Molecular Composition of Staufen2-Containing Ribonucleoproteins in Embryonic Rat Brain

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (α- and β-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs

Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions

Effect of solution saturation state and temperature on diopside dissolution

Steady-state dissolution rates of diopside are measured as a function of solution saturation state using a titanium flow-through reactor at pH 7.5 and temperature ranging from 125 to 175°C. Diopside dissolved stoichiometrically under all experimental conditions and rates were not dependent on sample history. At each temperature, rates continuously decreased by two orders of magnitude as equilibrium was approached and did not exhibit a dissolution plateau of constant rates at high degrees of undersaturation. The variation of diopside dissolution rates with solution saturation can be described equally well with a ion exchange model based on transition state theory or pit nucleation model based on crystal growth/dissolution theory from 125 to 175°C. At 175°C, both models over predict dissolution rates by two orders of magnitude indicating that a secondary phase precipitated in the experiments. The ion exchange model assumes the formation of a Si-rich, Mg-deficient precursor complex. Lack of dependence of rates on steady-state aqueous calcium concentration supports the formation of such a complex, which is formed by exchange of protons for magnesium ions at the surface. Fit to the experimental data yields [Formula: see text] where the Mg-H exchange coefficient, n = 1.39, the apparent activation energy, E(a )= 332 kJ mol(-1), and the apparent rate constant, k = 10(41.2 )mol diopside cm(-2 )s(-1). Fits to the data with the pit nucleation model suggest that diopside dissolution proceeds through retreat of steps developed by nucleation of pits created homogeneously at the mineral surface or at defect sites, where homogeneous nucleation occurs at lower degrees of saturation than defect-assisted nucleation. Rate expressions for each mechanism (i) were fit to [Formula: see text] where the step edge energy (α) for homogeneously nucleated pits were higher (275 to 65 mJ m(-2)) than the pits nucleated at defects (39 to 65 mJ m(-2)) and the activation energy associated with the temperature dependence of site density and the kinetic coefficient for homogeneously nucleated pits (E(b-homogeneous )= 2.59 × 10(-16 )mJ K(-1)) were lower than the pits nucleated at defects (E(b-defect assisted )= 8.44 × 10(-16 )mJ K(-1))

Suppression of Her2/neu expression through ILK inhibition is regulated by a pathway involving TWIST and YB-1

In a previous study it was found that the therapeutic effects of QLT0267, a small molecule inhibitor of integrin-linked kinase (ILK), were influenced by Her2/neu expression. To understand how inhibition or silencing of ILK influences Her2/neu expression, Her2/neu signaling was evaluated in six Her2/neu-positive breast cancer cell lines (LCC6Her2, MCF7Her2, SKBR3, BT474, JIMT-1 and KPL-4). Treatment with QLT0267 engendered suppression (32–87%) of total Her2/neu protein in these cells. Suppression of Her2/neu was also observed following small interfering RNA-mediated silencing of ILK expression. Time course studies suggest that ILK inhibition or silencing caused transient decreases in P-AKTser473, which were not temporally related to Her2/neu downregulation. Attenuation of ILK activity or expression was, however, associated with decreases in YB-1 (Y-box binding protein-1) protein and transcript levels. YB-1 is a known transcriptional regulator of Her2/neu expression, and in this study it is demonstrated that inhibition of ILK activity using QLT0267 decreased YB-1 promoter activity by 50.6%. ILK inhibition was associated with changes in YB-1 localization, as reflected by localization of cytoplasmic YB-1 into stress granules. ILK inhibition also suppressed TWIST (a regulator of YB-1 expression) protein expression. To confirm the role of ILK on YB-1 and TWIST, cells were engineered to overexpress ILK. This was associated with a fourfold increase in the level of YB-1 in the nucleus, and a 2- and 1.5-fold increase in TWIST and Her2/neu protein levels, respectively. Taken together, these data indicate that ILK regulates the expression of Her2/neu through TWIST and YB-1, lending support to the use of ILK inhibitors in the treatment of aggressive Her2/neu-positive tumors

Eggs in the Freezer: Energetic Consequences of Nest Site and Nest Design in Arctic Breeding Shorebirds

Birds construct nests for several reasons. For species that breed in the Arctic, the insulative properties of nests are very important. Incubation is costly there and due to an increasing surface to volume ratio, more so in smaller species. Small species are therefore more likely to place their nests in thermally favourable microhabitats and/or to invest more in nest insulation than large species. To test this hypothesis, we examined characteristics of nests of six Arctic breeding shorebird species. All species chose thermally favourable nesting sites in a higher proportion than expected on the basis of habitat availability. Site choice did not differ between species. Depth to frozen ground, measured near the nests, decreased in the course of the season at similar non-species-specific speeds, but this depth increased with species size. Nest cup depth and nest scrape depth (nest cup without the lining) were unrelated to body mass (we applied an exponent of 0.73, to account for metabolic activity of the differently sized species). Cup depth divided by diameter2 was used as a measure of nest cup shape. Small species had narrow and deep nests, while large species had wide shallow nests. The thickness of nest lining varied between 0.1 cm and 7.6 cm, and decreased significantly with body mass. We reconstruct the combined effect of different nest properties on the egg cooling coefficient using previously published quantitative relationships. The predicted effect of nest cup depth and lining depth on heat loss to the frozen ground did not correlate with body mass, but the sheltering effect of nest cup diameter against wind and the effects of lining material on the cooling coefficient increased with body mass. Our results suggest that small arctic shorebirds invest more in the insulation of their nests than large species

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Forster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.Peer reviewe

Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue

Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein - RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway

Dysregulation of autophagy and stress granule-related proteins in stress-driven Tau pathology

Imbalance of neuronal proteostasis associated with misfolding and aggregation of Tau protein is a common neurodegenerative feature in Alzheimer's disease (AD) and other Tauopathies. Consistent with suggestions that lifetime stress may be an important AD precipitating factor, we previously reported that environmental stress and high glucocorticoid (GC) levels induce accumulation of aggregated Tau; however, the molecular mechanisms for such process remain unclear. Herein, we monitor a novel interplay between RNA-binding proteins (RBPs) and autophagic machinery in the underlying mechanisms through which chronic stress and high GC levels impact on Tau proteostasis precipitating Tau aggregation. Using molecular, pharmacological and behavioral analysis, we demonstrate that chronic stress and high GC trigger mTOR-dependent inhibition of autophagy, leading to accumulation of Tau aggregates and cell death in P301L-Tau expressing mice and cells. In parallel, we found that environmental stress and GC disturb cellular homeostasis and trigger the insoluble accumulation of different RBPs, such as PABP, G3BP1, TIA-1, and FUS, shown to form stress granules (SGs) and Tau aggregation. Interestingly, an mTOR-driven pharmacological stimulation of autophagy attenuates the GC-driven accumulation of Tau and SG-related proteins as well as the related cell death, suggesting a critical interface between autophagy and the response of the SG-related protein in the neurodegenerative potential of chronic stress and GC. These studies provide novel insights into the RNA-protein intracellular signaling regulating the precipitating role of environmental stress and GC on Tau-driven brain pathology.We would like to thank Professor Juergen Gotz, (University of Queensland, Australia) for the kind offer of eGFP-P301LTau SH-SY5Y cells and Dr. Bruno Almeida for his technical assistance. J.M.S. was granted with a PhD fellowship (SRFH/BD/88932/2012) by Portuguese Foundation for Science & Technology (FCT); I.S. is holder of FCT Investigator grants (IF/01799/2013), C.D. is a recipient of PhD fellowship of PHDoc program and co-tutelle PhD student of UMinho-UPMC universities. This work was funded by FCT research grants "PTDC/SAU-NMC/113934/2009" (I.S.), the Portuguese North Regional Operational Program (ON. 2) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER) as well as the Project Estrategico co-funded by FCT (PEst-C/SAU/LA0026/2013) and the European Regional Development Fund COMPETE (FCOMP-01-0124-FEDER-037298) as well as the project NORTE-01-0145-FEDER000013, supported by the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). In addition, this work was partly funded by Canon Foundation in Europe. This work has been also funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145FEDER-007038. This study was also supported to BW by grants from NIH (AG050471, NS089544, and ES020395), the BrightFocus Foundation, the Alzheimer Association and the Cure Alzeimer Foundation. Human brain tissue was generously provided by the National Institute of Aging Boston University AD Center (P30AG13846).info:eu-repo/semantics/publishedVersio

How ice grows from premelting films and water droplets

Close to the triple point, the surface of ice is covered by a thin liquid layer (so-called quasi-liquid layer) which crucially impacts growth and melting rates. Experimental probes cannot observe the growth processes below this layer, and classical models of growth by vapor deposition do not account for the formation of premelting films. Here, we develop a mesoscopic model of liquid-film mediated ice growth, and identify the various resulting growth regimes. At low saturation, freezing proceeds by terrace spreading, but the motion of the buried solid is conveyed through the liquid to the outer liquid-vapor interface. At higher saturations water droplets condense, a large crater forms below, and freezing proceeds undetectably beneath the droplet. Our approach is a general framework that naturally models freezing close to three phase coexistence and provides a first principle theory of ice growth and melting which may prove useful in the geosciences.Comment: 32 pages, 10 figure