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LSSS 2017-2018

2017LSSS2018

Life Sciences Seminar Series

 

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Ulrike Kutay

ETH Zurich, Switzerland

Cellular reorganization for open mitosis

Selected Publications

Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins.Linder MI, Köhler M, Boersema P, Weberruss M, Wandke C, Marino J, Ashiono C, Picotti P, Antonin W, Kutay U
Dev Cell 2017 Oct 23; 43(2):141-156.e7

Abstract

During interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle. Here, we show that both CDK1 and polo-like kinase 1 (PLK1) support mitotic NPC disintegration by hyperphosphorylation of Nup98, the gatekeeper nucleoporin, and Nup53, a central nucleoporin linking the inner NPC scaffold to the pore membrane. Multisite phosphorylation of Nup53 critically contributes to its liberation from its partner nucleoporins, including the pore membrane protein NDC1. Initial steps of NPC disassembly in semi-permeabilized cells can be reconstituted by a cocktail of mitotic kinases including cyclinB-CDK1, NIMA, and PLK1, suggesting that the unzipping of nucleoporin interactions by protein phosphorylation is an important principle underlying mitotic NE permeabilization.

Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology.Pawar S, Ungricht R, Tiefenboeck P, Leroux JC, Kutay U
Elife 2017 Aug 14; 6

Abstract

Newly synthesized membrane proteins are targeted to the inner nuclear membrane (INM) by diffusion within the membrane system of the endoplasmic reticulum (ER), translocation through nuclear pore complexes (NPCs) and retention on nuclear partners. Using a visual in vitro assay we previously showed that efficient protein targeting to the INM depends on nucleotide hydrolysis. We now reveal that INM targeting is GTP-dependent. Exploiting in vitro reconstitution and in vivo analysis of INM targeting, we establish that Atlastins, membrane-bound GTPases of the ER, sustain the efficient targeting of proteins to the INM by their continued activity in preserving ER topology. When ER topology is altered, the long-range diffusional exchange of proteins in the ER network and targeting efficiency to the INM are diminished. Highlighting the general importance of proper ER topology, we show that Atlastins also influence NPC biogenesis and timely exit of secretory cargo from the ER.

Advanced Cell Classifier: User-Friendly Machine-Learning-Based Software for Discovering Phenotypes in High-Content Imaging Data.Piccinini F, Balassa T, Szkalisity A, Molnar C, Paavolainen L, Kujala K, Buzas K, Sarazova M, Pietiainen V, Kutay U, Smith K, Horvath P
Cell Syst 2017 Jun 28; 4(6):651-655.e5

Abstract

High-content, imaging-based screens now routinely generate data on a scale that precludes manual verification and interrogation. Software applying machine learning has become an essential tool to automate analysis, but these methods require annotated examples to learn from. Efficiently exploring large datasets to find relevant examples remains a challenging bottleneck. Here, we present Advanced Cell Classifier (ACC), a graphical software package for phenotypic analysis that addresses these difficulties. ACC applies machine-learning and image-analysis methods to high-content data generated by large-scale, cell-based experiments. It features methods to mine microscopic image data, discover new phenotypes, and improve recognition performance. We demonstrate that these features substantially expedite the training process, successfully uncover rare phenotypes, and improve the accuracy of the analysis. ACC is extensively documented, designed to be user-friendly for researchers without machine-learning expertise, and distributed as a free open-source tool at www.cellclassifier.org.

Poly(A)-specific ribonuclease is a nuclear ribosome biogenesis factor involved in human 18S rRNA maturation.Montellese C, Montel-Lehry N, Henras AK, Kutay U, Gleizes PE, O'Donohue MF
Nucleic Acids Res 2017 Jun 20; 45(11):6822-6836

Abstract

The poly-A specific ribonuclease (PARN), initially characterized for its role in mRNA catabolism, supports the processing of different types of non-coding RNAs including telomerase RNA. Mutations in PARN are linked to dyskeratosis congenita and pulmonary fibrosis. Here, we show that PARN is part of the enzymatic machinery that matures the human 18S ribosomal RNA (rRNA). Consistent with its nucleolar steady-state localization, PARN is required for 40S ribosomal subunit production and co-purifies with 40S subunit precursors. Depletion of PARN or expression of a catalytically-compromised PARN mutant results in accumulation of 3΄ extended 18S rRNA precursors. Analysis of these processing intermediates reveals a defect in 3΄ to 5΄ trimming of the internal transcribed spacer 1 (ITS1) region, subsequent to endonucleolytic cleavage at site E. Consistent with a function of PARN in exonucleolytic trimming of 18S-E pre-rRNA, recombinant PARN can process the corresponding ITS1 RNA fragment in vitro. Trimming of 18S-E pre-rRNA by PARN occurs in the nucleus, upstream of the final endonucleolytic cleavage by the endonuclease NOB1 in the cytoplasm. These results identify PARN as a new component of the ribosome biogenesis machinery in human cells. Defects in ribosome biogenesis could therefore underlie the pathologies linked to mutations in PARN.

Mechanisms and functions of nuclear envelope remodelling.Ungricht R, Kutay U
Nat Rev Mol Cell Biol 2017 Apr; 18(4):229-245

Abstract

As a compartment border, the nuclear envelope (NE) needs to serve as both a protective membrane shell for the genome and a versatile communication interface between the nucleus and the cytoplasm. Despite its important structural role in sheltering the genome, the NE is a dynamic and highly adaptable boundary that changes composition during differentiation, deforms in response to mechanical challenges, can be repaired upon rupture and even rapidly disassembles and reforms during open mitosis. NE remodelling is fundamentally involved in cell growth, division and differentiation, and if perturbed can lead to devastating diseases such as muscular dystrophies or premature ageing.

Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis.Bammert L, Jonas S, Ungricht R, Kutay U
Nucleic Acids Res 2016 Nov 16; 44(20):9803-9820

Abstract

Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S ribosomal subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5'ETS and ITS1 regions of the ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S ribosomal subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth.

Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing.Larburu N, Montellese C, O'Donohue MF, Kutay U, Gleizes PE, Plisson-Chastang C
Nucleic Acids Res 2016 Sep 30; 44(17):8465-78

Abstract

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3'-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.

Cellular Reorganization during Mitotic Entry.Champion L, Linder MI, Kutay U
Trends Cell Biol 2017 Jan; 27(1):26-41

Abstract

The preparation of eukaryotic cells for division requires an extensive cellular reorganization, affecting cytoskeletal elements, chromatin, and organelles. These drastic changes in cellular architecture ensure the proper segregation of chromosomes and inheritance of organelles. The morphological alterations occurring during mitotic entry are tightly coordinated with the cell cycle, mainly through the action of mitotic kinases. Conversely, the fidelity of these processes impacts mitotic progression and is important for organismal homeostasis and cell fate. Here, we provide an overview of major architectural changes observed during early mitosis and review recent progress in understanding their regulatory mechanisms, focusing on processes accompanying mitotic cell rounding and restructuring of organelles in mammalian cells.

Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription.Kang J, Kusnadi EP, Ogden AJ, Hicks RJ, Bammert L, Kutay U, Hung S, Sanij E, Hannan RD, Hannan KM, Pearson RB
Oncotarget 2016 Aug 2; 7(31):48887-48904

Abstract

Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer.

Editorial overview: The cell nucleus: Plastic, elastic and fantastic.Cohen-Fix O, Kutay U
Curr Opin Cell Biol 2016 Jun; 40:iv-v

Abstract

A Wounded Nucleus Needs ESCRT Service.Ungricht R, Kutay U
Dev Cell 2016 May 9; 37(3):202-4

Abstract

Two recent publications in Science report on frequent rupturing events of the nuclear envelope induced by migration of cells through narrow openings. Nuclear envelope "wounds" are repaired by the ESCRT-III machinery to limit DNA damage, nuclear fragmentation, and cell death.

An In Vitro Assay to Study Targeting of Membrane Proteins to the Inner Nuclear Membrane.Ungricht R, Pawar S, Kutay U
Methods Mol Biol 2016; 1411:461-77

Abstract

Newly synthesized membrane proteins are inserted into the endoplasmic reticulum (ER) from where they are constantly sorted to various cellular compartments. To analyze and visualize sorting of membrane proteins to the inner nuclear membrane (INM), we developed a trap-release system that uncouples membrane integration into the ER from transport. This assay allows the simultaneous release of a large pool of an INM-destined membrane protein from the ER and microscopy-based monitoring of targeting to the INM. The use of semi-permeabilized HeLa cells further enables the identification and characterization of essential requirements of the targeting process. This protocol provides a detailed description of reporter construction, in vitro reconstitution, and visualization of trafficking.

Genome-wide RNAi Screening Identifies Protein Modules Required for 40S Subunit Synthesis in Human Cells.Badertscher L, Wild T, Montellese C, Alexander LT, Bammert L, Sarazova M, Stebler M, Csucs G, Mayer TU, Zamboni N, Zemp I, Horvath P, Kutay U
Cell Rep 2015 Dec 29; 13(12):2879-91

Abstract

Ribosome biogenesis is a highly complex process requiring many assisting factors. Studies in yeast have yielded comprehensive knowledge of the cellular machinery involved in this process. However, many aspects of ribosome synthesis are different in higher eukaryotes, and the global set of mammalian ribosome biogenesis factors remains unexplored. We used an imaging-based, genome-wide RNAi screen to find human proteins involved in 40S ribosomal subunit biogenesis. Our analysis identified ∼ 300 factors, many part of essential protein modules such as the small subunit (SSU) processome, the eIF3 and chaperonin complexes, and the ubiquitin-proteasome system. We demonstrate a role for the vertebrate-specific factor RBIS in ribosome synthesis, uncover a requirement for the CRL4 E3 ubiquitin ligase in nucleolar ribosome biogenesis, and reveal that intracellular glutamine synthesis supports 40S subunit production.

The NF45/NF90 Heterodimer Contributes to the Biogenesis of 60S Ribosomal Subunits and Influences Nucleolar Morphology.Wandrey F, Montellese C, Koos K, Badertscher L, Bammert L, Cook AG, Zemp I, Horvath P, Kutay U
Mol Cell Biol 2015 Oct; 35(20):3491-503

Abstract

The interleukin enhancer binding factors ILF2 (NF45) and ILF3 (NF90/NF110) have been implicated in various cellular pathways, such as transcription, microRNA (miRNA) processing, DNA repair, and translation, in mammalian cells. Using tandem affinity purification, we identified human NF45 and NF90 as components of precursors to 60S (pre-60S) ribosomal subunits. NF45 and NF90 are enriched in nucleoli and cosediment with pre-60S ribosomal particles in density gradient analysis. We show that association of the NF45/NF90 heterodimer with pre-60S ribosomal particles requires the double-stranded RNA binding domains of NF90, while depletion of NF45 and NF90 by RNA interference leads to a defect in 60S biogenesis. Nucleoli of cells depleted of NF45 and NF90 have altered morphology and display a characteristic spherical shape. These effects are not due to impaired rRNA transcription or processing of the precursors to 28S rRNA. Consistent with a role of the NF45/NF90 heterodimer in nucleolar steps of 60S subunit biogenesis, downregulation of NF45 and NF90 leads to a p53 response, accompanied by induction of the cyclin-dependent kinase inhibitor p21/CIP1, which can be counteracted by depletion of RPL11. Together, these data indicate that NF45 and NF90 are novel higher-eukaryote-specific factors required for the maturation of 60S ribosomal subunits.

Establishment of NE asymmetry—targeting of membrane proteins to the inner nuclear membrane.Ungricht R, Kutay U
Curr Opin Cell Biol 2015 Jun; 34:135-41

Abstract

The inner nuclear membrane (INM) represents a specialized subdomain of the endoplasmic reticulum (ER). The INM houses a unique set of integral membrane proteins that perform key functions in the organization of intranuclear architecture, control of gene expression and coupling of the nucleus to the cytoskeleton. However, the molecular mechanism of membrane protein sorting from the ER to the INM has remained enigmatic. Recently, novel approaches combining visual kinetic assays and computational modeling were used to define the requirements of trafficking to the INM in human cells. These studies reveal that nuclear retention, diffusional mobility in the ER as well as the number and architecture of NPCs are major determinants of INM targeting, collectively lending support to a diffusion-retention-based mechanism.

Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane.Ungricht R, Klann M, Horvath P, Kutay U
J Cell Biol 2015 Jun 8; 209(5):687-703

Abstract

Newly synthesized membrane proteins are constantly sorted from the endoplasmic reticulum (ER) to various membranous compartments. How proteins specifically enrich at the inner nuclear membrane (INM) is not well understood. We have established a visual in vitro assay to measure kinetics and investigate requirements of protein targeting to the INM. Using human LBR, SUN2, and LAP2β as model substrates, we show that INM targeting is energy-dependent but distinct from import of soluble cargo. Accumulation of proteins at the INM relies on both a highly interconnected ER network, which is affected by energy depletion, and an efficient immobilization step at the INM. Nucleoporin depletions suggest that translocation through nuclear pore complexes (NPCs) is rate-limiting and restricted by the central NPC scaffold. Our experimental data combined with mathematical modeling support a diffusion-retention-based mechanism of INM targeting. We experimentally confirmed the sufficiency of diffusion and retention using an artificial reporter lacking natural sorting signals that recapitulates the energy dependence of the process in vivo.