Karsten Weis
Swiss Federal Institute of Technology (ETH), Biology, Faculty Member
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Molecular biological investigations of HIV have made fundamental contributions to our understanding of eukaryotic biology. These studies elucidated new paradigms in transcription, RNA and protein export from the nucleus to the cytoplasm,... more
Molecular biological investigations of HIV have made fundamental contributions to our understanding of eukaryotic biology. These studies elucidated new paradigms in transcription, RNA and protein export from the nucleus to the cytoplasm, cellular activation, morphology and vesicular trafficking.
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Import of proteins into the nucleus is a two-step process, involving nuclear localization sequence (NLS)-dependent docking of the substrate at the nuclear envelope followed by translocation through the nuclear pore. A recombinant human... more
Import of proteins into the nucleus is a two-step process, involving nuclear localization sequence (NLS)-dependent docking of the substrate at the nuclear envelope followed by translocation through the nuclear pore. A recombinant human protein, hSRP1alpha, bound in vitro specifically and directly to substrates containing either a simple or bipartite NLS motif. hSRP1alpha promoted docking of import substrates to the nuclear
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Mitosis is triggered in vertebrate cells by the cyclin B1-Cdc2 complex. The activation of this complex at the end of G2 phase is accompanied by its translocation from the cytoplasm to the nucleus. We used digitonin-permeabilized human... more
Mitosis is triggered in vertebrate cells by the cyclin B1-Cdc2 complex. The activation of this complex at the end of G2 phase is accompanied by its translocation from the cytoplasm to the nucleus. We used digitonin-permeabilized human cells to analyze the mechanism by which cyclin B1-Cdc2 is imported into the nucleus. Cyclin B1-Cdc2 import was not blocked by inhibitors of the importin alpha-dependent import pathway or by dominant negative versions of the GTPase Ran or importin beta. However, the rate of cyclin B1 import was decreased by immunodepletion of importin beta from cytosol. Purified importin beta promoted cyclin B1 import in the absence of cytosol or Ran and in the presence of the dominant negative Ran mutant. We conclude that cyclin B1 import is mediated by an unusual importin beta-dependent mechanism that does not require Ran.
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Research Interests: Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology, Genetics, and 54 moreMarine Biology, Neuroscience, Environmental Science, Geophysics, Physics, Materials Science, Quantum Physics, Developmental Biology, Immunology, Climate Change, Molecular Biology, Structural Biology, Genomics, RNA, Computational Biology, Transcriptomics, Molecular Evolution, Biotechnology, Systems Biology, Cancer, Biology, Metabolomics, Cell Cycle, Proteomics, Ecology, Drug Discovery, Evolution, Nanotechnology, Astrophysics, Neurobiology, Medicine, Multidisciplinary, Palaeobiology, Functional Genomics, Nature, Macromolecular X-Ray Crystallography, Signal Transduction, Astronomy, DNA, Crystal structure, Humans, Escherichia coli, Animals, Cell Signalling, Medical Research, Protein Secondary Structure Prediction, Amino Acid Profile, Protein Conformation, Amino Acid Sequence, Recombinant Proteins, Protein Binding, Earth Science, Conformational Change, and Nuclear localization signal
In recent years, our understanding of macromolecular transport processes across the nuclear envelope has grown dramatically, and a large number of soluble transport receptors mediating either nuclear import or nuclear export have been... more
In recent years, our understanding of macromolecular transport processes across the nuclear envelope has grown dramatically, and a large number of soluble transport receptors mediating either nuclear import or nuclear export have been identified. Most of these receptors belong to one large family of proteins, all of which share homology with the protein import receptor importin beta (also named karyopherin beta). Members of this family have been classified as importins or exportins on the basis of the direction they carry their cargo. To date, the family includes 14 members in the yeast Saccharomyces cerevisiae and at least 22 members in humans. Importins and exportins are regulated by the small GTPase Ran, which is thought to be highly enriched in the nucleus in its GTP-bound form. Importins recognize their substrates in the cytoplasm and transport them through nuclear pores into the nucleus. In the nucleoplasm, RanGTP binds to importins, inducing the release of import cargoes. In ...
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Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) translocation that fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene product, PML. The involvement of RAR alpha is particularly intriguing in view... more
Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) translocation that fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene product, PML. The involvement of RAR alpha is particularly intriguing in view of the efficient therapeutic effect of retinoic acid (RA) in this disease. In this report, we show that PML is specifically localized within a discrete subnuclear compartment corresponding to nuclear bodies recognized by patient autoimmune sera. In APL cells, the PML-RAR alpha hybrid displays an abnormal localization and directs RXR and other nuclear antigens into aberrant structures that are tightly bound to chromatin. This suggests that the hybrid could exert a dominant negative effect by diverting a subset of proteins from their natural sites of action. Interestingly, treatment of APL cells with RA induces a complete relocalization of each of these proteins. We propose that the beneficial role of RA in promoting myeloid differentiation in APL might be related to its ability to restore a normal subnuclear organization.
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Research Interests: RNA, Kinetics, Fluorescence Microscopy, Biological Chemistry, Confocal Microscopy, and 23 moreBiological Sciences, DNA, Humans, Animals, Biological, Polymerase Chain Reaction, Alternative splicing, Plasmids, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Immunoprecipitation, CHEMICAL SCIENCES, Cell nucleus, HeLa cells, Transfection, Alternative Splicing, Glutathione Transferase, Amino Acid Sequence, Recombinant Proteins, Protein Binding, Hydrolysis, Protein Biosynthesis, Oligonucleotides, and Adenosine Triphosphate
... 2000). About a third of the nucleoporins belong to the so-called FG Nup subclass, defined by the presence of domains rich in ... 2004). Nup159 is an FG-containing Nup that localizes to the cytoplasmic fibrils of the NPC and is... more
... 2000). About a third of the nucleoporins belong to the so-called FG Nup subclass, defined by the presence of domains rich in ... 2004). Nup159 is an FG-containing Nup that localizes to the cytoplasmic fibrils of the NPC and is required for mRNA export (Gorsch et al. ...
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Research Interests: Macromolecular X-Ray Crystallography, In Situ Hybridization, Cell and Molecular Biology, Biological Sciences, Saccharomyces cerevisiae, and 10 moreCrystal structure, Mutation, Nuclear pore complex, Plasmids, Cell nucleus, Protein Secondary Structure Prediction, Protein Conformation, Recombinant Proteins, Cytoplasm, and Site-directed Mutagenesis
Beads coated with the guanine nucleotide exchange factor RCC1 and a kinesin motor protein are sufficient to induce mitotic spindle assembly in Xenopus egg cytoplasm.
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Single-particle tracking has been applied to study chromatin motion in live cells, revealing a wealth of dynamical behavior of the genomic material once believed to be relatively static throughout most of the cell cycle. Here we used the... more
Single-particle tracking has been applied to study chromatin motion in live cells, revealing a wealth of dynamical behavior of the genomic material once believed to be relatively static throughout most of the cell cycle. Here we used the dual-color three-dimensional (3D) double-helix point spread function microscope to study the correlations of movement between two fluorescently labeled gene loci on either the same or different budding yeast chromosomes. We performed fast (10 Hz) 3D tracking of the two copies of the GAL locus in diploid cells in both activating and repressive conditions. As controls, we tracked pairs of loci along the same chromosome at various separations, as well as transcriptionally orthogonal genes on different chromosomes. We found that under repressive conditions, the GAL loci exhibited significantly higher velocity cross-correlations than they did under activating conditions. This relative increase has potentially important biological implications, as it migh...
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mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure... more
mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread ...
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RNA localization is of critical importance in many fundamental cell biological and developmental processes by regulating the spatial control of gene expression. To investigate how spindle-localized RNAs might influence mitosis, we... more
RNA localization is of critical importance in many fundamental cell biological and developmental processes by regulating the spatial control of gene expression. To investigate how spindle-localized RNAs might influence mitosis, we comprehensively surveyed all messenger RNAs (mRNAs) that bound to microtubules during metaphase in both Xenopus laevis egg extracts and mitotic human cell extracts. We identify conserved classes of mRNAs that are enriched on microtubules in both human and X. laevis. Active mitotic translation occurs on X. laevis meiotic spindles, and a subset of microtubule-bound mRNAs (MT-mRNAs) associate with polyribosomes. Although many MT-mRNAs associate with polyribosomes, we find that active translation is not required for mRNA localization to mitotic microtubules. Our results represent the first genome-wide survey of mRNAs localized to a specific cytoskeletal component and suggest that microtubule localization of specific mRNAs is likely to function in mitotic regulation and mRNA segregation during cell division.
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DEAD-box ATPases/helicases are a large family of enzymes (>35 in humans) involved in almost all aspects of RNA metabolism including ribosome biogenesis, RNA splicing, export, translation,... more
DEAD-box ATPases/helicases are a large family of enzymes (>35 in humans) involved in almost all aspects of RNA metabolism including ribosome biogenesis, RNA splicing, export, translation, and decay. Many members of this family are ATP-dependent RNA-binding proteins that interact with the RNA phosphodiester backbone and promote structural remodeling of target complexes through ATP binding and hydrolysis. Here, we describe the methods used in our laboratory to characterize the DEAD-box ATPase Dbp5 of Saccharomyces cerevisiae. Dbp5 is essential for the process of mRNA export in budding yeast and highly conserved orthologs can be found in all eukaryotes. Specifically, we describe enzyme assays to measure the catalytic activity of Dbp5 in association with RNA and known binding partners, as well as assays developed to measure the binding affinities and release kinetics of RNA and adenosine nucleotides from Dbp5. These assays have provided important information that has shaped our current models of Dbp5 function in mRNA export and should be useful for the characterization of other DEAD-box family members.
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Nuclear export of mRNA in eukaryotic cells is mediated by soluble transport factors and components of the nuclear pore complex (NPC). The cytoplasmically oriented nuclear pore protein Nup159 plays a critical role in mRNA export through... more
Nuclear export of mRNA in eukaryotic cells is mediated by soluble transport factors and components of the nuclear pore complex (NPC). The cytoplasmically oriented nuclear pore protein Nup159 plays a critical role in mRNA export through its conserved N-terminal domain (NTD). Here, we report the crystal structure of the Nup159 NTD, refined to 2.5 A. The structure reveals an unusually asymmetric seven-bladed beta-propeller that is structurally conserved throughout eukarya. Using structure-based conservation analysis, we have targeted specific surface residues for mutagenesis. Residue substitutions in a conserved loop of the NTD abolish in vitro binding to Dbp5, a DEAD box helicase required for mRNA export. In vivo, these mutations cause Dbp5 mislocalization and block mRNA export. These findings suggest that the Nup159 NTD functions in mRNA export as a binding platform, tethering shuttling Dbp5 molecules at the nuclear periphery and locally concentrating this mRNA remodeling factor at t...
Research Interests: Macromolecular X-Ray Crystallography, In Situ Hybridization, Cell and Molecular Biology, Biological Sciences, Saccharomyces cerevisiae, and 10 moreCrystal structure, Mutation, Nuclear pore complex, Plasmids, Cell nucleus, Protein Secondary Structure Prediction, Protein Conformation, Recombinant Proteins, Cytoplasm, and Site-directed Mutagenesis
Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. About 30 different proteins (nucleoporins, nups) arrange around a central eightfold rotational axis to build the modular NPC.... more
Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. About 30 different proteins (nucleoporins, nups) arrange around a central eightfold rotational axis to build the modular NPC. Nup188 and Nup192 are related and evolutionary conserved, large nucleoporins that are part of the NPC scaffold. Here we determine the structure of Nup188. The protein folds into an extended stack of helices where an N-terminal 130 kDa segment forms an intricate closed ring, while the C-terminal region is a more regular, superhelical structure. Overall, the structure has distant similarity with flexible S-shaped nuclear transport receptors (NTRs). Intriguingly, like NTRs, both Nup188 and Nup192 specifically bind FG-repeats and are able to translocate through NPCs by facilitated diffusion. This blurs the existing dogma of a clear distinction between stationary nups and soluble NTRs and suggests an evolutionary relationship between the NPC and the soluble nuclear transport machinery. DOI:http://dx.doi.org/10.7554/eLife.00745.001.
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The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane... more
The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.
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The small GTPase Ran controls cellular processes by interacting with members of the importin beta family that bind specifically to the GTP-bound form of Ran, and this regulates the interaction between importin beta-like proteins and... more
The small GTPase Ran controls cellular processes by interacting with members of the importin beta family that bind specifically to the GTP-bound form of Ran, and this regulates the interaction between importin beta-like proteins and cellular factors. The structures of RanGDP and RanGTP are markedly different, and major structural changes are found in the switch I and switch II regions and in the C-terminal extension of Ran. Here, we show that a deletion mutant of Ran, lacking the entire C-terminal extension, termed Ran Core, can bind to importin beta in its GDP-bound form with high affinity. The ability of Ran CoreGDP to dissociate cargo from importin beta results in an import block in digitonin-permeabilized cells and leads to microtubule aster formation in mitotic Xenopus egg extract. As for importin beta, also transportin, importin 7 and exportin-t can no longer discriminate efficiently between the two nucleotide-bound forms of Ran Core. In contrast, a significant reduction in affinity of the RanGDP-binding protein NTF2 for Ran CoreGDP is observed, indicating that the switch regions have changed conformation in the Ran Core mutant. Our results demonstrate that the C terminus of Ran is a major determinant of the state of Ran, and that removal of this allows the GDP-bound form to adopt a GTP-like conformation, thereby creating a constitutively active protein.
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The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic... more
The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vit...
Research Interests: Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology, Genetics, and 58 moreMarine Biology, Neuroscience, Environmental Science, Geophysics, Physics, Materials Science, Quantum Physics, Developmental Biology, Immunology, Climate Change, Molecular Biology, Structural Biology, Genomics, RNA, Computational Biology, Transcriptomics, Kinetics, Biotechnology, Systems Biology, Cancer, Biology, Metabolomics, Cell Cycle, Proteomics, Ecology, Drug Discovery, Evolution, Parallel Processing, Nanotechnology, Astrophysics, Neurobiology, Medicine, Multidisciplinary, Palaeobiology, Functional Genomics, Nature, Fluorescence Resonance Energy Transfer, Signal Transduction, Astronomy, DNA, Mitosis, Fluorescence Lifetime, Xenopus, Humans, Meiosis, Animals, Cell Signalling, Medical Research, Microtubules, Chromatin, Nuclear envelope, Guanosine Triphosphate, HeLa cells, Oocytes, Cell Cycle Proteins, Earth Science, Biosensing Techniques, and Somatic Cells
Regulation of microtubule dynamics and organization in mitosis by a number of microtubule-associated proteins (MAPs) is required for proper bipolar spindle assembly, yet the precise mechanisms by which many MAPs function are poorly... more
Regulation of microtubule dynamics and organization in mitosis by a number of microtubule-associated proteins (MAPs) is required for proper bipolar spindle assembly, yet the precise mechanisms by which many MAPs function are poorly understood. One interesting class of MAPs is known to localize to the nucleus during interphase yet fulfill important spindle functions during mitosis. We have identified Xenopus nuclear factor 7 (Xnf7), a developmental regulator of dorsal-ventral patterning, as a microtubule-binding protein that also associates with the nuclear import receptor importin alpha/beta. Xnf7 localized to interphase nuclei and metaphase spindles both in Xenopus egg extracts and cultured cells. Xnf7-depleted spindles were hypersensitive to microtubule-depolymerizing agents. Functional characterization of Xnf7 revealed that it binds directly to microtubules, exhibits RING-finger-dependent E3-ubiquitin-ligase activity, and has C-terminal-dependent microtubule-bundling activity. The minimal microtubule-bundling domain of Xnf7 was sufficient to rescue the spindle-hypersensitivity phenotype. Thus, we have identified Xnf7 as a nuclear MAP whose microtubule-bundling activity, but not E3-ligase activity, contributes to microtubule organization and spindle integrity. Characterization of the multiple activities of Xnf7 may have implications for understanding human diseases caused by mutations in related proteins.