TY  - JOUR
AB  - The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.
AU  - Chen, Y.-L.
AU  - Jones, A.
AU  - Crawford, A.*
AU  - Sattler, M.
AU  - Ettinger, A.
AU  - Torres-Padilla, M.E.
C1  - 70546
C2  - 55665
TI  - Determinants of minor satellite RNA function in chromosome segregation in mouse embryonic stem cells.
JO  - J. Cell Biol.
VL  - 223
IS  - 7
PY  - 2024
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.
AU  - Wiese, C.*
AU  - Abele, M.*
AU  - Al, B.*
AU  - Altmann, M.
AU  - Steiner, A.*
AU  - Kalbfuß, N.*
AU  - Strohmayr, A.*
AU  - Ravikumar, R.*
AU  - Park, C.H.*
AU  - Brunschweiger, B.*
AU  - Meng, C.*
AU  - Facher, E.*
AU  - Ehrhardt, D.W.*
AU  - Falter-Braun, P.
AU  - Wang, Z.Y.*
AU  - Ludwig, C.*
AU  - Assaad, F.F.*
C1  - 70362
C2  - 55530
CY  - 950 Third Ave, 2nd Flr, New York, Ny 10022 Usa
TI  - Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis.
JO  - J. Cell Biol.
VL  - 223
IS  - 5
PB  - Rockefeller Univ Press
PY  - 2024
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Skin homeostasis is maintained by stem cells, which must communicate to balance their regenerative behaviors. Yet, how adult stem cells signal across regenerative tissue remains unknown due to challenges in studying signaling dynamics in live mice. We combined live imaging in the mouse basal stem cell layer with machine learning tools to analyze patterns of Ca2+ signaling. We show that basal cells display dynamic intercellular Ca2+ signaling among local neighborhoods. We find that these Ca2+ signals are coordinated across thousands of cells and that this coordination is an emergent property of the stem cell layer. We demonstrate that G2 cells are required to initiate normal levels of Ca2+ signaling, while connexin43 connects basal cells to orchestrate tissue-wide coordination of Ca2+ signaling. Lastly, we find that Ca2+ signaling drives cell cycle progression, revealing a communication feedback loop. This work provides resolution into how stem cells at different cell cycle stages coordinate tissue-wide signaling during epidermal regeneration.
AU  - Moore, J.L.*
AU  - Bhaskar, D.*
AU  - Gao, F.*
AU  - Matte-Martone, C.*
AU  - Du, S.*
AU  - Lathrop, E.*
AU  - Ganesan, S.*
AU  - Shao, L.*
AU  - Norris, R.*
AU  - Campamà Sanz, N.*
AU  - Annusver, K.*
AU  - Kasper, M.*
AU  - Cox, A.*
AU  - Hendry, C.*
AU  - Rieck, B.
AU  - Krishnaswamy, S.*
AU  - Greco, V.*
C1  - 67791
C2  - 54269
CY  - 950 Third Ave, 2nd Flr, New York, Ny 10022 Usa
TI  - Cell cycle controls long-range calcium signaling in the regenerating epidermis.
JO  - J. Cell Biol.
VL  - 222
IS  - 7
PB  - Rockefeller Univ Press
PY  - 2023
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is under debate. Here, we use FIB-SEM to image islet β cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules, and microtubules of seven β cells, and generate a comprehensive spatial map of microtubule-organelle interactions. We find that microtubules form nonradial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane, where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus their supportive role in insulin secretion.
AU  - Müller, A.
AU  - Schmidt, D.*
AU  - Xu, C.S.*
AU  - Pang, S.*
AU  - D'Costa, J.V.
AU  - Kretschmar, S.*
AU  - Münster, C.
AU  - Kurth, T.*
AU  - Jug, F.*
AU  - Weigert, M.*
AU  - Hess, H.F.*
AU  - Solimena, M.
C1  - 60907
C2  - 49741
CY  - 950 Third Ave, 2nd Flr, New York, Ny 10022 Usa
TI  - 3D FIB-SEM reconstruction of microtubule-organelle interaction in whole primary mouse β cells.
JO  - J. Cell Biol.
VL  - 220
IS  - 2
PB  - Rockefeller Univ Press
PY  - 2021
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Glucose homeostasis and growth essentially depend on the hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand-receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have resolved only site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we present the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin-site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis for a comprehensive description of ligand-receptor interactions that ultimately will inform new approaches to structure-based drug design.
AU  - Gutmann, T.
AU  - Schäfer, I.B.*
AU  - Poojari, C.*
AU  - Brankatschk, B.
AU  - Vattulainen, I.*
AU  - Strauss, M.*
AU  - Coskun, Ü.
C1  - 57353
C2  - 47737
CY  - 950 Third Ave, 2nd Flr, New York, Ny 10022 Usa
TI  - Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain.
JO  - J. Cell Biol.
VL  - 219
IS  - 1
PB  - Rockefeller Univ Press
PY  - 2020
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Numb functions as an oncosuppressor by inhibiting Notch signaling and stabilizing p53. This latter effect depends on the interaction of Numb with Mdm2, the E3 ligase that ubiquitinates p53 and commits it to degradation. In breast cancer (BC), loss of Numb results in a reduction of p53-mediated responses including sensitivity to genotoxic drugs and maintenance of homeostasis in the stem cell compartment. In this study, we show that the Numb-Mdm2 interaction represents a fuzzy complex mediated by a short Numb sequence encompassing its alternatively spliced exon 3 (Ex3), which is necessary and sufficient to inhibit Mdm2 and prevent p53 degradation. Alterations in the Numb splicing pattern are critical in BC as shown by increased chemoresistance of tumors displaying reduced levels of Ex3-containing isoforms, an effect that could be mechanistically linked to diminished p53 levels. A reduced level of Ex3-less Numb isoforms independently predicts poor outcome in BCs harboring wild-type p53. Thus, we have uncovered an important mechanism of chemoresistance and progression in p53-competent BCs.
AU  - Colaluca, I.N.*
AU  - Basile, A.*
AU  - Freiburger, L.
AU  - D'Uva, V.*
AU  - Disalvatore, D.*
AU  - Vecchi, M.*
AU  - Confalonieri, S.*
AU  - Tosoni, D.*
AU  - Cecatiello, V.*
AU  - Malabarba, M.G.*
AU  - Yang, C.J.*
AU  - Kainosho, M.*
AU  - Sattler, M.
AU  - Mapelli, M.*
AU  - Pece, S.*
AU  - di Fiore, P.P.*
C1  - 52983
C2  - 44265
CY  - New York
SP  - 745-762
TI  - A Numb-Mdm2 fuzzy complex reveals an isoform-specific involvement of Numb in breast cancer.
JO  - J. Cell Biol.
VL  - 217
IS  - 2
PB  - Rockefeller Univ Press
PY  - 2018
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VAN IMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VAN IMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.
AU  - Conic, S.*
AU  - Desplancq, D.*
AU  - Ferrand, A.*
AU  - Fischer, V.*
AU  - Heyer, V.*
AU  - Martin, B.R.S.*
AU  - Pontabry, J.
AU  - Oulad-Abdelghani, M.*
AU  - Babu, K.N.*
AU  - Wright, G.D.*
AU  - Molina, N.*
AU  - Weiss, E.*
AU  - Tora, L.*
C1  - 53423
C2  - 44726
CY  - New York
SP  - 1537-1552
TI  - Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.
JO  - J. Cell Biol.
VL  - 217
IS  - 4
PB  - Rockefeller Univ Press
PY  - 2018
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Insulin receptor (IR) signaling plays a critical role in the regulation of metabolism and growth in multicellular organisms. IRs are unique among receptor tyrosine kinases in that they exist exclusively as covalent (αβ)homodimers at the cell surface. Transmembrane signaling by the IR can therefore not be based on ligand-induced dimerization as such but must involve structural changes within the existing receptor dimer. In this study, using glycosylated full-length human IR reconstituted into lipid nanodiscs, we show by single-particle electron microscopy that insulin binding to the dimeric receptor converts its ectodomain from an inverted U-shaped conformation to a T-shaped conformation. This structural rearrangement of the ectodomain propagates to the transmembrane domains, which are well separated in the inactive conformation but come close together upon insulin binding, facilitating autophosphorylation of the cytoplasmic kinase domains.
AU  - Gutmann, T.
AU  - Kim, K.H.*
AU  - Grzybek, M.
AU  - Walz, T.*
AU  - Coskun, Ü.
C1  - 53058
C2  - 44288
SP  - 1643-1649
TI  - Visualization of ligand-induced transmembrane signaling in the full-length human insulin receptor.
JO  - J. Cell Biol.
VL  - 217
IS  - 5
PY  - 2018
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - T helper cell subsets orchestrate context- and pathogen-specific responses of the immune system. They mostly do so by secreting specific cytokines that attract or induce activation and differentiation of other immune or nonimmune cells. The differentiation of T helper 1 (Th1), Th2, T follicular helper, Th17, and induced regulatory T cell subsets from naive T cells depends on the activation of intracellular signal transduction cascades. These cascades originate from T cell receptor and costimulatory receptor engagement and also receive critical input from cytokine receptors that sample the cytokine milieu within secondary lymphoid organs. Signal transduction then leads to the expression of subset-specifying transcription factors that, in concert with other transcription factors, up-regulate downstream signature genes. Although regulation of transcription is important, recent research has shown that posttranscriptional and posttranslational regulation can critically shape or even determine the outcome of Th cell differentiation. In this review, we describe how specific microRNAs, long noncoding RNAs, RNA-binding proteins, and ubiquitin-modifying enzymes regulate their targets to skew cell fate decisions.
AU  - Hoefig, K.P.
AU  - Heissmeyer, V.
C1  - 53450
C2  - 44870
CY  - 950 Third Ave, 2nd Flr, New York, Ny 10022 Usa
SP  - 2615-2631
TI  - Posttranscriptional regulation of T helper cell fate decisions.
JO  - J. Cell Biol.
VL  - 217
IS  - 8
PB  - Rockefeller Univ Press
PY  - 2018
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Type II isoforms of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA-II) contain a phosphorylatable epitope within the inhibitory domain of RII subunits (pRII) with still unclear function. In vitro, RII phosphorylation occurs in the absence of cAMP, whereas staining of cells with pRII-specific antibodies revealed a cAMP-dependent pattern. In sensory neurons, we found that increased pRII immunoreactivity reflects increased accessibility of the already phosphorylated RII epitope during cAMP-induced opening of the tetrameric RII2:C-2 holoenzyme. Accordingly, induction of pRII by cAMP was sensitive to novel inhibitors of dissociation, whereas blocking catalytic activity was ineffective. Also in vitro, cAMP increased the binding of pRII antibodies to RII2:C-2 holoenzymes. Identification of an antibody specific for the glycine-rich loop of catalytic subunits facing the pRII-epitope confirmed activity-dependent binding with similar kinetics, proving that the reassociation is rapid and precisely controlled. Mechanistic modeling further supported that RII phosphorylation precedes cAMP binding and controls the inactivation by modulating the reassociation involving the coordinated action of phosphodiesterases and phosphatases.
AU  - Isensee, J.*
AU  - Kaufholz, M.*
AU  - Knape, M.J.*
AU  - Hasenauer, J.
AU  - Hammerich, H.*
AU  - Gonczarowska-Jorge, H.*
AU  - Zahedi, R.P.*
AU  - Schwede, F.*
AU  - Herberg, F.W.*
AU  - Hucho, T.*
C1  - 53833
C2  - 45068
CY  - 950 Third Ave, 2nd Flr, New York, Ny 10022 Usa
SP  - 2167-2184
TI  - PKA-RII subunit phosphorylation precedes activation by cAMP and regulates activity termination.
JO  - J. Cell Biol.
VL  - 217
IS  - 6
PB  - Rockefeller Univ Press
PY  - 2018
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Crown. In mammals, histone H1 consists of a family of related proteins, including five replication-dependent (H1.1-H1.5) and two replication-independent (H1.10 and H1.0) subtypes, all expressed in somatic cells. To systematically study the expression and function of H1 subtypes, we generated knockin mouse lines in which endogenous H1 subtypes are tagged. We focused on key developmental periods when epigenetic reprogramming occurs: early mouse embryos and primordial germ cell development. We found that dynamic changes in H1 subtype expression and localization are tightly linked with chromatin remodeling and might be crucial for transitions in chromatin structure during reprogramming. Although all somatic H1 subtypes are present in the blastocyst, each stage of preimplantation development is characterized by a different combination of H1 subtypes. Similarly, the relative abundance of somatic H1 subtypes can distinguish male and female chromatin upon sex differentiation in developing germ cells. Overall, our data provide new insights into the chromatin changes underlying epigenetic reprogramming. We suggest that distinct H1 subtypes may mediate the extensive chromatin remodeling occurring during epigenetic reprogramming and that they may be key players in the acquisition of cellular totipotency and the establishment of specific cellular states.
AU  - Izzo, A.
AU  - Ziegler-Birling, C.*
AU  - Hill, P.W.S.*
AU  - Brondani, L.*
AU  - Hajkova, P.*
AU  - Torres-Padilla, M.E.
AU  - Schneider, R.
C1  - 52110
C2  - 43737
CY  - New York
SP  - 3017-3028
TI  - Dynamic changes in H1 subtype composition during epigenetic reprogramming.
JO  - J. Cell Biol.
VL  - 216
IS  - 10
PB  - Rockefeller Univ Press
PY  - 2017
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3',5'-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 10(5) GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces "molecule noise." Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons.
AU  - Pichlo, M.*
AU  - Bungert-Plümke, S.*
AU  - Weyand, I.*
AU  - Seifert, R.*
AU  - Bönigk, W.*
AU  - Strünker, T.*
AU  - Kashikar, N.D.*
AU  - Goodwin, N.*
AU  - Müller, A.*
AU  - Pelzer, P.*
AU  - Van, Q.*
AU  - Enderlein, J.*
AU  - Klemm, C.*
AU  - Krause, E.*
AU  - Trötschel, C.*
AU  - Poetsch, A.*
AU  - Kremmer, E.
AU  - Kaupp, U.B.*
C1  - 31955
C2  - 34902
CY  - New York
SP  - 541-557
TI  - High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor.
JO  - J. Cell Biol.
VL  - 206
IS  - 4
PB  - Rockefeller Univ Press
PY  - 2014
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - The assembly and composition of ribonucleic acid (RNA)-transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.
AU  - Heym, R.G.
AU  - Zimmermann, D.*
AU  - Edelmann, F.
AU  - Israel, L.*
AU  - Ökten, Z.*
AU  - Kovar, D.R.*
AU  - Niessing, D.
C1  - 28861
C2  - 33547
SP  - 971-984
TI  - In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation.
JO  - J. Cell Biol.
VL  - 203
IS  - 6
PY  - 2013
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Macrophages are long-lived target cells for HIV infection and are considered viral reservoirs. HIV assembly in macrophages occurs in virus-containing compartments (VCCs) in which virions accumulate and are stored. The regulation of the trafficking and release of these VCCs remains unknown. Using high resolution light and electron microscopy of HIV-1-infected primary human macrophages, we show that the spatial distribution of VCCs depended on the microtubule network and that VCC-limiting membrane was closely associated with KIF3A+ microtubules. Silencing KIF3A strongly decreased virus release from HIV-1-infected macrophages, leading to VCC accumulation intracellularly. Time-lapse microscopy further suggested that VCCs and associated KIF3A move together along microtubules. Importantly, KIF3A does not play a role in HIV release from T cells that do not possess VCCs. These results reveal that HIV-1 requires the molecular motor KIF3 to complete its cycle in primary macrophages. Targeting this step may lead to novel strategies to eliminate this viral reservoir.
AU  - Gaudin, R.*
AU  - Cunha de Alencar, B.*
AU  - Jouve, M.*
AU  - Bèrre, S.*
AU  - Le Bouder, E.*
AU  - Schindler, M.
AU  - Varthaman, A.*
AU  - Gobert, F.-X.*
AU  - Benaroch, P.*
C1  - 11041
C2  - 30474
SP  - 467-479
TI  - Critical role for the kinesin KIF3A in the HIV life cycle in primary human macrophages.
JO  - J. Cell Biol.
VL  - 199
IS  - 3
PB  - Rockefeller Univ. Press
PY  - 2012
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Systematic affinity purification combined with mass spectrometry analysis of N- and C-tagged cytoplasmic Hsp70/Hsp110 chaperones was used to identify new roles of Hsp70/Hsp110 in the cell. This allowed the mapping of a chaperone-protein network consisting of 1,227 unique interactions between the 9 chaperones and 473 proteins and highlighted roles for Hsp70/Hsp110 in 14 broad biological processes. Using this information, we uncovered an essential role for Hsp110 in spindle assembly and, more specifically, in modulating the activity of the widely conserved kinesin-5 motor Cin8. The role of Hsp110 Sse1 as a nucleotide exchange factor for the Hsp70 chaperones Ssa1/Ssa2 was found to be required for maintaining the proper distribution of kinesin-5 motors within the spindle, which was subsequently required for bipolar spindle assembly in S phase. These data suggest a model whereby the Hsp70-Hsp110 chaperone complex antagonizes Cin8 plus-end motility and prevents premature spindle elongation in S phase.
AU  - Makhnevych, T.*
AU  - Wong, P.
AU  - Pogoutse, O.*
AU  - Vizeacoumar, F.J.*
AU  - Greenblatt, J.F.*
AU  - Emili, A.*
AU  - Houry, W.A.*
C1  - 8630
C2  - 30263
SP  - 623-636
TI  - Hsp110 is required for spindle length control.
JO  - J. Cell Biol.
VL  - 198
IS  - 4
PB  - Rockefeller Univ. Press
PY  - 2012
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Whether or not metazoan replication initiates at random or specific but flexible sites is an unsolved question. The lack of sequence specificity in origin recognition complex (ORC) DNA binding complicates genome-scale chromatin immunoprecipitation (ChIP)-based studies. Epstein-Barr virus (EBV) persists as chromatinized minichromosomes that are replicated by the host replication machinery. We used EBV to investigate the link between zones of pre-replication complex (pre-RC) assembly, replication initiation, and micrococcal nuclease (MNase) sensitivity at different cell cycle stages in a genome-wide fashion. The dyad symmetry element (DS) of EBV's latent origin, a well-established and very efficient pre-RC assembly region, served as an internal control. We identified 64 pre-RC zones that correlate spatially with 57 short nascent strand (SNS) zones. MNase experiments revealed that pre-RC and SNS zones were linked to regions of increased MNase sensitivity, which is a marker of origin strength. Interestingly, although spatially correlated, pre-RC and SNS zones were characterized by different features. We propose that pre-RCs are formed at flexible but distinct sites, from which only a few are activated per single genome and cell cycle.
AU  - Papior, P.
AU  - Arteaga-Salas, J.M.*
AU  - Günther, T.*
AU  - Grundhoff, A.*
AU  - Schepers, A.
C1  - 8629
C2  - 30353
SP  - 509-528
TI  - Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus.
JO  - J. Cell Biol.
VL  - 198
IS  - 4
PB  - Rockefeller Univ. Press
PY  - 2012
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - The position of the centrosome ahead of the nucleus has been considered crucial for coordinating neuronal migration in most developmental situations. The proximity of the centrosome has also been correlated with the site of axonogenesis in certain differentiating neurons. Despite these positive correlations, accumulating experimental findings appear to negate a universal role of the centrosome in determining where an axon forms, or in leading the migration of neurons. To further examine this controversy in an in vivo setting, we have generated cell type-specific multi-cistronic gene expression to monitor subcellular dynamics in the developing zebrafish cerebellum. We show that migration of rhombic lip-derived neurons is characterized by a centrosome that does not persistently lead the nucleus, but which is instead regularly overtaken by the nucleus. In addition, axonogenesis is initiated during the onset of neuronal migration and occurs independently of centrosome proximity. These in vivo data reveal a new temporal orchestration of organelle dynamics and provide important insights into the variation in intracellular processes during vertebrate brain differentiation.
AU  - Distel, M.
AU  - Hocking, J.C.
AU  - Volkmann, K.
AU  - Köster, R.W.
C1  - 5767
C2  - 27728
SP  - 875-890
TI  - The centrosome neither persistently leads migration nor determines the site of axonogenesis in migrating neurons in vivo.
JO  - J. Cell Biol.
VL  - 191
IS  - 4
PB  - Rockefeller Univ. Press
PY  - 2010
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Type V myosin (MyoV)-dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2-tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail-lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain.
AU  - Heuck, A.
AU  - Fetka, I.*
AU  - Brewer, D.N.*
AU  - Hüls, D.
AU  - Munson, M.*
AU  - Jansen, R.P.*
AU  - Niessing, D.
C1  - 5545
C2  - 27601
SP  - 497-510
TI  - The structure of the Myo4p globular tail and its function in ASH1 mRNA localization.
JO  - J. Cell Biol.
VL  - 189
IS  - 3
PB  - Rockefeller Univ. Press
PY  - 2010
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Nucleosomal incorporation of specialized histone variants is an important mechanism to generate different functional chromatin states. Here, we describe the identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y. Their messenger RNAs are found in certain human cell lines, in addition to several normal and malignant human tissues. In keeping with their primate specificity, H3.X and H3.Y are detected in different brain regions. Transgenic H3.X and H3.Y proteins are stably incorporated into chromatin in a similar fashion to the known H3 variants. Importantly, we demonstrate biochemically and by mass spectrometry that endogenous H3.Y protein exists in vivo, and that stress stimuli, such as starvation and cellular density, increase the abundance of H3.Y-expressing cells. Global transcriptome analysis revealed that knockdown of H3.Y affects cell growth and leads to changes in the expression of many genes involved in cell cycle control. Thus, H3.Y is a novel histone variant involved in the regulation of cellular responses to outside stimuli.
AU  - Wiedemann, S.M.*
AU  - Mildner, S.N.*
AU  - Bönisch, C.*
AU  - Israel, L.*
AU  - Maiser, A.*
AU  - Matheisl, S.*
AU  - Straub, T.*
AU  - Merkl, R.*
AU  - Leonhardt, H.*
AU  - Kremmer, E.
AU  - Schermelleh, L.*
AU  - Hake, S.B.*
C1  - 4142
C2  - 27965
SP  - 777-791
TI  - Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y.
JO  - J. Cell Biol.
VL  - 190
IS  - 5
PB  - Rockfeller Univ. Press
PY  - 2010
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Cyclin-dependent kinases (Cdks) fulfill key functions in many cellular processes, including cell cycle progression and cytoskeletal dynamics. A limited number of Cdk substrates have been identified with few demonstrated to be regulated by Cdk-dependent phosphorylation. We identify on protein expression arrays novel cyclin E-Cdk2 substrates, including SIRT2, a member of the Sirtuin family of NAD(+)-dependent deacetylases that targets alpha-tubulin. We define Ser-331 as the site phosphorylated by cyclin E-Cdk2, cyclin A-Cdk2, and p35-Cdk5 both in vitro and in cells. Importantly, phosphorylation at Ser-331 inhibits the catalytic activity of SIRT2. Gain- and loss-of-function studies demonstrate that SIRT2 interfered with cell adhesion and cell migration. In postmitotic hippocampal neurons, neurite outgrowth and growth cone collapse are inhibited by SIRT2. The effects provoked by SIRT2, but not those of a nonphosphorylatable mutant, are antagonized by Cdk-dependent phosphorylation. Collectively, our findings identify a posttranslational mechanism that controls SIRT2 function, and they provide evidence for a novel regulatory circuitry involving Cdks, SIRT2, and microtubules.
AU  - Pandithage, R.*
AU  - Lilischkis, R.*
AU  - Harting, K.*
AU  - Wolf, A.*
AU  - Jedamzik, B.*
AU  - Luscher-Firzlaff, J.*
AU  - Vervoorts, J.*
AU  - Lasonder, E.*
AU  - Kremmer, E.
AU  - Knöll, B.*
AU  - Lüscher, B.*
C1  - 2070
C2  - 25702
SP  - 915-929
TI  - The regulation of SIRT2 function by cyclin-dependent kinases affects cell motility.
JO  - J. Cell Biol.
VL  - 180
IS  - 5
PB  - Rockefeller Univ. Press
PY  - 2008
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - Chlamydiae replicate in a vacuole within epithelial cells and commonly induce cell damage and a deleterious inflammatory response of unknown molecular pathogenesis. The chlamydial protease-like activity factor (CPAF) translocates from the vacuole to the cytosol, where it cleaves several cellular proteins. CPAF is synthesized as an inactive precursor that is processed and activated during infection. Here, we show that CPAF can be activated in uninfected cells by experimentally induced oligomerization, reminiscent of the activation mode of initiator caspases. CPAF activity induces proteolysis of cellular substrates including two novel targets, cyclin B1 and PARP, and indirectly results in the processing of pro-apoptotic BH3-only proteins. CPAF activation induces striking morphological changes in the cell and, later, cell death. Biochemical and ultrastructural analysis of the cell death pathway identify the mechanism of cell death as nonapoptotic. Active CPAF in uninfected human cells thus mimics many features of chlamydial infection, implicating CPAF as a major factor of chlamydial pathogenicity, Chlamydia-associated cell damage, and inflammation.
AU  - Paschen, S.A.*
AU  - Christian, J.G.
AU  - Vier, J.*
AU  - Schmidt, F.
AU  - Walch, A.K.
AU  - Ojcius, D.M.*
AU  - Häcker, G.*
C1  - 3270
C2  - 25382
SP  - 117-127
TI  - Cytopathicity of Chlamydia is largely reproduced by expression of a single chlamydial protease.
JO  - J. Cell Biol.
VL  - 182
IS  - 1
PB  - Rockefeller Univ. Press
PY  - 2008
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - The Notch ligands Dll1 and Dll3 are coexpressed in the presomitic mesoderm of mouse embryos. Despite their coexpression, mutations in Dll1 and Dll3 cause strikingly different defects. To determine if there is any functional equivalence, we replaced Dll1 with Dll3 in mice. Dll3 does not compensate for Dll1; DLL1 activates Notch in Drosophila wing discs, but DLL3 does not. We do not observe evidence for antagonism between DLL1 and DLL3, or repression of Notch activity in mice or Drosophila. In vitro analyses show that differences in various domains of DLL1 and DLL3 individually contribute to their biochemical nonequivalence. In contrast to endogenous DLL1 located on the surface of presomitic mesoderm cells, we find endogenous DLL3 predominantly in the Golgi apparatus. Our data demonstrate distinct in vivo functions for DLL1 and DLL3. They suggest that DLL3 does not antagonize DLL1 in the presomitic mesoderm and warrant further analyses of potential physiological functions of DLL3 in the Golgi network.
AU  - Geffers, I.*
AU  - Serth, K.*
AU  - Chapman, G.*
AU  - Jaekel, R.*
AU  - Schuster-Gossler, K.*
AU  - Cordes, R.*
AU  - Sparrow, D.B.*
AU  - Kremmer, E.
AU  - Dunwoodie, S.L.*
AU  - Klein, T.*
AU  - Gossler, A.*
C1  - 1431
C2  - 24803
SP  - 465-476
TI  - Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo.
JO  - J. Cell Biol.
VL  - 178
IS  - 3
PB  - Rockefeller Univ. Press
PY  - 2007
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen-binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference-mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required.
AU  - Spada, F.*
AU  - Haemmer, A.*
AU  - Kuch, D.*
AU  - Rothbauer, U.*
AU  - Schermelleh, L.*
AU  - Kremmer, E.
AU  - Carell, T.*
AU  - Längst, G.*
AU  - Leonhardt, H.*
C1  - 3176
C2  - 24795
SP  - 565-571
TI  - DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells.
JO  - J. Cell Biol.
VL  - 176
IS  - 5
PB  - Rockefeller Univ. Press
PY  - 2007
SN  - 0021-9525
ER  - 

TY  - JOUR
AU  - Hölzel, M.
AU  - Rohrmoser, M.
AU  - Schlee, M.
AU  - Grimm, T.
AU  - Harasim, T.
AU  - Malamoussi, A.
AU  - Gruber-Eber, A.
AU  - Kremmer, E.
AU  - Hiddemann, W.
AU  - Bornkamm, G.W.
AU  - Eick, D.
C1  - 667
C2  - 22763
SP  - 367-378
TI  - Mammalian WDR12 is a novel member of the Pes1-Bop1 complex and is required for ribosome biogenesis and cell proliferation.
JO  - J. Cell Biol.
VL  - 170
PY  - 2005
SN  - 0021-9525
ER  - 

TY  - JOUR
AU  - Cremer, M.*
AU  - Küppler, K.*
AU  - Wagler, B.*
AU  - Wizelman, L.*
AU  - von Hase, J.*
AU  - Weiland, Y.
AU  - Kreja, L.*
AU  - Diebold, J.*
AU  - Speicher, M.R.
AU  - Cremer, Th.*
C1  - 10105
C2  - 21584
SP  - 809-820
TI  - Inheritance of gene density-related higher order chromatin arrangements in normal and tumor cell nuclei. 
JO  - J. Cell Biol.
VL  - 162
PY  - 2003
SN  - 0021-9525
ER  - 

TY  - JOUR
AU  - Closs, E.I.
AU  - Murray, A.B.
AU  - Schmidt, J.
AU  - Schön, A.
AU  - Erfle, V.
AU  - Strauss, P.G.
C1  - 18272
C2  - 11493
TI  - C-fos Expression Precedes Osteogenic Differentiation of Cartilage Cells in vitro.
JO  - J. Cell Biol.
PY  - 1990
SN  - 0021-9525
ER  - 

TY  - JOUR
AB  - The cartilagenous tissue of mandibular condyles of newborn mice contains progenitor cells as well as young and mature chondrogenic cells. During in vitro cultivation of the tissue, progenitor cells undergo osteogenic differentiation and form new bone (Silberman, M., D. Lewison, H. Gonen, M.A. Lizarbe, and K. von der Mark, 1983. Anat. Rec. 206: 373-383). We have studied the expression of genes that typify osteogenic differentiation in mandibular condyles during in vitro cultivation. RNAs of the genes for collagen type I, osteonectin, alkaline phosphatase, and bone gla protein were sequentially expressed in progenitor cells and hypertrophic chondrocytes during culture. Osteopontin expression peaked in both the early and the late phase of the differentiation process. The data indicate a distinct sequence of expression of osteoblast-specific genes during osteogenic differentiation and new bone formation in mandibular condyles.
AU  - Strauß, P.G.
AU  - Closs, E.I.
AU  - Schmidt, J.
AU  - Erfle, V.F.
C1  - 41192
C2  - 36480
SP  - 1369-1378
TI  - Gene expression during osteogenic differentiation in mandibular condyles in vitro.
JO  - J. Cell Biol.
VL  - 110
IS  - 4
PY  - 1990
SN  - 0021-9525
ER  -