Author Correction: Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist.

Funder: Canadian Institutes of Health Research (Grant: FRN-142349) Natural Sciences and Engineering Research Council of Canada (Grant: RGPIN 05871-2014)Abstract: Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes

Teixobactin kills bacteria by a two-pronged attack on the cell envelope

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1–3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates

Teixobactin kills bacteria by a two-pronged attack on the cell envelope

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1–3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates

Cross-talk of calcium-dependent protein kinase and MAP kinase signaling

Plants use different signaling pathways to acclimate to changing environmental conditions. Fast changes in the concentration of free Ca2+ ions-so called Ca2+ signals-are among the first responses to many stress situations. These signals are decoded by different types of calcium-dependent protein kinases, which-together with mitogen-activated protein kinases (MAPK)-present two major pathways that are widely used to adapt the cellular metabolism to a changing environment. Ca2+-dependent protein kinase (CDPK) and MAPK pathways are known to be involved in signaling of abiotic and biotic stress in animal, yeast and plant cells. in many cases both pathways are activated in response to the same stimuli leading to the question of a potential cross-talk between those pathways. Cross-talk between Ca2+-dependent and MAPK signaling pathways has been elaborately studied in animal cells, but it has hardly been investigated in plants. Early studies of CDPKs involved in the biotic stress response in tobacco indicated a cross-talk of CDPK and MAPK activities, whereas a recent study in Arabidopsis revealed that CDPKs and MAPKs act differentially in innate immune signaling and showed no direct cross-talk between CDPK and MAPK activities. Similar results were also reported for CDPK and MAPK activities in the salt-stress response in Arabidopsis. Different modes of action are furthermore supported by the different subcellular localization of the involved kinases. in this review, we discuss recent findings on CDPK and MAPK signaling with respect to potential cross-talk and the subcellular localization of the involved components

Neuronal Growth Cone Size-Dependent and -Independent Parameters of Microtubule Polymerization

Migration and pathfinding of neuronal growth cones during neurite extension is critically dependent on dynamic microtubules. In this study we sought to determine, which aspects of microtubule polymerization relate to growth cone morphology and migratory characteristics. We conducted a multiscale quantitative microscopy analysis using automated tracking of microtubule plus ends in migrating growth cones of cultured murine dorsal root ganglion (DRG) neurons. Notably, this comprehensive analysis failed to identify any changes in microtubule polymerization parameters that were specifically associated with spontaneous extension vs. retraction of growth cones. This suggests that microtubule dynamicity is a basic mechanism that does not determine the polarity of growth cone response but can be exploited to accommodate diverse growth cone behaviors. At the same time, we found a correlation between growth cone size and basic parameters of microtubule polymerization including the density of growing microtubule plus ends and rate and duration of microtubule growth. A similar correlation was observed in growth cones of neurons lacking the microtubule-associated protein MAP1B. However, MAP1B-null growth cones, which are deficient in growth cone migration and steering, displayed an overall reduction in microtubule dynamicity. Our results highlight the importance of taking growth cone size into account when evaluating the influence on growth cone microtubule dynamics of different substrata, guidance factors or genetic manipulations which all can change growth cone morphology and size. The type of large scale multiparametric analysis performed here can help to separate direct effects that these perturbations might have on microtubule dynamics from indirect effects resulting from perturbation-induced changes in growth cone size.© 2018 Kiss, Fischer, Kleele, Misgeld and Props

PEX11 family members are membrane elongation factors that coordinate peroxisome proliferation and maintenance

Dynamic changes of membrane structure are intrinsic to organelle morphogenesis and homeostasis. Ectopic expression of proteins of the PEX11 family from yeast, plant or human lead to the formation of juxtaposed elongated peroxisomes (JEPs),which is evocative of an evolutionary conserved function of these proteins in membrane tubulation. Microscopic examinations reveal that JEPs are composed of independent elongated peroxisomes with heterogeneous distribution of matrix proteins. We established the homo- and heterodimerization properties of the human PEX11 proteins and their interaction with the fission factor hFis1, which is known to recruit the GTPase DRP1 to the peroxisomal membrane. We show that excess of hFis1 but not of DRP1 is sufficient to fragment JEPs into normal round-shaped organelles, and illustrate the requirement of microtubules for JEP formation. Our results demonstrate that PEX11-induced JEPs represent intermediates in the process of peroxisome membrane proliferation and that hFis1 is the limiting factor for progression. Hence, we propose a model for a conserved role of PEX11 proteins in peroxisome maintenance through peroxisome polarization, membrane elongation and segregation

The decapping activator HPat a novel factor co-purifying with GW182 from Drosophila cells

miRNAs post-transcriptionally regulate gene expression in many eukaryotes and thereby affect a wide range of biological processes. GW182 is a key factor in translation repression and mRNA degradation by miRNAs. In this study we investigate the potential interaction of GW182 and translation or mRNA degradation factors in Drosophila S2 cells. We have identified the decapping activator HP at as a novel factor co-purifying with GW182. Furthermore, we show that the C-terminal domain of GW182, important for gene silencing, is sufficient to form a complex with HP at. Our findings implicate a potential interaction of the miRNA effector component GW182 with the decapping machinery

Maneuvers on PCNA Rings during DNA Replication and Repair

DNA replication and repair are essential cellular processes that ensure genome duplication and safeguard the genome from deleterious mutations. Both processes utilize an abundance of enzymatic functions that need to be tightly regulated to ensure dynamic exchange of DNA replication and repair factors. Proliferating cell nuclear antigen (PCNA) is the major coordinator of faithful and processive replication and DNA repair at replication forks. Post-translational modifications of PCNA, ubiquitination and acetylation in particular, regulate the dynamics of PCNA-protein interactions. Proliferating cell nuclear antigen (PCNA) monoubiquitination elicits ‘polymerase switching’, whereby stalled replicative polymerase is replaced with a specialized polymerase, while PCNA acetylation may reduce the processivity of replicative polymerases to promote homologous recombination-dependent repair. While regulatory functions of PCNA ubiquitination and acetylation have been well established, the regulation of PCNA-binding proteins remains underexplored. Considering the vast number of PCNA-binding proteins, many of which have similar PCNA binding affinities, the question arises as to the regulation of the strength and sequence of their binding to PCNA. Here I provide an overview of post-translational modifications on both PCNA and PCNA-interacting proteins and discuss their relevance for the regulation of the dynamic processes of DNA replication and repair.© 2018 by the autho

Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2

Erythropoiesis requires erythropoietin (Epo) and stem cell factor (SCF) signaling via their receptors EpoR and c-Kit. EpoR ¿ like many other receptors involved in hematopoiesis¿ acts via the kinase Jak2. Deletion of EpoR or Jak2 causes embryonic lethality due to defective erythropoiesis. The contribution of distinct EpoR/Jak2-induced signaling pathways (MAPK, PI3K, Stat5) to functional erythropoiesis is incompletely understood. Here we demonstrate that expression of a constitutively activated Stat5a mutant (cS5) was sufficient to relieve the proliferation defect of Jak2-/- and EpoR-/- cells in an Epo-independent manner. Also tamoxifen-induced DNA binding of a Stat5a-ER* fusion construct enabled erythropoiesis in the absence of Epo. Furthermore, c-Kit could enhance signaling through the Jak2-Stat5 axis, particularly in lymphoid and myeloid progenitors. Although abundance of hematopoietic stem cells was 2.5-fold reduced in Jak2-/- fetal livers, transplantation of Jak2-/--cS5 fetal liver cells into irradiated mice gave rise to mature erythroid and myeloid cells of donor origin up to 6 months after transplantation. Cytokine- and c-Kit pathways do not function independently of each other in hematopoiesis, but cooperate to attain full Jak2/Stat5 activation. In conclusion, activated Stat5 is a critical downstream effector of Jak2 in erythro-/myelopoiesis, and Jak2 functionally links cytokine- with c-Kit-receptor tyrosine kinase signaling

Immunosorbent assay using gold colloid cluster technology for determination of IgEs in patients’ sera

This study focuses on the development of a sensitive and simple cluster-linked immunosorbent assay (CLISA) using gold colloidal cluster labeling for determination of proteins such as antigens (Ags) or antibodies (Abs). Abs for detection can be labeled with gold colloid clusters (GCCs). The Fc domain of the Abs binds to the clusters, and the Fab domain to the Ag on a nitrocellulose membrane or a microtiter plate as a support for dot-blotting. The signal of positive interaction between GCC-labeled Abs and its dotted Ag is detectable by the naked eye and can be quantified by comparison to a color scale prepared from a dilution series of known sample concentrations. The colored reaction product is stable for prolonged periods and does not fade, making this method a simple, fast, and convenient means for detection of Ag or Ab biorecognitions and an alternative to enzyme-linked immunosorbent assay. Several interactions between different Ags or Abs (eg, ß-lactoglobulin) and solutions avoiding gold colloidal cluster flocculation (eg, using protein G) were studied. CLISA was tested for other analytical purposes such as detection of IgEs in patients’ sera