Improved low-invasive mRNA electroporation method into immature mouse oocytes visualizes protein dynamics during development.

One of the major causes of oocyte quality deterioration, chromosome segregation abnormalities manifest mainly during meiosis I, which occurs before and during ovulation. However, currently, there is a technical limitation in the introduction of mRNA into premature oocytes without impairing embryonic developmental ability. In this study, we established a low-invasive electroporation (EP) method to introduce mRNA into pre-ovulatory, germinal vesicle (GV) mouse oocytes in an easier manner than the traditional microinjection method. The EP method with an optimized impedance value resulted in the efficient introduction of mRNAs encoding enhanced green fluorescent protein (EGFP) into the GV oocytes surrounded by cumulus cells at a survival rate of 95.0%. Furthermore, the introduction of histone H2B-EGFP mRNA into the GV oocytes labeled most of the oocytes without affecting the blastocyst development rate, indicating the feasibility of the visualization of oocyte chromosomal dynamics that enable us to assay chromosomal integrity in oocyte maturation and cell count in embryonic development. The establishment of this EP method offers extensive assays to select pre-implantation embryos and enables the surveying of essential factors for mammalian oocyte quality determination.

CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore.

Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes containing the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-associated network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly associates with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an additional putative DNA-binding protein. Our analysis indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.

Geometrical Constraints Greatly Hinder Formin mDia1 Activity.

Formins are one of the central players in the assembly of most actin networks in cells. The sensitivity of these processive molecular machines to mechanical tension is now well established. However, how the activity of formins is affected by geometrical constraints related to network architecture, such as filament cross-linking and formin spatial confinement, remains largely unknown. Combining microfluidics and micropatterning, we reconstituted in vitro mDia1 formin-elongated filament bundles induced by fascin, with different geometrical constraints on the formins, and measured the impact of these constraints on formin elongation rate and processivity. When filaments are not bundled, the anchoring details of formins have only a mild impact on their processivity and do not affect their elongation rate. When formins are unanchored, we show that filament bundling by fascin reduces both their elongation rate and their processivity. Strikingly, when filaments elongated by surface-anchored formins are cross-linked together, formin elongation rate immediately decreases and processivity is reduced up to 24-fold depending on the cumulative impact of formin rotational and translational freedom. Our results reveal an unexpected crosstalk between the constraints at the filament and the formin levels. We anticipate that in cells the molecular details of formin anchoring to the plasma membrane strongly modulate formin activity at actin filament barbed ends.

Preparation of an experimental mouse model lacking selenium-dependent glutathione peroxidase activities by feeding a selenium-deficient diet.

Relatively young (4-week-old) selenium deficient (SeD) mice, which lack the activity of selenium-dependent glutathione peroxidase (GSH-Px) isomers, were prepared using torula yeast-based SeD diet. Mice were fed the torula yeast-based SeD diet and ultra-pure water. Several different timings for starting the SeD diet were assessed. The weekly time course of liver comprehensive GSH-Px activity after weaning was monitored. Protein expression levels of GPx1 and 4 in the liver were measured by Western blot analysis. Gene expression levels of GPx1, 2, 3, 4, and 7 in the liver were measured by quantitative real-time PCR. Apoptotic activity of thymocytes after hydrogen peroxide (H2O2) exposure was compared. Thirty-day survival rates after whole-body X-ray irradiation were estimated. Pre-birth or right-after-birth starting of the SeD diet in dams was unable to lead to creation of SeD mice due to neonatal death. This suggests that Se is necessary for normal birth and healthy growing of mouse pups. Starting the mother on the SeD diet from 2 weeks after giving birth (SeD-trial-2w group) resulted in a usable SeD mouse model. The liver GSH-Px activity of the SeD-trial-2w group was almost none from 4 week olds, but the mice survived for more than 63 weeks. Protein and gene expression of GPx1 was suppressed in the SeD-trial-2w group, but that of GPx4 was not. The thymocytes of the SeD-trial-2w group were sensitive to H2O2-induced apoptosis. The SeD-trial-2w group was sensitive to whole-body X-ray irradiation compared with control mice. The SeD-trial-2w model may be a useful animal model for H2O2/hydroperoxide-induced oxidative stress.

Nuclear envelope localization of PIG-B is essential for GPI-anchor synthesis in Drosophila.

Membrane lipid biosynthesis is a complex process that takes place in various intracellular compartments. Glycosylphosphatidylinositol (GPI), a lipid involved in membrane anchoring of some proteins, is synthesized by the PIG enzymes. Most PIGs are localized to the endoplasmic reticulum (ER), but Drosophila PIG-B (DmPIG-B) is localized to the nuclear envelope (NE). To determine whether the NE localization of DmPIG-B is functionally important, we defined the determinants of localization and generated an ER-localized form, denoted DmPIG-B[ER]. The enzymatic activity of DmPIG-B[ER] was comparable to that of NE-localized DmPIG-B[NE]. Expression of DmPIG-B[ER] inefficiently rescued the lethality of the PIG-B mutant, whereas DmPIG-B[NE] rescued this lethality fully. DmPIG-B[ER] was preferentially degraded by lysosomes, suggesting that the NE localization is essential for function and stability of the protein. In addition, we found that the region of the ER proximal to the NE is the site of translation of GPI-anchored proteins and addition of GPI. Thus, the NE and proximal ER may provide a platform for efficient GPI anchoring.

The advantages of microfluidics to study actin biochemistry and biomechanics.

The regulated assembly of actin filaments is essential in nearly all cell types. Studying actin assembly dynamics can pose many technical challenges. A number of these challenges can be overcome by using microfluidics to observe and manipulate single actin filaments under an optical microscope. In particular, microfluidics can be tremendously useful for applying different mechanical stresses to actin filaments and determining how the physical context of the filaments affects their regulation by biochemical factors. In this review, we summarize the main features of microfluidics for the study of actin assembly dynamics, and we highlight some recent developments that have emerged from the combination of microfluidics and other techniques. We use two case studies to illustrate our points: the rapid assembly of actin filaments by formins and the disassembly of filaments by actin depolymerizing factor (ADF)/cofilin. Both of these protein families play important roles in cells. They regulate actin assembly through complex molecular mechanisms that are sensitive to the filaments' mechanical context, with multiple activities that need to be quantified separately. Microfluidics-based experiments have been extremely useful for gaining insight into the regulatory actions of these two protein families.

PIGN prevents protein aggregation in the endoplasmic reticulum independently of its function in the GPI synthesis.

Quality control of proteins in the endoplasmic reticulum (ER) is essential for ensuring the integrity of secretory proteins before their release into the extracellular space. Secretory proteins that fail to pass quality control form aggregates. Here we show the PIGN-1/PIGN is required for quality control in Caenorhabditis elegans and in mammalian cells. In C. elegans pign-1 mutants, several proteins fail to be secreted and instead form abnormal aggregation. PIGN-knockout HEK293 cells also showed similar protein aggregation. Although PIGN-1/PIGN is responsible for glycosylphosphatidylinositol (GPI)-anchor biosynthesis in the ER, certain mutations in C. elegans pign-1 caused protein aggregation in the ER without affecting GPI-anchor biosynthesis. These results show that PIGN-1/PIGN has a conserved and non-canonical function to prevent deleterious protein aggregation in the ER independently of the GPI-anchor biosynthesis. PIGN is a causative gene for some human diseases including multiple congenital seizure-related syndrome (MCAHS1). Two pign-1 mutations created by CRISPR/Cas9 that correspond to MCAHS1 also cause protein aggregation in the ER, implying that the dysfunction of the PIGN non-canonical function might affect symptoms of MCAHS1 and potentially those of other diseases.

Epithelial Tumors Originate in Tumor Hotspots, a Tissue-Intrinsic Microenvironment.

Malignant tumors are caused by uncontrolled proliferation of transformed mutant cells that have lost the ability to maintain tissue integrity. Although a number of causative genetic backgrounds for tumor development have been discovered, the initial steps mutant cells take to escape tissue integrity and trigger tumorigenesis remain elusive. Here, we show through analysis of conserved neoplastic tumor-suppressor genes (nTSGs) in Drosophila wing imaginal disc epithelia that tumor initiation depends on tissue-intrinsic local cytoarchitectures, causing tumors to consistently originate in a specific region of the tissue. In this "tumor hotspot" where cells constitute a network of robust structures on their basal side, nTSG-deficient cells delaminate from the apical side of the epithelium and begin tumorigenic overgrowth by exploiting endogenous Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling activity. Conversely, in other regions, the "tumor coldspot" nTSG-deficient cells are extruded toward the basal side and undergo apoptosis. When the direction of delamination is reversed through suppression of RhoGEF2, an activator of the Rho family small GTPases, and JAK/STAT is activated ectopically in these coldspot nTSG-deficient cells, tumorigenesis is induced. These data indicate that two independent processes, apical delamination and JAK/STAT activation, are concurrently required for the initiation of nTSG-deficient-induced tumorigenesis. Given the conservation of the epithelial cytoarchitecture, tumorigenesis may be generally initiated from tumor hotspots by a similar mechanism.

Adherens junction-associated pores mediate the intercellular transport of endosomes and cytoplasmic proteins.

Intercellular endosomes (IEs) are endocytosed vesicles shuttled through the adherens junctions (AJs) between two neighboring epidermal cells during Drosophila dorsal closure. The cell-to-cell transport of IEs requires DE-cadherin (DE-cad), microtubules (MTs) and kinesin. However, the mechanisms by which IEs can be transported through the AJs are unknown. Here, we demonstrate the presence of AJ-associated pores with MTs traversing through the pores. Live imaging allows direct visualization of IEs being transported through the AJ-associated pores. By using an optogenetic dimerization system, we observe that the dimerized IE-kinesin complexes move across AJs into the neighboring cell. The AJ-associated pores also allow intercellular movement of soluble proteins. Importantly, most epidermal cells form dorsoventral-oriented two-cell syncytia. Together, we present a model in which an AJ-associated pore mediates the intercellular transport of IEs and proteins between two cells in direct contact.

Analysis of redox states of protic and aprotic solutions irradiated by low linear energy transfer carbon-ion beams using a 2,2-diphenyl-1-picrylhydrazyl radical.

The quantitative evaluation of changes in the redox state induced by low linear energy transfer (LET) radiations such as the plateau region of heavy-ion beams via formation of reactive oxygen species is of considerable importance to eliminate the adverse effects of radiation therapy on normal tissues adjacent to a tumour. In this study, a 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙) was used as a redox probe to estimate the redox states of protic and aprotic solutions irradiated by low LET carbon-ion (C-ion) beams. The dose dependence of the decrease in the absorption band due to DPPH˙ (which was solubilised by ß-cyclodextrin (ß-CD) in water) after irradiation with low LET C-ion beams (13 keV µm-1) was similar to that after X-irradiation. Similar results were obtained when H2O was replaced with methanol or acetonitrile although the slope values of the plots of the absorbance changes vs. radiation doses were twice larger as compared to the case in ß-CD-containing H2O. Moreover, DPPH˙ was more susceptible to the C-ion beam than to X-rays in isopropyl myristate (IPM), which is one of the saturated fatty acid esters.

Localized hydroxyl radical generation at mmol/L and mol/L levels in water by photon irradiation.

The generation of localized hydroxyl radical (•OH) in aqueous samples by low linear energy transfer irradiation was investigated. Several concentrations of 5,5-dimethyl-1-pyrroline-N-oxid solution (from 0.5 to 1,680 mmol/L) were prepared and irradiated with an identical dose of X-ray or γ-ray. The density of •OH generation in aqueous solution was evaluated by the electron paramagnetic resonance spin-trapping technique using 5,5-dimethyl-1-pyrroline-N-oxid as an electron paramagnetic resonance spin-trapping agent. The relationship between the molecular density of 5,5-dimethyl-1-pyrroline-N-oxid in the samples and the concentration of 5,5-dimethyl-1-pyrroline-N-oxid-OH generated in the irradiated samples was analyzed. Two different characteristic linear trends were observed in the 5,5-dimethyl-1-pyrroline-N-oxid-OH/5,5-dimethyl-1-pyrroline-N-oxid plots, which suggested •OH generation in two fashions, i.e., mmol/L- and mol/L-level local concentrations. The dose, dose rate, and/or the energy of photon irradiation did not affect the shapes of the 5,5-dimethyl-1-pyrroline-N-oxid-OH/5,5-dimethyl-1-pyrroline-N-oxid plots. Moreover, the addition of 5 mmol/L caffeine could cancel the contribution of mmol/L-level •OH generation, leaving a trace of mol/L-level •OH generation. Thus, the localized mmol/L- and mol/L-level generations of •OH, which were independent of experimental parameters such as dose, dose rate, and/or the energy of photon of low linear energy transfer radiation, were established.

The Matrix Proteins Hasp and Hig Exhibit Segregated Distribution within Synaptic Clefts and Play Distinct Roles in Synaptogenesis.

The synaptic cleft is the space through which neurotransmitters convey neural information between two synaptic terminals. This space is presumably filled with extracellular matrix molecules involved in synaptic function or differentiation. However, little is known about the identities of the matrix components, and it remains unclear how these molecules organize the matrix in synaptic clefts. In this study, we identified Hasp, a Drosophila secretory protein containing CCP and WAP domains. Molecular genetic analysis revealed that Hasp diffuses extracellularly and is predominantly captured at synaptic clefts of cholinergic synapses. Furthermore, Hasp regulates levels of DLG and the nAChR subunits Dα6 and Dα7 at postsynaptic terminals. Hasp is required for trapping of another matrix protein, Hig, which is also secreted and diffused in the brain, at synaptic clefts of cholinergic synapses; however, Hig is dispensable for localization of Hasp at synaptic clefts. In addition, in the brains of triple mutants for the nAChR subunits Dα5, Dα6, and Dα7, the level of Hig, but not Hasp, was markedly reduced in synaptic regions, indicating that these nAChR subunits are required to anchor Hig to synaptic clefts. High-resolution microscopy revealed that Hasp and Hig exhibit segregated distribution within individual synaptic clefts, reflecting their differing roles in synaptogenesis. These data provide insight into how Hasp and Hig construct the synaptic cleft matrix and regulate the differentiation of cholinergic synapses, and also illuminate a previously unidentified architecture within synaptic clefts. SIGNIFICANCE STATEMENT: The synapse has been extensively studied because it is essential for neurotransmission. By contrast, the space between the synaptic terminals, the synaptic cleft, is still an undeveloped research area despite its ubiquity in synapses. In fruit fly brains, we obtained evidence that the matrix protein Hasp and the previously identified Hig, both of which are secreted extracellularly, localize predominantly to synaptic clefts of cholinergic synapses, and modulate the levels of nAChR subunits on postsynaptic membranes. However, Hasp and Hig play differential roles in matrix formation and exhibit segregated distribution within synaptic clefts. These results reveal the molecular mechanisms of synaptic matrix construction and illuminate a molecular architecture within synaptic clefts previously unrevealed in any animal species.

The two-row malting barley cultivar 'New Sachiho Golden' with null lipoxygenase-1 improves flavor stability in beer and was developed by marker-assisted selection.

Lipoxygenase-1 (LOX-1) null 'New Sachiho Golden' is a two-row malting barley (Hordeum vulgare L.) cultivar released in 2015 that was developed at the Tochigi Prefectural Agricultural Experimental Station by backcross breeding using the high-yield leading cultivar 'Sachiho Golden' as a recurrent parent and the LOX-1 null mutant 'Daikei LM1' as a non-recurrent parent. To develop 'New Sachiho Golden' we used a simple LOX activity assay and marker-assisted selection. This is the first LOX-1 null malting barley cultivar in Japan that is resistant to barley yellow mosaic virus (types I-III). Agronomic characteristics and malting qualities of 'New Sachiho Golden' were similar to those of 'Sachiho Golden', except that 'New Sachiho Golden' had no LOX activity in ungerminated grains and had clearly lower LOX activity during malting than 'Sachiho Golden'. The concentrations of a trans-2-nonenal (T2N) precursor in wort and beer made from 'New Sachiho Golden' were significantly lower than in those made from 'Sachiho Golden', both before and after storage.

Amplification of glutathione-mediated oxidative stress by catalase in an aqueous solution at hyperthermal temperatures.

The glutathione (GSH)-mediated superoxide (O2•-) generation in an aqueous solution and relation of hydrogen peroxide (H2O2) and effect of catalase were investigated. GSH-induced O2•- generation in hyperthermal temperatures was measured by the nitroblue tetrazolium (NBT) mehod. Heating an aqueous solution containing GSH caused superoxide from dissolved O2. H2O2 was generated simultaneously in this reaction mixture probably from the hydroperoxy radical (HO2•), which is equilibrated with O2•- in an aqueous condition, and then H2O2 consumed O2•-. Coexisting catalase in the reaction mixture, as a result, could increase O2•- generation. The catalase-exaggerated extracellular O2•- generation could give a harmful effect to living cells. This GSH-induced oxidative stress can be a part of mechanisms of hyperthermia therapy.

A facile and rapid access to resveratrol derivatives and their radioprotective activity.

A facile and rapid access to resveratrol derivatives has been achieved based on palladium-catalyzed oxidative Heck reaction of aryl boronic acids with styrenes followed by demethylation in moderate to good yields. A series of resveratrol derivatives with various functional groups has been synthesized easily. The radioprotective activity of synthesized compounds has also been evaluated using rat thymocytes. The results revealed that some resveratrol derivatives efficiently protected the thymocytes from radiation-induced apoptosis.

Non-invasive measurement of melanin-derived radicals in living mouse tail using X-band EPR.

The aim of this experiment is to measure in vivo generation of melanin-derived radicals non-invasively, as a quantifiable index of radio-biological effect. Melanin-derived radicals in a living intact mouse tail tip were non-invasively measured in very simple way using an X-band electron paramagnetic resonance spectrometer. Colored mouse strains, C57BL/6NCr, BDF1, and C3H/He, have clear EPR signal corresponding to melanin-derived radicals in the tail tip; however, albino mouse strains, BALB/cCr, ddY, ICR, have no EPR signals. An X-ray fraction of 2 Gy/day (1 Gy/min) was repeatedly irradiated to a C3H/He mouse tail skin every Monday to Friday for 4 weeks. In comparison to before starting irradiation, the C3H/He mouse tail skin became darker, like a suntan. The melanin-derived radicals in C3H/He mouse tail skin were increased in association with X-ray fractions. Melanin-derived radicals in mouse tail skin can be readily and chronologically measurable by using X-band EPR spectrometer, and can be a marker for a radiobiological effect in the skin.

Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila.

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex (pcx) encodes a multi-pass transmembrane protein of unknown function, widely found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. Here, we established that Pcx is a component of the N-signaling pathway. Pcx was required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that Pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function resulted in enlargement of the ER that was not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes was induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, we speculate that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. Our results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity.

Loss of axonal mitochondria promotes tau-mediated neurodegeneration and Alzheimer's disease-related tau phosphorylation via PAR-1.

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi-mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD-related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.

A structural perspective of the MAVS-regulatory mechanism on the mitochondrial outer membrane using bioluminescence resonance energy transfer.

In most eukaryotic cells, mitochondria have various essential roles for proper cell function, such as energy production, and in mammals mitochondria also act as a platform for antiviral innate immunity. Mitochondrial-mediated antiviral immunity depends on the activation of the cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathway, and on the participation of mitochondrial antiviral signaling (MAVS), which is localized on the mitochondrial outer membrane. After RNA virus infection, RLRs translocate to the mitochondrial surface to interact with MAVS, and the adaptor protein undergoes a conformational change that is essential for downstream signaling, although its structural features are poorly understood. Here we examined the MAVS-regulatory mechanism on the mitochondrial outer membrane using bioluminescence resonance energy transfer (BRET) in live cells. Using a combination of BRET and functional analysis, we found that the activated MAVS conformation is a highly ordered oligomer, at least more than three molecules per complex unit on the membrane. Hepatitis C virus NS3/4A protease and mitofusin 2, which are known MAVS inhibitors, interfere with MAVS homotypic oligomerization in a distinct manner, each differentially altering the active conformation of MAVS. Our results reveal structural features underlying the precise regulation of MAVS signaling on the mitochondrial outer membrane, and may provide insight into other signaling systems involving organelles.