Tissue flow regulates planar cell polarity independently of the Frizzled core pathway.

Planar cell polarity (PCP) regulates the orientation of external structures. A core group of proteins that includes Frizzled forms the heart of the PCP regulatory system. Other PCP mechanisms that are independent of the core group likely exist, but their underlying mechanisms are elusive. Here, we show that tissue flow is a mechanism governing core group-independent PCP on the Drosophila notum. Loss of core group function only slightly affects bristle orientation in the adult central notum. This near-normal PCP results from tissue flow-mediated rescue of random bristle orientation during the pupal stage. Manipulation studies suggest that tissue flow can orient bristles in the opposite direction to the flow. This process is independent of the core group and implies that the apical extracellular matrix functions like a "comb" to align bristles. Our results reveal the significance of cooperation between tissue dynamics and extracellular substances in PCP establishment.

Immunocytochemistry of phospholipase D1 and D2 in cultured cells.

Activation of Gq protein-coupled receptors triggers the phospholipase C (PLC) pathway, which yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DG kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA), which serves as another second messenger. Along with PLC-DGK pathway, PA is produced directly by the action of phospholipase D (PLD), which hydrolyzes the major membrane phospholipid: phosphatidylcholine (PC). PA is converted to DG by phosphatidic acid phosphatase, suggesting that PLD, together with DGK, is a key enzyme regulating DG and PA. PLD has been implicated in a broad range of cellular processes. However, cellular expression and subcellular localization of PLD remain elusive because of a lack of specific antibodies against PLDs. For this study, we raised specific antibodies against major mammalian PLD isoforms: PLD1 and PLD2. Immunocytochemical analysis using specific antibodies showed clearly that native PLD1 and PLD2 localize to distinct subcellular regions as dot-like structures in cultured cells. PLD1 predominantly localizes to the plasma membrane, whereas PLD2 mostly localizes within the cytoplasm. These findings suggest that PLD1 and PLD2 have different roles in the phosphoinositide signaling pathway in distinct subcellular regions.

A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation.

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.

Development of multi-channel apparatus for electron-atom Compton scattering to study the momentum distribution of atoms in a molecule.

We have developed multi-channel apparatus for electron-atom Compton scattering to study the momentum distribution of atoms in a molecule. It combines the features of both a spherical electron energy analyzer and a large-area position sensitive detector, thereby having an ability to cover almost completely the azimuthal angle range available for quasi-elastic electron Rutherford backscattering at an angle of 135°. Details and performance of the apparatus are reported, together with experimental results measured for Xe and CH4 at an incident electron energy of 2 keV. In particular, it is shown that the instrumental sensitivity is remarkably high, which has increased the signal count rate by nearly three orders of magnitude compared to existing setups. This technical progress would be useful for advancing atomic momentum spectroscopy studies.

Vps34 regulates myofibril proteostasis to prevent hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common heart disease with a prevalence of 1 in 500 in the general population. Several mutations in genes encoding cardiac proteins have been found in HCM patients, but these changes do not predict occurrence or prognosis and the molecular mechanisms underlying HCM remain largely elusive. Here we show that cardiac expression of vacuolar protein sorting 34 (Vps34) is reduced in a subset of HCM patients. In a mouse model, muscle-specific loss of Vps34 led to HCM-like manifestations and sudden death. Vps34-deficient hearts exhibited abnormal histopathologies, including myofibrillar disarray and aggregates containing αB-crystallin (CryAB). These features result from a block in the ESCRT-mediated proteolysis that normally degrades K63-polyubiquitinated CryAB. CryAB deposition was also found in myocardial specimens from a subset of HCM patients whose hearts showed decreased Vps34. Our results identify disruption of the previously unknown Vps34-CryAB axis as a potentially novel etiology of HCM.

Electron Momentum Spectroscopy Investigation of Molecular Conformations of Ethanol Considering Vibrational Effects.

The interpretation of experimental electron momentum distributions (EMDs) of ethanol, one of the simplest molecules having conformers, has confused researchers for years. High-level calculations of Dyson orbital EMDs by thermally averaging the gauche and trans conformers as well as molecular dynamical simulations failed to quantitatively reproduce the experiments for some of the outer valence orbitals. In this work, the valence shell electron binding energy spectrum and EMDs of ethanol are revisited by the high-sensitivity electron momentum spectrometer employing symmetric noncoplanar geometry at an incident energy of 1200 eV plus binding energy, together with a detailed analysis of the influence of vibrational motions on the EMDs for the two conformers employing a harmonic analytical quantum mechanical (HAQM) approach by taking into account all of the vibrational modes. The significant discrepancies between theories and experiments in previous works have now been interpreted quantitatively, indicating that the vibrational effect plays a significant role in reproducing the experimental results, not only through the low-frequency OH and CH3 torsion modes but also through other high-frequency ones. Rational explanation of experimental momentum profiles provides solid evidence that the trans conformer is slightly more stable than the gauche conformer, in accordance with thermodynamic predictions and other experiments. The case of ethanol demonstrates the significance of considering vibrational effects when performing a conformational study on flexible molecules using electron momentum spectroscopy.

Vibrational Effects on Electron Momentum Distributions of Outer-Valence Orbitals of Oxetane.

Vibrational effects on electron momentum distributions (EMDs) of outer-valence orbitals of oxetane are computed with a comprehensive consideration of all vibrational modes. It is found that vibrational motions influence EMDs of all outer-valence orbitals noticeably. The agreement between theoretical and experimental momentum profiles of the first five orbitals is greatly improved when including molecular vibrations in the calculation. In particular, the large turn-up at low momentum in the experimental momentum profile of the 3b1 orbital is well interpreted by vibrational effects, indicating that, besides the low-frequency ring-puckering mode, C-H stretching motion also plays a significant role in affecting EMDs of outer-valence orbitals of oxetane. The case of oxetane exhibits the significance of checking vibrational effects when performing electron momentum spectroscopy measurements.

INPP4B Is a PtdIns(3,4,5)P3 Phosphatase That Can Act as a Tumor Suppressor.

UNLABELLED: Inositol polyphosphate 4-phosphatase B (INPP4B) has been identified as a tumor suppressor mutated in human breast, ovary, and prostate cancers. The molecular mechanism underlying INPP4B's tumor-suppressive role is currently unknown. Here, we demonstrate that INPP4B restrains tumor development by dephosphorylating the PtdIns(3,4,5)P3 that accumulates in situations of PTEN deficiency. In vitro, INPP4B directly dephosphorylates PtdIns(3,4,5)P3. In vivo, neither inactivation of Inpp4b (Inpp4b(Δ/Δ)) nor heterozygous deletion of Pten (Pten(+/-)) in mice causes thyroid abnormalities, but a combination of these mutations induces malignant thyroid cancers with lung metastases. At the molecular level, simultaneous deletion of Inpp4b and Pten synergistically increases PtdIns(3,4,5)P3 levels and activates AKT downstream signaling proteins in thyroid cells. We propose that the PtdIns(3,4,5)P3 phosphatase activity of INPP4B can function as a "back-up" mechanism when PTEN is deficient, making INPP4B a potential novel therapeutic target for PTEN-deficient or PIK3CA-activated cancers. SIGNIFICANCE: Although INPP4B expression is reduced in several types of human cancers, our work on Inpp4B-deficient mice provides the first evidence that INPP4B is a bona fide tumor suppressor whose function is particularly important in situations of PTEN deficiency. Our biochemical data demonstrate that INPP4B directly dephosphorylates PtdIns(3,4,5)P3.

Molecular orbital imaging of the acetone S2 excited state using time-resolved (e, 2e) electron momentum spectroscopy.

We report a time-resolved (e, 2e) experiment on the deuterated acetone molecule in the S2 Rydberg state with a lifetime of 13.5 ps. The acetone S2 state was prepared by a 195 nm pump laser and probed with electron momentum spectroscopy using a 1.2 keV incident electron beam of 1 ps temporal width. In spite of the low data statistics as well as of the limited time resolution (±35 ps) due to velocity mismatch, the experimental results clearly demonstrate that electron momentum spectroscopy measurements of short-lived transient species are feasible, opening the door to time-resolved orbital imaging in momentum space.

The conserved discs-large binding partner Banderuola regulates asymmetric cell division in Drosophila.

BACKGROUND: Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood. RESULTS: To identify additional proteins required for ACD, we have carried out an in vivo live imaging RNAi screen for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. We identify Banderuola (Bnd), an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms. CONCLUSIONS: Bnd is a novel regulator of ACD in different types of cells. Our data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution.

Dachsous-dependent asymmetric localization of spiny-legs determines planar cell polarity orientation in Drosophila.

In Drosophila, planar cell polarity (PCP) molecules such as Dachsous (Ds) may function as global directional cues directing the asymmetrical localization of PCP core proteins such as Frizzled (Fz). However, the relationship between Ds asymmetry and Fz localization in the eye is opposite to that in the wing, thereby causing controversy regarding how these two systems are connected. Here, we show that this relationship is determined by the ratio of two Prickle (Pk) isoforms, Pk and Spiny-legs (Sple). Pk and Sple form different complexes with distinct subcellular localizations. When the amount of Sple is increased in the wing, Sple induces a reversal of PCP using the Ds-Ft system. A mathematical model demonstrates that Sple is the key regulator connecting Ds and the core proteins. Our model explains the previously noted discrepancies in terms of the differing relative amounts of Sple in the eye and wing.

Vibrational effects on valence electron momentum distributions of CH2F2.

We report an electron momentum spectroscopy study of vibrational effects on the electron momentum distributions for the outer valence orbitals of difluoromethane (CH2F2). The symmetric noncoplanar (e,2e) experiment has been performed at an incident electron energy of 1.2 keV. Furthermore, a theoretical calculation of the electron momentum distributions of the CH2F2 molecule has been carried out with vibrational effects being involved. It is shown from comparisons between experiment and theory that it is essential to take into account influences of the CH2 asymmetric stretching and CH2 rocking vibrational modes for a proper understanding of the electron momentum distribution of the 2b1 orbital having the CH-bonding character. The results of CH2F2and additional theoretical calculations for (CH3)2O and H2CO molecules strongly suggest that vibrational effects on electron momentum distributions tend to be appreciable for non-total symmetry molecular orbitals delocalized over some equivalent CH-bond sites.

Phosphatidylinositol 4-phosphate 5-kinase ß regulates growth cone morphology and semaphorin 3A-triggered growth cone collapse in mouse dorsal root ganglion neurons.

Growth cone motility and morphology, which are critical for axon guidance, are controlled through intracellular events such as actin cytoskeletal reorganization and vesicular trafficking. The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] has been implicated in regulation of these cellular processes in a diverse range of cell types. The main kinases involved in the production of PI(4,5)P2 are the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family, which consist of three isozymes, α, ß and γ. Here, we demonstrate the involvement of PIP5Kß in growth cone dynamics. Overexpression of a lipid kinase-deficient mutant of PIP5Kß (PIP5Kß-KD) in mouse dorsal root ganglion (DRG) neurons stimulated axon elongation and increased growth cone size, whereas wild-type PIP5Kß tended to show opposite effects. Furthermore, PIP5Kß-KD inhibited growth cone collapse of DRG neurons induced by semaphorin 3A (Sema3A). These results provide evidence that PIP5Kß negatively regulates axon elongation and growth cone size and is involved in the cellular signaling pathway for Sema3A-triggered repulsion in DRG neurons.

Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine.

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

Vibrational effects on valence electron momentum distributions of ethylene.

We report an electron momentum spectroscopy study of vibrational effects on the electron momentum distributions for the outer valence orbitals of ethylene (C(2)H(4)). The symmetric noncoplanar (e,2e) experiment has been conducted at an impact energy of 1.2 keV. Furthermore, a theoretical method of calculating electron momentum distributions for polyatomic molecules has been developed with vibrational effects being involved. It is shown from comparisons between experiment and theory that taking into account effects of the CH(2) asymmetric stretching and CH(2) rocking vibrational modes of C(2)H(4) is essential for a proper understanding of the electron momentum distribution of the 1b(3g) molecular orbital.

Phospholipase D2 activation by p38 MAP kinase is involved in neurite outgrowth.

p38 mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth. However, the underlying molecular mechanism(s) remains unclear. Here, we demonstrate that phospholipase D2 (PLD2) mediates p38 signaling in neurite outgrowth. Stimulation of rat pheochromocytoma PC12 cells with nerve growth factor activated PLD2 and augmented neurite outgrowth, both of which were inhibited by pharmacological suppression of p38. Overexpression of constitutively active MAP kinase kinase 6 (MKK6-CA) activated coexpressed PLD2 in PC12 and mouse neuroblastoma N1E-115 cells. Overexpression of wild-type PLD2 in these cells strongly augmented the neurite outgrowth induced by MKK6-CA, whereas lipase-deficient PLD2 suppressed it. These findings provide evidence that PLD2 functions as a downstream molecule of p38 in the neurite outgrowth signaling cascade.

O1s photoionization dynamics in oriented NO2.

We have performed extensive density functional theory (DFT) calculations, partial cross sections, dipole prepared continuum orbitals, dipole amplitudes and phase shifts, asymmetry parameters ß, and molecular frame photoelectron angular distributions, to elucidate the O1s photoionization dynamics of NO(2) molecule with emphasis on the shape resonances in the O1s ionization continuum. In the shape resonance region, the ß parameters and photoelectron angular distributions have been compared with our experimental results. Fairly good agreement between the theory and experiment has confirmed that the DFT level calculations can well describe the photoionization dynamics of the simple molecule such as NO(2). Interference due to equivalent atom photoionization is theoretically considered, and the possibility of detection of the effect in the two degenerate channels with different combinations of light polarization and photoemission direction is discussed.

Aging effects of major and trace elements in rat bones and their mutual correlations.

The concentrations of 29 major to trace elements in rat bones (femur) aging from 5 to 113 weeks old were determined. The samples were decomposed by high purity nitric acid and hydrogen peroxide. Nine elements (Na, Mg, P, K, Ca, Fe, Ni, Zn, and Sr) were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) and 20 elements (Li, B, Al, V, Cr, Mn, Cu, As, Se, Rb, Mo, Ag, Cd, Sb, Cs, Ba, W, Tl, Pb, and U) were determined by inductively coupled plasma mass spectrometry (ICP-MS). Aging effects on these elements and mutual elemental correlations were investigated. The concentrations of Ca, P and Na increased in the initial stage of 5-17 weeks and then maintained constant values, whereas those of Mg, K, Mn, Sr and Ba showed decreasing trends of differing patterns. Furthermore, Cu, Zn and Mo showed increasing trends for a whole range of ages. Selenium showed a remarkable increasing trend with a factor of 10. The values of Na, Mg, P, K, Ca, Mn, Cu, Zn, Se and Mo in the age-matched rats distributed narrow ranges, indicating that the metabolism of these elements in bone was well-established. By contrast, those of Al, V, Ni, Ag, Cd, Sb, W, Tl, and U were distributed across a broad range. The metabolism of these elements was not well-established. A cluster analysis was performed using an elemental correlation matrix.

N 1s photoelectron angular distributions from fixed-in-space NO2 molecules: stereodynamics and symmetry considerations.

Angular distributions of N 1s photoelectrons from fixed-in-space NO(2) molecules have been measured over the energy region of shape resonance and above. A multiple-coincidence velocity-map imaging technique for observation of molecular frame photoelectron angular distributions (MF-PADs) has been extended to nonlinear molecular targets. Density functional theory calculations have also been conducted to elucidate the photoionization dynamics and shape resonance in the N 1s photoionization of NO(2). Results show that the N 1s MF-PADs exhibit strong shape variation as a function of both photoelectron kinetic energy and symmetries of final states, whereas asymmetry parameters of laboratory frame PADs show a local minimum around the shape resonance region and increase monotonically as the photon energy increases. Over the shape resonance, the spatial shape of the photoelectron wave function with b(2)-symmetry closely resembles that of 5b(2)(∗) unoccupied molecular orbital of NO(2), although the MF-PAD pattern for b(2)-symmetry does not correspond directly to the 5b(2)(∗) orbital shape. At higher kinetic energy of 90 eV, MF-PADs become less structured, but still show a significant dependence on the symmetry of final states.

The PtdIns(3,4)P(2) phosphatase INPP4A is a suppressor of excitotoxic neuronal death.

Phosphorylated derivatives of phosphatidylinositol, collectively referred to as phosphoinositides, occur in the cytoplasmic leaflet of cellular membranes and regulate activities such as vesicle transport, cytoskeletal reorganization and signal transduction. Recent studies have indicated an important role for phosphoinositide metabolism in the aetiology of diseases such as cancer, diabetes, myopathy and inflammation. Although the biological functions of the phosphatases that regulate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) have been well characterized, little is known about the functions of the phosphatases regulating the closely related molecule phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)). Here we show that inositol polyphosphate phosphatase 4A (INPP4A), a PtdIns(3,4)P(2) phosphatase, is a suppressor of glutamate excitotoxicity in the central nervous system. Targeted disruption of the Inpp4a gene in mice leads to neurodegeneration in the striatum, the input nucleus of the basal ganglia that has a central role in motor and cognitive behaviours. Notably, Inpp4a(-/-) mice show severe involuntary movement disorders. In vitro, Inpp4a gene silencing via short hairpin RNA renders cultured primary striatal neurons vulnerable to cell death mediated by N-methyl-d-aspartate-type glutamate receptors (NMDARs). Mechanistically, INPP4A is found at the postsynaptic density and regulates synaptic NMDAR localization and NMDAR-mediated excitatory postsynaptic current. Thus, INPP4A protects neurons from excitotoxic cell death and thereby maintains the functional integrity of the brain. Our study demonstrates that PtdIns(3,4)P(2), PtdIns(3,4,5)P(3) and the phosphatases acting on them can have distinct regulatory roles, and provides insight into the unique aspects and physiological significance of PtdIns(3,4)P(2) metabolism. INPP4A represents, to our knowledge, the first signalling protein with a function in neurons to suppress excitotoxic cell death. The discovery of a direct link between PtdIns(3,4)P(2) metabolism and the regulation of neurodegeneration and involuntary movements may aid the development of new approaches for the treatment of neurodegenerative disorders.