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Protein kinases regulate a multitude of processes by reversible phosphorylation of target molecules. Induction of cell proliferation and differentiation are fundamental to development and rely on tightly controlled kinase activities. Vaccinia-Related Kinases (VRKs) have emerged as a multifunctional family of kinases with essential functions conserved, from nematodes and fruit flies, to humans. VRK substrates include chromatin and transcription factors, whereas deregulation of VRKs is implicated in sterility, cancer and neurological defects. In contrast to previous observations, we describe here that Caenorhabditis elegans VRK-1 is expressed in all cell types, including proliferating and post-mitotic cells. Despite the ubiquitous expression pattern, we find that vrk-1 mutants are particularly impaired in uterine development. Our data show that VRK-1 is required for uterine cell proliferation and differentiation. Moreover, the anchor cell, a specialized uterine cell, fails to fuse with neighboring cells to form the utse syncytium in vrk-1 mutants, thus providing further insight on the role of VRKs in organogenesis. ; We gratefully acknowledge funding from the Spanish Ministry of Economy and Competitiveness (BFU2013-42709P), the Autonomous Government of Andalusia (P08-CVI-3920) and the European Regional Development Fund to P.A. and a postdoctoral contract from the Autonomous Government of Andalusia to A.D. ; Peer reviewed

The Nuclear Pore Complex (NPC) is a fascinating structure whose functional relevance and complexity attract significant interest. Within the NPC, several different subcomplexes interact with each other to form a highly conserved and stable structure. One of these subcomplexes is the NUP107 complex, constituted by 7-9 members. A wide variety of functions have been ascribed to the NUP107 complex, ranging from NPC assembly to mRNA export to cell differentiation. Recently, genetic dissection of the NUP107 complex has provided novel insight to the assembly of the complex and has, moreover, revealed an unexpected connection with the mitotic spindle assembly checkpoint protein MAD1. ; The Spanish Ministry of Economy and Competitiveness (BFU2010-15478/BMC), the Autonomous Government of Andalusia (P08-CVI-3920) and the European Regional Development Fund are acknowledged for financial support. ; Peer Reviewed

The inner nuclear membrane proteins emerin and LEMD2 have both overlapping and separate functions in regulation of nuclear organization, gene expression and cell differentiation. We report here that emerin (EMR-1) and LEM domain protein 2 (LEM-2) are expressed in all tissues throughout Caenorhaditis elegans development but their relative distribution differs between cell types. The ratio of EMR-1 to LEM-2 is particularly high in contractile tissues, intermediate in neurons and hypodermis and lowest in intestine and germ line. We find that LEM-2 is recruited earlier than EMR-1 to reforming nuclear envelopes, suggesting the presence of separate mitotic membrane compartments and specific functions of each protein. Concordantly, we observe that nuclei of lem-2 mutant embryos, but not of emr-1 mutants, have reduced nuclear circularity. Finally, we uncover a so-far-unknown role of LEM-2 in nuclear separation and anchoring of microtubule organizing centers. ; This work was funded by the Spanish Ministry of Economy and Competitiveness [grant numbers BFU2010-15478 and BFU2013-42709P], and the Autonomous Government of Andalusia [grant number P08-CVI-3920]. ; Peer reviewed

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License. ; The precise developmentalmap of the Caenorhabditis elegans cell lineage, as well as a complete genome sequence and feasibility of genetic manipulation make this nematode species highly attractive to study the role of epigenetics during development. Genetic dissection of phenotypical traits, such as formation of egg-laying organs or starvation- resistant dauer larvae, has illustrated how chromatin modifiers may regulate specific cell-fate decisions and behavioral programs. Moreover, the transparent body of C. elegans facilitates non-invasive microscopy to study tissue-specific accumulation of heterochromatin at the nuclear periphery. We also review here recent findings on how small RNA molecules contribute to epigenetic control of gene expression that can be propagated for several generations and eventually determine longevity. ; This work was supported by the CNRS [to F.P.]; Fondation ARC [SFI20101201659 to F.P.]; Ligue Contre le Cancer [to F.P.]; the Spanish Ministry of Economy and Competitiveness [BFU2010-15478 to P.A.]; the Autonomous Government of Andalusia [P08-CVI-3920 to P.A.]; and the European Regional Development Fund. ; Peer Reviewed

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License. ; During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis. ; This work was supported by an ATIP grant from Centre National de la Recherche Scientifique and Human Frontiers Science Program Grant RGP0034/2010 to M.D. W.N. is the recipient of a PhD fellowship from the French Government. ; Peer Reviewed

11 pages, 5 figures, 1 table.-- PMID: 19337973 [PubMed]. ; The Vaccinia-Related Kinases (VRKs) branched off early from the family of casein kinase (CK) I and compose a relatively uncharacterized family of the kinome. The VRKs were discovered due to their close sequence relation to the vaccinia virus B1R serine/threonine kinase. They were first described in phosphorylation of transcription factors that led to the discovery of an autoregulatory mechanism between VRK and the tumor suppressor transcription factor p53. The relevance of VRKs has broadened recently by introduction of its members as essential regulators in cell signaling, nuclear envelope dynamics, chromatin modifications, apoptosis and cellular stress response. Several phosphorylation substrates have been described, as well as the first positive and negative regulators of VRK. We provide an overview of the VRKs across species and discuss the wide diversity of cellular and organismal requirements for this kinase family. ; Work in the Askjaer laboratory is supported by the Spanish Ministry of Science and Innovation (BFU-2007-60116) and the Andalusian Regional Government (P07-CVI-02697). ; Peer reviewed

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License. ; Nuclear pore complexes consist of several subcomplexes. The NUP107 complex is important for nucleocytoplasmic transport, nuclear envelope assembly, and kinetochore function. However, the underlying molecular mechanisms and the roles of individual complex members remain elusive. We report the first description of a genetic disruption of NUP107 in a metazoan. Caenorhabditis elegans NUP107/npp-5 mutants display temperature-dependent lethality. Surprisingly, NPP-5 is dispensable for incorporation of most nucleoporins into nuclear pores and for nuclear protein import. In contrast, NPP-5 is essential for proper kinetochore localization of NUP133/NPP-15, another NUP107 complex member, whereas recruitment of NUP96/NPP-10C and ELYS/MEL-28 is NPP-5 independent. We found that kinetochore protein NUF2/HIM-10 and Aurora B/AIR-2 kinase are less abundant on mitotic chromatin upon NPP-5 depletion. npp-5 mutants are hypersensitive to anoxia, suggesting that the spindle assembly checkpoint (SAC) is compromised. Indeed, NPP-5 interacts genetically and physically with SAC protein MAD1/MDF-1, whose nuclear envelope accumulation requires NPP-5. Thus our results strengthen the emerging connection between nuclear pore proteins and chromosome segregation. © 2012 Tatavarty et al. ; This work was funded by the Spanish Ministry of Science and Innovation (BFU-2007-60116, BFU-2010-15478), the Spanish National Research Council (200820I028), the European Regional Development Fund, and the Regional Government of Andalusia (P07-CVI-02697). In addition, we acknowledge the Fundación Ramón Areces for a fellowship to E.R. and the Junta de Andalucía for institutional support. Some nematode strains used in this work were provided by the National Bioresource Project for the Nematode C. elegans (directed by Shohei Mitani), the C. elegans Gene Knockout Consortium, and the Caenorhabditis Genetic Center (University of Minnesota, Minneapolis, MN), which is funded by the National Institutes of Health, National Center for Research Resources. ; Peer Reviewed

Nuclear envelope (NE) architecture and aging have been associated since the discovery that certain human progeria diseases are due to perturbations in processing of lamin A protein, generating alterations in NE morphology. However, whether changes in the NE are a causal effect of normal and premature aging is still controversial. Caenorhabditis elegans is a model organism where observations supporting both, dependent and independent roles of nuclear architecture in the aging process, have been reported. We found that the long-lived glp-1 mutant and dietary restriction delayed age-associated nuclear morphology changes. In addition, we observed that the long-lived mutant of the insulin/IGF receptor daf-2 delayed the age-dependent changes of nuclear architecture at 25 °C, as previously described. However, when daf-2 animals were incubated at 20 °C they remained long-lived, but nuclear appearance changed at similar rate as in the wild type. This supports the idea that both phenotypes, longevity and maintenance of nuclear architecture are tightly associated but can be separated and argues that nuclear morphology deterioration is not a cause of the natural aging process. ; This work was funded by the Autonomous Government of Andalusia (P07-CVI- 02697). In addition, we wish to acknowledge Fundación Ramón Areces for a fellowship to ER. Some nematode strains used in this work were provided by the ''Caenorhabditis Genetic Center'', which is funded by the NIH National Center for Research Resources (NCRR). ; Peer Reviewed

The factors that sequester transcriptionally repressed heterochromatin at the nuclear periphery are currently unknown. In a genome-wide RNAi screen, we found that depletion of S-adenosylmethionine (SAM) synthetase reduces histone methylation globally and causes derepression and release of heterochromatin from the nuclear periphery in Caenorhabditis elegans embryos. Analysis of histone methyltransferases (HMTs) showed that elimination of two HMTs, MET-2 and SET-25, mimics the loss of SAM synthetase, abrogating the perinuclear attachment of heterochromatic transgenes and of native chromosomal arms rich in histone H3 lysine 9 methylation. The two HMTs target H3K9 in a consecutive fashion: MET-2, a SETDB1 homolog, mediates mono- and dimethylation, and SET-25, a previously uncharacterized HMT, deposits H3K9me3. SET-25 colocalizes with its own product in perinuclear foci, in a manner dependent on H3K9me3, but not on its catalytic domain. This colocalization suggests an autonomous, self-reinforcing mechanism for the establishment and propagation of repeat-rich heterochromatin. ; This work was supported by the European Union Network of Excellence "Epigenome", the Novartis Research Foundation, the "Fondation Suisse de recherche sur les maladies musculaires", the Spanish Ministry of Science and Innovation (BFU2010-15478 to P.A.), and EMBO (ASTF 478-2011 to C.G.A.). ; Peer reviewed

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD. ; J.C.H. was supported by an FP7 Marie Curie Action Intra-European Fellowship and by a grant from the Foundation Suisse de Recherche sur les Maladies Musculaires. A.M. was funded by the same and by a Marie Heim-Vögtlin grant from the Swiss National Science Foundation. C.M.-J., R.R.-B., and P.A. thank the Spanish State Research Agency and the European Regional Development Fund (BFU2016-79313-P and MDM-2016- 0687) for support. This project received funding from the European Research Council under the European Union's Horizon 2020 Research and Innovation program (Epiherigans grant no. 743312 to S.M.G.). ; Peer reviewed

Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress and have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode. ; Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) and by the Japanese National Bioresource Project. We thank Cristina Cecchi, Amir Shapir, Paul Sternberg, Bart Braeckman, Chris Link, Simon Tuck, Keith Blackwell, José López-Barneo and LivOn Labs for strains and chemicals, Katie McCallum and Danielle Garsin for their help with skn-1 experiments and Elizabeth Veal and Michel Toledano for critical reading of the manuscript. Prof. Rafael Fernández-Chacón is deeply acknowledged for his continuous support. AMV was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-64408-P) and the Instituto de Salud Carlos III (PI11/00072, cofinanced by the Fondo Social Europeo) and is a member of the GENIE and EU-ROS Cost Action of the European Union. NJS was supported by a grant from the US National Institutes of Health National Institute for Arthritis and Musculoskeletal and Skin Diseases (AR-054342). CJG was funded by a Doctoral Training Studentship provided by the University of Nottingham. PA was supported by the Spanish Ministry of Economy and Competitiveness (BFU2013-42709P). JC is a member of the GENIE Cost action and was funded by Rioja Salud Foundation (Onco-2-2015). ; Peer Reviewed

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans loss-of-function mutants for the glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human, as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH-depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation. ; . The Spanish Ministry of Economy and Competitiveness supported EF-S and VG (BFU2016–78265-P), PA (BFU2016– 79313-P and MDM-2016–0687), and AM-V (BFU2015–64408-P). AM-V was also supported by the Instituto de Salud Carlos III (PI11/ 00072) and RPV-M (CPII16/00004, PI14/00949 and PI17/00011). All projects were cofinanced by the Fondo Social Europeo (FEDER). AM-V is a member of the GENIE and EU-ROS Cost Actions of the European Union and RPV-M is a Marie Curie Fellow (CIG322034, EU).