Items 1-20 of 96

    • Journal Article

      Disruption of Arabidopsis neutral ceramidases 1 and 2 results in specific sphingolipid imbalances triggering different phytohormone‐dependent plant cell death programmes 

      Zienkiewicz, Agnieszka; Gömann, Jasmin; König, Stefanie; Herrfurth, Cornelia; Liu, Yi‐Tse; Meldau, Dorothea; Feussner, Ivo
      New Phytologist p.1-19
      Sphingolipids act as regulators of programmed cell death (PCD) and the plant defence response. The homeostasis between long-chain base (LCB) and ceramide (Cer) seems to play an important role in executions of PCD. Therefore, deciphering the role of neutral ceramidases (NCER) is crucial to identify the sphingolipid compounds that trigger and execute PCD. We performed comprehensive sphingolipid and phytohormone analyses of Arabidopsis ncer mutants, combined with gene expression profiling and microscopic analyses. While ncer1 exhibited early leaf senescence (developmentally controlled PCD - dPCD) and an increase in hydroxyceramides, ncer2 showed spontaneous cell death (pathogen-triggered PCD-like - pPCD) accompanied by an increase in LCB t18:0 at 35 d, respectively. Loss of NCER1 function resulted in accumulation of jasmonoyl-isoleucine (JA-Ile) in the leaves, whereas disruption of NCER2 was accompanied by higher levels of salicylic acid (SA) and increased sensitivity to Fumonisin B1 (FB1 ). All mutants were also found to activate plant defence pathways. These data strongly suggest that NCER1 hydrolyses ceramides whereas NCER2 functions as a ceramide synthase. Our results reveal an important role of NCER in the regulation of both dPCD and pPCD via a tight connection between the phytohormone and sphingolipid levels in these two processes.
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    • Journal Article

      Conditional gene expression reveals stage‐specific functions of the unfolded protein response in the Ustilago maydis– maize pathosystem 

      Schmitz, Lara; Kronstad, James W.; Heimel, Kai
      Molecular Plant Pathology
      Ustilago maydis is a model organism for the study of biotrophic plant-pathogen interactions. The sexual and pathogenic development of the fungus are tightly connected since fusion of compatible haploid sporidia is prerequisite for infection of the host plant, maize (Zea mays). After plant penetration, the unfolded protein response (UPR) is activated and required for biotrophic growth. The UPR is continuously active throughout all stages of pathogenic development in planta. However, since development of UPR deletion mutants stops directly after plant penetration, the role of an active UPR at later stages of development remained to be determined. Here, we establish a gene expression system for U. maydis that uses endogenous, conditionally active promoters to either induce or repress expression of a gene of interest during different stages of plant infection. Integration of the expression constructs into the native genomic locus and removal of resistance cassettes were required to obtain a wild-type-like expression pattern. This indicates that genomic localization and chromatin structure are important for correct promoter activity and gene expression. By conditional expression of the central UPR regulator, Cib1, in U. maydis, we show that a functional UPR is required for continuous plant defence suppression after host infection and that U. maydis relies on a robust control system to prevent deleterious UPR hyperactivation.
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    • Journal Article

      A key role for foxQ2 in anterior head and central brain patterning in insects 

      Kitzmann, Peter; Weißkopf, Matthias; Schacht, Magdalena Ines; Bucher, Gregor
      Development 2017; 144(16) p.2969-2981
      Anterior patterning of animals is based on a set of highly conserved transcription factors but the interactions within the protostome anterior gene regulatory network (aGRN) remain enigmatic. Here, we identify the red flour beetle Tribolium castaneum ortholog of foxQ2 (Tc-foxQ2) as a novel upstream component of the aGRN. It is required for the development of the labrum and higher order brain structures, namely the central complex and the mushroom bodies. We reveal Tc-foxQ2 interactions by RNAi and heat shock-mediated misexpression. Surprisingly, Tc-foxQ2 and Tc-six3 mutually activate each other, forming a novel regulatory module at the top of the aGRN. Comparisons of our results with those of sea urchins and cnidarians suggest that foxQ2 has acquired more upstream functions in the aGRN during protostome evolution. Our findings expand the knowledge on foxQ2 gene function to include essential roles in epidermal development and central brain patterning.
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    • Journal Article

      Specific expression and function of the Six3 optix in Drosophila serially homologous organs 

      Al Khatib, Amer; Siomava, Natalia; Iannini, Antonella; Posnien, Nico; Casares, Fernando
      Biology Open 2017; 6(8) p.1155-1164
      Organ size and pattern results from the integration of two positional information systems. One global information system, encoded by the Hox genes, links organ type with position along the main body axis. Within specific organs, local information is conveyed by signaling molecules that regulate organ growth and pattern. The mesothoracic (T2) wing and the metathoracic (T3) haltere of Drosophila represent a paradigmatic example of this coordination. The Hox gene Ultrabithorax (Ubx), expressed in the developing T3, selects haltere identity by, among other processes, modulating the production and signaling efficiency of Dpp, a BMP2-like molecule that acts as a major regulator of size and pattern. However, the mechanisms of the Hox-signal integration in this well-studied system are incomplete. Here, we have investigated this issue by studying the expression and function of the Six3 transcription factor optix during Drosophila wing and haltere development. We find that in both organs, Dpp defines the expression domain of optix through repression, and that the specific position of this domain in wing and haltere seems to reflect the differential signaling profile among these organs. We show that optix expression in wing and haltere primordia is conserved beyond Drosophila in other higher diptera. In Drosophila, optix is necessary for the growth of wing and haltere. In the wing, optix is required for the growth of the most anterior/proximal region (the 'marginal cell') and for the correct formation of sensory structures along the proximal anterior wing margin; the halteres of optix mutants are also significantly reduced. In addition, in the haltere, optix is necessary for the suppression of sensory bristles.
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    • Journal Article

      An ancestral apical brain region contributes to the central complex under the control of foxQ2 in the beetle Tribolium 

      He, Bicheng; Buescher, Marita; Farnworth, Max Stephen; Strobl, Frederic; Stelzer, Ernst HK; Koniszewski, Nikolaus DB; Muehlen, Dominik; Bucher, Gregor
      eLife 2019; 8: Art. e49065
      The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.
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    • Journal Article

      yRACK1/Asc1 proxiOMICs—Towards Illuminating Ships Passing in the Night 

      Schmitt, Kerstin; Valerius, Oliver
      Cells 2019; 8(11): Art. 1384
      Diverse signals and stress factors regulate the activity and homeostasis of ribosomes in all cells. The Saccharomyces cerevisiae protein Asc1/yRACK1 occupies an exposed site at the head region of the 40S ribosomal subunit (hr40S) and represents a central hub for signaling pathways. Asc1 strongly affects protein phosphorylation and is involved in quality control pathways induced by translation elongation arrest. Therefore, it is important to understand the dynamics of protein formations in the Asc1 microenvironment at the hr40S. We made use of the in vivo protein-proximity labeling technique Biotin IDentification (BioID). Unbiased proxiOMICs from two adjacent perspectives identified nucleocytoplasmic shuttling mRNA-binding proteins, the deubiquitinase complex Ubp3-Bre5, as well as the ubiquitin E3 ligase Hel2 as neighbors of Asc1. We observed Asc1-dependency of hr40S localization of mRNA-binding proteins and the Ubp3 co-factor Bre5. Hel2 and Ubp3-Bre5 are described to balance the mono-ubiquitination of Rps3 (uS3) during ribosome quality control. Here, we show that the absence of Asc1 resulted in massive exposure and accessibility of the C-terminal tail of its ribosomal neighbor Rps3 (uS3). Asc1 and some of its direct neighbors together might form a ribosomal decision tree that is tightly connected to close-by signaling modules.
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    • Journal Article

      Evolution of intron splicing towards optimized gene expression is based on various Cis- and Trans-molecular mechanisms 

      Frumkin, Idan; Yofe, Ido; Bar-Ziv, Raz; Gurvich, Yonat; Lu, Yen-Yun; Voichek, Yoav; Towers, Ruth; Schirman, Dvir; Krebber, Heike; Pilpel, Yitzhak
      PLOS Biology 2019; 17(8): Art. e3000423
      Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when organisms adapt to new challenges? How does evolution optimize inefficiency of introns' splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron's splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein's domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands.
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    • Journal Article

      Topoisomerase IV can functionally replace all type 1A topoisomerases in Bacillus subtilis 

      Reuß, Daniel R.; Faßhauer, Patrick; Mroch, Philipp Joel; Ul-Haq, Inam; Koo, Byoung-Mo; Pöhlein, Anja; Gross, Carol A.; Daniel, Rolf; Brantl, Sabine; Stülke, Jörg
      Nucleic Acids Research 2019; 47(10) p.5231-5242
      DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases.
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    • Journal Article

      Structural basis for RNA translocation by DEAH-box ATPases 

      Hamann, Florian; Enders, Marieke; Ficner, Ralf
      Nucleic Acids Research 2019; 47(8) p.4349-4362
      DEAH-box adenosine triphosphatases (ATPases) play a crucial role in the spliceosome-mediated excision of pre-mRNA introns. Recent spliceosomal cryo-EM structures suggest that these proteins utilize translocation to apply forces on ssRNAs rather than direct RNA duplex unwinding to ensure global rearrangements. By solving the crystal structure of Prp22 in different adenosine nucleotide-free states, we identified two missing conformational snapshots of genuine DEAH-box ATPases that help to unravel the molecular mechanism of translocation for this protein family. The intrinsic mobility of the RecA2 domain in the absence of adenosine di- or triphosphate (ADP/ATP) and RNA enables DEAH-box ATPases to adopt different open conformations of the helicase core. The presence of RNA suppresses this mobility and stabilizes one defined open conformation when no adenosine nucleotide is bound. A comparison of this novel conformation with the ATP-bound state of Prp43 reveals that these ATPases cycle between closed and open conformations of the helicase core, which accommodate either a four- or five-nucleotide stack in the RNA-binding tunnel, respectively. The continuous repetition of these states enables these proteins to translocate in 3'-5' direction along an ssRNA with a step-size of one RNA nucleotide per hydrolyzed ATP. This ATP-driven motor function is maintained by a serine in the conserved motif V that senses the catalytic state and accordingly positions the RecA2 domain.
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    • Journal Article

      Membrane Lipids, Waxes and Oxylipins in the Moss Model Organism Physcomitrella patens 

      Resemann, Hanno C.; Lewandowska, Milena; Gömann, Jasmin; Feussner, Ivo
      Plant and Cell Physiology 2019; 60(6) p.1166-1175
      The moss Physcomitrella patens receives increased scientific interest since its genome was sequenced a decade ago. As a bryophyte, it represents the first group of plants that evolved in a terrestrial habitat still without a vascular system that developed later in tracheophytes. It is easily transformable via homologous recombination, which enables the formation of targeted loss-of-function mutants. Even though genetics, development and life cycle in Physcomitrella are well studied nowadays, research on lipids in Physcomitrella is still underdeveloped. This review aims on presenting an overview on the state of the art of lipid research with a focus on membrane lipids, surface lipids and oxylipins. We discuss in this review that Physcomitrella possesses very interesting features regarding its membrane lipids. Here, the presence of very-long-chain polyunsaturated fatty acids (VLC-PUFA) still shows a closer similarity to marine microalgae than to vascular plants. Unlike algae, Physcomitrella has a cuticle comparable to vascular plants composed of cutin and waxes. The presence of VLC-PUFA in Physcomitrella also leads to a greater variability of signaling lipids even though the phytohormone jasmonic acid is not present in this organism, which is different to vascular plants. In summary, the research on lipids in Physcomitrella is still in its infancy, especially considering membrane lipids. We hope that this review will help to promote the further advancement of lipid research in this important model organism in the future, so we can better understand how lipids are involved in the evolution of land plants.
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    • Journal Article

      COP9 Signalosome Interaction with UspA/Usp15 Deubiquitinase Controls VeA-Mediated Fungal Multicellular Development 

      Meister, Cindy; Thieme; Karl G.; Thieme, Sabine; Köhler, Anna M.; Schmitt, Kerstin; Valerius, Oliver; Braus, Gerhard H.
      Biomolecules 2019; 9(6): Art. 238
      COP9 signalosome (CSN) and Den1/A deneddylases physically interact and promote multicellular development in fungi. CSN recognizes Skp1/cullin-1/Fbx E3 cullin-RING ligases (CRLs) without substrate and removes their posttranslational Nedd8 modification from the cullin scaffold. This results in CRL complex disassembly and allows Skp1 adaptor/Fbx receptor exchange for altered substrate specificity. We characterized the novel ubiquitin-specific protease UspA of the mold Aspergillus nidulans, which corresponds to CSN-associated human Usp15 and interacts with six CSN subunits. UspA reduces amounts of ubiquitinated proteins during fungal development, and the uspA gene expression is repressed by an intact CSN. UspA is localized in proximity to nuclei and recruits proteins related to nuclear transport and transcriptional processing, suggesting functions in nuclear entry control. UspA accelerates the formation of asexual conidiospores, sexual development, and supports the repression of secondary metabolite clusters as the derivative of benzaldehyde (dba) genes. UspA reduces protein levels of the fungal NF-kappa B-like velvet domain protein VeA, which coordinates differentiation and secondary metabolism. VeA stability depends on the Fbx23 receptor, which is required for light controlled development. Our data suggest that the interplay between CSN deneddylase, UspA deubiquitinase, and SCF-Fbx23 ensures accurate levels of VeA to support fungal development and an appropriate secondary metabolism.
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    • Journal Article

      Formation and development of the male copulatory organ in the spider Parasteatoda tepidariorum involves a metamorphosis-like process 

      Quade, Felix Simon Christian; Holtzheimer, Jana; Frohn, Jasper; Töpperwien, Mareike; Salditt, Tim; Prpic, Nikola-Michael
      Scientific Reports 2019; 9(1): Art. 6945
      Spiders have evolved a unique male copulatory organ, the pedipalp bulb. The morphology of the bulb is species specific and plays an important role in species recognition and prezygotic reproductive isolation. Despite its importance for spider biodiversity, the mechanisms that control bulb development are virtually unknown. We have used confocal laser scanning microscopy (CLSM) and diffusible iodine-based contrast-enhanced micro computed tomography (dice-µCT) to study bulb development in the spider Parasteatoda tepidariorum. These imaging technologies enabled us to study bulb development in situ, without the use of destructive procedures for the first time. We show here that the inflated pedipalp tip in the subadult stage is filled with haemolymph that rapidly coagulates. Coagulation indicates histolytic processes that disintegrate tibia and tarsus, similar to histolytic processes during metamorphosis in holometabolous insects. The coagulated material contains cell inclusions that likely represent the cell source for the re-establishment of tarsus and tibia after histolysis, comparable to the histoblasts in insect metamorphosis. The shape of the coagulated mass prefigures the shape of the adult tarsus (cymbium) like a blueprint for the histoblasts. This suggests a unique role for controlled coagulation after histolysis in the metamorphosis-like morphogenesis of the male pedipalp.
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    • Journal Article

      Signal peptide peptidase activity connects the unfolded protein response to plant defense suppression by Ustilago maydis 

      Pinter, Niko; Hach, Christina Andrea; Hampel, Martin; Rekhter, Dmitrij; Zienkiewicz, Krzysztof; Feussner, Ivo; Poehlein, Anja; Daniel, Rolf; Finkernagel, Florian; Heimel, Kai
      PLOS Pathogens 2019; 15(4): Art. e1007734
      The corn smut fungus Ustilago maydis requires the unfolded protein response (UPR) to maintain homeostasis of the endoplasmic reticulum (ER) during the biotrophic interaction with its host plant Zea mays (maize). Crosstalk between the UPR and pathways controlling pathogenic development is mediated by protein-protein interactions between the UPR regulator Cib1 and the developmental regulator Clp1. Cib1/Clp1 complex formation results in mutual modification of the connected regulatory networks thereby aligning fungal proliferation in planta, efficient effector secretion with increased ER stress tolerance and long-term UPR activation in planta. Here we address UPR-dependent gene expression and its modulation by Clp1 using combinatorial RNAseq/ChIPseq analyses. We show that increased ER stress resistance is connected to Clp1-dependent alterations of Cib1 phosphorylation, protein stability and UPR gene expression. Importantly, we identify by deletion screening of UPR core genes the signal peptide peptidase Spp1 as a novel key factor that is required for establishing a compatible biotrophic interaction between U. maydis and its host plant maize. Spp1 is dispensable for ER stress resistance and vegetative growth but requires catalytic activity to interfere with the plant defense, revealing a novel virulence specific function for signal peptide peptidases in a biotrophic fungal/plant interaction.
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    • Journal Article

      Biallelic mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence 

      Bonnin, Edith; Cabochette, Pauline; Filosa, Alessandro; Jühlen, Ramona; Komatsuzaki, Shoko; Hezwani, Mohammed; Dickmanns, Achim; Martinelli, Valérie; Vermeersch, Marjorie; Supply, Lynn; et al.
      Martins, NunoPirenne, LaurenceRavenscroft, GianinaLombard, MarcusPort, SarahSpillner, ChristianeJanssens, SandraRoets, EllenVan Dorpe, JoLammens, MartinKehlenbach, Ralph HFicner, RalfLaing, Nigel GHoffmann, KatrinVanhollebeke, BenoitFahrenkrog, Birthe
      PLOS Genetics 2018; 14(12): Art. e1007845
      Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.
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    • Journal Article

      Targeting of LRRC59 to the Endoplasmic Reticulum and the Inner Nuclear Membrane. 

      Blenski, Marina; Kehlenbach, Ralph H.
      International Journal of Molecular Sciences 2019; 20(2): Art. 334
      LRRC59 (leucine-rich repeat-containing protein 59) is a tail-anchored protein with a single transmembrane domain close to its C-terminal end that localizes to the endoplasmic reticulum (ER) and the nuclear envelope. Here, we investigate the mechanisms of membrane integration of LRRC59 and its targeting to the inner nuclear membrane (INM). Using purified microsomes, we show that LRRC59 can be post-translationally inserted into ER-derived membranes. The TRC-pathway, a major route for post-translational membrane insertion, is not required for LRRC59. Like emerin, another tail-anchored protein, LRRC59 reaches the INM, as demonstrated by rapamycin-dependent dimerization assays. Using different approaches to inhibit importin α/β-dependent nuclear import of soluble proteins, we show that the classic nuclear transport machinery does not play a major role in INM-targeting of LRRC59. Instead, the size of the cytoplasmic domain of LRRC59 is an important feature, suggesting that targeting is governed by passive diffusion.
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    • Journal Article

      The green microalga Lobosphaera incisa harbours an arachidonate 15 S‐lipoxygenase 

      Djian, B.; Hornung, E.; Ischebeck, T.; Feussner, I.
      Plant Biology 2018; 21(S1) p.131-142
      The green microalga Lobosphaera incisa is an oleaginous eukaryotic alga that is rich in arachidonic acid (20:4). Being rich in this polyunsaturated fatty acid (PUFA), however, makes it sensitive to oxidation. In plants, lipoxygenases (LOXs) are the major enzymes that oxidise these molecules. • Here, we describe, to our best knowledge, the first characterisation of a cDNA encoding a LOX (LiLOX) from a green alga. To obtain first insights into its function, we expressed it in E. coli, purified the recombinant enzyme and analysed its enzyme activity. • The protein sequence suggests that LiLOX and plastidic LOXs from bryophytes and flowering plants may share a common ancestor. The fact that LiLOX oxidises all PUFAs tested with a consistent oxidation on the carbon n-6, suggests that PUFAs enter the substrate channel through their methyl group first (tail first). Additionally, LiLOX form the fatty acid hydroperoxide in strict S configuration. • LiLOX may represent a good model to study plastid LOX, because it is stable after heterologous expression in E. coli and highly active in vitro. Moreover, as the first characterised LOX from green microalgae, it opens the possibility to study endogenous LOX pathways in these organisms.
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    • Journal Article

      A perforated anodised aluminium slide for improved specimen clearing and imaging for confocal laser scanning microscopy 

      Quade, Felix S. C.; Preitz, Beate; Prpic, Nikola-Michael
      BMC Research Notes. 2018 Oct 10;11(1):716
      Objective The bleaching, clearing and handling of tiny specimens with soft tissue and cuticular components for confocal laser scanning microscopy is difficult, because after cuticle bleaching and tissue clearing the specimens are virtually invisible. We have adjusted the design of the specimen container described by Smolla et al. (Arthropod Struct Dev 43:175–81, 2014) to handle tiny specimens. Results We describe a perforated and anodised aluminium slide that was designed to hold the distal tips of the pedipalp appendages of the spider Parasteatoda tepidariorum during clearing, and that can then be used directly for confocal laser scanning microscopy. We believe that this slide design will be helpful for others who want to visualise specimens between 500 and 800 µm with confocal laser scanning microscopy.
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    • Journal Article

      The mitochondrial TMEM177 associates with COX20 during COX2 biogenesis. 

      Lorenzi, Isotta; Oeljeklaus, Silke; Aich, Abhishek; Ronsör, Christin; Callegari, Sylvie; Dudek, Jan; Warscheid, Bettina; Dennerlein, Sven; Rehling, Peter
      Biochimica et Biophysica Acta 2018; 1865(2) p.323-333
      The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the CuA-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network. Loss or increase in the amount of TMEM177 affects COX20 abundance leading to reduced or increased COX20 levels respectively. TMEM177 associates with newly synthesized COX2 and SCO2 in a COX20-dependent manner. Our data shows that by unbalancing the amount of TMEM177, newly synthesized COX2 accumulates in a COX20-associated state. We conclude that TMEM177 promotes assembly of COX2 at the level of CuA-site formation.
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      Cyanophage-encoded lipid desaturases: oceanic distribution, diversity and function 

      Roitman, Sheila; Hornung, Ellen; Flores-Uribe, José; Sharon, Itai; Feussner, Ivo; Béjà, Oded
      The ISME Journal 2018; 12(2) p.343-355
      Cyanobacteria are among the most abundant photosynthetic organisms in the oceans; viruses infecting cyanobacteria (cyanophages) can alter cyanobacterial populations, and therefore affect the local food web and global biochemical cycles. These phages carry auxiliary metabolic genes (AMGs), which rewire various metabolic pathways in the infected host cell, resulting in increased phage fitness. Coping with stress resulting from photodamage appears to be a central necessity of cyanophages, yet the overall mechanism is poorly understood. Here we report a novel, widespread cyanophage AMG, encoding a fatty acid desaturase (FAD), found in two genotypes with distinct geographical distribution. FADs are capable of modulating the fluidity of the host's membrane, a fundamental stress response in living cells. We show that both viral FAD (vFAD) families are Δ9 lipid desaturases, catalyzing the desaturation at carbon 9 in C16 fatty acid chains. In addition, we present a comprehensive fatty acid profiling for marine cyanobacteria, which suggests a unique desaturation pathway of medium- to long-chain fatty acids no longer than C16, in accordance with the vFAD activity. Our findings suggest that cyanophages are capable of fiddling with the infected host's membranes, possibly leading to increased photoprotection and potentially enhancing viral-encoded photosynthetic proteins, resulting in a new viral metabolic network.
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      Sumoylation Protects Against β-Synuclein Toxicity in Yeast. 

      Popova, Blagovesta; Kleinknecht, Alexandra; Arendarski, Patricia; Mischke, Jasmin; Wang, Dan; Braus, Gerhard H.
      Frontiers in molecular neuroscience 2018; 11 p.1-17: Art. 94
      Aggregation of α-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD). The budding yeast Saccharomyces cerevisiae serves as reference cell to study the interplay between αSyn misfolding, cytotoxicity and post-translational modifications (PTMs). The synuclein family includes α, β and γ isoforms. β-synuclein (βSyn) and αSyn are found at presynaptic terminals and both proteins are presumably involved in disease pathogenesis. Similar to αSyn, expression of βSyn leads to growth deficiency and formation of intracellular aggregates in yeast. Co-expression of αSyn and βSyn exacerbates the cytotoxicity. This suggests an important role of βSyn homeostasis in PD pathology. We show here that the small ubiquitin-like modifier SUMO is an important determinant of protein stability and βSyn-induced toxicity in eukaryotic cells. Downregulation of sumoylation in a yeast strain, defective for the SUMO-encoding gene resulted in reduced yeast growth, whereas upregulation of sumoylation rescued growth of yeast cell expressing βSyn. This corroborates a protective role of the cellular sumoylation machinery against βSyn-induced toxicity. Upregulation of sumoylation significantly reduced βSyn aggregate formation. This is an indirect molecular process, which is not directly linked to βSyn sumoylation because amino acid substitutions in the lysine residues required for βSyn sumoylation decreased aggregation without changing yeast cellular toxicity. αSyn aggregates are more predominantly degraded by the autophagy/vacuole than by the 26S ubiquitin proteasome system. We demonstrate a vice versa situation for βSyn, which is mainly degraded in the 26S proteasome. Downregulation of sumoylation significantly compromised the clearance of βSyn by the 26S proteasome and increased protein stability. This effect is specific, because depletion of functional SUMO did neither affect βSyn aggregate formation nor its degradation by the autophagy/vacuolar pathway. Our data support that cellular βSyn toxicity and aggregation do not correlate in their cellular impact as for αSyn but rather represent two distinct independent molecular functions and molecular mechanisms. These insights into the relationship between βSyn-induced toxicity, aggregate formation and degradation demonstrate a significant distinction between the impact of αSyn compared to βSyn on eukaryotic cells.
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