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[
International C. elegans Meeting,
2001]
Ran is a small GTPase implicated in multiple cellular processes throughout the cell cycle. During interphase Ran was first shown to regulate transport of macromolecules in and out of the cell nucleus. Ran probably does so by serving as a molecular switch that signals the subcellular localization to transport receptors. Later Ran was also demonstrated to have a strong effect on microtubule dynamics in mitotic cell extracts where it is required for formation of a bipolar spindle. Closing the cycle, Ran is finally required for formation of a nuclear envelope around chromatin in Xenopus extracts, most likely reflecting a role for Ran in regeneration of the nuclei after cell division. Strikingly, Ran presumably carries out all three functions by creating protein gradients around chromatin. While the function of Ran in nucleocytoplasmic trafficking is well-characterized from numerous biochemical and in vivo studies it remains to be precisely determined if and how Ran regulates the mitotic spindle apparatus and nuclear envelope assembly in living cells. To address these important issues we have investigated the effects of RNAi-mediated suppression of Ran expression in several transgenic C. elegans lines. We have generated C. elegans lines that express various GFP-tagged cellular reporter proteins in the germline enabling us to make detailed time lapse microscopy recordings of early embryos. From these studies we can now evaluate the effects of Ran depletion on the dynamics of chromatin, microtubules and the nuclear envelope during early embryogenesis. Embryos in which Ran expression is inhibited show strong abnormalities in pronuclear and nuclear appearance and in severe cases no nuclei can be detected. This strongly supports an essential role for Ran in generation of a closed nuclear envelope. Secondly, targeting Ran by RNAi prevents formation of a mitotic spindle while astral microtubules are unaffected, which again provides evidence in favor of recent in vitro observations. We are continuously generating suitable markers to study the effects of Ran on the cell cycle and are currently also disrupting the expression of a broad range of C. elegans genes whose homologues in other systems are known to interact with Ran.
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[
International Worm Meeting,
2005]
The nuclear envelope (NE) serves as an important barrier between the cell nucleus and the surrounding cytoplasm, preventing free diffusion of macromolecules between the two compartments. The NE consists of an outer and an inner nuclear membrane, which are joined at nuclear pore complexes (NPCs) to form aqueous channels in the NE. The NPCs are complex structures consisting of approximately 30 different proteins termed nucleoporins, each present in multiple copies. The function of the NPC in transport of proteins and RNAs in and out of the nucleus during interphase has been studied intensively and several nucleoporins have been assigned specific functions during nuclear import and/or export. However, much less is known about the formation of the NE including the NPCs. We have previously identified 20 C. elegans nucleoporins and could show that a subset of these is required for normal nuclear appearance (Galy et al. MBC, 14:5104-5115). We present here evidence that the essential nucleoporin CeNup155/NPP-8 is a key regulator of NE formation. Monitoring GFP::NPP-8 in living embryos revealed that NPP-8 is dynamically associated with the NE during interphase, soluble during mitosis and recruited back to the reforming NE early during anaphase. Depletion of CeNup155/NPP-8 by RNAi prevents the re-association of most nucleoporins with chromatin. Biochemical analysis using Xenopus cell-free extracts confirmed that also vertebrate Nup155 is essential for NE formation, specifically by preventing proper localisation of the nucleoporins Nup107 and POM121 that previously have been shown to regulate early steps of NE formation. Using electron microscopy we could show that Nup155 is required for enclosure of chromatin by a nuclear membrane both in vivo in C. elegans embryos and in vitro in Xenopus extracts. As a consequence of these defects the nuclear lamina fails to assemble and severe chromatin missegregation is observed. In conclusion, we have demonstrated that Nup155 carries out an essential and conserved function in NE and NPC formation. Peter Askjaer and Cerstin Franz contributed equally to this work.
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[
European Worm Meeting,
2002]
The small GTPase Ran has been found to play pivotal roles in several aspects of the cell cycle. We have investigated in real time the role of the Ran GTPase cycle in spindle formation and nuclear envelope regeneration in dividing Caenorhabditis elegans embryos. We found that Ran and its co-factors RanBP2, RanGAP and RCC1 are all essential for reformation of the nuclear envelope after division. Knocking down the expression of any of these component of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation, however, a bipolar spindle eventually forms. Irregular chromatin structures and chromatin bridges due to spindle failure and DNA condensation defects were frequently observed in embryos where the Ran cycle is perturbed. Finally, we have demonstrated that IMA-2, which is a homologue of vertebrate importin , is essential for spindle formation in early embryos, presumably acting downstream of Ran.
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[
MicroPubl Biol,
2019]
Several techniques are available for spatiotemporal control of genome recombination and gene expression in the nematode Caenorhabditis elegans. Here we report a novel tool to combine the powerful FLP-Frt and GAL4-UAS systems to increase their versality and to offer additional levels of control.FLP is an enzyme that catalyzes recombination between two short Frt DNA sequences and is frequently used to excise genomic fragments flanked by Frt sites, thereby either activating or knocking out gene expression, depending on the experimental design (Hubbard, 2014). Recently, we generated multiple strains that stably express FLP in different somatic tissues from single-copy transgenes and demonstrated that they in most cases induce recombination in ~100% of the cells of the expected tissue (Munoz-Jimenez et al., 2017). We subsequently constructed a strain for germline recombination to permanently knock out Frt-flanked genes or exons (Macas-Len and Askjaer, 2018).The GAL4-UAS system is based on the Saccharomyces cerevisiae Gal4p transcription factor and its cognate DNA target called upstream activating sequence (UAS). Typically, this bipartite system includes a series of driver strains expressing GAL4 in specific tissues and one or several strains with an effector gene downstream of UAS repeats. Wang and colleagues from the Sternberg laboratory recently optimized the GAL4-UAS system for C. elegans (cGAL) and reported several tissue-specific cGAL drivers (Wang et al., 2017). Moreover, they have developed a split cGAL toolkit where the DNA binding and activation domains are expressed as individual polypeptides, thereby enabling further fine-tuning of spatiotemporal control: only when and where the two components are co-expressed they will activate the UAS::effector transgene (Wang et al., 2018).
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[
International Worm Meeting,
2009]
The nuclear envelope (NE) is the physical barrier between the nucleus and the cytoplasm. In addition, the NE plays important roles in regulation of gene expression, nuclear stability and anchoring of the chromatin. Within the NE we find macrostructures called nuclear pore complexes (NPC), that are evolutionary conserved assemblies that allow the traffic of proteins and RNA between the nucleus and the cytoplasm. These NPCs are composed of multiple copies of around 30 different proteins called nucleoporins. These nucleoporins interact with each other to form in some cases subcomplexes, such as the NPC subcomplex Nup107-160, consisting of 9 different nucleoporins. The Nup107-160 subcomplex plays important roles in nucleocytoplasmic transport, chromatin organization and kinetochore function. One of the main interests in our lab is to understand the mechanisms underlying the function of the Nup107-160 subcomplex through the analysis of its individual members. Among metazoa, Caenorhabditis elegans is an attractive model system to dissect genetically the functions of the individual Nup107-160 subcomplex members. Mutant alleles have been isolated for several of the Nup107-160 subcomplex genes1. We have initiated our studies with the analysis of deletion alleles of Nup107/npp-5 (
ok1966,
tm3039) and Nup133/npp-15 (
ok1954). Using a broad variety of reporter strains we are investigating how these mutations affect to kinetochore functions, DNA segregation, nucleocytoplasmic transport and cellular response to stress during all stages of development. 1
http://celeganskoconsortium.omrf.org/ and
http://www.shigen.nig.ac.jp/. -
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
The nuclear envelope (NE) is an essential lipo-protein structure that serves numerous pivotal roles. These include compartmentalization, control of nuclear position and morphology, contribution to cell stability, chromatin organization and regulation of gene expression. The mechanism by which the NE controls gene expression is not well understood yet. Traditionally, the anchoring of chromatin to the nuclear periphery has been associated with silencing and heterochromatin formation suggesting that movement of genes from the nuclear interior to the periphery could serve as a way of transcriptional control. However, recent studies have shown that there is also actively transcribed chromatin at the NE, specially associated with the nuclear pore complexes. Also, mutations in NE proteins affect the activity of several transcription factors and histone modifiers. To unravel how the NE can regulate gene expression, we have developed tools to perform, for the first time in C. elegans, genome wide analysis using the DamID method. This method is based on the expression in vivo of chimeric proteins containing an adenine methyltransferase (Dam) from E. coli that is able to methylate the DNA in the vicinity of native binding sites of a chromatin protein. Using the MosSCI technique, we have created strains containing a single copy insertion of Dam fused to the NE proteins emerin/EMR-1 and lamin/LMN-1 and to the nucleoporin Nup98/NPP-10N that with the use of microarrays will provide us a better understanding of the role of the NE in chromatin organization and the regulation of gene expression.
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[
International Worm Meeting,
2011]
The nuclear envelope (NE) has emerged as an important structure that serves numerous pivotal roles in the cell including compartmentalization, control of nuclear position and morphology, contribution to cell stability, chromatin organization and regulation of gene expression. The mechanism by which the NE controls gene expression is not well understood yet. Traditionally, the anchoring of chromatin to the nuclear periphery has been associated with silencing and heterochromatin formation. However, recent studies have shown that there is also actively transcribed chromatin at the NE, specially associated with the nuclear pore complexes. To unravel how the NE can regulate gene expression, we have developed tools to perform genome wide analysis using the DamID method. This method is based on the expression in vivo of chimeric proteins containing an adenine methyltransferase (Dam) from E. coli that methylates the DNA in the vicinity of native binding sites of a chromatin-interacting protein. Using the MosSCI technique, we have created Caenorhabditis elegans strains containing single copy insertions of Dam fused to NE and nuclear pore proteins such as emerin/EMR-1, lamin/LMN-1 and Nup98/NPP-10N. Employing a genetically amenable model system enables us to analyze nuclear architecture across several mutant backgrounds, and we are currently exploring methods to control expression of the Dam fusion proteins in a temporal manner and in specific tissues. We have confirmed the correct expression and localization of our fusion proteins and we are currently analyzing DNA binding sites using whole-genome tiling arrays and qPCR.
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[
International Worm Meeting,
2011]
The nuclear envelope (NE) constitutes a physical barrier between the nucleoplasm and the cytoplasm in eukaryotic cells. The NE is composed by the nuclear lamina and inner and outer nuclear membranes, each enriched for numerous transmembrane and peripheral proteins. Within the NE nuclear pore complexes (NPC) composed of multiple copies of around 30 nucleoporins are responsible for transport in and out of the nucleus. In addition, the NE plays a critical role in organization of nuclear architecture and control of gene transcription by providing an anchoring surface for chromatin and transcription factors. Although the list of NE components has expanded considerably during recent years, it is conceivably that numerous, important, NE proteins remain to be identified. To identify novel NE proteins we have performed a genome-wide RNAi screen for genes that show synthetic lethality with mutations in genes encoding the nucleoporin Nup50/NPP-16 or the NE transmembrane protein LEM-2. NPP-16 plays an important role in resistance to anoxia (Hajeri et al, Mol Biol Cell 21, 712-24) but its function in NE assembly is unknown. LEM-2 shares redundant functions with emerin/EMR-1, another NE membrane protein, since simultaneous depletion of LEM-2 and EMR-1 causes severe cell division defects (Liu et al, PNAS 100, 4598-603). Our screen consisted in three steps: (1) genome-wide analysis in liquid cultures in 96-well plates; (2) rapid verification on single NGM plates; (3) quantitative analysis on NGM plates in triplicates. We are currently analyzing the strongest candidates by time-lapse and immunofluorescence microscopy to identify the precise cellular and developmental phenotypes. Moreover, we will present detailed descriptions of LEM-2 and NPP-16 in terms of expression pattern and dynamics during the cell cycle. Combined, our experiments provide novel insight into NE structure and function.
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[
European Worm Meeting,
2006]
Vincent Galy, Peter Askjaer, Cerstin Franz1, Carmen Lopez-Iglesias5, and Iain W. Mattaj1. The nuclear envelope (NE) of eukaryotic cells mediates nucleo-cytoplasmic transport and contributes to control of gene expression. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. Assembly of nuclear pore complexes (NPCs) and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes. Here we report the identification and characterization of MEL-28, a large conserved protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of
mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. Light microscopy and transmission electron microscopy analysis demonstrate that MEL-28 localizes at NPCs during interphase, at kinetochores in early to mid mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early assembling, stable NE component required for all aspects of NE assembly. Based on its dynamic localization, we suggest that MEL-28 may link chromatin to the assembling NE.
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[
Parasitol Today,
1993]
Arrested development dramatically alters the life history of some species of soil-transmitted nematodes and elicits profound variations in the epidemiology of the infections they cause. Here, Peter Hotez, John Hawdon and Gerhard Schad show how an understanding of the cellular and molecular bases of arrested development may lead to new approaches for the control of ancylostomiasis and related infections.