The presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

PURPOSE: Is the presence of cytoplasmic strings (CS) in human blastocysts associated with the probability of clinical pregnancy with fetal heart (CPFH) after transfer. METHODS: This case-control study involved 300 single blastocyst transfers. 150 of these resulted in a CPFH (cases) while 150 did not (controls). All embryos were cultured in Embryoscope+ and AI software (IVY) was used to select the blastocyst with the highest score from the cohort for transfer. An embryologist, blind to the transfer outcome, recorded the CS number, location, and duration of their activity. RESULTS: There was a significant difference in the number of blastocysts that contained CS, with 97.3% of women's blastocysts resulting in +CPFH containing the CS compared to 88.7% of blastocysts in women who did not have a pregnancy (p = 0.007, OR; 4.67, CI 95% 1.5-14.2). CS appeared 2.4 h earlier in embryo development in the +CPFH group compared to their negative counterparts (p = 0.007). There was a significant difference in the average number of CS/blastocyst with a higher number being present in those that achieved a clinical pregnancy (mean: 6.2, SD 2.9) compared to those that did not (mean: 4.6, SD 3.0) (p ≤ 0.0001). There was a significant increase in the number of vesicles seen traveling along the CS with more seen in the blastocysts resulting in a +CPFH (mean: 4.3 SD 2.1) compared to those in the -CPFH group (mean: 3.1, SD 2.1). CONCLUSION: This study has shown that the presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

Uncoupling of nucleo-cytoplasmic RNA export and localization during stress.

Eukaryotic cells contain sub-cellular compartments that are not membrane bound. Some structures are always present, such as nuclear speckles that contain RNA-binding proteins (RBPs) and poly(A)+ RNAs. Others, like cytoplasmic stress granules (SGs) that harbor mRNAs and RBPs, are induced upon stress. When we examined the formation and composition of nuclear speckles during stress induction with tubercidin, an adenosine analogue previously shown to affect nuclear speckle composition, we unexpectedly found that it also led to the formation of SGs and to the inhibition of several crucial steps of RNA metabolism in cells, thereby serving as a potent inhibitor of the gene expression pathway. Although transcription and splicing persisted under this stress, RBPs and mRNAs were mislocalized in the nucleus and cytoplasm. Specifically, lncRNA and RBP localization to nuclear speckles was disrupted, exon junction complex (EJC) recruitment to mRNA was reduced, mRNA export was obstructed, and cytoplasmic poly(A)+ RNAs localized in SGs. Furthermore, nuclear proteins that participate in mRNA export, such as nucleoporins and mRNA export adaptors, were mislocalized to SGs. This study reveals structural aspects of granule assembly in cells, and describes how the flow of RNA from the nucleus to the cytoplasm is severed under stress.

The membrane-distal regions of integrin α cytoplasmic domains contribute differently to integrin inside-out activation.

Functioning as signal receivers and transmitters, the integrin α/ß cytoplasmic tails (CT) are pivotal in integrin activation and signaling. 18 α integrin subunits share a conserved membrane-proximal region but have a highly diverse membrane-distal (MD) region at their CTs. Recent studies demonstrated that the presence of α CTMD region is essential for talin-induced integrin inside-out activation. However, it remains unknown whether the non-conserved α CTMD regions differently regulate the inside-out activation of integrin. Using αIIbß3, αLß2, and α5ß1 as model integrins and by replacing their α CTMD regions with those of α subunits that pair with ß3, ß2, and ß1 subunits, we analyzed the function of CTMD regions of 17 α subunits in talin-mediated integrin activation. We found that the α CTMD regions play two roles on integrin, which are activation-supportive and activation-regulatory. The regulatory but not the supportive function depends on the sequence identity of α CTMD region. A membrane-proximal tyrosine residue present in the CTMD regions of a subset of α integrins was identified to negatively regulate integrin inside-out activation. Our study provides a useful resource for investigating the function of α integrin CTMD regions.

The Angelman syndrome protein Ube3a/E6AP is required for Golgi acidification and surface protein sialylation.

Angelman syndrome (AS) is a severe disorder of postnatal brain development caused by neuron-specific loss of the HECT (homologous to E6AP carboxy terminus) domain E3 ubiquitin ligase Ube3a/E6AP. The cellular role of Ube3a remains enigmatic despite recent descriptions of synaptic and behavioral deficits in AS mouse models. Although neuron-specific imprinting is thought to limit the disease to the brain, Ube3a is expressed ubiquitously, suggesting a broader role in cellular function. In the current study, we demonstrate a profound structural disruption and cisternal swelling of the Golgi apparatus (GA) in the cortex of AS (UBE3A(m-/p+)) mice. In Ube3a knockdown cell lines and UBE3A(m-/p+) cortical neurons, the GA is severely under-acidified, leading to osmotic swelling. Both in vitro and in vivo, the loss of Ube3a and corresponding elevated pH of the GA is associated with a marked reduction in protein sialylation, a process highly dependent on intralumenal Golgi pH. Altered ion homeostasis of the GA may provide a common cellular pathophysiology underlying the diverse plasticity and neurodevelopmental deficits associated with AS.

The P-body component USP52/PAN2 is a novel regulator of HIF1A mRNA stability.

HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52 (ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3'-UTR (untranslated region)-dependent poly(A)-tail-length-independent destabilization in HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body (processing body) components and we confirmed further that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.

Ectopic formation of primordial germ cells by transplantation of the germ plasm: direct evidence for germ cell determinant in Xenopus.

In many animals, the germ line is specified by a distinct cytoplasmic structure called germ plasm (GP). GP is necessary for primordial germ cell (PGC) formation in anuran amphibians including Xenopus. However, it is unclear whether GP is a direct germ cell determinant in vertebrates. Here we demonstrate that GP acts autonomously for germ cell formation in Xenopus. EGFP-labeled GP from the vegetal pole was transplanted into animal hemisphere of recipient embryos. Cells carrying transplanted GP (T-GP) at the ectopic position showed characteristics similar to the endogenous normal PGCs in subcellular distribution of GP and presence of germ plasm specific molecules. However, T-GP-carrying-cells in the ectopic tissue did not migrate towards the genital ridge. T-GP-carrying cells from gastrula or tailbud embryos were transferred into the endoderm of wild-type hosts. From there, they migrated into the developing gonad. To clarify whether ectopic T-GP-carrying cells can produce functional germ cells, they were identified by changing the recipients, from the wild-type Xenopus to transgenic Xenopus expressing DsRed2. After transferring T-GP carrying cells labeled genetically with DsRed2 into wild-type hosts, we could find chimeric gonads in mature hosts. Furthermore, the spermatozoa and eggs derived from T-GP-carrying cells were fertile. Thus, we have demonstrated that Xenopus germ plasm is sufficient for germ cell determination.

Ubiquitin-proteasome-rich cytoplasmic structures in neutrophils of patients with Shwachman-Diamond syndrome.

BACKGROUND: Shwachman-Diamond syndrome is an autosomal recessive disorder in which severe bone marrow dysfunction causes neutropenia and an increased risk of leukemia. Recently, novel particulate cytoplasmic structures, rich in ubiquitinated and proteasomal proteins, have been detected in epithelial cells and neutrophils from patients with Helicobacter pylori gastritis and several epithelial neoplasms. DESIGN AND METHODS: Blood neutrophils from 13 cases of Shwachman-Diamond syndrome - ten with and three without SBDS gene mutation - and ten controls were investigated by confocal microscopy and ultrastructural immunocytochemistry using antibodies against ubiquitinated proteins, proteasomes, p62 protein, and Helicobacter pylori VacA, urease and outer membrane proteins. RESULTS: Many extensively disseminated particulate cytoplasmic structures, accounting for 22.78 ± 5.57% (mean ± standard deviation) of the total cytoplasm, were found in blood neutrophils from mutated Shwachman-Diamond syndrome patients. The particulate cytoplasmic structures showed immunoreactivity for polyubiquitinated proteins and proteasomes, but no reactivity for Helicobacter pylori products, which are present in particulate cytoplasmic structures of Helicobacter pylori-positive gastritis. Neutrophils from patients with Shwachman-Diamond syndrome frequently showed p62-positive autophagic vacuoles and apoptotic changes in 5% of cells. No particulate cytoplasmic structures were observed in most control neutrophils; however, in a few cells from two cases we noted focal development of minute particulate cytoplasmic structures, accounting for 0.74 ± 0.56% of the total cytoplasm (P<0.001 versus particulate cytoplasmic structures from mutated Shwachman-Diamond syndrome patients). Neutrophils from non-mutated Shwachman-Diamond-syndrome-like patients resembled controls in two cases, and a third case showed particulate cytoplasmic structure patterns intermediate between those in controls and those in mutated Shwachman-Diamond syndrome patients. CONCLUSIONS: Particulate cytoplasmic structures are a prominent feature of neutrophils from patients with Shwachman-Diamond syndrome. They may help us to understand the mechanism of granulocyte dysfunction and the neoplastic risk of the disease.

Post-transcriptional regulation in the myo1Δ mutant of Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae myosin type II-deficient (myo1Δ) strains remain viable and divide, despite the absence of a cytokinetic ring, by activation of the PKC1-dependent cell wall integrity pathway (CWIP). Since the myo1Δ transcriptional fingerprint is a subset of the CWIP fingerprint, the myo1Δ strain may provide a simplified paradigm for cell wall stress survival. RESULTS: To explore the post-transcriptional regulation of the myo1Δ stress response, 1,301 differentially regulated ribosome-bound mRNAs were identified by microarray analysis of which 204 were co-regulated by transcription and translation. Four categories of mRNA were significantly affected - protein biosynthesis, metabolism, carbohydrate metabolism, and unknown functions. Nine genes of the 20 CWIP fingerprint genes were post-transcriptionally regulated. Down and up regulation of selected ribosomal protein and cell wall biosynthesis mRNAs was validated by their distribution in polysomes from wild type and myo1Δ strains. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2α-P) and a reduction in the steady state levels of the translation initiation factor eIF4Gp in myo1Δ strains. Deletion of GCN2 in myo1Δ abolished eIF2αp phosphorylation, and showed a severe growth defect. The presence of P-bodies in myo1Δ strains suggests that the process of mRNA sequestration is active, however, the three representative down regulated RP mRNAs, RPS8A, RPL3 and RPL7B were present at equivalent levels in Dcp2p-mCh-positive immunoprecipitated fractions from myo1Δ and wild type cells. These same RP mRNAs were also selectively co-precipitated with eIF2α-P in myo1Δ strains. CONCLUSIONS: Quantitative analysis of ribosome-associated mRNAs and their polyribosome distributions suggests selective regulation of mRNA translation efficiency in myo1Δ strains. Inhibition of translation initiation factor eIF2α (eIF2α-P) in these strains was by Gcn2p-dependent phosphorylation. The increase in the levels of eIF2α-P; the genetic interaction between GCN2 and MYO1; and the reduced levels of eIF4Gp suggest that other signaling pathways, in addition to the CWIP, may be important for myo1Δ strain survival. Selective co-immunoprecipitation of RP mRNAs with eIF2α-P in myo1Δ strains suggests a novel mode of translational regulation. These results indicate that post-transcriptional control is important in the myo1Δ stress response and possibly other stresses in yeast.

Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella.

Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1beta and IL-18. However, the NLRs that respond to microbial pathogens in vivo are poorly defined. We show that the NLRs NLRP3 and NLRC4 both activate caspase-1 in response to Salmonella typhimurium. Responding to distinct bacterial triggers, NLRP3 and NLRC4 recruited ASC and caspase-1 into a single cytoplasmic focus, which served as the site of pro-IL-1beta processing. Consistent with an important role for both NLRP3 and NLRC4 in innate immune defense against S. typhimurium, mice lacking both NLRs were markedly more susceptible to infection. These results reveal unexpected redundancy among NLRs in host defense against intracellular pathogens in vivo.

Dynein and kinesin regulate stress-granule and P-body dynamics.

Stress granules (SGs) and P-bodies (PBs) are related cytoplasmic structures harboring silenced mRNAs. SGs assemble transiently upon cellular stress, whereas PBs are constitutive and are further induced by stress. Both foci are highly dynamic, with messenger ribonucleoproteins (mRNPs) and proteins rapidly shuttling in and out. Here, we show that impairment of retrograde transport by knockdown of mammalian dynein heavy chain 1 (DHC1) or bicaudal D1 (BicD1) inhibits SG formation and PB growth upon stress, without affecting protein-synthesis blockage. Conversely, impairment of anterograde transport by knockdown of kinesin-1 heavy chain (KIF5B) or kinesin light chain 1 (KLC1) delayed SG dissolution. Strikingly, SG dissolution is not required to restore translation. Simultaneous knockdown of dynein and kinesin reverted the effect of single knockdowns on both SGs and PBs, suggesting that a balance between opposing movements driven by these molecular motors governs foci formation and dissolution. Finally, we found that regulation of SG dynamics by dynein and kinesin is conserved in Drosophila.

The cytoplasmic structure hypothesis for ribosome assembly, vertical inheritance, and phylogeny.

Fundamental questions in evolution concern deep divisions in the living world and vertical versus horizontal information transfer. Two contrasting views are: (i) three superkingdoms Archaea, Eubacteria, and Eukarya based on vertical inheritance of genes encoding ribosomes; versus (ii) a prokaryotic/eukaryotic dichotomy with unconstrained horizontal gene transfer (HGT) among prokaryotes. Vertical inheritance implies continuity of cytoplasmic and structural information whereas HGT transfers only DNA. By hypothesis, HGT of the translation machinery is constrained by interaction between new ribosomal gene products and vertically inherited cytoplasmic structure made largely of preexisting ribosomes. Ribosomes differentially enhance the assembly of new ribosomes made from closely related genes and inhibit the assembly of products from more distal genes. This hypothesis suggests experiments for synthetic biology: the ability of synthetic genomes to "boot," i.e., establish hereditary continuity, will be constrained by the phylogenetic closeness of the cell "body" into which genomes are placed.

The chromatoid body of male germ cells: epigenetic control and miRNA pathway.

Differentiation of germ cells is characterized by a remarkable degree of cellular restructuring and gene regulation that involves complex events of genomic and epigenetic reorganization. The pathways that govern miRNAs have been shown to play an important role in the male germ cell lineage. The chromatoid body is a finely filamentous, lobulated perinuclear granule located in the cytoplasm of male germ cells. The role of the chromatoid body in the mouse has remained elusive for longtime, although it was proposed to be involved in RNA storing and metabolism. Recent findings show that the chromatoid body is related to the RNA processing body (P-body) of somatic cells and that it seems to operate as an intracellular nerve-center of the microRNA pathway. The role of the chromatoid body underscores the importance of posttranscriptional gene regulation and of the microRNA pathway in the control of postmeiotic male germ cell differentiation.

Localization and nucleotide specificity of Blastocystis succinyl-CoA synthetase.

The anaerobic lifestyle of the intestinal parasite Blastocystis raises questions about the biochemistry and function of its mitochondria-like organelles. We have characterized the Blastocystis succinyl-CoA synthetase (SCS), a tricarboxylic acid cycle enzyme that conserves energy by substrate-level phosphorylation. We show that SCS localizes to the enigmatic Blastocystis organelles, indicating that these organelles might play a similar role in energy metabolism as classic mitochondria. Although analysis of residues inside the nucleotide-binding site suggests that Blastocystis SCS is GTP-specific, we demonstrate that it is ATP-specific. Homology modelling, followed by flexible docking and molecular dynamics simulations, indicates that while both ATP and GTP fit into the Blastocystis SCS active site, GTP is destabilized by electrostatic dipole interactions with Lys 42 and Lys 110, the side-chains of which lie outside the nucleotide-binding cavity. It has been proposed that residues in direct contact with the substrate determine nucleotide specificity in SCS. However, our results indicate that, in Blastocystis, an electrostatic gatekeeper controls which ligands can enter the binding site.

P-body formation is a consequence, not the cause, of RNA-mediated gene silencing.

P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. We show that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, we show that releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity.

microRNA-mediated silencing inside P-bodies.

Cytoplasmic processing bodies, or P-bodies, contain a high concentration of enzymes and factors required for mRNA turnover and translational repression. Recent studies provide evidence that the mRNAs silenced by miRNAs are localized to P-bodies for storage or degradation, perhaps in adjacent subcompartments. mRNP remodeling, potentially induced by miRISC or RNA helicase activity, may cause the modification of the translation initiation complex at the 5' end of mRNA, following translational repression and localization to P-bodies. Further remodeling in P-bodies may facilitate access of the decapping complex to the cap structure, thus inducing mRNA degradation. However, with appropriate signals, stored mRNAs in P-bodies could be released and returned to the translational machinery through mechanisms requiring binding of regulatory proteins to the 3' UTR of mRNAs. Here a model is proposed to explain the repression and degradation stages of the mRNAs within PBs. This model includes preservation or disruption of a stable closed loop structure of the mRNAs, compartmentalization in PBs and mRNA escape triggered by additional binding proteins.

The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans.

In metazoans, microRNAs (miRNAs) carry out various regulatory functions through association with multiprotein miRNA-induced silencing complexes (miRISCs) that contain Dicer and Argonaute proteins. How miRNAs regulate the expression of their mRNA targets remains a major research question. We have identified the C. elegans ain-1 gene through a genetic suppressor screen and shown that it functions with the heterochronic genetic pathway that regulates developmental timing. Biochemical analysis indicates that AIN-1 interacts with protein complexes containing an Argonaute protein, Dicer, and miRNAs. AIN-1 shares homology with the candidate human neurological disease protein GW182, shown to localize in cytoplasmic processing bodies that are sites of mRNA degradation and storage. A functional AIN-1::GFP also localizes at the likely worm processing bodies. When coexpressed from transgenes, AIN-1 targets ALG-1 to the foci. These results suggest a model where AIN-1 regulates a subset of miRISCs by localization to the processing bodies, facilitating degradation or translational inhibition of mRNA targets.

CIITA leucine-rich repeats control nuclear localization, in vivo recruitment to the major histocompatibility complex (MHC) class II enhanceosome, and MHC class II gene transactivation.

The major histocompatibility complex (MHC) class II transactivator CIITA plays a pivotal role in the control of the cellular immune response through the quantitative regulation of MHC class II expression. We have analyzed a region of CIITA with similarity to leucine-rich repeats (LRRs). CIITA LRR alanine mutations abolish both the transactivation capacity of full-length CIITA and the dominant-negative phenotype of CIITA mutants with N-terminal deletions. We demonstrate direct interaction of CIITA with the MHC class II promoter binding protein RFX5 and could also detect novel interactions with RFXANK, NF-YB, and -YC. However, none of these interactions is influenced by CIITA LRR mutagenesis. On the other hand, chromatin immunoprecipitation shows that in vivo binding of CIITA to the MHC class II promoter is dependent on LRR integrity. LRR mutations lead to an impaired nuclear localization of CIITA, indicating that a major function of the CIITA LRRs is in nucleocytoplasmic translocation. There is, however, evidence that the CIITA LRRs are also involved more directly in MHC class II gene transactivation. CIITA interacts with a novel protein of 33 kDa in a manner sensitive to LRR mutagenesis. CIITA is therefore imported into the nucleus by an LRR-dependent mechanism, where it activates transcription through multiple protein-protein interactions with the MHC class II promoter binding complex.