TY - JOUR AB - Bacteria divide by binary fission. The protein machine responsible for this process is the divisome, a transient assembly of more than 30 proteins in and on the surface of the cytoplasmic membrane. Together, they constrict the cell envelope and remodel the peptidoglycan layer to eventually split the cell into two. For Escherichia coli, most molecular players involved in this process have probably been identified, but obtaining the quantitative information needed for a mechanistic understanding can often not be achieved from experiments in vivo alone. Since the discovery of the Z-ring more than 30 years ago, in vitro reconstitution experiments have been crucial to shed light on molecular processes normally hidden in the complex environment of the living cell. In this review, we summarize how rebuilding the divisome from purified components – or at least parts of it - have been instrumental to obtain the detailed mechanistic understanding of the bacterial cell division machinery that we have today. AU - Radler, Philipp AU - Loose, Martin ID - 14834 IS - 1 JF - European Journal of Cell Biology KW - Cell Biology KW - General Medicine KW - Histology KW - Pathology and Forensic Medicine SN - 0171-9335 TI - A dynamic duo: Understanding the roles of FtsZ and FtsA for Escherichia coli cell division through in vitro approaches VL - 103 ER - TY - JOUR AB - Small GTPases play essential roles in the organization of eukaryotic cells. In recent years, it has become clear that their intracellular functions result from intricate biochemical networks of the GTPase and their regulators that dynamically bind to a membrane surface. Due to the inherent complexities of their interactions, however, revealing the underlying mechanisms of action is often difficult to achieve from in vivo studies. This review summarizes in vitro reconstitution approaches developed to obtain a better mechanistic understanding of how small GTPase activities are regulated in space and time. AU - Loose, Martin AU - Auer, Albert AU - Brognara, Gabriel AU - Budiman, Hanifatul R AU - Kowalski, Lukasz M AU - Matijevic, Ivana ID - 12163 IS - 6 JF - FEBS Letters KW - Cell Biology KW - Genetics KW - Molecular Biology KW - Biochemistry KW - Structural Biology KW - Biophysics SN - 0014-5793 TI - In vitro reconstitution of small GTPase regulation VL - 597 ER - TY - JOUR AB - The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader. AU - Manavella, Pablo A AU - Godoy Herz, Micaela A AU - Kornblihtt, Alberto R AU - Sorenson, Reed AU - Sieburth, Leslie E AU - Nakaminami, Kentaro AU - Seki, Motoaki AU - Ding, Yiliang AU - Sun, Qianwen AU - Kang, Hunseung AU - Ariel, Federico D AU - Crespi, Martin AU - Giudicatti, Axel J AU - Cai, Qiang AU - Jin, Hailing AU - Feng, Xiaoqi AU - Qi, Yijun AU - Pikaard, Craig S ID - 12669 IS - 6 JF - The Plant Cell KW - Cell Biology KW - Plant Science SN - 1040-4651 TI - Beyond transcription: compelling open questions in plant RNA biology VL - 35 ER - TY - JOUR AB - Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing. AU - Cikes, Domagoj AU - Elsayad, Kareem AU - Sezgin, Erdinc AU - Koitai, Erika AU - Ferenc, Torma AU - Orthofer, Michael AU - Yarwood, Rebecca AU - Heinz, Leonhard X. AU - Sedlyarov, Vitaly AU - Darwish-Miranda, Nasser AU - Taylor, Adrian AU - Grapentine, Sophie AU - al-Murshedi, Fathiya AU - Abot, Anne AU - Weidinger, Adelheid AU - Kutchukian, Candice AU - Sanchez, Colline AU - Cronin, Shane J. F. AU - Novatchkova, Maria AU - Kavirayani, Anoop AU - Schuetz, Thomas AU - Haubner, Bernhard AU - Haas, Lisa AU - Hagelkruys, Astrid AU - Jackowski, Suzanne AU - Kozlov, Andrey AU - Jacquemond, Vincent AU - Knauf, Claude AU - Superti-Furga, Giulio AU - Rullman, Eric AU - Gustafsson, Thomas AU - McDermot, John AU - Lowe, Martin AU - Radak, Zsolt AU - Chamberlain, Jeffrey S. AU - Bakovic, Marica AU - Banka, Siddharth AU - Penninger, Josef M. ID - 12747 JF - Nature Metabolism KW - Cell Biology KW - Physiology (medical) KW - Endocrinology KW - Diabetes and Metabolism KW - Internal Medicine SN - 2522-5812 TI - PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing VL - 5 ER - TY - JOUR AB - Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis (Arabidopsis thaliana). To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize (Zea mays). We show that Clade IB ZmRALF2/3 mutations, but not Clade III ZmRALF1/5 mutations, cause cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located in the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution patterns leading to altered cell wall organization and thickness culminating in pollen tube burst. Clade IB, but not Clade III ZmRALFs, strongly interact as ligands with the pollen-specific Catharanthus roseus RLK1-like (CrRLK1L) receptor kinases Zea mays FERONIA-like (ZmFERL) 4/7/9, LORELEI-like glycosylphosphatidylinositol-anchor (LLG) proteins Zea mays LLG 1 and 2 (ZmLLG1/2) and Zea mays pollen extension-like (PEX) cell wall proteins ZmPEX2/4. Notably, ZmFERL4 outcompetes ZmLLG2 and ZmPEX2 outcompetes ZmFERL4 for ZmRALF2 binding. Based on these data, we suggest that Clade IB RALFs act in a dual role as cell wall components and extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in maize and probably other plants. AU - Zhou, Liang-Zi AU - Wang, Lele AU - Chen, Xia AU - Ge, Zengxiang AU - Mergner, Julia AU - Li, Xingli AU - Küster, Bernhard AU - Längst, Gernot AU - Qu, Li-Jia AU - Dresselhaus, Thomas ID - 14726 JF - The Plant Cell KW - Cell Biology KW - Plant Science SN - 1040-4651 TI - The RALF signaling pathway regulates cell wall integrity during pollen tube growth in maize ER - TY - JOUR AB - We developed LIONESS, a technology that leverages improvements to optical super-resolution microscopy and prior information on sample structure via machine learning to overcome the limitations (in 3D-resolution, signal-to-noise ratio and light exposure) of optical microscopy of living biological specimens. LIONESS enables dense reconstruction of living brain tissue and morphodynamics visualization at the nanoscale. AU - Danzl, Johann G AU - Velicky, Philipp ID - 14770 IS - 8 JF - Nature Methods KW - Cell Biology KW - Molecular Biology KW - Biochemistry KW - Biotechnology SN - 1548-7091 TI - LIONESS enables 4D nanoscale reconstruction of living brain tissue VL - 20 ER - TY - JOUR AB - Germ granules, condensates of phase-separated RNA and protein, are organelles that are essential for germline development in different organisms. The patterning of the granules and their relevance for germ cell fate are not fully understood. Combining three-dimensional in vivo structural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that the localization of RNA molecules to the periphery of the granules, where ribosomes are localized, depends on translational activity at this location. In addition, we find that the vertebrate-specific Dead end (Dnd1) protein is essential for nanos3 RNA localization at the condensates’ periphery. Accordingly, in the absence of Dnd1, or when translation is inhibited, nanos3 RNA translocates into the granule interior, away from the ribosomes, a process that is correlated with the loss of germ cell fate. These findings highlight the relevance of sub-granule compartmentalization for post-transcriptional control and its importance for preserving germ cell totipotency. AU - Westerich, Kim Joana AU - Tarbashevich, Katsiaryna AU - Schick, Jan AU - Gupta, Antra AU - Zhu, Mingzhao AU - Hull, Kenneth AU - Romo, Daniel AU - Zeuschner, Dagmar AU - Goudarzi, Mohammad AU - Gross-Thebing, Theresa AU - Raz, Erez ID - 14781 IS - 17 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - Spatial organization and function of RNA molecules within phase-separated condensates in zebrafish are controlled by Dnd1 VL - 58 ER - TY - JOUR AB - Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area before they become rapidly and continuously bent, and this mechanism is confirmed in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane. AU - Mund, Markus AU - Tschanz, Aline AU - Wu, Yu-Le AU - Frey, Felix F AU - Mehl, Johanna L. AU - Kaksonen, Marko AU - Avinoam, Ori AU - Schwarz, Ulrich S. AU - Ries, Jonas ID - 14788 IS - 3 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Clathrin coats partially preassemble and subsequently bend during endocytosis VL - 222 ER - TY - JOUR AB - Homeostatic balance in the intestinal epithelium relies on a fast cellular turnover, which is coordinated by an intricate interplay between biochemical signalling, mechanical forces and organ geometry. We review recent modelling approaches that have been developed to understand different facets of this remarkable homeostatic equilibrium. Existing models offer different, albeit complementary, perspectives on the problem. First, biomechanical models aim to explain the local and global mechanical stresses driving cell renewal as well as tissue shape maintenance. Second, compartmental models provide insights into the conditions necessary to keep a constant flow of cells with well-defined ratios of cell types, and how perturbations can lead to an unbalance of relative compartment sizes. A third family of models address, at the cellular level, the nature and regulation of stem fate choices that are necessary to fuel cellular turnover. We also review how these different approaches are starting to be integrated together across scales, to provide quantitative predictions and new conceptual frameworks to think about the dynamics of cell renewal in complex tissues. AU - Corominas-Murtra, Bernat AU - Hannezo, Edouard B ID - 12162 JF - Seminars in Cell & Developmental Biology KW - Cell Biology KW - Developmental Biology SN - 1084-9521 TI - Modelling the dynamics of mammalian gut homeostasis VL - 150-151 ER - TY - JOUR AB - Understanding complex living systems, which are fundamentally constrained by physical phenomena, requires combining experimental data with theoretical physical and mathematical models. To develop such models, collaborations between experimental cell biologists and theoreticians are increasingly important but these two groups often face challenges achieving mutual understanding. To help navigate these challenges, this Perspective discusses different modelling approaches, including bottom-up hypothesis-driven and top-down data-driven models, and highlights their strengths and applications. Using cell mechanics as an example, we explore the integration of specific physical models with experimental data from the molecular, cellular and tissue level up to multiscale input. We also emphasize the importance of constraining model complexity and outline strategies for crosstalk between experimental design and model development. Furthermore, we highlight how physical models can provide conceptual insights and produce unifying and generalizable frameworks for biological phenomena. Overall, this Perspective aims to promote fruitful collaborations that advance our understanding of complex biological systems. AU - Schwayer, Cornelia AU - Brückner, David ID - 14827 IS - 24 JF - Journal of Cell Science KW - Cell Biology SN - 0021-9533 TI - Connecting theory and experiment in cell and tissue mechanics VL - 136 ER - TY - JOUR AB - Nuclear pore complexes (NPCs) bridge the nucleus and the cytoplasm and are indispensable for crucial cellular activities, such as bidirectional molecular trafficking and gene transcription regulation. The discovery of long-lived proteins (LLPs) in NPCs from postmitotic cells raises the exciting possibility that the maintenance of NPC integrity might play an inherent role in lifelong cell function. Age-dependent deterioration of NPCs and loss of nuclear integrity have been linked to age-related decline in postmitotic cell function and degenerative diseases. In this review, we discuss our current understanding of NPC maintenance in proliferating and postmitotic cells, and how malfunction of nucleoporins (Nups) might contribute to the pathogenesis of various neurodegenerative and cardiovascular diseases. AU - Liu, Jinqiang AU - HETZER, Martin W ID - 11051 IS - 3 JF - Trends in Cell Biology KW - Cell Biology SN - 0962-8924 TI - Nuclear pore complex maintenance and implications for age-related diseases VL - 32 ER - TY - JOUR AB - Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1’s function is evolutionarily conserved in plants, as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by bridging autophagosomes with the multivesicular body-localized ESCRT-I component VPS23A, leading to the formation of amphisomes. Similar to CFS1-ATG8 interaction, disrupting the CFS1-VPS23A interaction blocks autophagic flux and renders plants sensitive to nitrogen starvation. Altogether, our results reveal a conserved vacuolar sorting hub that regulates autophagic flux in plants. AU - Zhao, Jierui AU - Bui, Mai Thu AU - Ma, Juncai AU - Künzl, Fabian AU - Picchianti, Lorenzo AU - De La Concepcion, Juan Carlos AU - Chen, Yixuan AU - Petsangouraki, Sofia AU - Mohseni, Azadeh AU - García-Leon, Marta AU - Gomez, Marta Salas AU - Giannini, Caterina AU - Gwennogan, Dubois AU - Kobylinska, Roksolana AU - Clavel, Marion AU - Schellmann, Swen AU - Jaillais, Yvon AU - Friml, Jiří AU - Kang, Byung-Ho AU - Dagdas, Yasin ID - 12121 IS - 12 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole VL - 221 ER - TY - JOUR AB - MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer’s DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer⋅miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways. AU - Zapletal, David AU - Taborska, Eliska AU - Pasulka, Josef AU - Malik, Radek AU - Kubicek, Karel AU - Zanova, Martina AU - Much, Christian AU - Sebesta, Marek AU - Buccheri, Valeria AU - Horvat, Filip AU - Jenickova, Irena AU - Prochazkova, Michaela AU - Prochazka, Jan AU - Pinkas, Matyas AU - Novacek, Jiri AU - Joseph, Diego F. AU - Sedlacek, Radislav AU - Bernecky, Carrie A AU - O’Carroll, Dónal AU - Stefl, Richard AU - Svoboda, Petr ID - 12143 IS - 21 JF - Molecular Cell KW - Cell Biology KW - Molecular Biology SN - 1097-2765 TI - Structural and functional basis of mammalian microRNA biogenesis by Dicer VL - 82 ER - TY - JOUR AB - Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration. AU - Hino, Naoya AU - Matsuda, Kimiya AU - Jikko, Yuya AU - Maryu, Gembu AU - Sakai, Katsuya AU - Imamura, Ryu AU - Tsukiji, Shinya AU - Aoki, Kazuhiro AU - Terai, Kenta AU - Hirashima, Tsuyoshi AU - Trepat, Xavier AU - Matsuda, Michiyuki ID - 12238 IS - 19 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - A feedback loop between lamellipodial extension and HGF-ERK signaling specifies leader cells during collective cell migration VL - 57 ER - TY - JOUR AB - Neurons extend axons to form the complex circuitry of the mature brain. This depends on the coordinated response and continuous remodelling of the microtubule and F-actin networks in the axonal growth cone. Growth cone architecture remains poorly understood at nanoscales. We therefore investigated mouse hippocampal neuron growth cones using cryo-electron tomography to directly visualise their three-dimensional subcellular architecture with molecular detail. Our data showed that the hexagonal arrays of actin bundles that form filopodia penetrate and terminate deep within the growth cone interior. We directly observed the modulation of these and other growth cone actin bundles by alteration of individual F-actin helical structures. Microtubules with blunt, slightly flared or gently curved ends predominated in the growth cone, frequently contained lumenal particles and exhibited lattice defects. Investigation of the effect of absence of doublecortin, a neurodevelopmental cytoskeleton regulator, on growth cone cytoskeleton showed no major anomalies in overall growth cone organisation or in F-actin subpopulations. However, our data suggested that microtubules sustained more structural defects, highlighting the importance of microtubule integrity during growth cone migration. AU - Atherton, Joseph AU - Stouffer, Melissa A AU - Francis, Fiona AU - Moores, Carolyn A. ID - 12283 IS - 7 JF - Journal of Cell Science KW - Cell Biology SN - 0021-9533 TI - Visualising the cytoskeletal machinery in neuronal growth cones using cryo-electron tomography VL - 135 ER - TY - JOUR AB - Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis. AU - Weier, Ann-Kathrin AU - Homrich, Mirka AU - Ebbinghaus, Stephanie AU - Juda, Pavel AU - Miková, Eliška AU - Hauschild, Robert AU - Zhang, Lili AU - Quast, Thomas AU - Mass, Elvira AU - Schlitzer, Andreas AU - Kolanus, Waldemar AU - Burgdorf, Sven AU - Gruß, Oliver J. AU - Hons, Miroslav AU - Wieser, Stefan AU - Kiermaier, Eva ID - 12122 IS - 12 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Multiple centrosomes enhance migration and immune cell effector functions of mature dendritic cells VL - 221 ER - TY - JOUR AB - Plant root architecture flexibly adapts to changing nitrate (NO3−) availability in the soil; however, the underlying molecular mechanism of this adaptive development remains under-studied. To explore the regulation of NO3−-mediated root growth, we screened for low-nitrate-resistant mutant (lonr) and identified mutants that were defective in the NAC transcription factor NAC075 (lonr1) as being less sensitive to low NO3− in terms of primary root growth. We show that NAC075 is a mobile transcription factor relocating from the root stele tissues to the endodermis based on NO3− availability. Under low-NO3− availability, the kinase CBL-interacting protein kinase 1 (CIPK1) is activated, and it phosphorylates NAC075, restricting its movement from the stele, which leads to the transcriptional regulation of downstream target WRKY53, consequently leading to adapted root architecture. Our work thus identifies an adaptive mechanism involving translocation of transcription factor based on nutrient availability and leading to cell-specific reprogramming of plant root growth. AU - Xiao, Huixin AU - Hu, Yumei AU - Wang, Yaping AU - Cheng, Jinkui AU - Wang, Jinyi AU - Chen, Guojingwei AU - Li, Qian AU - Wang, Shuwei AU - Wang, Yalu AU - Wang, Shao-Shuai AU - Wang, Yi AU - Xuan, Wei AU - Li, Zhen AU - Guo, Yan AU - Gong, Zhizhong AU - Friml, Jiří AU - Zhang, Jing ID - 12120 IS - 23 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - Nitrate availability controls translocation of the transcription factor NAC075 for cell-type-specific reprogramming of root growth VL - 57 ER - TY - JOUR AB - Reading, interpreting and crawling along gradients of chemotactic cues is one of the most complex questions in cell biology. In this issue, Georgantzoglou et al. (2022. J. Cell. Biol.https://doi.org/10.1083/jcb.202103207) use in vivo models to map the temporal sequence of how neutrophils respond to an acutely arising gradient of chemoattractant. AU - Stopp, Julian A AU - Sixt, Michael K ID - 12272 IS - 8 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Plan your trip before you leave: The neutrophils’ search-and-run journey VL - 221 ER - TY - JOUR AB - The T cell receptor (TCR) pathway receives, processes, and amplifies the signal from pathogenic antigens to the activation of T cells. Although major components in this pathway have been identified, the knowledge on how individual components cooperate to effectively transduce signals remains limited. Phase separation emerges as a biophysical principle in organizing signaling molecules into liquid-like condensates. Here, we report that phospholipase Cγ1 (PLCγ1) promotes phase separation of LAT, a key adaptor protein in the TCR pathway. PLCγ1 directly cross-links LAT through its two SH2 domains. PLCγ1 also protects LAT from dephosphorylation by the phosphatase CD45 and promotes LAT-dependent ERK activation and SLP76 phosphorylation. Intriguingly, a nonmonotonic effect of PLCγ1 on LAT clustering was discovered. Computer simulations, based on patchy particles, revealed how the cluster size is regulated by protein compositions. Together, these results define a critical function of PLCγ1 in promoting phase separation of the LAT complex and TCR signal transduction. AU - Zeng, Longhui AU - Palaia, Ivan AU - Šarić, Anđela AU - Su, Xiaolei ID - 10337 IS - 6 JF - Journal of Cell Biology KW - cell biology SN - 0021-9525 TI - PLCγ1 promotes phase separation of T cell signaling components VL - 220 ER - TY - JOUR AB - In order to combat molecular damage, most cellular proteins undergo rapid turnover. We have previously identified large nuclear protein assemblies that can persist for years in post-mitotic tissues and are subject to age-related decline. Here, we report that mitochondria can be long lived in the mouse brain and reveal that specific mitochondrial proteins have half-lives longer than the average proteome. These mitochondrial long-lived proteins (mitoLLPs) are core components of the electron transport chain (ETC) and display increased longevity in respiratory supercomplexes. We find that COX7C, a mitoLLP that forms a stable contact site between complexes I and IV, is required for complex IV and supercomplex assembly. Remarkably, even upon depletion of COX7C transcripts, ETC function is maintained for days, effectively uncoupling mitochondrial function from ongoing transcription of its mitoLLPs. Our results suggest that modulating protein longevity within the ETC is critical for mitochondrial proteome maintenance and the robustness of mitochondrial function. AU - Krishna, Shefali AU - Arrojo e Drigo, Rafael AU - Capitanio, Juliana S. AU - Ramachandra, Ranjan AU - Ellisman, Mark AU - HETZER, Martin W ID - 11052 IS - 21 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - Identification of long-lived proteins in the mitochondria reveals increased stability of the electron transport chain VL - 56 ER - TY - JOUR AB - During development, a single cell is transformed into a highly complex organism through progressive cell division, specification and rearrangement. An important prerequisite for the emergence of patterns within the developing organism is to establish asymmetries at various scales, ranging from individual cells to the entire embryo, eventually giving rise to the different body structures. This becomes especially apparent during gastrulation, when the earliest major lineage restriction events lead to the formation of the different germ layers. Traditionally, the unfolding of the developmental program from symmetry breaking to germ layer formation has been studied by dissecting the contributions of different signaling pathways and cellular rearrangements in the in vivo context of intact embryos. Recent efforts, using the intrinsic capacity of embryonic stem cells to self-assemble and generate embryo-like structures de novo, have opened new avenues for understanding the many ways by which an embryo can be built and the influence of extrinsic factors therein. Here, we discuss and compare divergent and conserved strategies leading to germ layer formation in embryos as compared to in vitro systems, their upstream molecular cascades and the role of extrinsic factors in this process. AU - Schauer, Alexandra AU - Heisenberg, Carl-Philipp J ID - 8966 JF - Developmental Biology KW - Developmental Biology KW - Cell Biology KW - Molecular Biology SN - 0012-1606 TI - Reassembling gastrulation VL - 474 ER - TY - JOUR AB - Genomic imprinting is an epigenetic mechanism that results in parental allele-specific expression of ~1% of all genes in mouse and human. Imprinted genes are key developmental regulators and play pivotal roles in many biological processes such as nutrient transfer from the mother to offspring and neuronal development. Imprinted genes are also involved in human disease, including neurodevelopmental disorders, and often occur in clusters that are regulated by a common imprint control region (ICR). In extra-embryonic tissues ICRs can act over large distances, with the largest surrounding Igf2r spanning over 10 million base-pairs. Besides classical imprinted expression that shows near exclusive maternal or paternal expression, widespread biased imprinted expression has been identified mainly in brain. In this review we discuss recent developments mapping cell type specific imprinted expression in extra-embryonic tissues and neocortex in the mouse. We highlight the advantages of using an inducible uniparental chromosome disomy (UPD) system to generate cells carrying either two maternal or two paternal copies of a specific chromosome to analyze the functional consequences of genomic imprinting. Mosaic Analysis with Double Markers (MADM) allows fluorescent labeling and concomitant induction of UPD sparsely in specific cell types, and thus to over-express or suppress all imprinted genes on that chromosome. To illustrate the utility of this technique, we explain how MADM-induced UPD revealed new insights about the function of the well-studied Cdkn1c imprinted gene, and how MADM-induced UPDs led to identification of highly cell type specific phenotypes related to perturbed imprinted expression in the mouse neocortex. Finally, we give an outlook on how MADM could be used to probe cell type specific imprinted expression in other tissues in mouse, particularly in extra-embryonic tissues. AU - Pauler, Florian AU - Hudson, Quanah AU - Laukoter, Susanne AU - Hippenmeyer, Simon ID - 9188 IS - 5 JF - Neurochemistry International KW - Cell Biology KW - Cellular and Molecular Neuroscience SN - 0197-0186 TI - Inducible uniparental chromosome disomy to probe genomic imprinting at single-cell level in brain and beyond VL - 145 ER - TY - JOUR AB - Background: The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. Results: Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Conclusions: The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins. AU - Rampelt, Heike AU - Sucec, Iva AU - Bersch, Beate AU - Horten, Patrick AU - Perschil, Inge AU - Martinou, Jean-Claude AU - van der Laan, Martin AU - Wiedemann, Nils AU - Schanda, Paul AU - Pfanner, Nikolaus ID - 8402 JF - BMC Biology KW - Biotechnology KW - Plant Science KW - General Biochemistry KW - Genetics and Molecular Biology KW - Developmental Biology KW - Cell Biology KW - Physiology KW - Ecology KW - Evolution KW - Behavior and Systematics KW - Structural Biology KW - General Agricultural and Biological Sciences SN - 1741-7007 TI - The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments VL - 18 ER - TY - JOUR AB - Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin-binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex, an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell-edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling.This article has an associated First Person interview with the first author of the paper. AU - Dimchev, Georgi A AU - Amiri, Behnam AU - Humphries, Ashley C. AU - Schaks, Matthias AU - Dimchev, Vanessa AU - Stradal, Theresia E. B. AU - Faix, Jan AU - Krause, Matthias AU - Way, Michael AU - Falcke, Martin AU - Rottner, Klemens ID - 8434 IS - 7 JF - Journal of Cell Science KW - Cell Biology SN - 0021-9533 TI - Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation VL - 133 ER - TY - JOUR AB - Background ESCRT-III is a membrane remodelling filament with the unique ability to cut membranes from the inside of the membrane neck. It is essential for the final stage of cell division, the formation of vesicles, the release of viruses, and membrane repair. Distinct from other cytoskeletal filaments, ESCRT-III filaments do not consume energy themselves, but work in conjunction with another ATP-consuming complex. Despite rapid progress in describing the cell biology of ESCRT-III, we lack an understanding of the physical mechanisms behind its force production and membrane remodelling. Results Here we present a minimal coarse-grained model that captures all the experimentally reported cases of ESCRT-III driven membrane sculpting, including the formation of downward and upward cones and tubules. This model suggests that a change in the geometry of membrane bound ESCRT-III filaments—from a flat spiral to a 3D helix—drives membrane deformation. We then show that such repetitive filament geometry transitions can induce the fission of cargo-containing vesicles. Conclusions Our model provides a general physical mechanism that explains the full range of ESCRT-III-dependent membrane remodelling and scission events observed in cells. This mechanism for filament force production is distinct from the mechanisms described for other cytoskeletal elements discovered so far. The mechanistic principles revealed here suggest new ways of manipulating ESCRT-III-driven processes in cells and could be used to guide the engineering of synthetic membrane-sculpting systems. AU - Harker-Kirschneck, Lena AU - Baum, Buzz AU - Šarić, Anđela ID - 10354 IS - 1 JF - BMC Biology KW - cell biology SN - 1741-7007 TI - Changes in ESCRT-III filament geometry drive membrane remodelling and fission in silico VL - 17 ER - TY - JOUR AB - Many adult tissues contain postmitotic cells as old as the host organism. The only organelle that does not turn over in these cells is the nucleus, and its maintenance represents a formidable challenge, as it harbors regulatory proteins that persist throughout adulthood. Here we developed strategies to visualize two classes of such long-lived proteins, histones and nucleoporins, to understand the function of protein longevity in nuclear maintenance. Genome-wide mapping of histones revealed specific enrichment of long-lived variants at silent gene loci. Interestingly, nuclear pores are maintained by piecemeal replacement of subunits, resulting in mosaic complexes composed of polypeptides with vastly different ages. In contrast, nondividing quiescent cells remove old nuclear pores in an ESCRT-dependent manner. Our findings reveal distinct molecular strategies of nuclear maintenance, linking lifelong protein persistence to gene regulation and nuclear integrity. AU - Toyama, Brandon H. AU - Arrojo e Drigo, Rafael AU - Lev-Ram, Varda AU - Ramachandra, Ranjan AU - Deerinck, Thomas J. AU - Lechene, Claude AU - Ellisman, Mark H. AU - HETZER, Martin W ID - 11061 IS - 2 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Visualization of long-lived proteins reveals age mosaicism within nuclei of postmitotic cells VL - 218 ER - TY - JOUR AB - Most neurons are not replaced during an animal’s lifetime. This nondividing state is characterized by extreme longevity and age-dependent decline of key regulatory proteins. To study the lifespans of cells and proteins in adult tissues, we combined isotope labeling of mice with a hybrid imaging method (MIMS-EM). Using 15N mapping, we show that liver and pancreas are composed of cells with vastly different ages, many as old as the animal. Strikingly, we also found that a subset of fibroblasts and endothelial cells, both known for their replicative potential, are characterized by the absence of cell division during adulthood. In addition, we show that the primary cilia of beta cells and neurons contains different structural regions with vastly different lifespans. Based on these results, we propose that age mosaicism across multiple scales is a fundamental principle of adult tissue, cell, and protein complex organization. AU - Arrojo e Drigo, Rafael AU - Lev-Ram, Varda AU - Tyagi, Swati AU - Ramachandra, Ranjan AU - Deerinck, Thomas AU - Bushong, Eric AU - Phan, Sebastien AU - Orphan, Victoria AU - Lechene, Claude AU - Ellisman, Mark H. AU - HETZER, Martin W ID - 11062 IS - 2 JF - Cell Metabolism KW - Cell Biology KW - Molecular Biology KW - Physiology SN - 1550-4131 TI - Age mosaicism across multiple scales in adult tissues VL - 30 ER - TY - THES AB - While cells of mesenchymal or epithelial origin perform their effector functions in a purely anchorage dependent manner, cells derived from the hematopoietic lineage are not committed to operate only within a specific niche. Instead, these cells are able to function autonomously of the molecular composition in a broad range of tissue compartments. By this means, cells of the hematopoietic lineage retain the capacity to disseminate into connective tissue and recirculate between organs, building the foundation for essential processes such as tissue regeneration or immune surveillance. Cells of the immune system, specifically leukocytes, are extraordinarily good at performing this task. These cells are able to flexibly shift their mode of migration between an adhesion-mediated and an adhesion-independent manner, instantaneously accommodating for any changes in molecular composition of the external scaffold. The key component driving directed leukocyte migration is the chemokine receptor 7, which guides the cell along gradients of chemokine ligand. Therefore, the physical destination of migrating leukocytes is purely deterministic, i.e. given by global directional cues such as chemokine gradients. Nevertheless, these cells typically reside in three-dimensional scaffolds of inhomogeneous complexity, raising the question whether cells are able to locally discriminate between multiple optional migration routes. Current literature provides evidence that leukocytes, specifically dendritic cells, do indeed probe their surrounding by virtue of multiple explorative protrusions. However, it remains enigmatic how these cells decide which one is the more favorable route to follow and what are the key players involved in performing this task. Due to the heterogeneous environment of most tissues, and the vast adaptability of migrating leukocytes, at this time it is not clear to what extent leukocytes are able to optimize their migratory strategy by adapting their level of adhesiveness. And, given the fact that leukocyte migration is characterized by branched cell shapes in combination with high migration velocities, it is reasonable to assume that these cells require fine tuned shape maintenance mechanisms that tightly coordinate protrusion and adhesion dynamics in a spatiotemporal manner. Therefore, this study aimed to elucidate how rapidly migrating leukocytes opt for an ideal migratory path while maintaining a continuous cell shape and balancing adhesive forces to efficiently navigate through complex microenvironments. The results of this study unraveled a role for the microtubule cytoskeleton in promoting the decision making process during path finding and for the first time point towards a microtubule-mediated function in cell shape maintenance of highly ramified cells such as dendritic cells. Furthermore, we found that migrating low-adhesive leukocytes are able to instantaneously adapt to increased tensile load by engaging adhesion receptors. This response was only occurring tangential to the substrate while adhesive properties in the vertical direction were not increased. As leukocytes are primed for rapid migration velocities, these results demonstrate that leukocyte integrins are able to confer a high level of traction forces parallel to the cell membrane along the direction of migration without wasting energy in gluing the cell to the substrate. Thus, the data in the here presented thesis provide new insights into the pivotal role of cytoskeletal dynamics and the mechanisms of force transduction during leukocyte migration. Thereby the here presented results help to further define fundamental principles underlying leukocyte migration and open up potential therapeutic avenues of clinical relevance. AU - Kopf, Aglaja ID - 6891 KW - cell biology KW - immunology KW - leukocyte KW - migration KW - microfluidics SN - 978-3-99078-002-2 TI - The implication of cytoskeletal dynamics on leukocyte migration ER - TY - JOUR AB - Mycobacterium tuberculosis can remain dormant in the host, an ability that explains the failure of many current tuberculosis treatments. Recently, the natural products cyclomarin, ecumicin, and lassomycin have been shown to efficiently kill Mycobacterium tuberculosis persisters. Their target is the N-terminal domain of the hexameric AAA+ ATPase ClpC1, which recognizes, unfolds, and translocates protein substrates, such as proteins containing phosphorylated arginine residues, to the ClpP1P2 protease for degradation. Surprisingly, these antibiotics do not inhibit ClpC1 ATPase activity, and how they cause cell death is still unclear. Here, using NMR and small-angle X-ray scattering, we demonstrate that arginine-phosphate binding to the ClpC1 N-terminal domain induces millisecond dynamics. We show that these dynamics are caused by conformational changes and do not result from unfolding or oligomerization of this domain. Cyclomarin binding to this domain specifically blocked these N-terminal dynamics. On the basis of these results, we propose a mechanism of action involving cyclomarin-induced restriction of ClpC1 dynamics, which modulates the chaperone enzymatic activity leading eventually to cell death. AU - Weinhäupl, Katharina AU - Brennich, Martha AU - Kazmaier, Uli AU - Lelievre, Joel AU - Ballell, Lluis AU - Goldberg, Alfred AU - Schanda, Paul AU - Fraga, Hugo ID - 8440 IS - 22 JF - Journal of Biological Chemistry KW - Cell Biology KW - Biochemistry KW - Molecular Biology SN - 0021-9258 TI - The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis VL - 293 ER - TY - JOUR AB - Neural progenitor cells (NeuPCs) possess a unique nuclear architecture that changes during differentiation. Nucleoporins are linked with cell-type-specific gene regulation, coupling physical changes in nuclear structure to transcriptional output; but, whether and how they coordinate with key fate-determining transcription factors is unclear. Here we show that the nucleoporin Nup153 interacts with Sox2 in adult NeuPCs, where it is indispensable for their maintenance and controls neuronal differentiation. Genome-wide analyses show that Nup153 and Sox2 bind and co-regulate hundreds of genes. Binding of Nup153 to gene promoters or transcriptional end sites correlates with increased or decreased gene expression, respectively, and inhibiting Nup153 expression alters open chromatin configurations at its target genes, disrupts genomic localization of Sox2, and promotes differentiation in vitro and a gliogenic fate switch in vivo. Together, these findings reveal that nuclear structural proteins may exert bimodal transcriptional effects to control cell fate. AU - Toda, Tomohisa AU - Hsu, Jonathan Y. AU - Linker, Sara B. AU - Hu, Lauren AU - Schafer, Simon T. AU - Mertens, Jerome AU - Jacinto, Filipe V. AU - HETZER, Martin W AU - Gage, Fred H. ID - 11067 IS - 5 JF - Cell Stem Cell KW - Cell Biology KW - Genetics KW - Molecular Medicine SN - 1934-5909 TI - Nup153 interacts with Sox2 to enable bimodal gene regulation and maintenance of neural progenitor cells VL - 21 ER - TY - JOUR AB - The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination. AU - Gustafson, Chelsea L. AU - Parsley, Nicole C. AU - Asimgil, Hande AU - Lee, Hsiau-Wei AU - Ahlbach, Christopher AU - Michael, Alicia Kathleen AU - Xu, Haiyan AU - Williams, Owen L. AU - Davis, Tara L. AU - Liu, Andrew C. AU - Partch, Carrie L. ID - 15155 IS - 4 JF - Molecular Cell KW - Cell Biology KW - Molecular Biology SN - 1097-2765 TI - A slow conformational switch in the BMAL1 transactivation domain modulates circadian rhythms VL - 66 ER - TY - JOUR AB - Repeated rounds of nuclear envelope (NE) rupture and repair have been observed in laminopathy and cancer cells and result in intermittent loss of nucleus compartmentalization. Currently, the causes of NE rupture are unclear. Here, we show that NE rupture in cancer cells relies on the assembly of contractile actin bundles that interact with the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex. We found that the loss of actin bundles or the LINC complex did not rescue nuclear lamina defects, a previously identified determinant of nuclear membrane stability, but did decrease the number and size of chromatin hernias. Finally, NE rupture inhibition could be rescued in cells treated with actin-depolymerizing drugs by mechanically constraining nucleus height. These data suggest a model of NE rupture where weak membrane areas, caused by defects in lamina organization, rupture because of an increase in intranuclear pressure from actin-based nucleus confinement. AU - Hatch, Emily M. AU - HETZER, Martin W ID - 11069 IS - 1 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Nuclear envelope rupture is induced by actin-based nucleus confinement VL - 215 ER - TY - JOUR AB - Aging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging. AU - Mertens, Jerome AU - Paquola, Apuã C.M. AU - Ku, Manching AU - Hatch, Emily AU - Böhnke, Lena AU - Ladjevardi, Shauheen AU - McGrath, Sean AU - Campbell, Benjamin AU - Lee, Hyungjun AU - Herdy, Joseph R. AU - Gonçalves, J. Tiago AU - Toda, Tomohisa AU - Kim, Yongsung AU - Winkler, Jürgen AU - Yao, Jun AU - HETZER, Martin W AU - Gage, Fred H. ID - 11079 IS - 6 JF - Cell Stem Cell KW - Cell Biology KW - Genetics KW - Molecular Medicine SN - 1934-5909 TI - Directly reprogrammed human neurons retain aging-associated transcriptomic signatures and reveal age-related nucleocytoplasmic defects VL - 17 ER - TY - JOUR AB - Aging is associated with the decline of protein, cell, and organ function. Here, we use an integrated approach to characterize gene expression, bulk translation, and cell biology in the brains and livers of young and old rats. We identify 468 differences in protein abundance between young and old animals. The majority are a consequence of altered translation output, that is, the combined effect of changes in transcript abundance and translation efficiency. In addition, we identify 130 proteins whose overall abundance remains unchanged but whose sub-cellular localization, phosphorylation state, or splice-form varies. While some protein-level differences appear to be a generic property of the rats’ chronological age, the majority are specific to one organ. These may be a consequence of the organ’s physiology or the chronological age of the cells within the tissue. Taken together, our study provides an initial view of the proteome at the molecular, sub-cellular, and organ level in young and old rats. AU - Ori, Alessandro AU - Toyama, Brandon H. AU - Harris, Michael S. AU - Bock, Thomas AU - Iskar, Murat AU - Bork, Peer AU - Ingolia, Nicholas T. AU - HETZER, Martin W AU - Beck, Martin ID - 11078 IS - 3 JF - Cell Systems KW - Cell Biology KW - Histology KW - Pathology and Forensic Medicine SN - 2405-4712 TI - Integrated transcriptome and proteome analyses reveal organ-specific proteome deterioration in old rats VL - 1 ER - TY - JOUR AB - Previously, we identified the nucleoporin gp210/Nup210 as a critical regulator of muscle and neuronal differentiation, but how this nucleoporin exerts its function and whether it modulates nuclear pore complex (NPC) activity remain unknown. Here, we show that gp210/Nup210 mediates muscle cell differentiation in vitro via its conserved N-terminal domain that extends into the perinuclear space. Removal of the C-terminal domain, which partially mislocalizes gp210/Nup210 away from NPCs, efficiently rescues the differentiation defect caused by the knockdown of endogenous gp210/Nup210. Unexpectedly, a gp210/Nup210 mutant lacking the NPC-targeting transmembrane and C-terminal domains is sufficient for C2C12 myoblast differentiation. We demonstrate that the endoplasmic reticulum (ER) stress-specific caspase cascade is exacerbated during Nup210 depletion and that blocking ER stress-mediated apoptosis rescues differentiation of Nup210-deficient cells. Our results suggest that the role of gp210/Nup210 in cell differentiation is mediated by its large luminal domain, which can act independently of NPC association and appears to play a pivotal role in the maintenance of nuclear envelope/ER homeostasis. AU - Gomez-Cavazos, J. Sebastian AU - HETZER, Martin W ID - 11075 IS - 6 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - The nucleoporin gp210/Nup210 controls muscle differentiation by regulating nuclear envelope/ER homeostasis VL - 208 ER - TY - JOUR AB - SNC1 (SUPPRESSOR OF NPR1, CONSTITUTIVE 1) is one of a suite of intracellular Arabidopsis NOD-like receptor (NLR) proteins which, upon activation, result in the induction of defense responses. However, the molecular mechanisms underlying NLR activation and the subsequent provocation of immune responses are only partially characterized. To identify negative regulators of NLR-mediated immunity, a forward genetic screen was undertaken to search for enhancers of the dwarf, autoimmune gain-of-function snc1 mutant. To avoid lethality resulting from severe dwarfism, the screen was conducted using mos4 (modifier of snc1, 4) snc1 plants, which display wild-type-like morphology and resistance. M2 progeny were screened for mutant, snc1-enhancing (muse) mutants displaying a reversion to snc1-like phenotypes. The muse9 mos4 snc1 triple mutant was found to exhibit dwarf morphology, elevated expression of the pPR2-GUS defense marker reporter gene and enhanced resistance to the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Via map-based cloning and Illumina sequencing, it was determined that the muse9 mutation is in the gene encoding the SWI/SNF chromatin remodeler SYD (SPLAYED), and was thus renamed syd-10. The syd-10 single mutant has no observable alteration from wild-type-like resistance, although the syd-4 T-DNA insertion allele displays enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Transcription of SNC1 is increased in both syd-4 and syd-10. These data suggest that SYD plays a subtle, specific role in the regulation of SNC1 expression and SNC1-mediated immunity. SYD may work with other proteins at the chromatin level to repress SNC1 transcription; such regulation is important for fine-tuning the expression of NLR-encoding genes to prevent unpropitious autoimmunity. AU - Johnson, Kaeli C.M. AU - Xia, Shitou AU - Feng, Xiaoqi AU - Li, Xin ID - 12196 IS - 8 JF - Plant and Cell Physiology KW - Cell Biology KW - Plant Science KW - Physiology KW - General Medicine SN - 0032-0781 TI - The chromatin remodeler SPLAYED negatively regulates SNC1-mediated immunity VL - 56 ER - TY - JOUR AB - The circadian clock orchestrates global changes in transcriptional regulation on a daily basis via the bHLH-PAS transcription factor CLOCK:BMAL1. Pathways driven by other bHLH-PAS transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for CLOCK:BMAL1. We show here that the cancer/testis antigen PASD1 fulfills this role to suppress circadian rhythms. PASD1 is evolutionarily related to CLOCK and interacts with the CLOCK:BMAL1 complex to repress transcriptional activation. Expression of PASD1 is restricted to germline tissues in healthy individuals but can be induced in cells of somatic origin upon oncogenic transformation. Reducing PASD1 in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. Our results describe a function for a germline-specific protein in regulation of the circadian clock and provide a molecular link from oncogenic transformation to suppression of circadian rhythms. AU - Michael, Alicia Kathleen AU - Harvey, Stacy L. AU - Sammons, Patrick J. AU - Anderson, Amanda P. AU - Kopalle, Hema M. AU - Banham, Alison H. AU - Partch, Carrie L. ID - 15160 IS - 5 JF - Molecular Cell KW - Cell Biology KW - Molecular Biology SN - 1097-2765 TI - Cancer/Testis antigen PASD1 silences the circadian clock VL - 58 ER - TY - JOUR AB - The nuclear pore complex (NPC) plays a critical role in gene expression by mediating import of transcription regulators into the nucleus and export of RNA transcripts to the cytoplasm. Emerging evidence suggests that in addition to mediating transport, a subset of nucleoporins (Nups) engage in transcriptional activation and elongation at genomic loci that are not associated with NPCs. The underlying mechanism and regulation of Nup mobility on and off nuclear pores remain unclear. Here we show that Nup50 is a mobile Nup with a pronounced presence both at the NPC and in the nucleoplasm that can move between these different localizations. Strikingly, the dynamic behavior of Nup50 in both locations is dependent on active transcription by RNA polymerase II and requires the N-terminal half of the protein, which contains importin α– and Nup153-binding domains. However, Nup50 dynamics are independent of importin α, Nup153, and Nup98, even though the latter two proteins also exhibit transcription-dependent mobility. Of interest, depletion of Nup50 from C2C12 myoblasts does not affect cell proliferation but inhibits differentiation into myotubes. Taken together, our results suggest a transport-independent role for Nup50 in chromatin biology that occurs away from the NPC. AU - Buchwalter, Abigail L. AU - Liang, Yun AU - HETZER, Martin W ID - 11082 IS - 16 JF - Molecular Biology of the Cell KW - Cell Biology KW - Molecular Biology SN - 1059-1524 TI - Nup50 is required for cell differentiation and exhibits transcription-dependent dynamics VL - 25 ER - TY - JOUR AB - In eukaryotic cells the nuclear genome is enclosed by the nuclear envelope (NE). In metazoans, the NE breaks down in mitosis and it has been assumed that the physical barrier separating nucleoplasm and cytoplasm remains intact during the rest of the cell cycle and cell differentiation. However, recent studies suggest that nonmitotic NE remodeling plays a critical role in development, virus infection, laminopathies, and cancer. Although the mechanisms underlying these NE restructuring events are currently being defined, one common theme is activation of protein kinase C family members in the interphase nucleus to disrupt the nuclear lamina, demonstrating the importance of the lamina in maintaining nuclear integrity. AU - Hatch, Emily AU - HETZER, Martin W ID - 11081 IS - 2 JF - Journal of Cell Biology KW - Cell Biology SN - 1540-8140 TI - Breaching the nuclear envelope in development and disease VL - 205 ER - TY - JOUR AB - Nuclear pore complex (NPC) proteins are known for their critical roles in regulating nucleocytoplasmic traffic of macromolecules across the nuclear envelope. However, recent findings suggest that some nucleoporins (Nups), including Nup98, have additional functions in developmental gene regulation. Nup98, which exhibits transcription-dependent mobility at the NPC but can also bind chromatin away from the nuclear envelope, is frequently involved in chromosomal translocations in a subset of patients suffering from acute myeloid leukemia (AML). A common paradigm suggests that Nup98 translocations cause aberrant transcription when they are recuited to aberrant genomic loci. Importantly, this model fails to account for the potential loss of wild type (WT) Nup98 function in the presence of Nup98 translocation mutants. Here we examine how the cell might regulate Nup98 nucleoplasmic protein levels to control transcription in healthy cells. In addition, we discuss the possibility that dominant negative Nup98 fusion proteins disrupt the transcriptional activity of WT Nup98 in the nucleoplasm to drive AML. AU - Franks, Tobias M. AU - HETZER, Martin W ID - 11083 IS - 3 JF - Trends in Cell Biology KW - Cell Biology SN - 0962-8924 TI - The role of Nup98 in transcription regulation in healthy and diseased cells VL - 23 ER - TY - JOUR AB - Protein turnover is an effective way of maintaining a functional proteome, as old and potentially damaged polypeptides are destroyed and replaced by newly synthesized copies. An increasing number of intracellular proteins, however, have been identified that evade this turnover process and instead are maintained over a cell's lifetime. This diverse group of long-lived proteins might be particularly prone to accumulation of damage and thus have a crucial role in the functional deterioration of key regulatory processes during ageing. AU - Toyama, Brandon H. AU - HETZER, Martin W ID - 11084 JF - Nature Reviews Molecular Cell Biology KW - Cell Biology KW - Molecular Biology SN - 1471-0072 TI - Protein homeostasis: Live long, won't prosper VL - 14 ER - TY - JOUR AB - The Nuclear Envelope (NE) contains over 100 different proteins that associate with nuclear components such as chromatin, the lamina and the transcription machinery. Mutations in genes encoding NE proteins have been shown to result in tissue-specific defects and disease, suggesting cell-type specific differences in NE composition and function. Consistent with these observations, recent studies have revealed unexpected functions for numerous NE associated proteins during cell differentiation and development. Here we review the latest insights into the roles played by the NE in cell differentiation, development, disease and aging, focusing primarily on inner nuclear membrane (INM) proteins and nuclear pore components. AU - Gomez-Cavazos, J Sebastian AU - HETZER, Martin W ID - 11089 IS - 6 JF - Current Opinion in Cell Biology KW - Cell Biology SN - 0955-0674 TI - Outfits for different occasions: tissue-specific roles of Nuclear Envelope proteins VL - 24 ER - TY - JOUR AB - Neoplastic cells are often characterized by specific morphological abnormalities of the nuclear envelope (NE), which have been used for cancer diagnosis for more than a century. The NE is a double phospholipid bilayer that encapsulates the nuclear genome, regulates all nuclear trafficking of RNAs and proteins and prevents the passive diffusion of macromolecules between the nucleoplasm and the cytoplasm. Whether there is a consequence to the proper functioning of the cell and loss of structural integrity of the nucleus remains unclear. Using live cell imaging, we characterize a phenomenon wherein nuclei of several proliferating human cancer cell lines become temporarily ruptured during interphase. Strikingly, NE rupturing was associated with the mislocalization of nucleoplasmic and cytoplasmic proteins and, in the most extreme cases, the entrapment of cytoplasmic organelles in the nuclear interior. In addition, we observed the formation of micronuclei-like structures during interphase and the movement of chromatin out of the nuclear space. The frequency of these NE rupturing events was higher in cells in which the nuclear lamina, a network of intermediate filaments providing mechanical support to the NE, was not properly formed. Our data uncover the existence of a NE instability that has the potential to change the genomic landscape of cancer cells. AU - Vargas, Jesse D. AU - Hatch, Emily M. AU - Anderson, Daniel J. AU - HETZER, Martin W ID - 11091 IS - 1 JF - Nucleus KW - Cell Biology SN - 1949-1034 TI - Transient nuclear envelope rupturing during interphase in human cancer cells VL - 3 ER - TY - JOUR AB - Nuclear pore complexes (NPCs) are built from ∼30 different proteins called nucleoporins or Nups. Previous studies have shown that several Nups exhibit cell-type-specific expression and that mutations in NPC components result in tissue-specific diseases. Here we show that a specific change in NPC composition is required for both myogenic and neuronal differentiation. The transmembrane nucleoporin Nup210 is absent in proliferating myoblasts and embryonic stem cells (ESCs) but becomes expressed and incorporated into NPCs during cell differentiation. Preventing Nup210 production by RNAi blocks myogenesis and the differentiation of ESCs into neuroprogenitors. We found that the addition of Nup210 to NPCs does not affect nuclear transport but is required for the induction of genes that are essential for cell differentiation. Our results identify a single change in NPC composition as an essential step in cell differentiation and establish a role for Nup210 in gene expression regulation and cell fate determination. AU - D'Angelo, Maximiliano A. AU - Gomez-Cavazos, J. Sebastian AU - Mei, Arianna AU - Lackner, Daniel H. AU - HETZER, Martin W ID - 11093 IS - 2 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - A change in nuclear pore complex composition regulates cell differentiation VL - 22 ER - TY - JOUR AB - Nuclear pore complexes (NPCs) assemble at the end of mitosis during nuclear envelope (NE) reformation and into an intact NE as cells progress through interphase. Although recent studies have shown that NPC formation occurs by two different molecular mechanisms at two distinct cell cycle stages, little is known about the molecular players that mediate the fusion of the outer and inner nuclear membranes to form pores. In this paper, we provide evidence that the transmembrane nucleoporin (Nup), POM121, but not the Nup107–160 complex, is present at new pore assembly sites at a time that coincides with inner nuclear membrane (INM) and outer nuclear membrane (ONM) fusion. Overexpression of POM121 resulted in juxtaposition of the INM and ONM. Additionally, Sun1, an INM protein that is known to interact with the cytoskeleton, was specifically required for interphase assembly and localized with POM121 at forming pores. We propose a model in which POM121 and Sun1 interact transiently to promote early steps of interphase NPC assembly. AU - Talamas, Jessica A. AU - HETZER, Martin W ID - 11094 IS - 1 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - POM121 and Sun1 play a role in early steps of interphase NPC assembly VL - 194 ER - TY - JOUR AB - As the gatekeepers of the eukaryotic cell nucleus, nuclear pore complexes (NPCs) mediate all molecular trafficking between the nucleoplasm and the cytoplasm. In recent years, transport-independent functions of NPC components, nucleoporins, have been identified including roles in chromatin organization and gene regulation. Here, we summarize our current view of the NPC as a dynamic hub for the integration of chromatin regulation and nuclear trafficking and discuss the functional interplay between nucleoporins and the nuclear genome. AU - Liang, Yun AU - HETZER, Martin W ID - 11096 IS - 1 JF - Current Opinion in Cell Biology KW - Cell Biology SN - 0955-0674 TI - Functional interactions between nucleoporins and chromatin VL - 23 ER - TY - JOUR AU - HETZER, Martin W AU - Cavalli, Giacomo ID - 11095 IS - 3 JF - Current Opinion in Cell Biology KW - Cell Biology SN - 0955-0674 TI - Editorial overview VL - 23 ER - TY - JOUR AU - HETZER, Martin W ID - 11098 IS - 2 JF - Aging KW - Cell Biology KW - Aging SN - 1945-4589 TI - The role of the nuclear pore complex in aging of post-mitotic cells VL - 2 ER - TY - JOUR AB - β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant. AU - Corazza, Alessandra AU - Rennella, Enrico AU - Schanda, Paul AU - Mimmi, Maria Chiara AU - Cutuil, Thomas AU - Raimondi, Sara AU - Giorgetti, Sofia AU - Fogolari, Federico AU - Viglino, Paolo AU - Frydman, Lucio AU - Gal, Maayan AU - Bellotti, Vittorio AU - Brutscher, Bernhard AU - Esposito, Gennaro ID - 8473 IS - 8 JF - Journal of Biological Chemistry KW - Cell Biology KW - Biochemistry KW - Molecular Biology SN - 0021-9258 TI - Native-unlike long-lived intermediates along the folding pathway of the amyloidogenic protein β2-Microglobulin revealed by real-time two-dimensional NMR VL - 285 ER - TY - JOUR AB - Over the last decade, the nuclear envelope (NE) has emerged as a key component in the organization and function of the nuclear genome. As many as 100 different proteins are thought to specifically localize to this double membrane that separates the cytoplasm and the nucleoplasm of eukaryotic cells. Selective portals through the NE are formed at sites where the inner and outer nuclear membranes are fused, and the coincident assembly of ∼30 proteins into nuclear pore complexes occurs. These nuclear pore complexes are essential for the control of nucleocytoplasmic exchange. Many of the NE and nuclear pore proteins are thought to play crucial roles in gene regulation and thus are increasingly linked to human diseases. AU - HETZER, Martin W AU - Wente, Susan R. ID - 11103 IS - 5 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - Border control at the nucleus: Biogenesis and organization of the nuclear membrane and pore complexes VL - 17 ER - TY - JOUR AB - Formation of the nuclear envelope (NE) around segregated chromosomes occurs by the reshaping of the endoplasmic reticulum (ER), a reservoir for disassembled nuclear membrane components during mitosis. In this study, we show that inner nuclear membrane proteins such as lamin B receptor (LBR), MAN1, Lap2β, and the trans-membrane nucleoporins Ndc1 and POM121 drive the spreading of ER membranes into the emerging NE via their capacity to bind chromatin in a collaborative manner. Despite their redundant functions, decreasing the levels of any of these trans-membrane proteins by RNAi-mediated knockdown delayed NE formation, whereas increasing the levels of any of them had the opposite effect. Furthermore, acceleration of NE formation interferes with chromosome separation during mitosis, indicating that the time frame over which chromatin becomes membrane enclosed is physiologically relevant and regulated. These data suggest that functionally distinct classes of chromatin-interacting membrane proteins, which are present at nonsaturating levels, collaborate to rapidly reestablish the nuclear compartment at the end of mitosis. AU - Anderson, Daniel J. AU - Vargas, Jesse D. AU - Hsiao, Joshua P. AU - HETZER, Martin W ID - 11106 IS - 2 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Recruitment of functionally distinct membrane proteins to chromatin mediates nuclear envelope formation in vivo VL - 186 ER - TY - JOUR AB - Nucleocytoplasmic transport occurs exclusively through nuclear pore complexes (NPCs) embedded in pores formed by inner and outer nuclear membrane fusion. The mechanism for de novo pore and NPC biogenesis remains unclear. Reticulons (RTNs) and Yop1/DP1 are conserved membrane protein families required to form and maintain the tubular endoplasmic reticulum (ER) and the postmitotic nuclear envelope. In this study, we report that members of the RTN and Yop1/DP1 families are required for nuclear pore formation. Analysis of Saccharomyces cerevisiae prp20-G282S and nup133Δ NPC assembly mutants revealed perturbations in Rtn1–green fluorescent protein (GFP) and Yop1-GFP ER distribution and colocalization to NPC clusters. Combined deletion of RTN1 and YOP1 resulted in NPC clustering, nuclear import defects, and synthetic lethality with the additional absence of Pom34, Pom152, and Nup84 subcomplex members. We tested for a direct role in NPC biogenesis using Xenopus laevis in vitro assays and found that anti-Rtn4a antibodies specifically inhibited de novo nuclear pore formation. We hypothesize that these ER membrane–bending proteins mediate early NPC assembly steps. AU - Dawson, T. Renee AU - Lazarus, Michelle D. AU - HETZER, Martin W AU - Wente, Susan R. ID - 11107 IS - 5 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - ER membrane–bending proteins are necessary for de novo nuclear pore formation VL - 184 ER - TY - JOUR AB - During mitosis in metazoans, segregated chromosomes become enclosed by the nuclear envelope (NE), a double membrane that is continuous with the endoplasmic reticulum (ER). Recent in vitro data suggest that NE formation occurs by chromatin-mediated reorganization of the tubular ER; however, the basic principles of such a membrane-reshaping process remain uncharacterized. Here, we present a quantitative analysis of nuclear membrane assembly in mammalian cells using time-lapse microscopy. From the initial recruitment of ER tubules to chromatin, the formation of a membrane-enclosed, transport-competent nucleus occurs within ∼12 min. Overexpression of the ER tubule-forming proteins reticulon 3, reticulon 4, and DP1 inhibits NE formation and nuclear expansion, whereas their knockdown accelerates nuclear assembly. This suggests that the transition from membrane tubules to sheets is rate-limiting for nuclear assembly. Our results provide evidence that ER-shaping proteins are directly involved in the reconstruction of the nuclear compartment and that morphological restructuring of the ER is the principal mechanism of NE formation in vivo. AU - Anderson, Daniel J. AU - HETZER, Martin W ID - 11111 IS - 5 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation VL - 182 ER - TY - JOUR AB - Nuclear pore complexes are large aqueous channels that penetrate the nuclear envelope, thereby connecting the nuclear interior with the cytoplasm. Until recently, these macromolecular complexes were viewed as static structures, the only function of which was to control the molecular trafficking between the two compartments. It has now become evident that this simplistic scenario is inaccurate and that nuclear pore complexes are highly dynamic multiprotein assemblies involved in diverse cellular processes ranging from the organization of the cytoskeleton to gene expression. In this review, we discuss the most recent developments in the nuclear-pore-complex field, focusing on the assembly, disassembly, maintenance and function of this macromolecular structure. AU - D’Angelo, Maximiliano A. AU - HETZER, Martin W ID - 11110 IS - 10 JF - Trends in Cell Biology KW - Cell Biology SN - 0962-8924 TI - Structure, dynamics and function of nuclear pore complexes VL - 18 ER - TY - JOUR AB - The nuclear envelope (NE) provides a selective barrier between the nuclear interior and the cytoplasm and constitutes a central component of intracellular architecture. During mitosis in metazoa, the NE breaks down leading to the complete mixing of the nuclear content with the cytosol. Interestingly, many NE components actively participate in mitotic progression. After chromosome segregation, the NE is reassembled around decondensing chromatin and the nuclear compartment is reestablished in the daughter cells. Here, we summarize recent progress in deciphering the molecular mechanisms underlying NE dynamics during cell division. AU - Kutay, Ulrike AU - HETZER, Martin W ID - 11109 IS - 6 JF - Current Opinion in Cell Biology KW - Cell Biology SN - 0955-0674 TI - Reorganization of the nuclear envelope during open mitosis VL - 20 ER - TY - JOUR AB - The nuclear envelope is a double-layered membrane that encloses the nuclear genome and transcriptional machinery. In dividing cells of metazoa, the nucleus completely disassembles during mitosis, creating the need to re-establish the nuclear compartment at the end of each cell division. Given the crucial role of the nuclear envelope in gene regulation and cellular organization, it is not surprising that its biogenesis and organization have become active research areas. We will review recent insights into nuclear membrane dynamics during the cell cycle. AU - Anderson, Daniel J AU - HETZER, Martin W ID - 11112 IS - 4 JF - Current Opinion in Cell Biology KW - Cell Biology SN - 0955-0674 TI - The life cycle of the metazoan nuclear envelope VL - 20 ER - TY - JOUR AB - The nuclear envelope (NE), a double membrane enclosing the nucleus of eukaryotic cells, controls the flow of information between the nucleoplasm and the cytoplasm and provides a scaffold for the organization of chromatin and the cytoskeleton. In dividing metazoan cells, the NE breaks down at the onset of mitosis and then reforms around segregated chromosomes to generate the daughter nuclei. Recent data from intact cells and cell-free nuclear assembly systems suggest that the endoplasmic reticulum (ER) is the source of membrane for NE assembly. At the end of mitosis, ER membrane tubules are targeted to chromatin via tubule ends and reorganized into flat nuclear membrane sheets by specific DNA-binding membrane proteins. In contrast to previous models, which proposed vesicle fusion to be the principal mechanism of NE formation, these new studies suggest that the nuclear membrane forms by the chromatin-mediated reshaping of the ER. AU - Anderson, Daniel J. AU - HETZER, Martin W ID - 11113 IS - 2 JF - Journal of Cell Science KW - Cell Biology SN - 0021-9533 TI - Shaping the endoplasmic reticulum into the nuclear envelope VL - 121 ER - TY - JOUR AB - The formation of the nuclear envelope (NE) around chromatin is a major membrane-remodelling event that occurs during cell division of metazoa. It is unclear whether the nuclear membrane reforms by the fusion of NE fragments or if it re-emerges from an intact tubular network of the endoplasmic reticulum (ER). Here, we show that NE formation and expansion requires a tubular ER network and occurs efficiently in the presence of the membrane fusion inhibitor GTPγS. Chromatin recruitment of membranes, which is initiated by tubule-end binding, followed by the formation, expansion and sealing of flat membrane sheets, is mediated by DNA-binding proteins residing in the ER. Thus, chromatin plays an active role in reshaping of the ER during NE formation. AU - Anderson, Daniel J. AU - HETZER, Martin W ID - 11115 IS - 10 JF - Nature Cell Biology KW - Cell Biology SN - 1465-7392 TI - Nuclear envelope formation by chromatin-mediated reorganization of the endoplasmic reticulum VL - 9 ER - TY - JOUR AB - Over the last years it has become evident that the nuclear envelope (NE) is more than a passive membrane barrier that separates the nucleus from the cytoplasm. The NE not only controls the trafficking of macromolecules between the nucleoplasm and the cytosol, but also provides anchoring sites for chromosomes and cytoskeleton to the nuclear periphery. Targeting of chromatin to the NE might actually be part of gene expression regulation in eukaryotes. Mutations in certain NE proteins are associated with a diversity of human diseases, including muscular dystrophy, neuropathy, lipodistrophy, torsion dystonia and the premature aging condition progeria. Despite the importance of the NE for cell division and differentiation, relatively little is known about its biogenesis and its role in human diseases. It is our goal to provide a comprehensive view of the NE and to discuss possible implications of NE-associated changes for gene expression, chromatin organization and signal transduction. AU - D’Angelo, M. A. AU - HETZER, Martin W ID - 11117 IS - 3 JF - Cellular and Molecular Life Sciences KW - Cell Biology KW - Cellular and Molecular Neuroscience KW - Pharmacology KW - Molecular Biology KW - Molecular Medicine SN - 1420-682X TI - The role of the nuclear envelope in cellular organization VL - 63 ER - TY - JOUR AB - The nuclear envelope (NE) is a highly specialized membrane that delineates the eukaryotic cell nucleus. It is composed of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) and, in metazoa, the lamina. The NE not only regulates the trafficking of macromolecules between nucleoplasm and cytosol but also provides anchoring sites for chromatin and the cytoskeleton. Through these interactions, the NE helps position the nucleus within the cell and chromosomes within the nucleus, thereby regulating the expression of certain genes. The NE is not static, rather it is continuously remodeled during cell division. The most dramatic example of NE reorganization occurs during mitosis in metazoa when the NE undergoes a complete cycle of disassembly and reformation. Despite the importance of the NE for eukaryotic cell life, relatively little is known about its biogenesis or many of its functions. We thus are far from understanding the molecular etiology of a diverse group of NE-associated diseases. AU - HETZER, Martin W AU - Walther, Tobias C. AU - Mattaj, Iain W. ID - 11120 JF - Annual Review of Cell and Developmental Biology KW - Cell Biology KW - Developmental Biology SN - 1081-0706 TI - Pushing the envelope: Structure, function, and dynamics of the nuclear periphery VL - 21 ER - TY - JOUR AB - The small GTPase Ran is a key regulator of nucleocytoplasmic transport during interphase. The asymmetric distribution of the GTP-bound form of Ran across the nuclear envelope — that is, large quantities in the nucleus compared with small quantities in the cytoplasm — determines the directionality of many nuclear transport processes. Recent findings that Ran also functions in spindle formation and nuclear envelope assembly during mitosis suggest that Ran has a general role in chromatin-centred processes. Ran functions in these events as a signal for chromosome position. AU - HETZER, Martin W AU - Gruss, Oliver J. AU - Mattaj, Iain W. ID - 11123 IS - 7 JF - Nature Cell Biology KW - Cell Biology SN - 1465-7392 TI - The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly VL - 4 ER - TY - JOUR AB - ICln is an ion channel identified by expression cloning using a cDNA library from Madin-Darby canine kidney cells. In all organisms tested so far, only one transcript for the ICln protein could be identified. Here we show that two splice variants of the ICln ion channel can be found in Caenorhabditis elegans. Moreover, we show that these two splice variants of the ICln channel protein, which we termed IClnN1 and IClnN2, can be functionally reconstituted and tested in an artificial lipid bilayer. In these experiments, the IClnN1-induced currents showed no voltage-dependent inactivation, whereas the IClnN2-induced currents fully inactivated at positive potentials. The molecular entity responsible for the voltage-dependent inactivation of IClnN2 is a cluster of positively charged amino acids encoded by exon 2a, which is absent in IClnN1. Our experiments suggest a mechanism of channel inactivation that is similar to the “ball and chain” model proposed for the Shaker potassium channel,i.e. a cluster of positively charged amino acids hinders ion permeation through the channel by a molecular and voltage-dependent interaction at the inner vestibulum of the pore. This hypothesis is supported by the finding that synthetic peptides with the same amino acid sequence as the positive cluster can transform the IClnN1-induced current to the current observed after reconstitution of IClnN2. Furthermore, we show that the nematode ICln gene is embedded in an operon harboring two additional genes, which we termed Nx and Ny. Co-reconstitution of Nx and IClnN2 and functional analysis of the related currents revealed a functional interaction between the two proteins, as evidenced by the fact that the IClnN2-induced current in the presence of Nx was no longer voltage-sensitive. The experiments described indicate that the genome organization in nematodes allows an effective approach for the identification of functional partner proteins of ion channels. AU - Fürst, Johannes AU - Ritter, Markus AU - Rudzki, Jakob AU - Danzl, Johann G AU - Gschwentner, Martin AU - Scandella, Elke AU - Jakab, Martin AU - König, Matthias AU - Oehl, Bernhard AU - Lang, Florian AU - Deetjen, Peter AU - Paulmichl, Markus ID - 13438 IS - 6 JF - Journal of Biological Chemistry KW - Cell Biology KW - Molecular Biology KW - Biochemistry SN - 0021-9258 TI - ICln Ion channel splice variants in Caenorhabditis elegans VL - 277 ER - TY - JOUR AB - Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97–Ufd1–Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97–p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation. AU - HETZER, Martin W AU - Meyer, Hemmo H. AU - Walther, Tobias C. AU - Bilbao-Cortes, Daniel AU - Warren, Graham AU - Mattaj, Iain W. ID - 11125 IS - 12 JF - Nature Cell Biology KW - Cell Biology SN - 1465-7392 TI - Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly VL - 3 ER - TY - JOUR AB - Nuclear import of the two uracil-rich small nuclear ribonucleoprotein (U snRNP) components U1A and U2B′′ is mediated by unusually long and complex nuclear localization signals (NLSs). Here we investigate nuclear import of U1A and U2B′′ in vitro and demonstrate that it occurs by an active, saturable process. Several lines of evidence suggest that import of the two proteins occurs by an import mechanism different to those characterized previously. No cross competition is seen with a variety of previously studied NLSs. In contrast to import mediated by members of the importin-β family of nucleocytoplasmic transport receptors, U1A/U2B′′ import is not inhibited by either nonhydrolyzable guanosine triphosphate (GTP) analogues or by a mutant of the GTPase Ran that is incapable of GTP hydrolysis. Adenosine triphosphate is capable of supporting U1A and U2B′′ import, whereas neither nonhydrolyzable adenosine triphosphate analogues nor GTP can do so. U1A and U2B′′ import in vitro does not require the addition of soluble cytosolic proteins, but a factor or factors required for U1A and U2B′′ import remains tightly associated with the nuclear fraction of conventionally permeabilized cells. This activity can be solubilized in the presence of elevated MgCl2. These data suggest that U1A and U2B′′ import into the nucleus occurs by a hitherto uncharacterized mechanism. AU - HETZER, Martin W AU - Mattaj, Iain W. ID - 11126 IS - 2 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - An Atp-dependent, Ran-independent mechanism for nuclear import of the U1a and U2b′′ spliceosome proteins VL - 148 ER - TY - JOUR AB - Nuclear formation in Xenopus egg extracts requires cytosol and is inhibited by GTPγS, indicating a requirement for GTPase activity. Nuclear envelope (NE) vesicle fusion is extensively inhibited by GTPγS and two mutant forms of the Ran GTPase, Q69L and T24N. Depletion of either Ran or RCC1, the exchange factor for Ran, from the assembly reaction also inhibits this step of NE formation. Ran depletion can be complemented by the addition of Ran loaded with either GTP or GDP but not with GTPγS. RCC1 depletion is only complemented by RCC1 itself or by RanGTP. Thus, generation of RanGTP by RCC1 and GTP hydrolysis by Ran are both required for the extensive membrane fusion events that lead to NE formation. AU - HETZER, Martin W AU - Bilbao-Cortés, Daniel AU - Walther, Tobias C AU - Gruss, Oliver J AU - Mattaj, Iain W ID - 11127 IS - 6 JF - Molecular Cell KW - Cell Biology KW - Molecular Biology SN - 1097-2765 TI - GTP hydrolysis by Ran is required for nuclear envelope assembly VL - 5 ER -