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

核孔复合体（NPC）是由约30个不同的核孔蛋白（Nups）的多个拷贝构建而成的~ 120mda结构。NPC穿过核膜，充当核细胞质运输的“看门人”。NPC的渗透性屏障由大约10个不同的“FG-Nups”组成，主要是含有丰富苯丙氨酸(F)和甘氨酸(G)结构域的无序Nups。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矩阵，其结构仍然难以捉摸。研究渗透性屏障的一种很有前途的方法是开发fg - nup的体外重建模型，该模型可以捕获功能性鼻咽癌的关键特征。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（NTRs）。我们正在开发一种微流体装置，可以通过液-液相分离（LLPS）解决fg - nps自组装后的动力学过程。我们已经证明，fg - nps可以在几分钟内短暂地形成类似液体的液滴状态（遵循LLPS），并逐渐变成更像固体的状态。尽管液体状态是短暂的，使用连续微流体混合器可以在液滴形成后立即探测渗透屏障特性，其时间分辨率在台式实验中是不可能的。使用该装置，我们发现液体状态令人惊讶地模仿了生理渗透性屏障的关键功能：大货物（> 4nm）需要功能性NTR：货物复合物才能进入液滴（Celetti等人，JCB 2019）。为了研究这种液体FG-Nup渗透屏障的功能，我们的目标是开发一个“多分析”平台，该平台将微流体与相干反斯托克斯拉曼光谱（CARS）和颗粒跟踪微流变学（PTM）相结合（Chatterjee et al., Adv. Sci. 2021），以研究富fg液滴的演化超分子结构和力学性能。同时，我们开发了一个多组分“贴纸和间隔”模型，该模型为预测具有特定相互作用位点的多价生物聚合物溶液的复杂相行为提供了理论框架，例如FG-Nups （Michels等人，biommacro . 2021）。总之，这些工具将提供一个连贯的画面来解释FG/FG/NTR混合物中同型和异型相互作用之间的平衡如何调节相行为，以及这与凝聚态的渗透势垒函数之间的关系。我们假设鼻咽癌的屏障特性受到NTRs和FG-Nup异质性的调节，这为鼻咽癌细胞渗透性的动态调节提供了一种可能的方法。最后，我们建立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到凝胶或淀粉样蛋白状态的蛋白，如FUS、Tau和α-突触核蛋白或其他与神经变性相关的蛋白。

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.