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

核孔复合体(NPC)是由30个不同核孔蛋白(NUP)的多个拷贝组成的~120个丙二醛结构。NPC穿越核膜，起着核质运输的“把关人”的作用。鼻咽癌的通透性屏障由10个不同的“FG核”组成，主要是含有富含苯丙氨酸(F)和甘氨酸(G)的结构域的无序核。这些FG结构域在NPC的中心区域以非共价方式相互作用形成超分子矩阵，其结构仍然难以捉摸。研究通透性屏障的一个有前景的方法是建立FG-NUP的体外重建模型，该模型捕捉到功能性鼻咽癌的关键特征。这包括对大于~4纳米的货物形成天然屏障，除非伴随有核运输受体(NTR)。我们正在开发一种微流控设备，它可以通过液-液相分离(LLP)在FG-NUPS自组装后解析动力学过程。我们已经证明，FG-NUP可以瞬时填充液滴状态(在LLP之后)，然后在几分钟内老化到更固态。即使液体状态是瞬变的，使用连续的微流体混合器也可以在液滴形成后立即探测渗透性屏障属性，时间分辨率在台式实验中是不可能的。使用这一设备，我们发现液体状态出人意料地模仿了生理渗透屏障的关键功能：大型货物(&gt；4 nm)需要一个功能性NTR：Cargo复合体才能进入液滴(Celeti等人，JCB 2019)。为了研究这种液体FG-Nup渗透性屏障如何发挥作用，我们的目标是开发一种“多分析”平台，将微流体与相干反斯托克斯拉曼光谱(CARS)和粒子跟踪微观流变学(PTM)相结合(Chatterjee等人，高级科学)。2021)，研究富FG液滴的超分子结构演变以及力学性能。同时，我们开发了一个多组分“粘合剂和间隔物”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一个理论框架，例如FG-NUPS(Michels等人，Biomacro)。2021年)。总而言之，这些工具将提供一幅连贯的图景，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，以及这如何与凝聚液体状态的渗透势垒函数相关。我们假设鼻咽癌的屏障特性受NTRs和FG-Nup异质性的调节，为动态调节鼻咽癌细胞的通透性提供了一种可能的手段。最后，我们建立的平台将作为一个通用的工具来研究相分离蛋白的LLP，特别是那些经历快速成熟到凝胶或淀粉样状态的LLP，例如FUS、Tau和α-突触核蛋白或其他与神经退化相关的蛋白质。

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.