The nuclear pore complex(NPC),the sole exchange channel between the nucleus and cytoplasm,is composed of several subcomplexes,among which the central barrier determines the permeability/selectivity of the NPC to domin...The nuclear pore complex(NPC),the sole exchange channel between the nucleus and cytoplasm,is composed of several subcomplexes,among which the central barrier determines the permeability/selectivity of the NPC to dominate the nucleocytoplasmic trafficking essential for many important signaling events in yeast and mammals.How plant NPC central barrier controls selective transport is a crucial question remaining to be elucidated.In this study,we uncovered that phase separation of the central barrier is critical for the permeability and selectivity of plant NPC in the regulation of various biotic stresses.Phenotypic assays of nup62 mutants and complementary lines showed that NUP62 positively regulates plant defense against Botrytis cinerea,one of the world’s most disastrous plant pathogens.Furthermore,in vivo imaging and in vitro biochemical evidence revealed that plant NPC central barrier undergoes phase separation to regulate selective nucleocytoplasmic transport of immune regulators,as exemplified by MPK3,essential for plant resistance to B.cinerea.Moreover,genetic analysis demonstrated that NPC phase separation plays an important role in plant defense against fungal and bacterial infection as well as insect attack.These findings reveal that phase separation of the NPC central barrier serves as an important mechanism to mediate nucleocytoplasmic transport of immune regulators and activate plant defense against a broad range of biotic stresses.展开更多
The nuclear pore complex(NPC),one of the largest protein complexes in eukaryotes,serves as a physical gate to regulate nucleocytoplasmic transport.Here,we determined the 8Åresolution cryo-electron microscopic(cry...The nuclear pore complex(NPC),one of the largest protein complexes in eukaryotes,serves as a physical gate to regulate nucleocytoplasmic transport.Here,we determined the 8Åresolution cryo-electron microscopic(cryo-EM)structure of the outer rings containing nuclear ring(NR)and cytoplasmic ring(CR)from the Xenopus laevis NPC,with local resolutions reaching 4.9Å.With the aid of AlphaFold2,we managed to build a pseudoatomic model of the outer rings,including the Y complexes and flanking components.In this most comprehensive and accurate model of outer rings to date,the almost complete Y complex structure exhibits much tighter interaction in the hub region.In addition to two copies of Y complexes,each asymmetric subunit in CR contains five copies of Nup358,two copies of the Nup214 complex,two copies of Nup205 and one copy of newly identified Nup93,while that in NR contains one copy of Nup205,one copy of ELYS and one copy of Nup93.These in-depth structural features represent a great advance in understanding the assembly of NPCs.展开更多
Nuclear pore complexes(NPCs)regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells.Intrinsically disordered Phe-Gly nucleoporins(FG-Nups)line the central conduit of NPCs to impart a...Nuclear pore complexes(NPCs)regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells.Intrinsically disordered Phe-Gly nucleoporins(FG-Nups)line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor(karyopherin;Kap).Here,we assess“Kap-centric”NPC models,which postulate that Kaps participate in establishing the selective barrier.We combine biomimetic nanopores,formed by tethering Nsp1 to the inner wall of a solid-state nanopore,with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores:one population that is transported across the pore in milliseconds,and a second population that is stably assembled within the FG mesh of the pore.Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh.Modeling reveals an accumulation of Kap95 near the pore wall,yielding a conductance decrease.We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that,even at high concentrations,Kaps only bind at most 8%of the FG-motifs in the nanopore,indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs.Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps,where a stable population of Kaps binds avidly to the NPC periphery,while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.展开更多
Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nu...Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex （NPC）, while most proteins are transported by energy driven transport mechanisms Active transport of viral proteins is mediated by nuclear localization signals （NLS）, which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral they contain short stretches of lysine or arginine residues. These signals are recognized proteins. Usually by the importin super-family （importin α and β） proteins that mediate the transport across the nuclear envelope through Ran-GTP In contrast, only one class of the leucine-rich nuclear export signal （NES） on viral proteins is known at present. Chromosome region maintenance 1 （CRM1） protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.展开更多
The pathophysiology of amyotrophic lateral sclerosis （ALS） is particularly challenging due to the heteroge- neity of its clinical presentation and the diversity of cellular, molecular and genetic peculiarities invol...The pathophysiology of amyotrophic lateral sclerosis （ALS） is particularly challenging due to the heteroge- neity of its clinical presentation and the diversity of cellular, molecular and genetic peculiarities involved. Molecular insights unveiled several novel genetic factors to be inherent in both familial and sporadic dis- ease entities, whose characterizations in terms of phenotype prediction, pathophysiological impact and putative prognostic value are a topic of current researches. However, apart from genetically well-defined high-confidence and other susceptibility loci, the role of DNA damage and repair strategies of the genome as a whole, either elicited as a direct consequence of the underlying genetic mutation or seen as an autono- mous parameter, in the initiation and progression of ALS, and the different cues involved in either process are still incompletely understood. This mini review summarizes current knowledge on DNA alterations and counteracting DNA repair strategies in ALS pathology and discusses the putative role of unconventional DNA entities including transposable elements and extrachromosomal circular DNA in the disease process. Focus is set on SODl-related pathophysiology, with extension to FUS, TDP-43 and C90RF72 mutations. Advancing our knowledge in the field will contribute to an improved understanding of this relentless dis- ease, for which therapeutic options others than symptomatic approaches are almost unavailable.展开更多
O-glycosylation of the nuclear pore complex(NPC)by O-linked N-acetylglucosamine(O-GlcNAc)is conserved within metazoans.Many nucleoporins(Nups)comprising the NPC are constitutively O-GlcNAcylated,but the functional rol...O-glycosylation of the nuclear pore complex(NPC)by O-linked N-acetylglucosamine(O-GlcNAc)is conserved within metazoans.Many nucleoporins(Nups)comprising the NPC are constitutively O-GlcNAcylated,but the functional role of this modification remains enigmatic.Weshowthat loss ofO-GlcNAc,induced by either inhibition ofO-GlcNAc transferase(OGT)or deletion of the gene encoding OGT,leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups.Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation.The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells.These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter.The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.展开更多
基金supported by funding from the National Natural Science Foundation of China(32250001 and 31830008 to D.X.,32150023 and 32125010 to P.L.,and 31901570 to D.W.)the National Key Research and Development Program of China(2019YFA0508403 to P.L.)+1 种基金the China Postdoctoral Science Foundation(2018M631447 to J.W.)a postdoctoral fellowship of the Tsinghua-Peking Joint Center for Life Sciences.
文摘The nuclear pore complex(NPC),the sole exchange channel between the nucleus and cytoplasm,is composed of several subcomplexes,among which the central barrier determines the permeability/selectivity of the NPC to dominate the nucleocytoplasmic trafficking essential for many important signaling events in yeast and mammals.How plant NPC central barrier controls selective transport is a crucial question remaining to be elucidated.In this study,we uncovered that phase separation of the central barrier is critical for the permeability and selectivity of plant NPC in the regulation of various biotic stresses.Phenotypic assays of nup62 mutants and complementary lines showed that NUP62 positively regulates plant defense against Botrytis cinerea,one of the world’s most disastrous plant pathogens.Furthermore,in vivo imaging and in vitro biochemical evidence revealed that plant NPC central barrier undergoes phase separation to regulate selective nucleocytoplasmic transport of immune regulators,as exemplified by MPK3,essential for plant resistance to B.cinerea.Moreover,genetic analysis demonstrated that NPC phase separation plays an important role in plant defense against fungal and bacterial infection as well as insect attack.These findings reveal that phase separation of the NPC central barrier serves as an important mechanism to mediate nucleocytoplasmic transport of immune regulators and activate plant defense against a broad range of biotic stresses.
基金Ministry of Science and Technology of China(2017YFA0504700 to FS and 2016YFA0500201 to CMZ)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB 37040102 to FS),and National Natural Science Foundation of China(31830020 to FS,31520103906 to CMZ)+2 种基金This work was also supported by grants from the National Science Fund for Distinguished Young Scholars(31925026 to FS)National Natural Science Foundation of China(31430051 to CMZ)National Key Research and Development Program of China(2016YFA0100501 to CMZ and 2018YFA0901102 to YZ).
文摘The nuclear pore complex(NPC),one of the largest protein complexes in eukaryotes,serves as a physical gate to regulate nucleocytoplasmic transport.Here,we determined the 8Åresolution cryo-electron microscopic(cryo-EM)structure of the outer rings containing nuclear ring(NR)and cytoplasmic ring(CR)from the Xenopus laevis NPC,with local resolutions reaching 4.9Å.With the aid of AlphaFold2,we managed to build a pseudoatomic model of the outer rings,including the Y complexes and flanking components.In this most comprehensive and accurate model of outer rings to date,the almost complete Y complex structure exhibits much tighter interaction in the hub region.In addition to two copies of Y complexes,each asymmetric subunit in CR contains five copies of Nup358,two copies of the Nup214 complex,two copies of Nup205 and one copy of newly identified Nup93,while that in NR contains one copy of Nup205,one copy of ELYS and one copy of Nup93.These in-depth structural features represent a great advance in understanding the assembly of NPCs.
基金funded by NWO-I programme“Projectruimte”,Grant No.16PR3242-1.
文摘Nuclear pore complexes(NPCs)regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells.Intrinsically disordered Phe-Gly nucleoporins(FG-Nups)line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor(karyopherin;Kap).Here,we assess“Kap-centric”NPC models,which postulate that Kaps participate in establishing the selective barrier.We combine biomimetic nanopores,formed by tethering Nsp1 to the inner wall of a solid-state nanopore,with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores:one population that is transported across the pore in milliseconds,and a second population that is stably assembled within the FG mesh of the pore.Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh.Modeling reveals an accumulation of Kap95 near the pore wall,yielding a conductance decrease.We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that,even at high concentrations,Kaps only bind at most 8%of the FG-motifs in the nanopore,indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs.Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps,where a stable population of Kaps binds avidly to the NPC periphery,while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.
基金The Startup Fund of the Hundred Talents Program of the Chinese Academy of Science (20071010-141)National Natural Science Foundation of China(30870120)+1 种基金Open Research Fund Program of the State Key Laboratory of Virology of China (2007003,2009007)Hubei Province Natural Science Foundation of Innovation Groups Project (2008CDA013)
文摘Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex （NPC）, while most proteins are transported by energy driven transport mechanisms Active transport of viral proteins is mediated by nuclear localization signals （NLS）, which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral they contain short stretches of lysine or arginine residues. These signals are recognized proteins. Usually by the importin super-family （importin α and β） proteins that mediate the transport across the nuclear envelope through Ran-GTP In contrast, only one class of the leucine-rich nuclear export signal （NES） on viral proteins is known at present. Chromosome region maintenance 1 （CRM1） protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.
基金supported by the Ministry for Economics,Sciences and Digital Society of Thuringia(TMWWDG),in the framework of the Pro Excellence Initiative Regener Aging(Regener Aging-FSU-I-03/14 to AK)the Interdisciplinary Center for Clinical Research(IZKF)Jena(Project FF01 to AK)
文摘The pathophysiology of amyotrophic lateral sclerosis （ALS） is particularly challenging due to the heteroge- neity of its clinical presentation and the diversity of cellular, molecular and genetic peculiarities involved. Molecular insights unveiled several novel genetic factors to be inherent in both familial and sporadic dis- ease entities, whose characterizations in terms of phenotype prediction, pathophysiological impact and putative prognostic value are a topic of current researches. However, apart from genetically well-defined high-confidence and other susceptibility loci, the role of DNA damage and repair strategies of the genome as a whole, either elicited as a direct consequence of the underlying genetic mutation or seen as an autono- mous parameter, in the initiation and progression of ALS, and the different cues involved in either process are still incompletely understood. This mini review summarizes current knowledge on DNA alterations and counteracting DNA repair strategies in ALS pathology and discusses the putative role of unconventional DNA entities including transposable elements and extrachromosomal circular DNA in the disease process. Focus is set on SODl-related pathophysiology, with extension to FUS, TDP-43 and C90RF72 mutations. Advancing our knowledge in the field will contribute to an improved understanding of this relentless dis- ease, for which therapeutic options others than symptomatic approaches are almost unavailable.
基金supported by a Discovery Grant(grant no.RGPIN/298406-2010)fromthe Natural Sciences and Engineering Research(NSERC),and the Canadian Institutes of Health Research(CIHR)(grant no.MOP-123341).Y.Z.thanks the CIHR for support through a postdoctoral fellowship.D.J.V.acknowledges the kind support of the Canada Research Chairs Program for a Tier I Canada Research Chair in Chemical Glycobiology and NSERC for support as an E.W.R.Steacie Memorial Fellow.N.Z.acknowledges the support from the National Heart Lung and Blood Institute(P01HL107153).
文摘O-glycosylation of the nuclear pore complex(NPC)by O-linked N-acetylglucosamine(O-GlcNAc)is conserved within metazoans.Many nucleoporins(Nups)comprising the NPC are constitutively O-GlcNAcylated,but the functional role of this modification remains enigmatic.Weshowthat loss ofO-GlcNAc,induced by either inhibition ofO-GlcNAc transferase(OGT)or deletion of the gene encoding OGT,leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups.Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation.The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells.These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter.The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.
