Peter Askjaer et al. (2001) International C. elegans Meeting "Investigation of the function of Ran by RNAi"

Iain W Mattaj, Eva Hannak, Anthony A Hyman, Peter Askjaer

[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.

Peter Askjaer et al. (2005) International Worm Meeting "CeNup155/NPP-8 is essential for an early step in nuclear envelope formation"

Peter Askjaer, Claude Antony, Wolfram Antonin, Carmen L Iglesias, Uta Haselmann, Iain W Mattaj, Cerstin Franz

[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.

Peter Askjaer et al. (2002) European Worm Meeting "The Ran GTPase cycle is essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos"

Anthony A Hyman, Peter Askjaer, Eva Hannak, Iain W Mattaj

[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.

Rodenas, Eduardo et al. (2009) International Worm Meeting "Dissecting the functions of the conserved Nup107-160 nuclear pore subcomplex."

Rodenas, Eduardo, Askjaer, Peter

[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/.

Gonzalez-Aguilera, Cristina et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Genome wide analysis of nuclear envelope proteins using DamID technique in C. elegans"

Gonzalez-Aguilera, Cristina, Askjaer, Peter

[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.

Gonzalez-Aguilera, Cristina et al. (2011) International Worm Meeting "Identification of new DNA targets of nuclear envelope proteins in C. elegans using the DamID technique."

Askjaer, Peter, Gonzalez-Aguilera, Cristina

[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.

Morales-Martinez, Adela et al. (2011) International Worm Meeting "Identification and characterization of new nuclear envelope proteins."

Ayuso, Cristina, Morales-Martinez, Adela, Askjaer, Peter

[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.

Vincent Galy et al. (2006) European Worm Meeting "MEL-28, a Novel Nuclear Envelope and Kinetochore Protein Essential for Zygotic Nuclear Envelope Assembly"

Carmen Lopez-Iglesias, Vincent Galy, Iain W. Mattaj, Cerstin Franz, Peter Askjaer

[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.

Morales Martinez, Adela et al. (2013) International Worm Meeting "The nuclear envelope protein LEM-2 is critical for nuclear positioning and centrosome attachment."

Morales Martinez, Adela, Ayuso, Cristina, Askjaer, Peter, Dobrzynska, Agnieszka

[International Worm Meeting, 2013]

LEM domain proteins (for LBR, Emerin and MAN1) are typically localized in the inner nuclear membrane where they serve as bridging molecules between the nuclear membranes, the nuclear lamina and chromatin proteins, such as BAF. In addition, LEM domain proteins regulate gene expression through specific interaction with transcription factors and chromatin modifying enzymes at the nuclear envelope. Mutations in LEM domain proteins or in interacting nuclear envelope proteins cause a wide range of severe human diseases, collectively known as laminopathies. Most laminopathies are still poorly understood and no efficient treatment has been identified. To explore the mechanisms behind LEM domain protein activities we are characterizing the C. elegans lem-2 gene, which encodes a protein homologous to vertebrate inner nuclear membrane proteins LEM2 and MAN1. Previous experiments have reported that LEM-2 shares redundant roles with emerin/EMR-1. We find that LEM-2 is expressed in all cell types but is enriched in the germ line and intestine relative to EMR-1. Moreover, LEM-2 is recruited significantly earlier than EMR-1 during nuclear envelope reassembly in telophase, suggesting that LEM-2 carries specific functions. Indeed, live imaging reveals that nuclear morphology is irregular in embryos lacking expression of LEM-2 and EMR-1 protein turnover is accelerated in lem-2 adults. The nuclear envelope is responsible for nucleus-centrosome attachment and alterations in the nuclear envelope can cause abnormal nuclear and centrosome positioning. In C. elegans nuclear positioning is mainly studied in mid-stage embryos and early larvae but we observe that mutation of lem-2 causes defects already in the second embryonic division. During AB division nuclei separate slower than in wild type embryos and nucleating centrosomes are observed at abnormal distance from nuclei. Thus, although expressed in the inner nuclear membrane our data suggest that LEM-2 is involved in the anchoring of centrosomes to the outer nuclear membrane. To identify potential interaction partners in this process, we are performing a genome-wide RNAi screen for enhancers of lem-2 phenotypes.

De La Cruz Ruiz, Patricia et al. (2021) International Worm Meeting "Heterochromatin protein 1 regulates longevity and the mitochondrial unfolded protein response"

De La Cruz Ruiz, Patricia, Askjaer, Peter, Artal Sanz, Marta

[International Worm Meeting, 2021]

Prohibitins (PHB-1 and PHB-2) form a large macromolecular structure at the mitochondrial inner membrane. Prohibitin deficiency shortens the lifespan of wild type animals, but dramatically extends the lifespan of a variety of metabolically compromised animals such as insulin/IGF-1 receptor (daf-2) mutants. This phenotype is accompanied by a differential induction of the mitochondrial Unfolded Protein Response (UPRmt), a stress protective mechanism that is attenuated in daf-2 mutants. Through a genome wide RNAi screen we identified Heterochromatin Protein 1 (HP1) as a new regulator of the UPRmt. HP1 proteins (HPL-1 and HPL-2 in C. elegans) bind histone H3 methylated on lysine 9 to maintain chromatin in a repressed state during development. We observed induction of the UPRmt in HP1 single and double mutants, as well as decreased basal and maximal respiration compared to wild type animals, suggesting a mitochondrial dysfunction. This is supported by an increased sensitivity of HP1 mutants to the antibiotic doxycycline, which specifically blocks mitochondrial translation. Furthermore, HP1 protein levels increased in response to phb-1 depletion, both, in otherwise wild type animals and in daf-2 mutants. Interestingly, hpl-1 mutants reduced the UPRmt triggered by phb-1 depletion, but suppressed the attenuated UPRmt in PHB-depleted daf-2 mutants. HPL-1 protein levels increase as animals age, while HPL-2 remains steady. Interestingly, hpl-1 deletion extended lifespan independently of phb-1. However, in daf-2 mutants, hpl-1 was required for the enhanced longevity conferred by PHB depletion. We are currently investigating which genes are targeted by HP1 proteins under conditions leading to opposing ageing phenotypes. Given the evolutionary conservation of ageing mechanisms and the proteins under study, research in short-lived model systems amenable to genetic dissection, such as C. elegans, is likely to be beneficial to human health.