FG Repeats Research Articles

Impact of distinct FG nucleoporin repeats on Nup98 self-association

Nucleoporins rich in phenylalanine/glycine (FG) residues form the permeability barrier within the nuclear pore complex and are implicated in several pathological cellular processes, including oncogenic fusion condensates. The self-association of FG-repeat proteins and interactions between FG-repeats play a critical role in these activities by forming hydrogel-like structures. Here we show that mutation of specific FG repeats of Nup98 can strongly decrease the protein’s self-association capabilities. We further present a cryo-electron microscopy structure of a Nup98 peptide fibril with higher stability per residue compared with previous Nup98 fibril structures. The high-resolution structure reveals zipper-like hydrophobic patches which contain a GLFG motif and are less compatible for binding to nuclear transport receptors. The identified distinct molecular properties of different regions of the nucleoporin may contribute to spatial variations in the self-association of FG-repeats, potentially influencing transport processes through the nuclear pore.

HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase2 that is assembled from cohesively interacting phenylalanine–glycine (FG) repeats3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a ‘self-translocating’ capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection.

CircAGFG1 Promotes Ovarian Cancer Progression Through the miR-409-3 p/ZEB1 Axis.

Circular RNAs (circRNAs) serve a crucial regulatory role in ovarian cancer (OC). Circular RNA ArfGAP with FG repeats 1 (circAGFG1) has been shown to be involved in promoting the progression of several cancers, containing triple-negative breast cancer, esophageal cancer and colorectal cancer. However, the function of circAGFG1 in OC is unclear. Quantitative real-time reverse transcription PCR (RT-qPCR) was conducted to determine the expression levels of circAGFG1 and miR-409-3p. The proliferation and metastasis of cells were determined by colony formation assays, EdU assays, transwell assays and wound healing assays. In addition, a dual-luciferase reporter assay was performed to validate the mechanism between circAGFG1, miR-409-3p, and ZEB1. Our data suggested that circAGFG1 was significantly overexpressed in OC tissues compared to normal ovarian epithelial tissues. Overexpression of circAGFG1 was correlated with intraperitoneal metastasis, tumor recurrence and advanced stage. Additionally, circAGFG1 overexpression revealed a poor prognosis in OC patients. Knockdown of circAGFG1 suppressed the proliferation, invasion and migration of OC cells. Mechanistically, circAGFG1 acted as a sponge of miR-409-3p to enhance the expression level of zinc finger E-box binding homeobox 1 (ZEB1), thereby conferring OC cell proliferation, invasion and migration. Importantly, re-expression of ZEB1 effectively reversed the effects of circAGFG1 knockdown on OC cells. In summary, our study indicated that circAGFG1 may act as a prognostic biomarker and potential therapeutic target for patients with OC.

CircAGFG1 absence decreases PKM2 expression to enhance oxaliplatin sensitivity in colorectal cancer in a miR-7-5p-dependent manner

Circular RNA (circRNA) ArfGAP with FG repeats 1 (circAGFG1) contributes to colorectal cancer (CRC) development. However, whether circAGFG1 regulates the resistance of CRC to oxaliplatin (L-OHP) remains unknown. CircAGFG1, microRNA-7-5p (miR-7-5p) and pyruvate kinase M2 (PKM2) RNA expression were quantified by quantitative real-time polymerase chain reaction. Protein expression was detected by western blot assay and immunohistochemistry assay. Glycolysis was analyzed through glucose uptake, lactate production and adenosine triphosphate (ATP) concentration assays. 50% inhibitory concentration of L-OHP was determined by cell counting kit-8 assay. Cell proliferation and apoptotic rate were analyzed by cell colony formation and flow cytometry analysis, respectively. Dual-luciferase reporter assay was used to identify the relationship among circAGFG1, miR-7- 5p and PKM2. The effect of circAGFG1 on L-OHP sensitivity in vivo was further evaluated by a xenograft model assay. CircAGFG1 and PKM2 expression were significantly increased, while miR-7-5p was decreased in L-OHP-resistant CRC tissues and cells. High circAGFG1 expression predicted a poor prognosis of CRC. CircAGFG1 knockdown or PKM2 depletion decreased glycolysis and cell proliferation and increased L-OHP sensitivity and cell apoptosis. PKM2 introduction rescued circAGFG1 silencing-induced effects in CRC cells. In terms of mechanism, circAGFG1 bound to miR-7-5p, which was identified to target PKM2. Also, circAGFG1 regulated PKM2 expression by interacting with miR-7-5p. Further, circAGFG1 knockdown improved the sensitivity of tumors to L-OHP in vivo. CircAGFG1 depletion inhibited L-OHP resistance by regulating the miR-7-5p/PKM2 pathway.

Super-resolution microscopy reveals focal organization of ER-associated Y-complexes inmitosis.

During mitotic entry of vertebrate cells, nuclear pore complexes (NPCs) are rapidly disintegrated. NPC disassembly is initiated by hyperphosphorylation of linker nucleoporins (Nups), which leads to the dissociation of FG repeat Nups and relaxation of the nuclear permeability barrier. However, less is known about disintegration of the huge nuclear and cytoplasmic rings, which are formed by annular assemblies of Y-complexes that are dissociated from NPCs as intact units. Surprisingly, we observe that Y-complex Nups display slower dissociation kinetics compared with other Nups during in vitro NPC disassembly, indicating a mechanistic difference in the disintegration of Y-based rings. Intriguingly, biochemical experiments reveal that a fraction of Y-complexes remains associated with mitotic ER membranes, supporting recent microscopic observations. Visualization of mitotic Y-complexes by super-resolution microscopy demonstrates that they form two classes of higher order assemblies: large clusters at kinetochores and small, focal ER-associated assemblies. These, however, lack features qualifying them as persisting ring-shaped subassemblies previously proposed to serve as structural templates for NPC reassembly during mitotic exit, which helps to refine current models of nuclear reassembly.

Nuclear export of pre-60S particles through the nuclear pore complex.

The nuclear pore complex (NPC) is the bidirectional gate that mediates the exchange of macromolecules or their assemblies between nucleus and cytoplasm1-3. The assembly intermediates of the ribosomal subunits, pre-60S and pre-40S particles, are among the largest cargoes of the NPC and the export of these gigantic ribonucleoproteins requires numerous export factors4,5. Here we report the cryo-electron microscopy structure of native pre-60S particles trapped in the channel of yeast NPCs. In addition to known assembly factors, multiple factors with export functions are also included in the structure. These factors in general bind to either the flexible regions or subunit interface of the pre-60S particle, and virtually form many anchor sites for NPC binding. Through interactions with phenylalanine-glycine (FG)repeats from various nucleoporins of NPC, these factors collectively facilitate the passage of the pre-60S particle through the central FG repeat network of the NPC. Moreover, in silico analysis of the axial and radial distribution of pre-60S particles within the NPC shows that a single NPC can take up to four pre-60S particles simultaneously, and pre-60S particles are enriched in theinner ring regions close to the wall of the NPC with the solvent-exposed surface facing the centre of the nuclear pore. Our data suggest a translocation model for the export of pre-60S particles through the NPC.

Improving the hole picture: towards a consensus on the mechanism of nuclear transport.

Nuclear pore complexes (NPCs) mediate the exchange of materials between the nucleoplasm and cytoplasm, playing a key role in the separation of nucleic acids and proteins into their required compartments. The static structure of the NPC is relatively well defined by recent cryo-EM and other studies. The functional roles of dynamic components in the pore of the NPC, phenylalanyl-glycyl (FG) repeat rich nucleoporins, is less clear because of our limited understanding of highly dynamic protein systems. These proteins form a 'restrained concentrate' which interacts with and concentrates nuclear transport factors (NTRs) to provide facilitated nucleocytoplasmic transport of cargoes. Very rapid on- and off-rates among FG repeats and NTRs supports extremely fast facilitated transport, close to the rate of macromolecular diffusion in cytoplasm, while complexes without specific interactions are entropically excluded, though details on several aspects of the transport mechanism and FG repeat behaviors remain to be resolved. However, as discussed here, new technical approaches combined with more advanced modeling methods will likely provide an improved dynamic description of NPC transport, potentially at the atomic level in the near future. Such advances are likely to be of major benefit in comprehending the roles the malfunctioning NPC plays in cancer, ageing, viral diseases, and neurodegeneration.

Barrier properties of Nup98 FG phases ruled by FG motif identity and inter-FG spacer length

Nup98 FG repeat domains comprise hydrophobic FG motifs linked through uncharged spacers. FG motifs capture nuclear transport receptors (NTRs) during nuclear pore complex (NPC) passage, confer inter-repeat cohesion, and condense the domains into a selective phase with NPC-typical barrier properties. We show that shortening inter-FG spacers enhances cohesion, increases phase density, and tightens such barrier - all consistent with a sieve-like phase. Phase separation tolerates mutating the Nup98-typical GLFG motifs, provided domain-hydrophobicity remains preserved. NTR-entry, however, is sensitive to (certain) deviations from canonical FG motifs, suggesting co-evolutionary adaptation. Unexpectedly, we observed that arginines promote FG-phase-entry apparently also by hydrophobic interactions/ hydrogen-bonding and not just through cation-π interactions. Although incompatible with NTR·cargo complexes, a YG phase displays remarkable transport selectivity, particularly for engineered GFPNTR-variants. GLFG to FSFG mutations make the FG phase hypercohesive, precluding NTR-entry. Extending spacers relaxes this hypercohesion. Thus, antagonism between cohesion and NTR·FG interactions is key to transport selectivity.

Single-molecule studies of nuclear transport using biomimetic nuclear pore complexes.

The nuclear pore complex (NPC) is the gatekeeper of the nucleus that regulates the flow of all molecules across the nuclear envelope. Remarkably, this ∼40 nm wide pore is capable of efficient and fast transport while remaining highly selective. The dense central channel of the NPC is filled with a dynamic mesh of intrinsically disordered proteins containing FG repeats (FG-Nups). While small molecules can freely pass this barrier, large biomolecules (>40 kDa) require specific transporter proteins. Our progress in understanding nuclear transport is hindered by the lack of experimental techniques that can probe the structure and dynamics of the disordered proteins and transport receptors inside the NPC channel with sufficient spatiotemporal resolution. We present two bottom-up approaches for constructing biomimetic NPCs that enable us to study different aspects of nuclear transport on the single-molecule level. First, we combine solid-state nanopores with optical detection in zero-mode waveguides. Biomimetic NPCs are constructed by coating nanopores with FG-Nups, allowing us to visualize the translocation of single fluorescently labelled molecules with ultimate sensitivity and specificity. Secondly, we implement a single-molecule FRET assay to study the structural dynamics within the central channel based on a circular DNA origami whose inside is lined with FG-Nups. Our multifaceted approach will provide direct mechanistic and structural insights into the molecular principles that govern nuclear transport.

A simple thermodynamic description of phase separation of Nup98 FG domains

The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules but allows facilitated passage of nuclear transport receptors that shuttle cargoes into or out of nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains, including foremost the charge-depleted FG domain of Nup98. We found that Nup98 FG domains show an LCST-type phase separation, and we provide comprehensive and orthogonal experimental datasets for a quantitative description of this behaviour. A derived thermodynamic model correlates saturation concentration with repeat number, temperature, and ionic strength. It allows estimating the enthalpy, entropy, and ΔG (0.2 kJ/mol, 0.1 kB·T) contributions per repeat to phase separation and inter-repeat cohesion. While changing the cohesion strength strongly impacts the strictness of barrier, these numbers provide boundary conditions for in-depth modelling not only of barrier assembly but also of NPC passage.

Circular RNA circ-AGFG1 contributes to esophageal squamous cell carcinoma progression and glutamine catabolism by targeting microRNA-497-5p/solute carrier family 1 member 5 axis.

Circular RNAs (circRNAs) have been shown to play important regulatory roles in human malignancies. However, the role of circRNA ArfGAP with FG repeats 1 (circ-AGFG1) in esophageal squamous cell carcinoma (ESCC) progression and its associated mechanism are still largely undefined. Cell proliferation was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-ethynyl-2'-deoxyuridine assay. Cell apoptosis was assessed by flow cytometry analysis. Transwell assay and wound healing assay were used to analyze cell invasion and migration abilities. The uptake of glutamine and the production of α-ketoglutarate and glutamate were analyzed using Glutamine Determination Kit, α-ketoglutarate Assay Kit and Glutamate Determination Kit. A xenograft tumor model was used to analyze the biological role of circ-AGFG1 in vivo . The interaction between microRNA-497-5p (miR-497-5p) and circ-AGFG1 or solute carrier family 1 member 5 (SLC1A5) was verified by dual-luciferase reporter assay. Circ-AGFG1 expression was upregulated in ESCC tissues and cell lines. Circ-AGFG1 silencing suppressed the proliferation, migration, invasion and glutaminolysis and triggered the apoptosis of ESCC cells. Circ-AGFG1 knockdown significantly slowed down tumor growth in vivo . Circ-AGFG1 acted as a sponge for miR-497-5p, and miR-497-5p interacted with the 3' untranslated region (3'UTR) of SLC1A5. miR-497-5p silencing largely abolished circ-AGFG1 silencing-induced effects in ESCC cells. miR-497-5p overexpression-mediated influences in ESCC cells were largely reversed by the addition of SLC1A5 expressing plasmid. Circ-AGFG1 could upregulate SLC1A5 expression by sponging miR-497-5p. In summary, circ-AGFG1 acted as an oncogene to elevate the malignant potential and promote the glutamine catabolism of ESCC cells by targeting the miR-497-5p/SLC1A5 axis.

AML-158 Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation

Rearrangements of the nucleoporin 98 gene (NUP98) define a high-risk subset of childhood acute myeloid leukemia (AML). The resulting fusion oncoproteins (FOs) involve the N-terminal, intrinsically disordered region of NUP98, and the C-terminal portion of one of more than 30 identified fusion partners. Approximately one third of fusion partners have DNA-binding homeodomains, and the remaining partners have other domains involved in gene regulation. NUP98 FOs have long been known to localize in nuclear puncta. Here, we investigated whether these puncta form by liquid-liquid phase separation (LLPS) and how they might contribute to cell transformation. We first focused on the NUP98::HOXA9 (NHA9) FO, in which the HOXA9 fusion partner includes a DNA-binding homeodomain. We expressed GFP-tagged NHA9 in HEK293T cells and characterized the resulting FO-associated nuclear puncta. We also investigated lentiviral FO expression in mouse hematopoietic stem and progenitor cells (HSPCs), studying localization using confocal imaging, self-renewal using colony forming unit assays, and gene expression using RNA sequencing. We next mutated the phenylalanine glycine (FG) repeats of NUP98 to disrupt interactions with other FOs and interacting proteins or mutated the homeodomain of HOXA9 to disrupt DNA binding. Finally, we investigated the applicability of our findings to other NUP98 FOs with and without homeodomains. NHA9 localizes in nuclear puncta in HEK293T and HSPCs, and mutation of FG repeats or the HOXA9 homeodomain abrogates puncta formation. Furthermore, expression of NHA9 confers self-renewal, but cell transformation does not occur in FO mutants that fail to form puncta. Upregulation of NUP98 FO target genes, including the HOXA cluster, occurred with expression of NHA9 and transforming, puncta-positive mutants but not in mutant cells that did not form puncta or transform HSPCs. Expression of additional NUP98 FOs (including NUP98::KDM5A, NUP98::LNP1, and NUP98::PRRX1) led to both puncta formation and cell transformation. Finally, cells from a NUP98::KDM5A patient derived xenograft confirmed puncta formation with endogenous FO expression. Taken together, these results demonstrate that NUP98 FOs undergo LLPS, show that phase separation is critical for cell transformation and gene expression changes, and provide rationale to further explore the exploitation of NUP98 FO-associated puncta for therapeutical benefit.

A unified statistical potential reveals that amino acid stickiness governs nonspecific recruitment of client proteins into condensates.

Membraneless organelles are cellular compartments that form by liquid–liquid phase separation of one or more components. Other molecules, such as proteins and nucleic acids, will distribute between the cytoplasm and the liquid compartment in accordance with the thermodynamic drive to lower the free energy of the system. The resulting distribution colocalizes molecular species to carry out a diversity of functions. Two factors could drive this partitioning: the difference in solvation between the dilute versus dense phase and intermolecular interactions between the client and scaffold proteins. Here, we develop a set of knowledge‐based potentials that allow for the direct comparison between stickiness, which is dominated by desolvation energy, and pairwise residue contact propensity terms. We use these scales to examine experimental data from two systems: protein cargo dissolving within phase‐separated droplets made from FG repeat proteins of the nuclear pore complex and client proteins dissolving within phase‐separated FUS droplets. These analyses reveal a close agreement between the stickiness of the client proteins and the experimentally determined values of the partition coefficients (R > 0.9), while pairwise residue contact propensities between client and scaffold show weaker correlations. Hence, the stickiness of client proteins is sufficient to explain their differential partitioning within these two phase‐separated systems without taking into account the composition of the condensate. This result implies that selective trafficking of client proteins to distinct membraneless organelles requires recognition elements beyond the client sequence composition.StatementEmpirical potentials for amino acid stickiness and pairwise residue contact propensities are derived. These scales are unique in that they enable direct comparison of desolvation versus contact terms. We find that partitioning of a client protein to a condensate is best explained by amino acid stickiness.

Selective Transport in the Nuclear Pore Complex

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic exchange controlling the flow of molecules into and out of the nucleus. The selective filter properties of NPCs enable translocation of specific molecules known as nuclear transport factors (NTRs) and their cargo. Central to this selectivity barrier is a group of largely intrinsically disordered nucleoporins (Nups) that contain multiple phenylalanine‐glycine repeats, termed FG Nups. The interactions between FG Nups and NTRs enable NTRs to translocate rapidly yet selectively through the NPC in what is referred to as the “transport paradox”. FG Nups in the NPC have a small folded anchor region tethering them to the NPC outer ring and otherwise are fully disordered, random coil polymers that remain predominantly disordered while engaged to NTRs. FG Nups interact with NTRs using mainly their FG motifs and minimally their intervening spacer residues. The overall enthalpy of the interaction increases as multivalency increases the frequency of individually weak FG‐NTR contacts. Tight binding is limited by an entropy penalty that disfavors simultaneous engagements of FG motifs. Small angle neutron scattering (SANS) shows that the entropy loss is partly due to the local rigidity of an FG motif in the interacting state(Fig 1). All‐atom molecular dynamics (MD) simulation indicates that spacers between the FG motifs behave as “entropic springs”, disfavoring any static binding of the FG repeats. The dynamics of FG Nups enables the FG motifs to slide along the hydrophobic patches of NTRs enabling FG motifs to be easily displaced by other competing FG motifs (Fig. 2). This explanation provides a simple hypothesis for the rapid exchange of FG motif contacts during transport, focusing on the entropic exclusion of non‐NTRs, and ‘solubilization’ of NTR complexes, in contrast to possible condensate formation which would provide a compartmentalization of components. These results reveal fundamental aspects of the functioning mechanisms underlying NPC transport at high structural resolution, something lacking in current models of nuclear transport.1. Sparks, S., et al., Analysis of Multivalent IDP Interactions: Stoichiometry, Affinity, and Local Concentration Effect Measurements. Methods Mol Biol, 2020. 2141: p. 463‐475.2. Sparks, S., et al., Deciphering the “Fuzzy” Interaction of FG Nucleoporins and Transport Factors Using Small‐Angle Neutron Scattering. Structure, 2018. 26(3): p. 477‐484 e4.3. Hayama, R., et al., Thermodynamic characterization of the multivalent interactions underlying rapid and selective translocation through the nuclear pore complex. J Biol Chem, 2018. 293(12): p. 4555‐4563.4. Hough, L.E., et al., The molecular mechanism of nuclear transport revealed by atomic‐scale measurements. Elife, 2015. 4.5. Raveh, B., et al., Slide‐and‐exchange mechanism for rapid and selective transport through the nuclear pore complex. Proc Natl Acad Sci U S A, 2016. 113(18): p. E2489‐97.

Atomic resolution dynamics of cohesive interactions in phase-separated Nup98 FG domains

Cohesive FG domains assemble into a condensed phase forming the selective permeability barrier of nuclear pore complexes. Nanoscopic insight into fundamental cohesive interactions has long been hampered by the sequence heterogeneity of native FG domains. We overcome this challenge by utilizing an engineered perfectly repetitive sequence and a combination of solution and magic angle spinning NMR spectroscopy. We map the dynamics of cohesive interactions in both phase-separated and soluble states at atomic resolution using TROSY for rotational correlation time (TRACT) measurements. We find that FG repeats exhibit nanosecond-range rotational correlation times and remain disordered in both states, although FRAP measurements show slow translation of phase-separated FG domains. NOESY measurements enable the direct detection of contacts involved in cohesive interactions. Finally, increasing salt concentration and temperature enhance phase separation and decrease local mobility of FG repeats. This lower critical solution temperature (LCST) behaviour indicates that cohesive interactions are driven by entropy.

Single-molecule studies of nuclear transport using biomimetic nuclear pore complexes

The nuclear pore complex (NPC) is the gatekeeper of the nucleus that regulates the flow of all molecules across the nuclear envelope. Remarkably, this ∼40 nm wide pore is capable of efficient and fast transport while remaining highly selective. The dense central channel of the NPC is filled with a dynamic spaghetti-like mesh of intrinsically disordered proteins containing FG repeats (FG-Nups). While small molecules can freely pass this barrier, large biomolecules (>40 kDa) require specific transporter proteins.

The role of phase separation by NUP98 fusion oncoproteins in leukemia

NUP98 fusion oncoproteins (FOs) are hallmarks of high-risk acute leukemia in children. NUP98 FOs universally contain the intrinsically disordered N-terminal region of NUP98, which harbors multiple “sticky” phenylalanine-glycine (FG) motifs. NUP98 fusion partners include a diverse set of genes that predominantly encode transcriptional or epigenetic regulators bearing folded DNA or chromatin-binding domains. Here, we focused on NUP98-HOXA9 (NHX9), an FO linking the N-terminal FG repeat domains of NUP98 (NUP98-FG) and the DNA-binding homeodomain (HD) of HOXA9.

Characterizing Binding Interactions That Are Essential for Selective Transport through the Nuclear Pore Complex.

Specific macromolecules are rapidly transported across the nuclear envelope via the nuclear pore complex (NPC). The selective transport process is facilitated when nuclear transport receptors (NTRs) weakly and transiently bind to intrinsically disordered constituents of the NPC, FG Nups. These two types of proteins help maintain the selective NPC barrier. To interrogate their binding interactions in vitro, we deployed an NPC barrier mimic. We created the stationary phase by covalently attaching fragments of a yeast FG Nup called Nsp1 to glass coverslips. We used a tunable mobile phase containing NTR, nuclear transport factor 2 (NTF2). In the stationary phase, three main factors affected binding: the number of FG repeats, the charge of fragments, and the fragment density. We also identified three main factors affecting binding in the mobile phase: the avidity of the NTF2 variant for Nsp1, the presence of nonspecific proteins, and the presence of additional NTRs. We used both experimentally determined binding parameters and molecular dynamics simulations of Nsp1FG fragments to create an agent-based model. The results suggest that NTF2 binding is negatively cooperative and dependent on the density of Nsp1FG molecules. Our results demonstrate the strengths of combining experimental and physical modeling approaches to study NPC-mediated transport.

New Family Members of FG Repeat Proteins and Their Unexplored Roles During Phase Separation.

The condensation and compartmentalization of biomacromolecules in the cell are driven by the process of phase separation. The main effectors of phase separation are intrinsically disordered proteins, which include proteins with a phenylalanine-glycine (FG) repeat domain. Our understanding of the biological function of FG repeat proteins during phase separation has been mainly derived from recent research on a member of the nuclear pore complex proteins, nucleoporins containing FG repeat domain (FG-NUPs). FG-NUPs form meshwork structures by inter- and intra-molecular FG domain interactions, which confine the nucleo-cytoplasmic exchange. Whereas FG-NUPs localize in the nuclear membrane, other FG repeat proteins reside in the cytoplasm and the nucleoplasm, and the biological function of the FG repeat domain of these proteins is not well described. In the present review, we list the FG repeat proteins that are known to phase separate in the cell, and review their biological functions. We extract the unraveled features of FG repeat proteins as an activator of barrier formation and homotypic cell-cell interactions. Understanding the regulatory mechanisms of FG repeat proteins will provide a potential delivery tool for therapeutic reagents.

Recapitulation of selective nuclear import and export with a perfectly repeated 12mer GLFG peptide

The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules while allowing facilitated passage of importins and exportins, which in turn shuttle cargo into or out of cell nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains. NPCs contain several distinct FG domains, each comprising variable repeats. Nevertheless, we now found that sequence heterogeneity is no fundamental requirement for barrier function. Instead, we succeeded in engineering a perfectly repeated 12mer GLFG peptide that self-assembles into a barrier of exquisite transport selectivity and fast transport kinetics. This barrier recapitulates RanGTPase-controlled importin- and exportin-mediated cargo transport and thus represents an ultimately simplified experimental model system. An alternative proline-free sequence forms an amyloid FG phase. Finally, we discovered that FG phases stain bright with ‘DNA-specific’ DAPI/ Hoechst probes, and that such dyes allow for a photo-induced block of nuclear transport.