Effete,a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.     

Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes

The vitrification procedure effects on molecular and cytoskeletal components and on developmental ability of in vitro matured prepubertal ovine oocytes were evaluated. MII oocytes were divided into three groups: (1) vitrified in cryoloops (VTR); (2) exposed to vitrification solutions and rehydrated without being plunged into liquid nitrogen (EXP); (3) without further treatment as a control (CTR). Two hours after treatment, membrane integrity, assessed by propidium iodide/Hoechst staining, was lower in VTR and EXP than in CTR (70.6%, 88.5% and 95.2%, respectively). Cleavage rate after fertilization was statistically different among all groups (21.4%, 45.4% and 82.8% for VTR, EXP and CTR groups respectively; P<0.01). Blastocyst rate in VTR (0.0%) and EXP (2.8%) groups was lower (P<0.01) than in CTR (22.8%). Maturation promoting factor activity was lower (P<0.01) in VTR and EXP groups compared with CTR at both 0 h (82.2%, 83.6% and 100%, respectively) and 2 h (60% and 53.9% and 100%, respectively) after warming. Immediately after warming VTR and EXP oocytes showed a lower rate of normal spindle and chromosome configuration compared to CTR (59.1%, 48.0% and 83.3%, respectively; P<0.01). After 2 h of culture in standard conditions the percentage of oocytes with normal spindle and chromosome organization decreased in both VTR and EXP groups compared to CTR (36.4%, 42.8% versus 87.5%, respectively). In conclusion the exposition to the tested cryoprotectant solution and the vitrification in cryoloops modified cytoskeletal components and alter biochemical pathways that compromise the developmental capacity of prepubertal in vitro matured ovine oocytes.     

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.     

Nicotiana attenuata SIPK,WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway

Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glucose (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants.Jasmonic acid (JA) and some of its precursors and derivatives are signal molecules that function as essential mediators of the plant''s wound, antiherbivore, and antipathogen responses, as well as in growth and development (Farmer, 1994; Creelman and Mullet, 1997; Turner et al., 2002). In unelicited mature leaves, JA is maintained at very low levels, however, upon specific stimulations, its biosynthesis is induced within a few minutes (Glauser et al., 2008). This rapid biosynthetic response must result from the activation of constitutively expressed JA biosynthesis enzymes in unelicited tissue by substrate availability and/or posttranslational modifications. At present, little is known about the molecular mechanisms that activate JA biosynthetic enzymes.According to the canonical mechanism for JA biosynthesis (Vick and Zimmerman, 1983), free α-linolenic acid (18:3^Δ9,12,15, 18:3) forms 13(S)-hydroperoxyoctadecatrienoic acid [13S-(OOH)-18:3] by the action of 13-lipoxygenase (13-LOX) in plastids. 13S-(OOH)-18:3 is converted by allene oxide synthase (AOS) into a highly unstable allene oxide intermediate that is processed by allene oxide cyclase (AOC) to yield (9S,13S)-12-oxo-phytodienoic acid (OPDA). OPDA is transported from the plastid into the peroxisome where it is reduced by the action of OPDA reductase 3 (OPR3) and after three cycles of β-oxidation, (3R,7S)-JA is formed. Due to the large number of enzymes and different cellular compartments involved in JA biosynthesis, it is expected that the pathway is regulated at multiple steps. Resolution of the structures of the tomato (Solanum lycopersicum) OPR3 and Arabidopsis (Arabidopsis thaliana) AOC2 and ACX1 has provided insights into potential regulatory mechanisms for these enzymes (e.g. oligomerization and phosphorylation; Pedersen and Henriksen, 2005; Breithaupt et al., 2006; Hofmann et al., 2006).The identification of two Arabidopsis plastidial glycerolipases, DAD1 and DGL (Ishiguro et al., 2001; Hyun et al., 2008), has provided genetic evidence for the importance of the release of trienoic fatty acids (FAs) from plastidial lipids in the activation of JA biosynthesis. Recently, some oxylipins have been found esterified to galactolipids in Arabidopsis leaves and hence it is possible that in this species preformed precursors could also supply the JA biosynthesis pathway after their release from lipids (Stelmach et al., 2001; Hisamatsu et al., 2003; Buseman et al., 2006). However, lipid-bound oxylipins are not formed in the leaves of all plant families (Böttcher and Weiler, 2007).In Nicotiana attenuata, wound-induced JA production is amplified by the application of lepidopteran larvae (e.g. Manduca sexta) oral secretions (OS) to mechanical wounds. Major elicitors of the OS-mediated response are FA-amino acid conjugates (FACs) that are sufficient to enhance JA production in leaves of this plant species (Halitschke et al., 2001). Recently, several regulatory factors with a potential function upstream of JA biosynthesis have been identified (Ludwig et al., 2005; Takabatake et al., 2006; Schweighofer et al., 2007; Takahashi et al., 2007); however, how these regulators affect JA biosynthesis is at present unknown. For example, wounding and herbivory in Nicotina spp. and tomato activate the mitogen-activated protein kinases salicylate-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK; Seo et al., 1999; Kandoth et al., 2007; Wu et al., 2007). When SIPK and WIPK expression is silenced in tobacco (Nicotiana tabacum), the plants accumulate 60% to 70% less JA than wild type after wounding or OS elicitation (Seo et al., 2007; Wu et al., 2007). Another regulatory component that affects JA production in N. attenuata is Nonexpressor of PR-1 (NPR1), an essential component of the salicylic acid (SA) signal transduction pathway first identified in Arabidopsis (Cao et al., 1994). N. attenuata NPR1-silenced plants accumulate 60% to 70% lower JA levels after elicitation than wild type (Rayapuram and Baldwin, 2007). NPR1 interacts with the JA and ethylene signaling cascades, and a cytosolic role for this factor in the regulation of JA-dependent responses/biosynthesis has been proposed (Spoel et al., 2003).In contrast to the mechanisms acting upstream of JA biosynthesis, the mechanisms mediating downstream JA responses are better characterized (Kazan and Manners, 2008; Browse, 2009). Among the best-characterized regulators of these responses is CORONATIVE INSENSITIVE1 (COI1), a gene that participates in jasmonate perception (Xie et al., 1998) and regulates gene expression through its interaction with the JASMONATE ZIM-DOMAIN repressors (Chini et al., 2007; Thines et al., 2007).To understand the early processes regulating the activation of JA biosynthesis by wounding and FAC elicitation in N. attenuata leaves, we quantified the initial rates of accumulation of plastid-derived JA precursors after these stimuli in wild type and four JA-deficient genotypes previously described: ir-sipk, ir-wipk, ir-npr1, and ir-coi1 (Rayapuram and Baldwin, 2007; Paschold et al., 2008; Meldau et al., 2009). We show that SIPK, WIPK, NPR1, and FACs contribute to the activation of de novo JA biosynthesis by affecting diverse early enzymatic steps in this pathway. The identification of a plastidial glycerolipase A₁ type I family protein (GLA1) essential for JA biosynthesis pointed to this enzyme as one potential target of some of these activating mechanisms.     

Egg trading in the simultaneously hermaphroditic polychaete worm Ophryotrocha gracilis (Huth)

Ophryotrocha gracilis (Huth 1934) is a simultaneously hermaphroditicpolychaete worm with external fertilization and a brief protandrousphase. The mating system of this species seems to meet conditionsleading to the establishment of egg-trading behavior. Experimentsshowed that mating occurs in pairs composed of two simultaneoushermaphrodites; sex roles are sequentially alternated and self-fertilizationis avoided. Egg reciprocation is kept evolutionarily stableby laying eggs in multiple, small-sized egg clutches and reducingthe reproductive success of pairs in which one of the two partnersdoes not reciprocate egg exchange. The frequency of ovigeroushermaphrodites in mass cultures of O. gracilis is about 50".Such a high mate availability preadapts hermaphrodites of O.gracilis to change partners very frequently and to reduce investmentin parental care, contrary to what is observed in another egg-trading,simultaneously hermaphroditic species, O. diadema. Laboratorypopulations of O. diadema have a frequency of only 17% ovigeroushermaphrodites     

AKTIP interacts with ESCRT I and is needed for the recruitment of ESCRT III subunits to the midbody

To complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission.