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

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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.