microfluidic device that allows resolving kinetic processes after FG-Nups self-assemble via liquid-liquid phase separation (LLPS). We have shown that FG-Nups can transiently populate a liquid like droplet state (following LLPS) that ages into more solid like state over minutes. Even though the liquid state is transient, the use of consecutive microfluidic mixers allows probing of the permeability barrier properties immediately after droplet formation with a time resolution that is impossible in benchtop experiments. Using this device, we have found that the liquid state surprisingly mimics the key functionality of a physiological permeability barrier: large cargoes (> 4nm) require a functional NTR:cargo complex to enter the droplet (Celetti et al., JCB 2019). To investigate how this liquid FG-Nup permeability barrier functions, we aim to develop a “multi-analytic” platform that integrates microfluidics with coherent anti-Stokes Raman spectroscopy (CARS) and particle tracking microrheology (PTM) (Chatterjee et al., Adv. Sci. 2021), to study the evolving supramolecular structure as well as the mechanical properties of FG-rich droplets. At the same time, we have developed a multi component “stickers and spacers” model that provides a theoretical framework for predicting the complex phase behavior of solutions of multivalent biopolymers with specific interacting sites, such as FG-Nups (Michels et al., Biomacro. 2021). Together, these tools will provide a coherent picture to explain how the balance between homo- and heterotypic interactions in FG/FG/NTR mixtures modulates the phase behavior and how this relates to the permeability barrier function of the condensed liquid state. We hypothesize that barrier properties of the NPC are modulated by NTRs and FG-Nup heterogeneity, providing a possible means for dynamic tuning of NPC permeability in cells. Finally, the platform we build will serve as a general tool to study LLPS of phase separating proteins, particularly those that undergo rapid maturation to gel or amyloid like states, such as e.g. FUS, Tau and α-synuclein or other proteins associated to neurodegeneration.

核孔复合物（NPC）是由~30种不同核孔蛋白（Nups）的多个拷贝构建的~120 MDa结构。NPC穿过核膜并作为核质运输的“看门人”。NPC的渗透性屏障由~ 10种不同的“FG-Nup”组成，主要是含有富含苯丙氨酸（F）和甘氨酸（G）的结构域的无序Nup。这些FG结构域非共价相互作用，在NPC的中心区域形成超分子基质，其结构仍然难以捉摸。研究渗透性屏障的一种有前途的方法是开发捕获功能性NPC的关键特征的FG-Nups的体外重建模型。这包括对大于~ 4 nm的货物形成天然屏障，除非伴有核转运受体（NTR）。我们正在开发一种微流体装置，该装置允许通过液-液相分离（LLPS）在FG-Nups自组装后解析动力学过程。我们已经表明，FG-Nups可以短暂地填充液体状液滴状态（在LLPS之后），其在几分钟内老化成更像固体的状态。即使液体状态是短暂的，连续的微流体混合器的使用允许在液滴形成后立即探测渗透性屏障性质，其时间分辨率在台式实验中是不可能的。使用该装置，我们已经发现液态令人惊讶地模拟生理渗透屏障的关键功能：大的货物（> 4 nm）需要功能性NTR：货物复合物进入液滴（Celetti等人，JCB 2019）。为了研究这种液体FG-Nup渗透性屏障如何起作用，我们的目标是开发一种“多分析”平台，其将微流体与相干反斯托克斯拉曼光谱（汽车）和粒子跟踪微流变学（PTM）集成（Chatterjee等人，高级科学2021），以研究富FG液滴的演变的超分子结构以及机械性质。同时，我们已经开发了多组分“粘着剂和间隔剂”模型，该模型提供了用于预测具有特定相互作用位点的多价生物聚合物的溶液的复杂相行为的理论框架，例如FG-NUPS（Michels等人，Biomacro。2021年）。总之，这些工具将提供一个连贯的图片来解释如何在FG/FG/NTR混合物的同型和异型相互作用之间的平衡调制的相行为，以及这是如何涉及到的凝聚液体状态的渗透性屏障功能。我们假设NPC的屏障特性受到NTR和FG-Nup异质性的调节，这为细胞中NPC渗透性的动态调节提供了一种可能的手段。最后，我们构建的平台将作为研究相分离蛋白的LLPS的通用工具，特别是那些经历快速成熟为凝胶或淀粉样状态的蛋白，例如FUS，Tau和α-突触核蛋白或其他与神经变性相关的蛋白。