Abi mutants in Dictyostelium reveal specific roles for the SCAR/WAVE complex in cytokinesis

Actin polymerization drives multiple cell processes involving movement and shape change. SCAR/WAVE proteins connect signaling to actin polymerization through the activation of the Arp2/3 complex. SCAR/WAVE is normally found in a complex with four other proteins: PIR121, Nap1, Abi2,and HSPC300 (Figure S1A available online) [1-3]. However,there is no consensus as to whether the complex functions as an unchanging unit or if it alters its composition in response to stimulation, as originally proposed by Edenet al. [1]. It also is unclear whether complex members exclusively regulate SCAR/WAVEs or if they have additional targets [4-6]. Here, we analyze the roles of the unique Dictyostelium Abi. We find that abiA null mutants show less severe defects in motility than do scar null cells, indicating--unexpectedly--that SCAR retains partial activity in the absence of Abi. Furthermore, abiA null mutants have a serious defect in cytokinesis, which is not seen in other SCAR complex mutants and is seen only when SCAR itself is present. Detailed examination reveals that normal cytokinesis requires SCAR activity, apparently regulated through multiple pathways.     

The Endoplasmic Reticulum Is a Reservoir for WAVE/SCAR Regulatory Complex Signaling in the Arabidopsis Leaf

During plant cell morphogenesis, signal transduction and cytoskeletal dynamics interact to locally organize the cytoplasm and define the geometry of cell expansion. The WAVE/SCAR (for WASP family verprolin homologous/suppressor of cyclic AMP receptor) regulatory complex (W/SRC) is an evolutionarily conserved heteromeric protein complex. Within the plant kingdom W/SRC is a broadly used effector that converts Rho-of-Plants (ROP)/Rac small GTPase signals into Actin-Related Protein2/3 and actin-dependent growth responses. Although the components and biochemistry of the W/SRC pathway are well understood, a basic understanding of how cells partition W/SRC into active and inactive pools is lacking. In this paper, we report that the endoplasmic reticulum (ER) is an important organelle for W/SRC regulation. We determined that a large intracellular pool of the core W/SRC subunit NAP1, like the known positive regulator of W/SRC, the DOCK family guanine nucleotide-exchange factor SPIKE1 (SPK1), localizes to the surface of the ER. The ER-associated NAP1 is inactive because it displays little colocalization with the actin network, and ER localization requires neither activating signals from SPK1 nor a physical association with its W/SRC-binding partner, SRA1. Our results indicate that in Arabidopsis (Arabidopsis thaliana) leaf pavement cells and trichomes, the ER is a reservoir for W/SRC signaling and may have a key role in the early steps of W/SRC assembly and/or activation.The W/SRC (for WASP family verprolin homologous/suppressor of cAMP receptor regulatory complex) and Actin-Related Protein (ARP)2/3 complex are part of an evolutionarily conserved Rho-of-Plants (ROP)/Rac small GTPase signal transduction cascade that controls actin-dependent morphogenesis in a wide variety of tissues and developmental contexts (Smith and Oppenheimer, 2005; Szymanski, 2005; Yalovsky et al., 2008). Many of the components and regulatory relationships among the complexes were discovered based on the stage-specific cell-swelling and -twisting phenotypes of the distorted class of Arabidopsis (Arabidopsis thaliana) trichome mutants (Szymanski et al., 1999; Zhang et al., 2005, 2008; Djakovic et al., 2006; Le et al., 2006; Uhrig et al., 2007). However, in both maize (Zea mays) and Arabidopsis, W/SRC and/or ARP2/3 are required for normal pavement cell morphogenesis (Frank and Smith, 2002; Mathur et al., 2003b; Brembu et al., 2004). Compared with other Arabidopsis pavement cell mutants, the shape defects of the distorted group are relatively mild. However, the distorted mutants and spike1 (spk1) differ from most other morphology mutants in that they display gaps in the shoot epidermis, most frequently at the interface of pavement cells and stomata (Qiu et al., 2002; Le et al., 2003; Li et al., 2003; Mathur et al., 2003b; Zhang et al., 2005; Djakovic et al., 2006). The cell gaps may reflect either uncoordinated growth between neighboring cells or defective cortical actin-dependent secretion of polysaccharides and/or proteins that promote cell-cell adhesion (Smith and Oppenheimer, 2005; Hussey et al., 2006; Leucci et al., 2007).In tip-growing cells, there is a strict requirement for actin to organize the trafficking and secretion activities of the cell to restrict growth to the apex. In Arabidopsis, the W/SRC-ARP2/3 pathway is not an essential tip growth component, because null alleles of both W/SRC and ARP2/3 subunits do not cause noticeable pollen tube or root hair phenotypes (Le et al., 2003; Djakovic et al., 2006). However, reverse genetic analysis of the W/SRC subunit BRK1 and ARP2/3 in the tip-growing protonemal cells of Physcomitrella patens revealed the obvious importance of this pathway (Harries et al., 2005; Perroud and Quatrano, 2008). Along similar lines, in two different legume species, W/SRC subunits are required for a normal root nodulation response to symbiotic bacteria (Yokota et al., 2009; Miyahara et al., 2010), indicating a conditional importance for this pathway in root hair growth. These genetic studies centered on the W/SRC and ARP2/3 pathways, in addition to those that involve a broader collection of actin-based morphology mutants (Smith and Oppenheimer, 2005; Blanchoin et al., 2010), are defining important cytoskeletal proteins and new interactions with the endomembrane system during morphogenesis. However, it is not completely clear how unstable actin filaments and actin bundle networks dictate the growth patterns of cells (Staiger et al., 2009).The difficulty of understanding the functions of specific actin arrays can be explained, in part, by the fact that plant cells that employ a diffuse growth mechanism have highly unstable cortical actin filaments and large actin bundles that do not have a geometry that obviously relates to the direction of growth or a specific subcellular activity (Blanchoin et al., 2010). This is in contrast to the cortical endocytic actin patches in yeast (Saccharomyces cerevisiae; Evangelista et al., 2002; Kaksonen et al., 2003) and cortical meshworks in the lamellipodia of crawling cells (Pollard and Borisy, 2003) that reveal subcellular locations where actin works to locally control membrane dynamics. In thick-walled plant cells, the magnitude of the forces that accompany turgor-driven cell expansion exceed those that could be generated by actin polymerization by orders of magnitude (Szymanski and Cosgrove, 2009). Localized cell wall loosening or the assembly of an anisotropic cell wall generates asymmetric yielding responses to turgor-induced stress (Baskin, 2005; Cosgrove, 2005). Therefore, the actin-based control of cell boundary dynamics is indirect, and the actin cytoskeleton influences cell shape change, in part, by actin and/or myosin-dependent trafficking of hormone transporters (Geldner et al., 2001) and organelles (Prokhnevsky et al., 2008), including those that control the localized delivery of protein complexes and polysaccharides that pattern the cell wall (Leucci et al., 2007; Gutierrez et al., 2009). In this scheme for actin-based growth control, the actin network dynamically rearranges at spatial scales that span from approximately 1- to 10-µm subcellular domains that may locally position organelles (Cleary, 1995; Gibbon et al., 1999; Szymanski et al., 1999) to the more than 100-µm actin bundle networks that operate at the spatial scales of entire cells (Gutierrez et al., 2009; Dyachok et al., 2011). It is clear from the work of several laboratories that the W/SRC and ARP2/3 protein complexes are required to organize cortical actin and actin bundle networks in trichomes (Szymanski et al., 1999; Le et al., 2003; Deeks et al., 2004; Zhang et al., 2005) and cylindrical epidermal cells (Mathur et al., 2003b; Dyachok et al., 2008, 2011). A key challenge now is to understand how plant cells deploy these approximately 10- to 20-nm heteromeric protein complexes to influence the patterns of growth at cellular scales.The genetic and biochemical control of ARP2/3 is complicated, but this is a tractable problem in plants, because the pathway is relatively simple compared with most other species in which it has been characterized. For example, in organisms ranging from yeast to humans, there are multiple types of ARP2/3 activators, protein complexes, and pathways that activate ARP2/3 (Welch and Mullins, 2002; Derivery and Gautreau, 2010). However, the maize and Arabidopsis genomes encode only WAVE/SCAR homologous proteins that can potently activate ARP2/3 (Frank et al., 2004; Basu et al., 2005). Detailed genetic and biochemical analyses of the WAVE/SCAR gene family in Arabidopsis demonstrated that the plant activators function interchangeably within the context of the W/SRC and define the lone pathway for ARP2/3 activation (Zhang et al., 2008). Bioinformatic analyses are consistent with a prominent role for W/SRC in the angiosperms, because in general, WASH complex subunits, which are structurally similar to WAVE/SCAR proteins, are largely absent from the higher plant genomes, while WAVE/SCAR genes are highly conserved (Kollmar et al., 2012).The components and regulatory schemes of the W/SRC-ARP2/3 pathway in Arabidopsis and P. patens are conserved compared with vertebrate species that employ these same protein complexes (Szymanski, 2005). For example, mutant complementation tests indicate that human W/SRC and ARP2/3 complex subunits can substitute for the Arabidopsis proteins (Mathur et al., 2003b). Furthermore, biochemical assays of Arabidopsis W/SRC (Basu et al., 2004; El-Assal et al., 2004; Frank et al., 2004; Le et al., 2006; Uhrig et al., 2007) and ARP2/3 assembly (Kotchoni et al., 2009) have shown that the binary interactions among W/SRC subunits and ARP2/3 complex assembly mechanisms are indistinguishable from those that have been observed for human W/SRC (Gautreau et al., 2004) and yeast ARP2/3 (Winter et al., 1999). After an initial period of controversy concerning the biochemical control of W/SRC, it is now apparent that vertebrate W/SRC (Derivery et al., 2009; Ismail et al., 2009), like the ARP2/3 complex (Machesky et al., 1999), is intrinsically inactive and requires positive regulation by Rac and other factors to fully activate ARP2/3 (Ismail et al., 2009; Lebensohn and Kirschner, 2009; Chen et al., 2010). Although overexpression of dominant negative ROP mutants causes trichome swelling and a reduced trichome branch number (Fu et al., 2002), the involvement of ROPs in trichome morphogenesis has been difficult to prove with a loss-of-function ROP allele because so many ROPs are expressed in this cell type (Marks et al., 2009). Existing reports on ROP loss-of-function mutants demonstrate the importance of pavement cell morphogenesis but do not document a trichome phenotype (Fu et al., 2005; Xu et al., 2010). A recent report describes a clever strategy to generate ROP loss-of-function lines that used the ectopic expression of ROP-specific bacterial toxins. There was a strong association between inducible expression of the toxins and the appearance of trichomes with severe trichome swelling and reduced branch number phenotypes (Singh et al., 2012). Although the exact mechanism of ROP-dependent control of W/SRC remains to be determined, the results described above in combination with the detection of direct interactions between the ROPGEF SPK1, active forms of ROP, and W/SRC subunits (Basu et al., 2004, 2008; Uhrig et al., 2007) strongly suggest that W/SRC is a ROP effector complex.The major challenge in the field now is to better understand the cellular control of W/SRC and how the complex is partitioned into active and inactive pools. In mammalian cells that crawl on a solid substrate, current models propose that a cytosolic pool of inactive WAVE/SCAR proteins and W/SRC is locally recruited and activated at specific plasma membrane surfaces in response to signals from some unknown Rac guanine nucleotide-exchange factor (GEF), protein kinase, and/or lipid kinase (Oikawa et al., 2004; Lebensohn and Kirschner, 2009; Chen et al., 2010). However, in Drosophila melanogaster neurons (Bogdan and Klämbt, 2003) and cultured human melanoma cells (Steffen et al., 2004), there are large pools of W/SRC with a perinuclear or organelle-like punctate localization that has no obvious relationship to cell shape or motility, raising uncertainty about the cellular mechanisms of W/SRC activation and the importance of different subcellular pools of the complex.In plants, cell fractionation experiments indicate that SCAR1 and ARP2/3 have an increased association with membranes compared with their animal counterparts (Dyachok et al., 2008; Kotchoni et al., 2009). In tip-growing moss protonemal cells, both the W/SRC subunit BRK1 and ARP2/3 localize to a population of unidentified organelles within the apical zone (Perroud and Quatrano, 2008). Similar live-cell imaging experiments in Arabidopsis reported a plasma membrane localization for SCAR1 and BRK1 in a variety of shoot epidermal and root cortex, and their accumulation at young trichome branch tips and at three-way cell wall junctions may define subcellular domains for W/SRC-ARP2/3-dependent actin filament nucleation at the plasma membrane (Dyachok et al., 2008). However, to our knowledge, active W/SRC, defined here as the fraction of W/SRC that colocalizes with ARP2/3 or actin, has not been reported in plants, and the plasma membrane is not necessarily the only organelle involved in W/SRC regulation. For example, the reported accumulation of BRK1 and SCAR1 at three-way cell wall junctions has a punctate appearance at the cell cortex that may not simply correspond to the plasma membrane (Dyachok et al., 2008). Also, in young stage 4 trichomes, there was an uncharacterized pool of intracellular SCAR1, but not BRK1, that localized to relatively large punctate structures (Dyachok et al., 2008). The endoplasmic reticulum (ER) may also be involved in W/SRC regulation. The ER-localized DOCK family ROPGEF SPK1 (Zhang et al., 2010) physically associates with multiple W/SRC proteins (Uhrig et al., 2007; Basu et al., 2008) and, based on genetic criteria, is an upstream, positive regulator of the W/SRC-ARP2/3 pathway (Basu et al., 2008). In the leaf, one function of SPK1 is to promote normal trafficking between the ER and Golgi; however, arp2/3 mutants do not share ER-stress phenotypes with spk1 (Zhang et al., 2010), making it unclear if SPK1 and the ER are directly involved in W/SRC signaling.This paper focuses on the localization and control of the W/SRC subunit NAP1/GNARLED/NAPP/HEM1/2. Arabidopsis NAP1 directly interacts with the ROP/Rac effector subunit SRA1/PIROGI/KLUNKER/PIRP (Basu et al., 2004; El-Assal et al., 2004; Uhrig et al., 2007). Based on the equally severe syndrome of nap1 and arp2/3 null phenotypes, and double mutant analyses, the only known function of NAP1 is to positively regulate ARP2/3 (Brembu et al., 2004; Deeks et al., 2004; El-Din El-Assal et al., 2004; Li et al., 2004). The vertebrate SRA1-NAP1 dimer is important for W/SRC assembly (Gautreau et al., 2004) and forms an extended physical surface that trans-inhibits the C-terminal ARP2/3-activating domain of WAVE/SCAR (Chen et al., 2010). The plant NAP1 and SRA1 proteins share end-to-end amino acid conservation with their vertebrate homologs and may form a heterodimer with similar functions (Basu et al., 2004; El-Assal et al., 2004; Uhrig et al., 2007). We report here that Arabidopsis NAP1 is strongly associated with ER membranes. In a detailed series of localization experiments, we detect a complicated intracellular distribution of NAP1 among the ER, the nucleus, and unidentified submicrometer punctae. A large pool of ER-associated NAP1 is inactive, based on the low level of colocalization with actin.Its accumulation on the ER does not require activating signals from either SPK1 or SRA1. These data indicate that the ER is a reservoir for W/SRC signaling and suggest that early steps in the positive regulation of NAP1 and the W/SRC occur on the ER surface.     

STAT3 but Not STAT1 Is Required for Astrocyte Differentiation

The JAK-STAT signaling pathway has been implicated in astrocyte differentiation. Both STAT1 and STAT3 are expressed in the central nervous system and are thought to be important for glial differentiation, as mainly demonstrated in vitro; however direct in vivo evidence is missing. We investigated whether STAT1 and STAT3 are essential for astrocyte development by testing the STAT responsiveness of astrocyte progenitors. STAT3 was absent in the ventricular zone where glial progenitors are born but begins to appear at the marginal zone at E16.5. At E18.5, both phospho-STAT1 and phospho-STAT3 were present in glial fibrillary acidic protein (GFAP)-expressing white matter astrocytes. Overexpression of STAT3 by electroporation of chicks in ovo induced increased numbers of astrocyte progenitors in the spinal cord. Likewise, elimination of STAT3 in Stat3 conditional knockout (cKO) mice resulted in depletion of white matter astrocytes. Interestingly, elimination of STAT1 in Stat1 null mice did not inhibit astrocyte differentiation and deletion of Stat1 failed to aggravate the glial defects in Stat3 cKO mice. Measuring the activity of STAT binding elements and the gfap promoter in the presence of various STAT mutants revealed that transactivation depended on the activity of STAT3 not STAT1. No synergistic interaction between STAT1 and STAT3 was observed. Cortical progenitors of Stat1 null; Stat3 cKO mice generated astrocytes when STAT3 or the splice variant Stat3β was supplied, but not when STAT1 was introduced. Together, our results suggest that STAT3 is necessary and sufficient for astrocyte differentiation whereas STAT1 is dispensable.     

The Signature 3-O-Sulfo Group of the Anticoagulant Heparin Sequence Is Critical for Heparin Binding to Antithrombin but Is Not Required for Allosteric Activation

Heparin and heparan sulfate glycosaminoglycans allosterically activate the serpin, antithrombin, by binding through a specific pentasaccharide sequence containing a critical 3-O-sulfo group. To elucidate the role of the 3-O-sulfo group in the activation mechanism, we compared the effects of deleting the 3-O-sulfo group or mutating the Lys¹¹⁴ binding partner of this group on antithrombin-pentasaccharide interactions by equilibrium binding and rapid kinetic analyses. Binding studies over a wide range of ionic strength and pH showed that loss of the 3-O-sulfo group caused a massive ∼60% loss in binding energy for the antithrombin-pentasaccharide interaction due to the disruption of a cooperative network of ionic and nonionic interactions. Despite this affinity loss, the 3-O-desulfonated pentasaccharide retained the ability to induce tryptophan fluorescence changes and to enhance factor Xa reactivity in antithrombin, indicative of normal conformational activation. Rapid kinetic studies showed that loss of the 3-O-sulfo group affected both the ability of the pentasaccharide to recognize native antithrombin and its ability to preferentially bind and stabilize activated antithrombin. By contrast, mutation of Lys¹¹⁴ solely affected the preferential interaction of the pentasaccharide with activated antithrombin. These findings demonstrate that the 3-O-sulfo group functions as a key determinant of heparin pentasaccharide activation of antithrombin both by contributing to the Lys¹¹⁴-independent recognition of native antithrombin and by triggering a Lys¹¹⁴-dependent induced fit interaction with activated antithrombin that locks the serpin in the activated state.Antithrombin, a member of the serpin superfamily of protein protease inhibitors, is the principal physiologic regulator of blood coagulation proteases in vertebrates (1, 2). Deficiencies of this blood plasma protein thus increase the risk of thrombotic disease (3), and complete deficiency appears to be incompatible with life (4). Antithrombin regulates the activity of its major target proteases, thrombin, factor Xa, and factor IXa, by inactivation of the enzymes through a branched pathway suicide substrate mechanism of inhibition that is characteristic of serpin-protease reactions (5). In this mechanism, the protease initially recognizes an exposed reactive loop of the serpin as a normal substrate and proceeds to cleave the loop and form the usual acyl-intermediate. However, once this cleavage has occurred, the serpin is induced to undergo a massive conformational change in which the N-terminal part of the reactive loop inserts into the major β-sheet of the serpin, causing the acyl-linked protease to be dragged to the opposite end of the protein inhibitor and inactivated through conformational distortion (6–8).In contrast to the rapid rates at which many serpins inactivate their target proteases, antithrombin inhibits coagulation proteases at slow nonphysiologic rates. However, these inhibition rates increase up to several thousand-fold in the presence of the sulfated glycosaminoglycans, heparin and heparan sulfate, which thereby act as efficient anticoagulants (9). The accelerating effects of heparin and heparan sulfate on antithrombin-protease reactions are dependent on the binding of a sequence-specific pentasaccharide to the serpin (10, 11), present in about one-third of naturally occurring heparin chains and in a much smaller fraction of heparan sulfate chains (12). A characteristic structural marker of the specific antithrombin-binding pentasaccharide region is the 3-O-sulfonated central glucosamine residue (Fig. 1), which is absent or rare in other parts of the heparin molecule (10). The x-ray structure of the antithrombin-pentasaccharide complex and mutagenesis studies have shown that antithrombin basic residues in the N-terminal region, helix A, helix D, and the loop preceding helix D form a positively charged site to which the negatively charged pentasaccharide binds (13). Lys¹¹⁴, Lys¹²⁵, and Arg¹²⁹ are the most important of these residues, with Lys¹¹⁴, the binding partner of the 3-O-sulfo group, contributing the greatest binding energy (9, 14).Open in a separate windowFIGURE 1.Structures of the natural (1) and 3-O-desulfonated (2) heparin pentasaccharides. Pentasaccharide structures are shown from nonreducing to reducing ends with the saccharides designated DEFGH for historical reasons. The 3-O-sulfo group is designated with an asterisk in 1.In this work, we have studied the interaction of a variant pentasaccharide lacking the 3-O-sulfo group with antithrombin. This variant has been previously reported to poorly bind antithrombin and to have greatly reduced anticoagulant activity, suggesting an essential role of the 3-O-sulfo group in binding and activating antithrombin (15–18). However, quantitative studies were done at physiologic ionic strength and pH, where binding to antithrombin was extremely weak, and the extent of inhibitor activation was difficult to quantify. We show, by analyzing pentasaccharide binding to and activation of α- and β-glycoforms of antithrombin over a wide range of ionic strength and pH, that deletion of the 3-O-sulfo group results in tremendous ∼10⁴ to 10⁵-fold losses in affinity for antithrombin under physiologic conditions due to the disruption of a cooperative network of ionic and nonionic interactions that stabilize the complex. Despite these large losses in affinity and in contrast to past reports (16), loss of the 3-O-sulfo group does not affect the ability of the pentasaccharide to induce conformational activation of antithrombin. Rapid kinetic studies of the variant pentasaccharide interaction with antithrombin reveal marked effects of 3-O-desulfonation on both initial binding and subsequent conformational activation steps. These effects contrast with the effects of mutating the Lys¹¹⁴ binding partner of the 3-O-sulfo group, which exclusively involve the second conformational activation step (14). Together, our findings show that the 3-O-sulfo group is of greater quantitative importance for the antithrombin-pentasaccharide interaction than any other pentasaccharide sulfo group or mutated antithrombin investigated so far due to its dual role in recognizing the native antithrombin conformation as well as in preferentially binding and stabilizing the activated antithrombin conformation. In the latter role, our results suggest that the 3-O-sulfo group must engage Lys¹¹⁴ to position pentasaccharide and antithrombin residues for an induced fit interaction that enhances affinity and locks antithrombin in the activated state.     

CAMKII Activation Is Not Required for Maintenance of Learning-Induced Enhancement of Neuronal Excitability

Pyramidal neurons in the piriform cortex from olfactory-discrimination trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the post-burst after-hyperpolarization (AHP) which is generated by repetitive spike firing. AHP reduction is due to decreased conductance of a calcium-dependent potassium current, the sI_AHP. We have previously shown that learning-induced AHP reduction is maintained by persistent protein kinase C (PKC) and extracellular regulated kinase (ERK) activation. However, the molecular machinery underlying this long-lasting modulation of intrinsic excitability is yet to be fully described. Here we examine whether the CaMKII, which is known to be crucial in learning, memory and synaptic plasticity processes, is instrumental for the maintenance of learning-induced AHP reduction. KN93, that selectively blocks CaMKII autophosphorylation at Thr286, reduced the AHP in neurons from trained and control rat to the same extent. Consequently, the differences in AHP amplitude and neuronal adaptation between neurons from trained rats and controls remained. Accordingly, the level of activated CaMKII was similar in pirifrom cortex samples taken form trained and control rats. Our data show that although CaMKII modulates the amplitude of AHP of pyramidal neurons in the piriform cortex, its activation is not required for maintaining learning-induced enhancement of neuronal excitability.     

The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability,but Not for DNA Methylation in Neurospora crassa

LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to DNA damaging agents. MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued DNA damage-hypersensitivity of Δmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Δmus-30 is partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Δmus-30 strains. We found that MUS-30-deficient cells are not defective for DSB repair, and we observed a negative genetic interaction between Δmus-30 and Δmei-3, the Neurospora RAD51 homolog required for homologous recombination. Together, our findings suggest that MUS-30, an LSH/DDM1 homolog, is required to prevent DNA damage arising from toxic base excision repair intermediates. Overall, our study provides important new information about the functions of the LSH/DDM1 family of enzymes.     

Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC in vitro and in vivo. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells.     

XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA ends in vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNA in vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4 in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF- or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.     

Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.     

MicroRNA-223 Regulates Granulopoiesis but Is Not Required for HSC Maintenance in Mice

MIR233 is genetically or epigenetically silenced in a subset of acute myeloid leukemia (AML). MIR223 is normally expressed throughout myeloid differentiation and highly expressed in hematopoietic stem cells (HSCs). However, the contribution of MIR223 loss to leukemic transformation and HSC function is largely unknown. Herein, we characterize HSC function and myeloid differentiation in Mir223 deficient mice. We show that Mir223 loss results in a modest expansion of myeloid progenitors, but is not sufficient to induce a myeloproliferative disorder. Loss of Mir223 had no discernible effect on HSC quiescence, long-term repopulating activity, or self-renewal capacity. These results suggest that MIR223 loss is likely not an initiating event in AML but may cooperate with other AML associated oncogenes to induce leukemogenesis.     

Activation of the NLRP3 Inflammasome Complex is Not Required for Stress-Induced Death of Pancreatic Islets

Loss of pancreatic beta cells is a feature of type-2 diabetes. High glucose concentrations induce endoplasmic reticulum (ER) and oxidative stress-mediated apoptosis of islet cells in vitro. ER stress, oxidative stress and high glucose concentrations may also activate the NLRP3 inflammasome leading to interleukin (IL)-1β production and caspase-1 dependent pyroptosis. However, whether IL-1β or intrinsic NLRP3 inflammasome activation contributes to beta cell death is controversial. This possibility was examined in mouse islets. Exposure of islets lacking functional NLRP3 or caspase-1 to H₂O_2, rotenone or thapsigargin induced similar cell death as in wild-type islets. This suggests that oxidative or ER stress do not cause inflammasome-mediated cell death. Similarly, deficiency of NLRP3 inflammasome components did not provide any protection from glucose, ribose or gluco-lipotoxicity. Finally, genetic activation of NLRP3 specifically in beta cells did not increase IL-1β production or cell death, even in response to glucolipotoxicity. Overall, our results show that glucose-, ER stress- or oxidative stress-induced cell death in islet cells is not dependent on intrinsic activation of the NLRP3 inflammasome.     

Dimerization of the Pseudomonas aeruginosa Translocator Chaperone PcrH Is Required for Stability,Not Function

Type III secretion systems rely on hydrophobic translocator proteins that form a pore in the host cell membrane to deliver effector proteins into targeted host cells. These translocator proteins are stabilized in the cytoplasm and targeted for export with the help of specific chaperone proteins. In Pseudomonas aeruginosa, the chaperone of the pore-forming translocator proteins is PcrH. Although all translocator chaperones dimerize, the location of the dimerization interface is in dispute. Moreover, it has been reported that interfering with dimerization interferes with chaperone function. However, binding of P. aeruginosa chaperone PcrH to its cognate secretion substrate, PopD, results in dissociation of the PcrH dimer in vitro, arguing that dimerization of PcrH is likely not important for substrate binding or targeting translocators for export. We demonstrate that PcrH dimerization occurs in vivo in P. aeruginosa and used a genetic screen to identify a dimerization mutant of PcrH. The mutant protein is fully functional in that it can both stabilize PopB and PopD in the cytoplasm and promote their export via the type III secretion system. The location of the mutation suggests that the dimerization interface of PcrH mirrors that of the Yersinia homolog SycD and not the dimerization interface that had previously been reported for PcrH based on crystallographic evidence. Finally, we present data that the dimerization mutant of PcrH is less stable than the wild-type protein in P. aeruginosa, suggesting that the function of dimerization is stabilization of PcrH in the absence of its cognate cargo.     

Lymphotoxin, but Not TNF, Is Required for Prion Invasion of Lymph Nodes

Neuroinvasion and subsequent destruction of the central nervous system by prions are typically preceded by a colonization phase in lymphoid organs. An important compartment harboring prions in lymphoid tissue is the follicular dendritic cell (FDC), which requires both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance. However, prions are still detected in TNFR1^−/− lymph nodes despite the absence of mature FDCs. Here we show that TNFR1-independent prion accumulation in lymph nodes depends on LTβR signaling. Loss of LTβR signaling, but not of TNFR1, was concurrent with the dedifferentiation of high endothelial venules (HEVs) required for lymphocyte entry into lymph nodes. Using luminescent conjugated polymers for histochemical PrP^Sc detection, we identified PrP^Sc deposits associated with HEVs in TNFR1^−/− lymph nodes. Hence, prions may enter lymph nodes by HEVs and accumulate or replicate in the absence of mature FDCs.     

Abi, Sra1, and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions

BACKGROUND: In animal cells, GTPase signaling pathways are thought to generate cellular protrusions by modulating the activity of downstream actin-regulatory proteins. Although the molecular events linking activation of a GTPase to the formation of an actin-based process with a characteristic morphology are incompletely understood, Rac-GTP is thought to promote the activation of SCAR/WAVE, whereas Cdc42 is thought to initiate the formation of filopodia through WASP. SCAR and WASP then activate the Arp2/3 complex to nucleate the formation of new actin filaments, which through polymerization exert a protrusive force on the membrane. RESULTS: Using RNAi to screen for genes regulating cell form in an adherent Drosophila cell line, we identified a set of genes, including Abi/E3B1, that are absolutely required for the formation of dynamic protrusions. These genes delineate a pathway from Cdc42 and Rac to SCAR and the Arp2/3 complex. Efforts to place Abi in this signaling hierarchy revealed that Abi and two components of a recently identified SCAR complex, Sra1 (p140/PIR121/CYFIP) and Kette (Nap1/Hem), protect SCAR from proteasome-mediated degradation and are critical for SCAR localization and for the generation of Arp2/3-dependent protrusions. CONCLUSIONS: In Drosophila cells, SCAR is regulated by Abi, Kette, and Sra1, components of a conserved regulatory SCAR complex. By controlling the stability, localization, and function of SCAR, these proteins may help to ensure that Arp2/3 activation and the generation of actin-based protrusions remain strictly dependant on local GTPase signaling.     

ESCRT-0 Is Not Required for Ectopic Notch Activation and Tumor Suppression in Drosophila

Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis.     

Syntaxin Binding Protein 1 Is Not Required for Allergic Inflammation via IgE-Mediated Mast Cell Activation

Mast cells play a central role in both innate and acquired immunity. When activated by IgE-dependent FcεRI cross-linking, mast cells rapidly initiate a signaling cascade and undergo an extensive release of their granule contents, including inflammatory mediators. Some SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor) proteins and SM (Sec1/Munc18) family proteins are involved in mast cell degranulation. However, the function of syntaxin binding protein 1 (STXBP1), a member of SM family, in mast cell degranulation is currently unknown. In this study, we examined the role of STXBP1 in IgE-dependent mast cell activation. Liver-derived mast cells (LMCs) from wild-type and STXBP1-deficient mice were cultured in vitro for the study of mast cell maturation, degranulation, cytokine and chemokine production, as well as MAPK, IκB-NFκB, and NFAT signaling pathways. In addition, in vivo models of passive cutaneous anaphylaxis and late-phase IgE-dependent inflammation were conducted in mast cell deficient W^sh mice that had been reconstituted with wild-type or STXBP1-deficient mast cells. Our findings indicate that STXBP1 is not required for any of these important functional mechanisms in mast cells both in vitro and in vivo. Our results demonstrate that STXBP1 is dispensable during IgE-mediated mast cell activation and in IgE-dependent allergic inflammatory reactions.     

Apolipoprotein E but Not B Is Required for the Formation of Infectious Hepatitis C Virus Particles

Our previous studies have found that hepatitis C virus (HCV) particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infectivity and production. Studies by others, however, suggested that both microsomal transfer protein (MTP) and apoB are important for HCV production. To define the roles of apoB and apoE in the HCV life cycle, we developed a single-cycle HCV growth assay to determine the correlation of HCV assembly with apoB and apoE expression, as well as the influence of MTP inhibitors on the formation of HCV particles. The small interfering RNA (siRNA)-mediated knockdown of apoE expression remarkably suppressed the formation of HCV particles. However, apoE expressed ectopically could restore the defect of HCV production posed by the siRNA-mediated knockdown of endogenous apoE expression. In contrast, apoB-specific antibodies and siRNAs had no significant effect on HCV infectivity and production, respectively, suggesting that apoB does not play a significant role in the HCV life cycle. Additionally, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked secretion of apoB-containing lipoproteins but did not affect HCV production unless apoE expression and secretion were inhibited. At higher concentrations, however, MTP inhibitors blocked apoE expression and secretion and consequently suppressed the formation of HCV particles. Furthermore, apoE was found to be sensitive to trypsin digestion and to interact with NS5A in purified HCV particles and HCV-infected cells, as demonstrated by coimmunoprecipitation. Collectively, these findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.Hepatitis C virus (HCV) is the leading cause of chronic viral hepatitis, affecting approximately 170 million people worldwide (8, 40). HCV coinfection with human immunodeficiency virus (HIV) is also common, occurring overall in 25 to 30% of HIV-positive persons (1). Individuals with chronic HCV infection are at high risk for the development of cirrhosis and hepatocellular carcinoma. A pegylated interferon and ribavirin combination is the standard therapy to treat hepatitis C but suffers from limited efficacy (<50% antiviral response among patients infected with the dominant genotype 1 HCV) and severe side effects (18, 27). More efficacious and safer antiviral drugs for effective treatment of hepatitis C are urgently needed. A thorough understanding of the HCV life cycle will likely provide novel targets for antiviral drug discovery and development to control HCV infection.HCV is an enveloped RNA virus containing a single-stranded, positive-sense RNA genome and is classified as a Hepacivirus in the Flaviviridae family (11, 33). The viral RNA genome carries a single open reading frame flanked by untranslated regions (UTRs) at both the 5′ and 3′ ends. The 5′ and 3′ UTRs contain cis-acting RNA elements important for the initiation of HCV polyprotein translation and viral RNA replication (24). Upon translation, the HCV polyprotein precursor is proteolytically processed by cellular peptidases and viral proteases into at least 10 different viral proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Studies with subgenomic HCV RNAs demonstrated that the NS3 to NS5B proteins, in association with intracellular membranes and cellular proteins, are essential and sufficient for HCV RNA replication in the cell (5, 14, 25). The newly synthesized HCV proteins and RNA genome are assembled to form progeny HCV particles by undetermined mechanisms.Our earlier work found that infectious HCV particles are highly enriched in apolipoprotein E (apoE), which is a major determinant of HCV infectivity and production in cell culture (10). ApoE-specific monoclonal antibodies (MAbs) effectively neutralized HCV infectivity, in a dose-dependent manner. The knockdown of apoE expression by specific small interfering RNA (siRNA) remarkably suppressed HCV production, suggesting that apoE is also important for the formation of infectious particles and/or egression (10). However, studies by others suggested that HCV assembly and production are dependent on microsomal transfer protein (MTP) and apolipoprotein B (apoB), both of which are essential components required for the assembly and secretion of very-low-density lipoproteins (VLDLs) (19, 21). In those studies, both apoB-specific siRNAs and MTP inhibitors were found to suppress HCV production (19, 21). It was speculated that HCV shares the same assembly and secretion pathway with VLDLs.To define the roles of apoB and apoE in the formation of HCV particles and egression, we developed a single-cycle HCV growth assay. Using this assay system, we have demonstrated that apoE but not apoB is required for the infectivity and formation of infectious HCV particles. First of all, apoB-specific MAb and polyclonal antibodies did not affect HCV infection. Additionally, apoE-specific siRNA potently inhibited the formation of infectious HCV particles, whereas HCV production was unaffected by the siRNA-mediated knockdown of apoB expression. Furthermore, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked apoB secretion but did not significantly affect HCV production prior to the blockage of apoE expression/secretion. At higher concentrations, however, both MTP inhibitors blocked apoE secretion and consequently suppressed the formation of infectious HCV particles. To further understand the role of apoE in HCV assembly, we carried out coimmunoprecipitation (co-IP) experiments and found that apoE-specific MAb pulled down NS5A but not other HCV proteins from lysed HCV particles, suggesting a specific interaction between apoE and NS5A during the formation of infectious HCV particles. Collectively, our findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.     

Salicylic Acid Is Not the Translocated Signal Responsible for Inducing Systemic Acquired Resistance but Is Required in Signal Transduction

Infection of plants by necrotizing pathogens can induce broad-spectrum resistance to subsequent pathogen infection. This systemic acquired resistance (SAR) is thought to be triggered by a vascular-mobile signal that moves throughout the plant from the infected leaves. A considerable amount of evidence suggests that salicylic acid (SA) is involved in the induction of SAR. Because SA is found in phloem exudate of infected cucumber and tobacco plants, it has been proposed as a candidate for the translocated signal. To determine if SA is the mobile signal, grafting experiments were performed using transgenic plants that express a bacterial SA-degrading enzyme. We show that transgenic tobacco root-stocks, although unable to accumulate SA, were fully capable of delivering a signal that renders nontransgenic scions resistant to further pathogen infection. This result indicated that the translocating, SAR-inducing signal is not SA. Reciprocal grafts demonstrated that the signal requires the presence of SA in tissues distant from the infection site to induce systemic resistance.