A network of interactions among seminal proteins underlies the long-term postmating response in Drosophila

Despite the importance of seminal proteins in fertility and their capacity to alter mated females'' physiology, the molecular pathways and networks through which they act have not been well characterized. Drosophila seminal fluid includes proteins that fall into biochemical classes conserved from insects to mammals, making it an excellent model with which to address this question. Drosophila seminal fluid also contains a “sex peptide” (SP, Acp70A) that plays a major role in regulating egg production and mating behavior in females for several days after mating. This long-term postmating response (LTR) initially requires the association of SP with sperm. The LTR also requires members of the conserved seminal protein classes (two lectins, a protease, and a cysteine-rich secretory protein). Here, we show that these seminal proteins function interdependently, regulating a three-step cascade (first, at the level of seminal protein transfer to the female; second, at the level of stability; and third, at the level of localization within females), leading to the normal localization of SP to sperm-storage organs. This localization is, in turn, necessary for successful induction of the LTR. The requirements for manifestation of the LTR in Drosophila establish the paradigm that multiple seminal proteins can exert their actions through a multistep, multicomponent network of interactions.     

Blanks, a nuclear siRNA/dsRNA-binding complex component, is required for Drosophila spermiogenesis

Small RNAs and a diverse array of protein partners control gene expression in eukaryotes through a variety of mechanisms. By combining siRNA affinity chromatography and mass spectrometry, we have identified the double-stranded RNA-binding domain protein Blanks to be an siRNA- and dsRNA-binding protein from Drosophila S2 cells. We find that Blanks is a nuclear factor that contributes to the efficiency of RNAi. Biochemical fractionation of a Blanks-containing complex shows that the Blanks complex is unlike previously described RNA-induced silencing complexes and associates with the DEAD-box helicase RM62, a protein previously implicated in RNA silencing. In flies, Blanks is highly expressed in testes tissues and is necessary for postmeiotic spermiogenesis, but loss of Blanks is not accompanied by detectable transposon derepression. Instead, genes related to innate immunity pathways are up-regulated in blanks mutant testes. These results reveal Blanks to be a unique component of a nuclear siRNA/dsRNA-binding complex that contributes to essential RNA silencing-related pathways in the male germ line.     

Dynein light chain 1 is required for autophagy,protein clearance,and cell death in Drosophila

Autophagy is a catabolic pathway that is important for turnover of long-lived proteins and organelles, and has been implicated in cell survival, tumor progression, protection from infection, neurodegeneration, and cell death. Autophagy and caspases are required for type II autophagic cell death of Drosophila larval salivary glands during development, but the mechanisms that regulate these degradation pathways are not understood. We conducted a forward genetic screen for genes that are required for salivary gland cell death, and here we describe the identification of Drosophila dynein light chain 1 (ddlc1) as a gene that is required for type II cell death. Autophagy is attenuated in ddlc1 mutants, but caspases are active in these cells. ddlc1 mutant salivary glands develop large fibrillar protein inclusions that stain positive for amyloid-specific dyes and ubiquitin. Ectopic expression of Atg1 is sufficient to induce autophagy, clear protein inclusions, and rescue degradation of ddlc1 mutant salivary glands. Furthermore, ddlc1 mutant larvae have decreased motility, and mutations in ddlc1 enhance the impairment of motility that is observed in a Drosophila model of neurodegenerative disease. Significantly, this decrease in larval motility is associated with decreased clearance of protein with polyglutamine expansion, the accumulation of p62 in neurons and muscles, and fewer synaptic boutons. These results indicate that DDLC1 is required for protein clearance by autophagy that is associated with autophagic cell death and neurodegeneration.     

GTP-induced conformational changes in septins and implications for function

Septins constitute a group of GTP-binding proteins involved in cytokinesis and other essential cellular functions. They form heterooligomeric complexes that polymerize into nonpolar filaments and are dynamic during different stages of the cell cycle. Posttranslational modifications and interacting partners are widely accepted regulators of septin filament function, but the contribution of nucleotide is undefined due to a lack of detailed structural information. Previous low-resolution structures showed that the G domain assembles into a linear polymer with 2 different interfaces involving the N and C termini and the G binding sites. Here we report the crystal structure of SEPT2 bound to GppNHp at 2.9 Å resolution. GTP binding induces conformational changes in the switch regions at the G interfaces, which are transmitted to the N-terminal helix and also affect the NC interface. Biochemical studies and sequence alignment suggest that a threonine, which is conserved in certain subgroups of septins, is responsible for GTP hydrolysis. Although this threonine is not present in yeast CDC3 and CDC11, its mutation in CDC10 and CDC12 induces temperature sensitivity. Highly conserved contact residues identified in the G interface are shown to be necessary for Cdc3–10, but not Cdc11–12, heterodimer formation and cell growth in yeast. Based on our findings, we propose that GTP binding/hydrolysis and the nature of the nucleotide influence the stability of interfaces in heterooligomeric and polymeric septins and are required for proper septin filament assembly/disassembly. These data also offer a first rationale for subdividing human septins into different functional subgroups.     

ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm

The developing endosperm of rice (Oryza sativa, Os) synthesizes a large amount of storage proteins on the rough (r)ER. The major storage proteins, glutelins and prolamins, contain either intra or intermolecular disulfide bonds, and oxidative protein folding is necessary for the sorting of the proteins to the protein bodies. Here, we investigated an electron transfer pathway for the formation of protein disulfide bonds in the rER of the rice endosperm, focusing on the roles of the thiol-disulfide oxidoreductase, OsEro1. Confocal microscopic analysis revealed that N-glycosylated OsEro1 is localized to the rER membrane in the subaleurone cells, and that targeting of OsEro1 to the rER membrane depends on the N-terminal region from Met-1 to Ser-55. The RNAi knockdown of OsERO1 inhibited the formation of native disulfide bonds in the glutelin precursors (proglutelins) and promoted aggregation of the proglutelins through nonnative intermolecular disulfide bonds in the rER. Inhibition of the formation of native disulfide bonds was also observed in the seeds of the esp2 mutant, which lacks protein disulfide isomerase-like (PDIL)1;1, but shows enhanced OsEro1 expression. We detected the generation of H₂O₂ in the rER of the WT subaleurone cells, whereas the rER-derived H₂O₂ levels decreased markedly in EM49 homozygous mutant seeds, which have fewer sulfhydryl groups than the WT seeds. Together, we propose that the formation of native disulfide bonds in proglutelins depends on an electron transfer pathway involving OsEro1 and OsPDIL.     

CD36 folding revealed by conformational epitope expression is essential for cytoadherence of Plasmodium falciparum-infected red blood cells

CD36 is a membrane glycoprotein and a putative scavenger receptor expressed by several cell types. In capillary endothelial cells, it mediates the adherence of erythrocytes infected with Plasmodium falciparum. The CD36 sequence contains two hydrophobic domains located at the amino-and carboxyl-termini of the protein, but the topology of this protein and the functional significance of these domains are still not clearly defined. We generated soluble CD36-IgG chimeric molecules by fusion of the extracellular domains of CD36 with human immunoglobulin domains. The construct containing the N-terminal hydrophobic domain of CD36 was completely retained intracellularly as membrane-associated molecule, suggesting that the N-terminal hydrophobic domain of the CD36 is a real transmembrane domain and that CD36 has hairpin topology. A small amount of the CD36-IgG chimeric construct lacking both transmembrane domains escaped retention, was correctly processed, and accumulated in the extracellular medium as a soluble molecule. This CD36-IgG construct failed to bind Plasmodium falciparum-infected erythrocytes. Using monoclonal antibodies specific for either conformational or structural epitopes, we demonstrate that failure of this CD36-IgG construct to bind infected erythrocytes was due to incorrect folding of the soluble chimeric molecule.     

SNMP is a signaling component required for pheromone sensitivity in Drosophila

The only known volatile pheromone in Drosophila, 11-cis-vaccenyl acetate (cVA), mediates a variety of behaviors including aggregation, mate recognition, and sexual behavior. cVA is detected by a small set of olfactory neurons located in T1 trichoid sensilla on the antennae of males and females. Two components known to be required for cVA reception are the odorant receptor Or67d and the extracellular pheromone-binding protein LUSH. Using a genetic screen for cVA-insensitive mutants, we have identified a third component required for cVA reception: sensory neuron membrane protein (SNMP). SNMP is a homolog of CD36, a scavenger receptor important for lipoprotein binding and uptake of cholesterol and lipids in vertebrates. In humans, loss of CD36 is linked to a wide range of disorders including insulin resistance, dyslipidemia, and atherosclerosis, but how CD36 functions in lipid transport and signal transduction is poorly understood. We show that SNMP is required in pheromone-sensitive neurons for cVA sensitivity but is not required for sensitivity to general odorants. Using antiserum to SNMP infused directly into the sensillum lymph, we show that SNMP function is required on the dendrites of cVA-sensitive neurons; this finding is consistent with a direct role in cVA signal transduction. Therefore, pheromone perception in Drosophila should serve as an excellent model to elucidate the role of CD36 members in transmembrane signaling.     

Seminal vesicle protein SVS2 is required for sperm survival in the uterus

In mammals, sperm migrate through the female reproductive tract to reach the egg; however, our understanding of this journey is highly limited. To shed light on this process, we focused on defining the functions of seminal vesicle secretion 2 (SVS2). SVS2^−/− male mice produced sperm but were severely subfertile, and formation of a copulatory plug to cover the female genital opening did not occur. Surprisingly, even when artificial insemination was performed with silicon as a substitute for the plug, sperm fertility in the absence of SVS2 remained severely reduced because the sperm were already dead in the uterus. Thus, our results provide evidence that the uterus induces sperm cell death and that SVS2 protects sperm from uterine attack.Fluids secreted from male accessory sex organs are believed to regulate fertility efficiency through the control of sperm functions such as motility and fertilizing ability in vivo (1, 2). Notably, the induced ability of fertilization-competent (i.e., capacitated) rabbit sperm is reverted to an incompetent state when it is mixed with seminal plasma; this phenomenon, discovered by Chang and Bedford (2, 3), is referred to as “decapacitation.” Subsequent to their findings, many studies have focused on identifying the decapacitation factor in the seminal plasma using in vitro assays (4–6); the seminal plasma is believed to stabilize sperm plasma membranes and prevent uterine sperm from undergoing premature capacitation and acrosomal reaction, an exocytotic process that occurs immediately before sperm–egg fusion (7, 8).In mammals the seminal vesicle is an accessory organ within the male reproductive tract, and its secretion influences sperm fertility and embryo development via oviductal expression of cytokines (9). Seminal vesicle secretion 2 (SVS2), a major component of the seminal vesicle secretions, inhibits sperm fertility in vitro, and homologous genes are conserved among many species (10, 11). As described previously (10), SVS2 binds to sperm in the uterus but not to sperm in the oviduct. In addition, SVS2 binds to ganglioside GM1 as its receptor on the sperm membrane, resulting in the control of sperm fertility (11). To study the role of SVS2 in vivo, we here focused on defining a role for SVS2 in in vivo fertilization.     

Comparison on serum biomarkers for anovulatory and ovulatory dysfunctional uterine bleeding in Lizu females

Objective:To screen,identify,and compare the serum biomarkers between anovulatory dysfunctional uterine bleeding(ADUB)and ovulatory dysfunctional uterine bleeding(ODUB)in Lizu females.Methods:The subjects included 128 ADLB patients,63 ODUB patients,and 93controls.The serum and supernate of the subjects'mense were collected and stored at-80°C until use.Differential proteins in the sera of three groups were screened using surface-enhaneed laser desorption ionization time-of-flight mass spectrometry.The screened proteins were then identified by tricine-SDS-PAGE gel and spectrometry.Protein expression levels in the menses of ADUB,ODUB,and control subjects were determined using ELISA,RT-PCR,and Western blotting.SPSS 14.1 was used for statistical analysis and chart drawing(a=0.05),Results:Three differentia)protein peaks with peak values of 11.80,13.59,and 14.68 km/z were,screened and identified as serum amyploid protein A(SAA),vascular endothelial growth factor,and vitamin K epoxide reductase,respectively.The SAA was highly expressed in the menses of ADUB and ODUB patients but poorly expressed in the controls.The vascular endothelial growth factor was highly expressed in the menses of ODUB and controls but poorly expressed in ADUB patients.Meanwhile,the vitamin K epoxide reductase was highly expressed in the menses of ADUB and control subjects but poorly expressed in ODUB patients.Conclusions:The SAA is the common serum biomarker of ADUB and ODUB.ADUB may be related to angiogenesis impairment,whereas ODUB may be associated with blood coagulation disruption.     

Nucleoporin MOS7/Nup88 is required for mitosis in gametogenesis and seed development in Arabidopsis

Angiosperm reproduction is characterized by alternate diploid sporophytic and haploid gametophytic generations. Gametogenesis shares similarities with that of animals except for the formation of the gametophyte, whereby haploid cells undergo several rounds of postmeiotic mitosis to form gametes and the accessory cells required for successful reproduction. The mechanisms regulating gametophyte development in angiosperms are incompletely understood. Here, we show that the nucleoporin Nup88-homolog MOS7 (Modifier of Snc1,7) plays a crucial role in mitosis during both male and female gametophyte formation in Arabidopsis thaliana. Using a mutagenesis screen, we identify the mos7-5 mutant allele, which causes ovule and pollen abortion in MOS7/mos7-5 heterozygous plants, and preglobular stage embryonic lethality in homozygous mos7-5 seeds. During interphase, we show that MOS7 is localized to the nuclear membrane but, like many nucleoporins, is associated with the spindle apparatus during mitosis. We detect interactions between MOS7 and several nucleoporins known to control spindle dynamics, and find that in pollen from MOS7/mos7-5 heterozygotes, abortion is accompanied by a failure of spindle formation, cell fate specification, and phragmoplast activity. Most intriguingly, we show that following gamete formation by MOS7/mos7-5 heterozygous spores, inheritance of either the MOS7 or the mos7-5 allele by a given gamete does not correlate with its respective survival or abortion. Instead, we suggest a model whereby MOS7, which is highly expressed in the Pollen- and Megaspore Mother Cells, enacts a dosage-limiting effect on the gametes to enable their progression through subsequent mitoses.A most striking difference between the developmental approaches of plants and animals is the intervention of mitotic divisions between meiosis and gamete formation in plants (1). Following meiosis, successive mitotic divisions of haploid cells produce both mature gametes and complex gametophytic structures encasing them, facilitating fertilization. The regulation of gametophyte formation is not completely understood, but is characterized by elegant cell, nuclear and organelle migration, led by microtubule activity (2, 3).Arabidopsis thaliana female gametophyte formation begins with meiosis of the diploid megaspore mother cell (MMC), forming four haploid megaspores. Female Gametogenesis stages (FG) follow, with megaspores migrating to the micropylar end of the gametophyte (FG1) (4). Three megaspores degenerate, one undergoes mitosis and nuclei migrate to opposite poles, likely facilitated by development of a large central vacuole (FG2). Two additional mitotic divisions generate the eight-nuclear FG5 female gametophyte. Nuclei migrate according to their cell-fate and simultaneous cytokinesis (cellularization) takes place, followed by polar nuclei fusion to form the homo-diploid central cell (FG6). At the micropylar pole lie the synergid cells and egg, and three antipodal cells are located at the opposite pole, which degenerate, marking formation of the mature female gametophyte (FG7) (5).Male gametogenesis also involves precise nuclear migration and both symmetric and asymmetric cell divisions. Meiosis of the diploid pollen mother cell (PMC) produces a tetrad of haploid microspores (6). Unlike female megaspores, however, and reminiscent of mammalian spermatogenesis, all four microspores survive to undergo asymmetric mitotic division, Pollen Mitosis I (PMI), each producing a generative cell engulfed in the cytoplasm of a vegetative cell. The generative cell undergoes a second mitosis (PMII) to form two identical sperm cells. The vegetative nucleus gives rise to the pollen tube (7).Around half of the genes so far identified as functioning in gametogenesis regulate both female and male gametophyte formation, and are involved in common cellular processes that occur in both such as mitosis, vacuole formation, cellularization, nuclear migration and cell expansion (8). To identify genes involved in gametogenesis, we performed a mutagenesis screen for lines showing seed and ovule lethality. We identified mos7-5, a MOS7 (Modifier Of Snc1,7) mutant. MOS7 is homologous to human and Drosophila melanogaster nucleoporin protein Nup88. Nucleoporins comprise nuclear pore complexes (NPCs) which traffic proteins and RNA between the nucleus and cytoplasm (9). Previously identified mos7 mutant alleles were mos7-1, a hypomorphic loss-of-function allele that revealed the importance of MOS7-mediated nucleocytoplasmic passage of defense proteins for plant innate immunity (10); mos7-2 and mos 7–4, both embryonic lethal when homozygous (10). Thus, MOS7, as well as an immune function, likely has a developmental function that is currently unknown, although alterations in human Nup88 expression result in multipolar spindles and promote carcinogenesis (11), so MOS7 may have a role in plant cell division.     

Homeobox gene distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system

Vertebrate Dlx genes have been implicated in the differentiation of multiple neuronal subtypes, including cortical GABAergic interneurons, and mutations in Dlx genes have been linked to clinical conditions such as epilepsy and autism. Here we show that the single Drosophila Dlx homolog, distal-less, is required both to specify chemosensory neurons and to regulate the morphologies of their axons and dendrites. We establish that distal-less is necessary for development of the mushroom body, a brain region that processes olfactory information. These are important examples of distal-less function in an invertebrate nervous system and demonstrate that the Drosophila larval olfactory system is a powerful model in which to understand distal-less functions during neurogenesis.     

Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis

Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1β and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-α and -β) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.     

Asymmetric receptor contact is required for tyrosine autophosphorylation of fibroblast growth factor receptor in living cells

Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.     

Lipoprotein LptE is required for the assembly of LptD by the beta-barrel assembly machine in the outer membrane of Escherichia coli

Most Gram-negative bacteria contain lipopolysaccharide (LPS), a glucosamine-based phospholipid, in the outer leaflet of the outer membrane (OM). LPS is unique to the bacterial OM and, in most cases, essential for cell viability. Transport of LPS from its site of synthesis to the cell surface requires eight essential proteins, MsbA and LptABCDEFG. Although the key players have been identified, the mechanism of LPS transport and assembly is not clear. The stable LptD/E complex is present at the OM and functions in the final stages of LPS assembly. Here, we have identified the mutant allele lptE6, which causes a two-amino-acid deletion in the lipoprotein LptE that affects its interaction with LptD. Highly specific suppressor mutations were isolated not only in lptD but also in bamA, which encodes the central component of the β-barrel assembly machine. We show that lptE6 and both suppressor mutations affect the assembly of the LptD/E complex and suggest that the lipoprotein LptE interacts with LptD while this protein is being assembled by the β-barrel assembly machine.