RESUMEN
In recent years, the convergence of multiple technologies and experimental approaches has led to the expanded use of cultured Drosophila cells as a model system. Their ease of culture and maintenance, susceptibility to RNA interference, and imaging characteristics have led to extensive use in both traditional experimental approaches and high-throughput RNAi screens. Here we describe Drosophila S2 cell culture and preparation for live-cell and fixed-cell fluorescence microscopy and scanning electron microscopy.
Asunto(s)
Citoesqueleto , Drosophila , Animales , Técnicas de Cultivo de Célula , Línea Celular , Drosophila melanogaster/genética , Microtúbulos , Interferencia de ARNRESUMEN
We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.
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The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Convalecencia , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Adulto , Anciano , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , COVID-19/genética , COVID-19/inmunología , Chlorocebus aethiops , Clonación Molecular , Mapeo Epitopo , Epítopos/genética , Epítopos/inmunología , Femenino , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células VeroRESUMEN
Ubiquitylation is a protein modification implicated in several cellular processes. This process is reversible by the action of deubiquinating enzymes (DUBs). USP45 is a ubiquitin specific protease about which little is known, aside from roles in DNA damage repair and differentiation of the vertebrate retina. Here, by using mass spectrometry we have identified Spindly as a new target of USP45. Our data show that Spindly and USP45 are part of the same complex and that their interaction specifically depends on the catalytic activity of USP45. In addition, we describe the type of ubiquitin chains associated with the complex that can be cleaved by USP45, with a preferential activity on K48 ubiquitin chain type and potentially K6. Here, we also show that Spindly is mono-ubiquitylated and this can be specifically removed by USP45 in its active form but not by the catalytic inactive form. Lastly, we identified a new role for USP45 in cell migration, similar to that which was recently described for Spindly.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Mapas de Interacción de Proteínas , Proteasas Ubiquitina-Específicas/metabolismo , Línea Celular Tumoral , Humanos , Ubiquitina/metabolismo , Proteasas Ubiquitina-Específicas/genética , UbiquitinaciónRESUMEN
Dynein is the sole processive minus-end-directed microtubule motor found in animals. It has roles in cell division, membrane trafficking, and cell migration. Together with dynactin, dynein regulates centrosomal orientation to establish and maintain cell polarity, controls focal adhesion turnover and anchors microtubules at the leading edge. In higher eukaryotes, dynein/dynactin requires additional components such as Bicaudal D to form an active motor complex and for regulating its cellular localization. Spindly is a protein that targets dynein/dynactin to kinetochores in mitosis and can activate its motility in vitro However, no role for Spindly in interphase dynein/dynactin function has been found. We show that Spindly binds to the cell cortex and microtubule tips and colocalizes with dynein/dynactin at the leading edge of migrating U2OS cells and primary fibroblasts. U2OS cells that lack Spindly migrated slower in 2D than control cells, although centrosome polarization appeared to happen properly in the absence of Spindly. Re-expression of Spindly rescues migration, but the expression of a mutant, which is defective for dynactin binding, failed to rescue this defect. Taken together, these data demonstrate that Spindly plays an important role in mediating a subset of dynein/dynactin's function in cell migration.
RESUMEN
Spindly was originally identified as a specific regulator of Dynein activity at the kinetochore. In early prometaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. Here, we present loss- and gain-of-function studies of Spindly using transgenic RNAi in Drosophila. Knock-down of Spindly in the female germ line results in mitotic arrest during embryonic cleavage divisions. We investigated the requirements of Spindly protein domains for its localisation and function, and found that the carboxy-terminal region controls Spindly localisation in a cell-type specific manner. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells, we altered Spindly protein levels in migrating cells and found that ovarian border cell migration is sensitive to the levels of Spindly protein. Our study uncovers novel functions of Spindly and a differential, functional requirement for its carboxy-terminal region in Drosophila.
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Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell's center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.
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Fertilidad/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Oocitos/fisiología , Animales , División Celular Asimétrica/genética , Segregación Cromosómica/fisiología , Citoplasma/fisiología , Femenino , Meiosis/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oocitos/metabolismo , Cuerpos Polares/metabolismo , Embarazo , Polos del Huso/metabolismoRESUMEN
To change shape, divide, form junctions, and migrate, cells reorganize their cytoskeletons in response to changing mechanical environments [1-4]. Actin cytoskeletal elements, including myosin II motors and actin crosslinkers, structurally remodel and activate signaling pathways in response to imposed stresses [5-9]. Recent studies demonstrate the importance of force-dependent structural rearrangement of α-catenin in adherens junctions [10] and vinculin's molecular clutch mechanism in focal adhesions [11]. However, the complete landscape of cytoskeletal mechanoresponsive proteins and the mechanisms by which these elements sense and respond to force remain to be elucidated. To find mechanosensitive elements in mammalian cells, we examined protein relocalization in response to controlled external stresses applied to individual cells. Here, we show that non-muscle myosin II, α-actinin, and filamin accumulate to mechanically stressed regions in cells from diverse lineages. Using reaction-diffusion models for force-sensitive binding, we successfully predicted which mammalian α-actinin and filamin paralogs would be mechanoaccumulative. Furthermore, a "Goldilocks zone" must exist for each protein where the actin-binding affinity must be optimal for accumulation. In addition, we leveraged genetic mutants to gain a molecular understanding of the mechanisms of α-actinin and filamin catch-bonding behavior. Two distinct modes of mechanoaccumulation can be observed: a fast, diffusion-based accumulation and a slower, myosin II-dependent cortical flow phase that acts on proteins with specific binding lifetimes. Finally, we uncovered cell-type- and cell-cycle-stage-specific control of the mechanosensation of myosin IIB, but not myosin IIA or IIC. Overall, these mechanoaccumulative mechanisms drive the cell's response to physical perturbation during proper tissue development and disease.
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Citoesqueleto de Actina/metabolismo , Actinina/metabolismo , Filaminas/metabolismo , Miosina Tipo II/metabolismo , Animales , Células HEK293 , Células HeLa , Humanos , Ratones , Células 3T3 NIHRESUMEN
PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Fosfoserina/metabolismo , Secuencia de Aminoácidos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Semivida , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/química , Interfase/efectos de los fármacos , Mitógenos/farmacología , Datos de Secuencia Molecular , Oncogenes , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In recent years, the convergence of multiple technologies and experimental approaches has led to the expanded use of cultured Drosophila cells as a model system. Their ease of culture and maintenance, susceptibility to RNA interference, and imaging characteristics have led to extensive use in both traditional experimental approaches as well as high-throughput RNAi screens. Here we describe Drosophila S2 cell culture and preparation for live-cell and fixed-cell fluorescence microscopy and scanning electron microscopy.
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Citoesqueleto/metabolismo , Drosophila melanogaster/citología , Microscopía Electrónica de Rastreo/métodos , Microscopía Fluorescente/métodos , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular , Drosophila melanogaster/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Polilisina/farmacología , Coloración y Etiquetado , Propiedades de Superficie , TransfecciónRESUMEN
The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome.
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Aurora Quinasa A/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Huso Acromático/metabolismo , Insuficiencia Suprarrenal/enzimología , Insuficiencia Suprarrenal/metabolismo , Animales , Ciclo Celular/fisiología , Células Cultivadas , Drosophila melanogaster , Acalasia del Esófago/enzimología , Acalasia del Esófago/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitosis/fisiología , Unión ProteicaRESUMEN
Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.
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Ciclo Celular , Miosina Tipo II/metabolismo , FosforilaciónRESUMEN
Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein-tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.
Asunto(s)
Citocinesis/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Cinesinas/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Isoformas de Proteínas/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Línea Celular , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Humanos , Cinesinas/genética , Microscopía Fluorescente/métodos , Microtúbulos/ultraestructura , Miosinas/ultraestructura , Isoformas de Proteínas/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.
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Citofotometría/métodos , Iluminación , Microscopía por Video/métodos , Algoritmos , Animales , Línea Celular , Citofotometría/instrumentación , Drosophila melanogaster , Procesamiento Automatizado de Datos , Análisis de Fourier , Procesamiento de Imagen Asistido por Computador , Cinesinas/metabolismo , Microscopía Fluorescente/métodos , Microscopía por Video/instrumentación , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
The eukaryotic spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores and prevents anaphase onset until all kinetochores are aligned on the metaphase plate. In higher eukaryotes, cytoplasmic dynein is involved in silencing the SAC by removing the checkpoint proteins Mad2 and the Rod-Zw10-Zwilch complex (RZZ) from aligned kinetochores (Howell, B.J., B.F. McEwen, J.C. Canman, D.B. Hoffman, E.M. Farrar, C.L. Rieder, and E.D. Salmon. 2001. J. Cell Biol. 155:1159-1172; Wojcik, E., R. Basto, M. Serr, F. Scaerou, R. Karess, and T. Hays. 2001. Nat. Cell Biol. 3:1001-1007). Using a high throughput RNA interference screen in Drosophila melanogaster S2 cells, we have identified a new protein (Spindly) that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, cells arrest in metaphase with high levels of kinetochore-bound Mad2 and RZZ. We also identified a human homologue of Spindly that serves a similar function. However, dynein's nonkinetochore functions are unaffected by Spindly depletion. Our findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores.
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Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Cinetocoros/metabolismo , Huso Acromático/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular , Drosophila melanogaster/fisiología , Proteínas Mad2 , Metafase , Complejos Multiproteicos/metabolismoRESUMEN
Despite the apparent overall structural stability of the nuclear pore complex during interphase, at least two nucleoporins have been shown to move dynamically on and off the pore. It is not yet certain what contribution nucleoporin mobility makes to the process of nuclear transport or how such mobility is regulated. Previously, we showed that Nup98 dynamically interacts with the NPC as well as bodies within the nucleus in a transcription-dependent manner. We have extended our studies of dynamics to include Nup153, another mobile nucleoporin implicated in RNA export. In both cases, we found that although only one domain is essential for NPC localization, other regions of the protein significantly affect the stability of association with the pore. Interestingly, like Nup98, the exchange of Nup153 on and off the pore is inhibited when transcription by Pol I and Pol II is blocked. We have mapped the regions required to link Nup98 and Nup153 mobility to transcription and found that the requirements differ depending on which polymerases are inhibited. Our data support a model whereby transcription of RNA is coupled to nucleoporin mobility, perhaps ultimately linking transport of RNAs to a cycle of remodeling at the nuclear pore basket.
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Proteínas de Complejo Poro Nuclear/química , Transcripción Genética , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Cricetinae , ADN/química , Dactinomicina/farmacología , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Luz , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Estructura Terciaria de Proteína , ARN/química , Factores de TiempoRESUMEN
Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four-amino acid segment, whereas hGle1B has a COOH-terminal 43-amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP-hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39-amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.
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Núcleo Celular/metabolismo , Citoplasma/metabolismo , Carioferinas/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Transporte Biológico Activo , Proteínas Portadoras , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Carioferinas/química , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Poli A/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/química , Homología de Secuencia de AminoácidoRESUMEN
The vertebrate nuclear pore is an enormous structure that spans the double membrane of the nuclear envelope. In yeast, most nucleoporins are found symmetrically on both the nuclear and cytoplasmic sides of the structure. However, in vertebrates most nucleoporins have been localized exclusively to one side of the nuclear pore. Herein, we show, by immunofluorescence and immunoelectron microscopy, that Nup98 is found on both sides of the pore complex. Additionally, we find that the pore-targeting domain of Nup98 interacts directly with the cytoplasmic nucleoporin Nup88, a component of the Nup214, Nup88, Nup62 subcomplex. Nup98 was previously described to interact with the nuclear-oriented Nup160, 133, 107, 96 complex through direct binding to Nup96. Interestingly, the same site within Nup98 is involved in binding to both Nup88 and Nup96. Autoproteolytic cleavage of the Nup98 C terminus is required for both of these binding interactions. When cleavage is blocked by a point mutation, a minimal eight amino acids downstream of the cleavage site is sufficient to prevent most binding to either Nup96 or Nup88. Thus, Nup98 interacts with both faces of the nuclear pore, a localization in keeping with its previously described nucleocytoplasmic shuttling activity.
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Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Complejo Poro Nuclear/biosíntesis , Poro Nuclear/metabolismo , Animales , Sitios de Unión , Células COS , ADN/metabolismo , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Proteínas de Complejo Poro Nuclear/metabolismo , Mutación Puntual , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Nup98 is a component of the nuclear pore that plays its primary role in the export of RNAs. Nup98 is expressed in two forms, derived from alternate mRNA splicing. Both forms are processed into two peptides through autoproteolysis mediated by the C-terminal domain of hNup98. The three-dimensional structure of the C-terminal domain reveals a novel protein fold, and thus a new class of autocatalytic proteases. The structure further reveals that the suggested nucleoporin RNA binding motif is unlikely to bind to RNA. The C terminus also contains sequences that target hNup98 to the nuclear pore complex. Noncovalent interactions between the C-terminal domain and the cleaved peptide tail are visible and suggest a model for cleavage-dependent targeting of hNup98 to the nuclear pore.
Asunto(s)
Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Evolución Molecular , Citometría de Flujo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Electricidad Estática , LevadurasRESUMEN
Nucleoporin 98 (Nup98), a glycine-leucine-phenylalanine-glycine (GLFG) amino acid repeat-containing nucleoporin, plays a critical part in nuclear trafficking. Injection of antibodies to Nup98 into the nucleus blocks the export of most RNAs. Nup98 contains binding sites for several transport factors; however, the mechanism by which this nucleoporin functions has remained unclear. Multiple subcellular localizations have been suggested for Nup98. Here we show that Nup98 is indeed found both at the nuclear pore complex and within the nucleus. Inside the nucleus, Nup98 associates with a novel nuclear structure that we term the GLFG body because the GLFG domain of Nup98 is required for targeting to this structure. Photobleaching of green fluorescent protein-Nup98 in living cells reveals that Nup98 is mobile and moves between these different localizations. The rate of recovery after photobleaching indicates that Nup98 interacts with other, less mobile, components in the nucleoplasm. Strikingly, given the previous link to nuclear export, the mobility of Nup98 within the nucleus and at the pore is dependent on ongoing transcription by RNA polymerases I and II. These data give rise to a model in which Nup98 aids in direction of RNAs to the nuclear pore and provide the first potential mechanism for the role of a mobile nucleoporin.