RESUMEN
Nuclear pore complexes (NPCs) conduct nucleocytoplasmic transport through an FG domain-controlled barrier. We now explore how surface-features of a mobile species determine its NPC passage rate. Negative charges and lysines impede passage. Hydrophobic residues, certain polar residues (Cys, His), and, surprisingly, charged arginines have striking translocation-promoting effects. Favorable cation-π interactions between arginines and FG-phenylalanines may explain this apparent paradox. Application of these principles to redesign the surface of GFP resulted in variants that show a wide span of transit rates, ranging from 35-fold slower than wild-type to â¼500 times faster, with the latter outpacing even naturally occurring nuclear transport receptors (NTRs). The structure of a fast and particularly FG-specific GFPNTR variant illustrates how NTRs can expose multiple regions for binding hydrophobic FG motifs while evading non-specific aggregation. Finally, we document that even for NTR-mediated transport, the surface-properties of the "passively carried" cargo can strikingly affect the translocation rate.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Dominios Proteicos , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Propiedades de SuperficieRESUMEN
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.
Asunto(s)
Proteínas de la Cápside , Cápside , Glicina , VIH-1 , Proteínas de Complejo Poro Nuclear , Poro Nuclear , Fenilalanina , Humanos , Transporte Activo de Núcleo Celular , Cápside/química , Cápside/metabolismo , Glicina/metabolismo , VIH-1/química , VIH-1/genética , VIH-1/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/virología , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Permeabilidad , Fenilalanina/metabolismo , Solubilidad , Internalización del Virus , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismoRESUMEN
Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from frog to human. We also report a simplified assay that directly tracks postmitotic NPC assembly with added fluorophore-labeled anti-Nup nanobodies. During interphase, NPCs are inserted into a pre-existing nuclear envelope. Monitoring this process is challenging because newly assembled NPCs are indistinguishable from pre-existing ones. We overcame this problem by inserting Xenopus-derived NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We further asked whether anti-Nup nanobodies could serve as NPC assembly inhibitors. Using a selection strategy against conserved epitopes, we obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93 α-solenoid domain in recruiting Nup358 and the Nup214·88·62 complex, as well as for Nup155 and the Nup98 autoproteolytic domain in NPC scaffold assembly. The latter suggests a checkpoint linking pore formation to the assembly of the Nup98-dominated permeability barrier.
Asunto(s)
Proteínas de Complejo Poro Nuclear , Poro Nuclear , Anticuerpos de Dominio Único , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Humanos , Anticuerpos de Dominio Único/metabolismo , Animales , Xenopus , Xenopus laevis , Células HeLaRESUMEN
Nuclear pore complexes (NPCs) maintain a permeability barrier between the nucleus and the cytoplasm through FG-repeat-containing nucleoporins (Nups). We previously proposed a "selective phase model" in which the FG repeats interact with one another to form a sieve-like barrier that can be locally disrupted by the binding of nuclear transport receptors (NTRs), but not by inert macromolecules, allowing selective passage of NTRs and associated cargo. Here, we provide direct evidence for this model in a physiological context. By using NPCs reconstituted from Xenopus laevis egg extracts, we show that Nup98 is essential for maintaining the permeability barrier. Specifically, the multivalent cohesion between FG repeats is required, including cohesive FG repeats close to the anchorage point to the NPC scaffold. Our data exclude alternative models that are based solely on an interaction between the FG repeats and NTRs and indicate that the barrier is formed by a sieve-like FG hydrogel.
Asunto(s)
Transporte Activo de Núcleo Celular , Modelos Biológicos , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Xenopus laevis/metabolismo , Animales , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/química , Permeabilidad , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismoRESUMEN
Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Mutación/inmunología , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/inmunología , Animales , COVID-19/virología , Camélidos del Nuevo Mundo/inmunología , Camélidos del Nuevo Mundo/virología , Línea Celular , Escherichia coli/virología , Femenino , Humanos , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Monoclonal immunoglobulins are widely successful as therapeutics and have also been effective in treating COVID-19. However, their production in mammalian cells is expensive and cannot be scaled to meet the demand in a global pandemic. Camelid VHH antibodies (also called nanobodies), however, can be manufactured cost-efficiently in bacteria or yeast. Here we highlight our progress in developing nanobodies that effectively neutralize SARS-CoV-2 and its variants.
RESUMEN
Nuclear pore complexes (NPCs) provide a selective passageway for receptor-mediated active transport between nucleus and cytoplasm, while maintaining the distinct molecular compositions of both compartments at large. In this review we discuss how NPCs gain a remarkable sorting selectivity from non-globular FG domains and their phase separation into dense polymer meshworks. The resulting sieve-like FG hydrogels are effective barriers to normal macromolecules but are at the same time highly permeable to shuttling nuclear transport receptors, which bind to FG motifs as well as to their designated cargoes. Phase separation driven by disordered protein domains was recently also recognized as being pivotal to the formation of membraneless organelles, making it an important emerging principle in cell biology.
Asunto(s)
Núcleo Celular/metabolismo , Poro Nuclear/metabolismo , Orgánulos/metabolismo , Transporte Activo de Núcleo Celular , Animales , HumanosRESUMEN
Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by <25 nm where conventional scanning failed to acquire sufficient signal.
Asunto(s)
Algoritmos , Colorantes Fluorescentes/química , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Microscopía Fluorescente/métodos , Animales , Células Cultivadas , Fluorescencia , Láseres de Colorantes , Compuestos Orgánicos/química , Fotoblanqueo , Xenopus laevisRESUMEN
Bowen-Conradi syndrome (BCS) is a severe genetic disorder that is characterised by various developmental abnormalities, bone marrow failure and early infant death. This disease is caused by a single mutation leading to the aspartate 86 to glycine (D86G) exchange in the essential nucleolar RNA methyltransferase EMG1. EMG1 is required for the synthesis of the small ribosomal subunit and is involved in modification of the 18S ribosomal RNA. Here, we identify the pre-ribosomal factors NOP14, NOC4L and UTP14A as members of a nucleolar subcomplex that contains EMG1 and is required for its recruitment to nucleoli. The BCS mutation in EMG1 leads to reduced nucleolar localisation, accumulation of EMG1D86G in nuclear foci and its proteasome-dependent degradation. We further show that EMG1 can be imported into the nucleus by the importins (Imp) Impα/ß or Impß/7. Interestingly, in addition to its role in nuclear import, binding of the Impß/7 heterodimer can prevent unspecific aggregation of both EMG1 and EMG1D86G on RNAs in vitro, indicating that the importins act as chaperones by binding to basic regions of the RNA methyltransferase. Our findings further indicate that in BCS, nuclear disassembly of the import complex and release of EMG1D86G lead to its nuclear aggregation and degradation, resulting in the reduced nucleolar recruitment of the RNA methyltransferase and defects in the biogenesis of the small ribosomal subunit.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Retardo del Crecimiento Fetal/genética , Metiltransferasas/genética , Proteínas Nucleares/genética , Trastornos Psicomotores/genética , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Retardo del Crecimiento Fetal/patología , Células HeLa , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Trastornos Psicomotores/patología , ARN Ribosómico 18S/genética , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
The controlled functionalization of surfaces with proteins is crucial for many analytical methods in life science research and biomedical applications. Here, a coating for silica-based surfaces is established which enables stable and selective immobilization of proteins with controlled orientation and tunable surface density. The coating is reusable, retains functionality upon long-term storage in air, and is applicable to surfaces of complex geometry. The protein anchoring method is validated on planar surfaces, and then a method is developed to measure the anchoring process in real time using silicon nitride solid-state nanopores. For surface attachment, polyhistidine tags that are site specifically introduced into recombinant proteins are exploited, and the yeast nucleoporin Nsp1 is used as model protein. Contrary to the commonly used covalent thiol chemistry, the anchoring of proteins via polyhistidine tag is reversible, permitting to take proteins off and replace them by other ones. Such switching in real time in experiments on individual nanopores is monitored using ion conductivity. Finally, it is demonstrated that silica and gold surfaces can be orthogonally functionalized to accommodate polyhistidine-tagged proteins on silica but prevent protein binding to gold, which extends the applicability of this surface functionalization method to even more complex sensor devices.
Asunto(s)
Técnicas Biosensibles/métodos , Proteínas/química , Nanoporos , Unión ProteicaRESUMEN
Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules î¶30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin ß-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like ß-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability.
Asunto(s)
Núcleo Celular/metabolismo , Glicina/química , Hidrogeles/análisis , Microdominios de Membrana/química , Poro Nuclear/metabolismo , Fenilalanina/química , Xenopus , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Núcleo Celular/química , Núcleo Celular/ultraestructura , Glicina/metabolismo , Hidrogeles/química , Hidrogeles/metabolismo , Microdominios de Membrana/metabolismo , Microdominios de Membrana/fisiología , Datos de Secuencia Molecular , Poro Nuclear/química , Poro Nuclear/fisiología , Proteínas de Complejo Poro Nuclear/análisis , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Permeabilidad , Fenilalanina/metabolismo , Secuencias Repetitivas de Aminoácido , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Xenopus/metabolismo , beta Carioferinas/análisis , beta Carioferinas/metabolismoRESUMEN
Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-ß and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis.
Asunto(s)
Actinas/metabolismo , Núcleo Celular/metabolismo , Citocinesis/fisiología , Cinesinas/metabolismo , Meiosis/fisiología , Oocitos/metabolismo , Xenopus laevis/metabolismo , Actomiosina/metabolismo , Animales , Cromatografía de Afinidad , Femenino , Técnica del Anticuerpo Fluorescente , Immunoblotting , Microtúbulos/metabolismo , Oocitos/citología , Faloidina/metabolismo , Ploidias , Proteómica , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en Tándem , Xenopus laevis/crecimiento & desarrolloRESUMEN
Global nuclear condensation, culminating in enucleation during terminal erythropoiesis, is poorly understood. Proteomic examination of extruded erythroid nuclei from fetal liver revealed a striking depletion of most nuclear proteins, suggesting that nuclear protein export had occurred. Expression of the nuclear export protein, Exportin 7 (Xpo7), is highly erythroid-specific, induced during erythropoiesis, and abundant in very late erythroblasts. Knockdown of Xpo7 in primary mouse fetal liver erythroblasts resulted in severe inhibition of chromatin condensation and enucleation but otherwise had little effect on erythroid differentiation, including hemoglobin accumulation. Nuclei in Xpo7-knockdown cells were larger and less dense than normal and accumulated most nuclear proteins as measured by mass spectrometry. Strikingly,many DNA binding proteins such as histones H2A and H3 were found to have migrated into the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in Xpo7-knockdown cells. Thus, terminal erythroid maturation involves migration of histones into the cytoplasm via a process likely facilitated by Xpo7.
Asunto(s)
Eritroblastos/citología , Eritroblastos/metabolismo , Histonas/sangre , Carioferinas/sangre , Proteína de Unión al GTP ran/sangre , Animales , Núcleo Celular/metabolismo , Citosol/metabolismo , Eritropoyesis/genética , Eritropoyesis/fisiología , Técnicas de Silenciamiento del Gen , Carioferinas/antagonistas & inhibidores , Carioferinas/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/sangre , Proteína de Unión al GTP ran/antagonistas & inhibidores , Proteína de Unión al GTP ran/genéticaRESUMEN
Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.
Asunto(s)
Proteína Básica de Mielina/metabolismo , Vaina de Mielina/metabolismo , Transición de Fase , Secuencia de Aminoácidos , Amiloide/metabolismo , Animales , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Membranas/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteína Básica de Mielina/química , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP-exportin-cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismo , Secuencia de Aminoácidos , Citoplasma/metabolismo , Humanos , Carioferinas/química , MicroARNs/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , ARN de Transferencia/metabolismo , Proteína de Unión al GTP ran/química , Proteína de Unión al GTP ran/genéticaRESUMEN
The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections.
Asunto(s)
COVID-19 , Anticuerpos de Dominio Único , Animales , Cricetinae , Humanos , SARS-CoV-2 , Aerosoles y Gotitas Respiratorias , Glicoproteína de la Espiga del Coronavirus , Técnicas de Cultivo de Célula , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
Nuclear pore complexes control the exchange of macromolecules between the cytoplasm and the nucleus. A selective permeability barrier that arises from a supramolecular assembly of intrinsically unfolded nucleoporin domains rich in phenylalanine-glycine dipeptides (FG domains) fills the nuclear pore. There is increasing evidence that selective transport requires cohesive FG domain interactions. To understand the functional roles of cohesive interactions, we studied monolayers of end-grafted FG domains as a bottom-up nanoscale model system of the permeability barrier. Based on detailed physicochemical analysis of the model films and comparison of the data with polymer theory, we propose that cohesiveness is tuned to promote rapid assembly of the permeability barrier and to generate a stable and compact pore-filling meshwork with a small mesh size. Our results highlight the functional importance of weak interactions, typically a few kBT per chain, and contribute important information to understand the mechanism of size-selective transport.
Asunto(s)
Dipéptidos/química , Proteínas de Complejo Poro Nuclear/química , Poro Nuclear/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Cinética , Permeabilidad , Unión Proteica , Estructura Terciaria de Proteína , Secuencias Repetitivas de AminoácidoRESUMEN
The permeability barrier of nuclear pore complexes (NPCs) controls all nucleo-cytoplasmic exchange. It is freely permeable for small molecules. Objects larger than approximately 30 kDa can efficiently cross this barrier only when bound to nuclear transport receptors (NTRs) that confer translocation-promoting properties. We had shown earlier that the permeability barrier can be reconstituted in the form of a saturated FG/FxFG repeat hydrogel. We now show that GLFG repeats, the other major FG repeat type, can also form highly selective hydrogels. While supporting massive, reversible importin-mediated cargo influx, FG/FxFG, GLFG or mixed hydrogels remained firm barriers towards inert objects that lacked nuclear transport signals. This indicates that FG hydrogels immediately reseal behind a translocating species and thus possess 'self-healing' properties. NTRs not only left the barrier intact, they even tightened it against passive influx, pointing to a role for NTRs in establishing and maintaining the permeability barrier of NPCs.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Secuencias Repetitivas de Aminoácido , Glicina/química , Humanos , Hidrogeles/química , Leucina/química , Fenilalanina/químicaRESUMEN
Nuclear pore complexes (NPCs) restrict uncontrolled nucleocytoplasmic fluxes of inert macromolecules but permit facilitated translocation of nuclear transport receptors and their cargo complexes. We probed the passive barrier of NPCs and observed sieve-like properties with a dominating mesh or channel radius of 2.6 nm, which is narrower than proposed earlier. A small fraction of diffusion channels has a wider opening, explaining the very slow passage of larger molecules. The observed dominant passive diameter approximates the distance of adjacent hydrophobic clusters of FG repeats, supporting the model that the barrier is made of FG repeat domains cross-linked with a spacing of an FG repeat unit length. Wheat germ agglutinin and the dominant-negative importin beta(45-462) fragment were previously regarded as selective inhibitors of facilitated NPC passage. We now observed that they do not distinguish between the passive and the facilitated mode. Instead, their inhibitory effect correlates with the size of the NPC-passing molecule. They have little effect on small species, inhibit the passage of green fluorescent protein-sized objects >10-fold and virtually block the translocation of larger ones. This suggests that passive and facilitated NPC passage proceed through one and the same permeability barrier.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Poro Nuclear/química , Poro Nuclear/metabolismo , Células HeLa , Humanos , Hidrogeles/química , Hidrogeles/metabolismo , Carioferinas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismoRESUMEN
Actin is a major cytoskeletal element and is normally kept cytoplasmic by exportin 6 (Exp6)-driven nuclear export. Here, we show that Exp6 recognizes actin features that are conserved from yeast to human. Surprisingly however, microinjected actin was not exported from Xenopus laevis oocyte nuclei, unless Exp6 was co-injected, indicating that the pathway is inactive in this cell type. Indeed, Exp6 is undetectable in oocytes, but is synthesized from meiotic maturation onwards, which explains how actin export resumes later in embryogenesis. Exp6 thus represents the first example of a strictly developmentally regulated nuclear transport pathway. We asked why Xenopus oocytes lack Exp6 and observed that ectopic application of Exp6 renders the giant oocyte nuclei extremely fragile. This effect correlates with the selective disappearance of a sponge-like intranuclear scaffold of F-actin. These nuclei have a normal G2-phase DNA content in a volume 100,000 times larger than nuclei of somatic cells. Apparently, their mechanical integrity cannot be maintained by chromatin and the associated nuclear matrix, but instead requires an intranuclear actin-scaffold.