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In dry southwestern South Tyrol, Italy, rock glaciers are dominant landforms of the high-mountain cryosphere. Their spatial distribution and degree of activity hold critical information on the current state of discontinuous permafrost and consequently on the response potential to climate warming. Traditional geomorphologic mapping, however, owing to the qualitative expert-based nature, typically displays a high degree of uncertainty and variability among operators with respect to the dynamic classification of intact (permafrost-bearing) and relict (permafrost-devoid) rock glaciers. This limits the reliability of geomorphologic rock glacier inventories for basic and applied purposes. To address this limitation, (i) we conduct a systematic evaluation of the improvements that synthetic aperture radar interferometry (InSAR) can afford to the detection and dynamic classification of rock glaciers and (ii) build an integrated inventory that combines the strengths of geomorphologic- and InSAR-based approaches. To exploit fully InSAR-based information towards a better understanding of the topo-climatic conditions that sustain creeping permafrost, we further explore how velocity and the spatial distribution of moving areas (MAs) within rock glaciers may vary as a function of simple topographic variables known to exert first-order controls on incoming solar radiation, such as elevation and aspect. Starting from a geomorphologic inventory (n=789), we characterize the kinematics of InSAR-based MAs and the relevant hosting rock glaciers on 36 Sentinel-1 interferograms in the 2018–2019 period. With respect to the original inventory, InSAR analysis allowed us to identify 14 previously undetected rock glaciers. Further, it confirmed that 246 (76 %) landforms, originally interpreted as intact, do exhibit detectable movement (i.e., ≥1 cm yr -1) and that 270 (60 %) of the relict labeled counterparts do not, whereas 144 (18 %) were kinematically undefined due to decorrelation. Most importantly, InSAR proved critical for reclassifying 121 (15 %) rock glaciers, clarifying that 41 (13 %) of those interpreted as being intact do not exhibit detectable movement and that 80 (17 %) of the original relict ones do move. Reclassification (i) allowed us to identify a cluster of intact rock glaciers below 2000 m a.s.l. associated with positive mean annual air temperature (MAAT), and (ii) by increasing the altitudinal overlap between intact and relict rock glaciers, it depicts a broad transition belt in the aspect–elevation space, which varies from 50 m on west-facing slopes to 500 m on easterly ones. This finding deteriorates the significance of elevation and aspect as topographic proxies for modeling permafrost occurrence and highlights the importance of using InSAR to inform such models. From a process-oriented standpoint, InSAR information proves fundamental for imaging how this altitudinal transition manifests through changing rates and styles of rock glacier surface deformation. Specifically, we find that, as rock glaciers move faster, an increasingly larger proportion of their surface becomes kinematically involved (i.e., percent MA cover) and that this proportion increases with elevation up to 2600–2800 m, beyond which an inflection occurs and consistent average values are attained. Considering that the inflection falls between the -1 and -2 °C MAAT – the lower boundary for discontinuous permafrost – and is independent of slope gradient, we conclude that this altitudinal pattern represents a geomorphic signature: the dynamic expression of increasing permafrost distribution, from sporadic to discontinuous. [ABSTRACT FROM AUTHOR]

We performed an extensive analysis of C-band SAR datasets provided by the European Space Agency (ESA) satellites ERS-1/2, Envisat ASAR, and Sentinel-1 in the period 1992–2020 aiming at reconstructing the multi-decadal spatial and temporal evolution of the surface displacements at the Brienz/Brinzauls landslide complex, located in canton Graubünden (Switzerland). To this end, we analyzed about 1000 SAR images by applying differential interferometry (InSAR), multitemporal stacking, and persistent scatterer interferometry (PSI) approaches. Moreover, we jointly considered digital image correlation (DIC) on high-resolution multi-temporal digital terrain models (DTM) generated from airborne surveys and InSAR results to compute 3-D surface deformation fields. The extensive network of GNSS stations across the Brienz landslide complex allowed us to extensively validate the deformation results obtained in our remote sensing analyses. Here, we illustrate the limitations occurring when relying on InSAR and/or PSI measurements for the analysis and interpretation of complex landslide scenarios, especially in cases of relevant spatial and temporal heterogeneities of the deformation field. The joint use of InSAR and DIC can deliver a better picture of the evolution of the deformation field, however, not for all displacement components. Since InSAR, PSI and DIC measurements are nowadays routinely used in the framework of local investigations, as well as in regional, national, and/or continental monitoring programs, our results are of major importance for users aiming at a comprehensive understanding of these datasets in landslide scenarios. [ABSTRACT FROM AUTHOR]

Microplastic and nanoplastic research has proliferated in recent years in response to the escalating plastic pollution crisis. However, a lack of optimised methods for sampling and sample processing has potential implications for contaminating samples resulting in an overestimation of the quantity of microplastics and nanoplastics present in environmental samples. In response, a series of recommendations have been made, but most have not been quantified or validated sources of contamination. In the present study, we investigated sources of plastic contamination in common laboratory procedures including water sources (e.g., Milli-Q), consumables (e.g., unburnt glassware), airflow (e.g., fume hood) and dust. Using flow cytometry, we identified water, air flow and dust as sources of significant contamination. Milli-Q and reverse osmosis were the least contaminated sources when compared with tap water. Interestingly, current recommendations are to use glass consumables in replacement of plastic consumables, however, we have identified glassware and glass consumables as a significant source of contamination. Current best practice is to cover the glass tube with aluminium foil to reduce airborne contamination, but we found fresh aluminium foil to be a significant source of contamination, bringing light to the limitations foil has as a contamination control measure. Lastly, we identified significant quantities of microplastics and nanoplastics present in dust collected within the laboratory, suggesting this is a widespread and underestimated source of contamination. We have provided validated sources of contamination for both consumables and common laboratory procedures and provided mitigation strategies based on these. Additional recommendations include the appropriate design of experimental controls to quantify levels of introduced contamination based on methods and the detection techniques utilised. The application of these mitigation strategies and appropriate experimental design will allow for more accurate estimations on the level of microplastic and nanoplastic contamination within environmental samples. [Display omitted] • Any experimental work adds microplastic and nanoplastic contamination. • Mitigation strategies to minimise contamination depends on detection techniques. • Plasticware consumables outperform glassware consumables. • Biological safety cabinets did not significantly reduce contamination. • Aluminium foil introduces contamination to samples. [ABSTRACT FROM AUTHOR]

The region of Tajikistan where the Rogun Hydropower Project is currently under construction has experienced large and catastrophic slope failures in the past, often triggered by earthquakes. Co-seismic slope failures are thus common and pose a high hazard potential; however, to date, no specific analysis of slope activity in this area has been presented in international journals. Here, we present an inventory of active landslides identified through satellite imagery analysis and in particular by exploiting space-borne differential radar interferometry. Surface displacements provide the basis for the detection of active slope instabilities, which are then further classified by using geomorphological indicators visible in optical satellite imagery. Additionally, the proximity of active landslides to tectonic lineaments, as well as regional seismicity, is analysed to investigate potential relationships and to provide an integrated river damming hazard potential. The results show that approximately 31% of all detected landslides would have a high damming hazard potential upon catastrophic failure, highlighting the importance of such phenomena for efficient long-term land use planning and management of hydropower plants. [ABSTRACT FROM AUTHOR]

This study reports major, minor, and trace element data and Sr isotope ratios for 11 uranium ore (uraninite, UO 2+x ) samples and one processed uranium ore concentrate (UOC) from various U.S. deposits. The uraninite investigated represent ores formed via different modes of mineralization (e.g., high- and low-temperature) and within various geological contexts, which include magmatic pegmatites, metamorphic rocks, sandstone-hosted, and roll front deposits. In situ trace element data obtained by laser ablation-ICP-MS and bulk sample Sr isotopic ratios for uraninite samples investigated here indicate distinct signatures that are highly dependent on the mode of mineralization and host rock geology. Relative to their high-temperature counterparts, low-temperature uranium ores record high U/Th ratios (>1000), low total rare earth element (REE) abundances (<1 wt%), high contents (>300 ppm) of first row transition metals (Sc, Ti, V, Cr, Mn, Co, Ni), and radiogenic 87 Sr/ 86 Sr ratios (>0.7200). Comparison of chondrite normalized REE patterns between uraninite and corresponding processed UOC from the same locality indicates identical patterns at different absolute concentrations. This result ultimately confirms the importance of establishing geochemical signatures of raw, uranium ore materials for attribution purposes in the forensic analysis of intercepted nuclear materials. [ABSTRACT FROM AUTHOR]

Shc (Src homology and collagen homology) proteins are considered prototypical signalling adaptors in mammalian cells. Consisting of four unique members, ShcA, B, C and D, and multiple splice isoforms, the family is represented in nearly every cell type in the body, where it engages in an array of fundamental processes to transduce environmental stimuli. Two decades of investigation have begun to illuminate the mechanisms of the flagship ShcA protein, whereas much remains to be learned about the newest discovery, ShcD. It is clear, however, that the distinctive modular architecture of Shc proteins, their promiscuous phosphotyrosine-based interactions with a multitude of membrane receptors, involvement in central cascades including MAPK (mitogen-activated protein kinase) and Akt, and unconventional contributions to oxidative stress and apoptosis all require intricate regulation, and underlie diverse physiological function. From early cardiovascular development and neuronal differentiation to lifespan determination and tumorigenesis, Shc adaptors have proven to bemore ubiquitous, versatile and dynamic than their structures alone suggest. [ABSTRACT FROM AUTHOR]

The glomerular filtration barrier in the kidney is formed in part by a specialized intercellular junction known as the slit diaphragm, which connects adjacent actin-based foot processes of kidney epithelial cells (podocytes). Mutations affecting a number of slit diaphragm proteins, including nephrin (encoded by NPHS1), lead to renal disease owing to disruption of the filtration barrier and rearrangement of the actin cytoskeleton, although the molecular basis for this is unclear. Here we show that nephrin selectively binds the Src homology 2 (SH2)/SH3 domain-containing Nck adaptor proteins, which in turn control the podocyte cytoskeleton in vivo. The cytoplasmic tail of nephrin has multiple YDxV sites that form preferred binding motifs for the Nck SH2 domain once phosphorylated by Src-family kinases. We show that this Nck–nephrin interaction is required for nephrin-dependent actin reorganization. Selective deletion of Nck from podocytes of transgenic mice results in defects in the formation of foot processes and in congenital nephrotic syndrome. Together, these findings identify a physiological signalling pathway in which nephrin is linked through phosphotyrosine-based interactions to Nck adaptors, and thus to the underlying actin cytoskeleton in podocytes. Simple and widely expressed SH2/SH3 adaptor proteins can therefore direct the formation of a specialized cellular morphology in vivo. [ABSTRACT FROM AUTHOR]

The development of the cardiovascular system and the development of the early hematopoietic systems are closely related, and both require signaling through the Tie2 receptor tyrosine kinase. Although endothelial cells and hematopoietic cells as well as their precursors share common gene expression patterns during development, it remains completely unknown how Tie2 signaling coordinately regulates cardiovascular development and early hematopoiesis in vivo. We show here that mice with a targeted mutation in tyrosine residue 1100 in the carboxyl-terminal tail of Tie2 display defective cardiac development and impaired hematopoietic and endothelial cell development in the paraaortic splanchnopleural mesoderm similar to that seen in Tie2-null mutant mice. Surprisingly, however, unlike Tie2-null mutant mice, mice deficient in signaling through this tyrosine residue show a normal association of perivascular cells with nascent blood vessels. These studies are the first to demonstrate the physiological importance of a single tyrosine residue in Tie2, and they suggest that multiple tyrosine residues in the receptor may coordinate cardiovascular development and early hematopoietic development. [ABSTRACT FROM AUTHOR]

Tie2/Tek is an endothelial cell receptor tyrosine kinase that induces signal transduction pathways involved in cell migration upon angiopoietin-1 (Ang1) stimulation. To address the importance of the various tyrosine residues of Tie2 in signal transduction, we generated a series of Tie2 mutants and examined their signaling properties. Using this approach in conjunction with a phosphorylation state-specific antibody, we identified tyrosine residue 1106 on Tie2 as an Ang1-dependent autophosphorylation site that mediates binding and phosphorylation of the downstream-of-kinase-related (Dok-R) docking protein. This tyrosine residue is contained within a unique interaction motif for the phosphotyrosine binding domain of Dok-R, and the pleckstrin homology domain of Dok-R further contributes to Tie2 binding in a phosphatidylinositol 3'-kinase-dependent manner. Introduction of a Tie2 mutant lacking tyrosine residue 1106 into endothelial cells interferes with Dok-R phosphorylation in response to Ang1. Furthermore, this mutant is unable to restore the migration potential of endothelial cells derived from mice lacking Tie2. Together, these findings demonstrate that tyrosine residue 1106 on Tie2 is critical for coupling downstream cell migration signal transduction pathways with Ang1 stimulation in endothelial cells. [ABSTRACT FROM AUTHOR]

Tek/Tie-2 is an endothelial cell (EC)-specific receptor tyrosine kinase that plays a critical role in angiogenesis via its regulation by the angiopoietin family of growth factor ligands. Angiopoietin-1 (Angi) can promote EC migration; however, the signaling mechanisms underlying this process remain elusive. Here we demonstrate that Dok-R/Dok-2 can associate with Tek in ECs following Angi stimulation, resulting in tyrosine phosphorylation of Dok-R and the subsequent recruitment of Nck and the p21-activating kinase (Pak/Paki) to the activated receptor. Ang1-mediated migration is increased upon Dok-R overexpression and this requires a functional Nck binding site on Dok-R. Localization of this Dok-R-Nck-Pak complex to the activated Tek receptor at the cellular membrane is coincident with activation of Pak kinase. The ability of Dok-R to bind Nck is required for maximal activation of Pak and overexpression of Pak results in increased Ang1-mediated cell motility. Our study outlines a novel signaling pathway underlying Angi-driven cell migration that involves Dok-R and its recruitment of Nck and the subsequent activation of Pak. [ABSTRACT FROM AUTHOR]

Angiogenesis is required for normal embryonic vascular development and aberrant angiogenesis contributes to several diseases, including cancer, diabetes and tissue ischaemia. What are the molecular mechanisms that regulate this important process? The Tie family of receptors and their ligands, the angiopoietins, are beginning to provide insight into how vessels make decisions such as whether to grow or regress ? processes that are important not only during development but throughout an organism's life. [ABSTRACT FROM AUTHOR]

Tek/Tie2 is an endothelial cell-specific receptor tyrosine kinase that has been shown to play a role in vascular development of the mouse. Targeted mutagenesis of both Tek and its agonistic ligand, Angiopoietin-1, result in embryonic lethality, demonstrating that the signal transduction pathway(s) mediated by this receptor are crucial for normal embryonic development. In an attempt to identify downstream signaling partners of the Tek receptor, we have used the yeast two-hybrid system to identify phosphotyrosine-dependent interactions. Using this approach, we have identified a novel docking molecule called Dok-R, which has sequence and structural homology to p62dok and IRS-3. Mapping of the phosphotyrosine-interaction domain within Dok-R shows that Dok-R interacts with Tek through a PTB domain. Dok-R is coexpressed with Tek in a number of endothelial cell lines. We show that coexpression of Dok-R with activated Tek results in tyrosine phosphorylation of Dok-R and that rasGAP and Nck coimmunoprecipitate with phosphorylated Dok-R. Furthermore, Dok-R is constitutively bound to Crk presumably through the proline rich tail of Dok-R. The cloning of Dok-R represents the first downstream substrate of the activated Tek receptor, and suggests that Tek can signal through a multitude of pathways. [ABSTRACT FROM AUTHOR]

Abstract The inflammatory response is essential for eradication of lipopolysaccharide (LPS) presenting microbial invaders but requires exquisite regulation to prevent detrimental vascular inflammation. Endothelial cells play active roles in both the initiation of inflammation, through the detection of LPS by Toll-like Receptor 4 (TLR4), and the resolution of inflammation, through the actions of the receptor tyrosine kinase, Tie2. The process by which Tie2 attenuates LPS-TLR4 driven inflammation is poorly understood. To investigate the effects of Tie2 on TLR4 signalling, Nf-κB activation was monitored in cells expressing Tie2 mutants harboring tyrosine (Y) to phenylalanine (F) substitutions in the cytoplasmic domain. Tie2 attenuated LPS induced Nf-κB activation in a manner requiring Tie2 kinase activation, the carboxy-terminal tyrosine residue Y1100 and downstream Erk1/2 signalling. Tyrosine 1100 was also required for the Tie2 dependent decrease in expression of the TLR4 signalling proteins, TRAF6 and IRAK1 and stabilization of the Nf-κB inhibitor, IκBα. In contrast, upregulation of known TLR4 antagonist miRNA-146b-5p required all three tyrosine phosphorylation sites in Tie2. Finally, we confirmed in an in vivo model that activation of Tie2 signalling reduces LPS mediated inflammation. Our results show that Y1100 initiated Erk1/2 signalling is essential for the anti-inflammatory effect of Tie2 on TLR4 mediated inflammation. Graphical abstract Unlabelled Image Highlights • Tie2 tyrosine kinase activity reduces endotoxin induced activation of Nf-κB. • Attenuation of Nf-κB activity depends on phosphorylation of Tie2 Y1100. • Tie2 suppression of Nf-κB activation is Erk1/2 dependent. • Tie2 regulation of miRNA-146b-5p in endothelial cells requires Erk1/2 activation. • Tie2 activation reduces markers of LPS-TLR4 mediated inflammation in vivo. [ABSTRACT FROM AUTHOR]

Shc family signalling adaptors connect activated transmembrane receptors to proximal effectors, and most also contain a sequence involved in clathrin-mediated receptor endocytosis. Notably, this AP2 adaptin-binding motif (AD) is absent from the ShcD (also known as Shc4) homolog, which also uniquely promotes ligand-independent phosphorylation of the epidermal growth factor receptor (EGFR). We now report that cultured cells expressing ShcD exhibit reduced EGF uptake, commensurate with a decrease in EGFR surface presentation. Under basal conditions, ShcD colocalises with the EGFR and facilitates its phosphorylation, ubiquitylation and accumulation in juxtanuclear vesicles identified as Rab11-positive endocytic recycling compartments. Accordingly, ShcD also functions as a constitutive binding partner for the E3 ubiquitin ligase Cbl. EGFR phosphorylation and focal accumulation likewise occur upon ShcD co-expression in U87 glioma cells. Loss of ShcD phosphotyrosine-binding function or insertion of the ShcA AD sequence each restore ligand acquisition through distinct mechanisms. The AD region also contains a nuclear export signal, indicating its multifunctionality. Overall, ShcD appears to possess several molecular permutations that actively govern the EGFR, which may have implications in development and disease. [ABSTRACT FROM AUTHOR]

The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis. [ABSTRACT FROM AUTHOR]

Shc family proteins serve as phosphotyrosine adaptor molecules in various receptor-mediated signaling pathways. In mammals, three distinct Shc genes have been described that encode proteins characterized by two phosphotyrosine-interaction modules, an amino-terminal phosphotyrosine binding (PTB) domain and a carboxy-terminal Src homology 2 domain. Here, we report the analysis of an uncharacterized fourth Shc family protein, ShcD/Shc4, that is expressed in adult brain and skeletal muscle. Consistent with this expression pattern, we find that ShcD can associate via its PTB domain with the phosphorylated muscle-specific kinase (MuSK) receptor tyrosine kinase and undergo tyrosine phosphorylation downstream of activated MuSK. Interestingly, additional sites of tyrosine phosphorylation, including a novel Grb2 binding site, are present on ShcD that are not found in other Shc family proteins. Activation of MuSK upon agrin binding at the neuromuscular junction (NMJ) induces clustering and tyrosine phosphorylation of acetylcholine receptors (AChRs) required for synaptic transmission. ShcD is coexpressed with MuSK in the postsynaptic region of the NMJ, and in cultured myotubes stimulated with agrin, expression of ShcD appears to be important for early tyrosine phosphorylation of the AChR. Thus, we have characterized a new member of the Shc family of docking proteins, which may mediate a specific aspect of signaling downstream of the MuSK receptor. [ABSTRACT FROM AUTHOR]

Podocytes are key components of the kidney blood filtration barrier, and their ability to withstand hemodynamic strain is proposed to be closely tied to their unique and flexible cytoarchitecture. However, the mechanisms that control such mechanotransduction are poorly understood. We have previously established that tyrosine phosphorylation of the transmembrane protein nephrin promotes recruitment of the Nck1/2 cytoskeletal adaptor proteins and downstream actin remodeling.Wenow reveal that Nck integrates nephrin with the Hippo kinase cascade through association with the adaptor protein WTIP. Using mutational analysis, we show that Nck sequesters WTIP and its binding partner Lats1 to phosphorylated nephrin, resulting in decreased phospho-activation of Lats1.We further demonstrate that, coincident with nephrin dephosphorylation in a transient model of podocyte injury in mice, Lats1 is rapidly activated, and this precedes significant down-regulation of the transcription regulator Yap. Moreover, we show reduced levels of Yap protein in mice with chronic disruption of nephrin phospho-signaling. Together, these findings support the existence of a dynamic molecular link between nephrin signaling and the canonical Hippo pathway in podocytes, which may facilitate the conversion of mechanical cues to biochemical signals promoting podocyte viability. [ABSTRACT FROM AUTHOR]

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependentphosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury. [ABSTRACT FROM AUTHOR]

Active rock glaciers represent the best visual expression of mountain permafrost that can be mapped and monitored directly using remotely sensed data. Active rock glaciers are bodies that consist of a perennially frozen ice/rock mixture and express a distinct flow-like morphology indicating downslope permafrost creep movement. Annual rates of motion have ranged from a few millimeters to several meters per year, varying within the annual cycle, from year to year, as well as at the decennial time scale. During the last decade, in situ observations in the European Alps have shown that active rock glaciers are responding almost synchronously to inter-annual and decennial changes in ground temperature, suggesting that the relative changes of their kinematics are a general indicator of the evolution of mountain permafrost conditions. Here, we used satellite radar interferometry (InSAR) to monitor the rate of motion of various active rock glaciers in the Swiss Alps, Qeqertarsuaq (Western Greenland), and the semiarid Andes of South America. Velocity time series computed with Sentinel-1 SAR images, regularly acquired since 2014, every six days over Europe and Greenland and every 12 days over the Andes, show annual fluctuations, with higher velocities at the end of the summer. A JERS-1 image pair of 1996 and stacks of very high-resolution SAR images from TerraSAR-X and Cosmo-SkyMed from 2008 to 2017 were analyzed using InSAR and offset tracking over the Western Swiss Alps in order to extend the main observation period of our study. A quantitative assessment of the accuracy of InSAR and offset tracking was performed by comparison with in situ methods. Our results for the three different study regions demonstrate that Sentinel-1 InSAR can complement worldwide in situ measurements of active rock glacier kinematics. [ABSTRACT FROM AUTHOR]

Background: Mammalian Shc (Src homology and collagen) proteins comprise a family of four phosphotyrosine adaptor molecules which exhibit varied spatiotemporal expression and signaling functions. ShcD is the most recently discovered homologue and it is highly expressed in the developing central nervous system (CNS) and adult brain. Presently however, its localization within specific cell types of mature neural structures has yet to be characterized.Results: In the current study, we examine the expression profile of ShcD in the adult rat CNS using immunohistochemistry, and compare with those of the neuronally enriched ShcB and ShcC proteins. ShcD shows relatively widespread distribution in the adult brain and spinal cord, with prominent levels of staining throughout the olfactory bulb, as well as in sub-structures of the cerebellum and hippocampus, including the subgranular zone. Co-localization studies confirm the expression of ShcD in mature neurons and progenitor cells. ShcD immunoreactivity is primarily localized to axons and somata, consistent with the function of ShcD as a cytoplasmic adaptor. Regional differences in expression are observed among neural Shc proteins, with ShcC predominating in the hippocampus, cerebellum, and some fiber tracts. Interestingly, ShcD is uniquely expressed in the olfactory nerve layer and in glomeruli of the main olfactory bulb.Conclusions: Together our findings suggest that ShcD may provide a distinct signaling contribution within the olfactory system, and that overlapping expression of ShcD with other Shc proteins may allow compensatory functions in the brain. [ABSTRACT FROM AUTHOR]

Mutations in the transmembrane protein nephrin (encoded by NPHS1) underlie nearly half of all cases of congenital nephrotic syndrome (CNS), which is caused by aberrations in the blood filtering function of glomerular podocytes. Nephrin directly contributes to the structure of the filtration barrier, and it also serves as a signaling scaffold in podocytes, undergoing tyrosine phosphorylation on its cytoplasmic tail to recruit intracellular effector proteins. Nephrin phosphorylation is lost in several human and experimental models of glomerular disease, and genetic studies have confirmed its importance in maintenance of the filtration barrier. To date, however, the effect of CNS-associated NPHS1 variants on nephrin phosphorylation remains to be determined, which hampers genotype-phenotype correlations. Here, we have characterized a novel nephrin sequence variant, A419T, which is expressed along with C623F in a patient presenting with CNS. Nephrin localization is altered in kidney biopsies, and we further demonstrate reduced surface expression and ER retention of A419T and C623F in cultured cells. Moreover, we show that both mutations impair nephrin tyrosine phosphorylation, and they exert dominant negative effects on wildtype nephrin signaling. Our findings thus reveal that missense mutations in the nephrin extracellular region can impact nephrin signaling, and they uncover a potential pathomechanism to explain the spectrum of clinical severity seen with mild NPHS1 mutations. [ABSTRACT FROM AUTHOR]

Proteins of the Src homology and collagen (Shc) family are typically involved in signal transduction events involving Ras/ MAPK and PI3K/Akt pathways. In the nervous system, they function proximal to the neurotrophic factors that regulate cell survival, differentiation, and neuron-specific characteristics. The least characterized homolog, ShcD, is robustly expressed in the developing and mature nervous system, but its contributions to neural cell circuitry are largely uncharted. We now report that ShcD binds to active Ret, TrkA, and TrkB neurotrophic factor receptors predominantly via its phosphotyrosine-binding (PTB) domain. However, in contrast to the conventional Shc adaptors, ShcD suppresses distal phosphorylation of the Erk MAPK. Accordingly, genetic knock-out of mouse ShcD enhances Erk phosphorylation in the brain. In cultured cells, this capacity is tightly aligned to phosphorylation of ShcD CH1 region tyrosine motifs, which serve as docking platforms for signal transducers, such as Grb2. Erk suppression is relieved through independent mutagenesis of the PTB domain and the CH1 tyrosine residues, and successive substitution of these tyrosines breaks the interaction between ShcD and Grb2, thereby promoting TrkB-Grb2 association. Erk phosphorylation can also be restored in the presence of wild type ShcD through Grb2 overexpression. Conversely, mutation of the ShcD SH2 domain results in enhanced repression of Erk. Although the SH2 domain is a less common binding interface in Shc proteins, we demonstrate that it associates with the Ptpn11 (Shp2) phosphatase, which in turn regulatesShcDtyrosine phosphorylation. Wetherefore propose a model whereby ShcD competes with neurotrophic receptors for Grb2 binding and opposes activation of the MAPK cascade. [ABSTRACT FROM AUTHOR]

In addition to the infectious consequences of immunodeficiency, patients with Wiskott-Aldrich syndrome (WAS) often suffer from poorly understood exaggerated immune responses that result in autoimmunity and elevated levels of serum IgE. Here, we have shown that WAS patients and mice deficient in WAS protein (WASP) frequently develop IgE-mediated reactions to common food allergens. WASP-deficient animals displayed an adjuvant-free IgE-sensitization to chow antigens that was most pronounced for wheat and soy and occurred under specific pathogen-free as well as germ-free housing conditions. Conditional deletion of Was in FOXP3+ Tregs resulted in more severe Th2-type intestinal inflammation than that observed in mice with global WASP deficiency, indicating that allergic responses to food allergens are dependent upon loss of WASP expression in this immune compartment. While WASP-deficient Tregs efficiently contained Th1- and Th17-type effector differentiation in vivo, they failed to restrain Th2 effector responses that drive allergic intestinal inflammation. Loss of WASP was phenotypically associated with increased GATA3 expression in effector memory FOXP3+ Tregs, but not in naive-like FOXP3+ Tregs, an effect that occurred independently of increased IL-4 signaling. Our results reveal a Treg-specific role for WASP that is required for prevention of Th2 effector cell differentiation and allergic sensitization to dietary antigens. [ABSTRACT FROM AUTHOR]

Development of the cardiovascular system is critically dependent on the ability of endothelial cells (ECs) to reorganize their intracellular actin architecture to facilitate migration, adhesion, and morphogenesis. Nck family cytoskeletal adaptors function as key mediators of actin dynamics in numerous cell types, though their role in EC biology remains largely unexplored. Here, we demonstrate an essential requirement for Nck within ECs. Mouse embryos lacking endothelial Nck1/2 expression develop extensive angiogenic defects that result in lethality at about embryonic day 10. Mutant embryos show immature vascular networks, with decreased vessel branching, aberrant perivascular cell recruitment, and reduced cardiac trabeculation. Strikingly, embryos deficient in endothelial Nck also fail to undergo the endothelial-to-mesenchymal transition (EnMT) required for cardiac valve morphogenesis, with loss of Nck disrupting expression of major EnMT markers, as well as suppressing mesenchymal outgrowth. Furthermore, we show that Nck-null ECs are unable to migrate downstream of vascular endothelial growth factor and angiopoietin-1, and they exhibit profound perturbations in cytoskeletal patterning, with disorganized cellular projections, impaired focal adhesion turnover, and disrupted actin-based signaling. Our collective findings thereby reveal a crucial role for Nck as a master regulator within the endothelium to control actin cytoskeleton organization, vascular network remodeling, and EnMT during cardiovascular development. [ABSTRACT FROM AUTHOR]

Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have previously shown that high glucose level exposure and diabetes increased the expression of SHP-1 causing podocyte apoptosis. SHP-1 possesses two SH2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown if SHP-1 interacts with nephrin and whether its elevated expression affects nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Co-expression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. Single tyrosine to phenylalanine mutation revealed that rat nephrin Tyr1127 and Tyr1152 are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2+/C96Y) compared to control littermate mice (Ins2+/+), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased co-localization of SHP-1 with nephrin in diabetic mice compared to control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin causing decreased nephrin phosphorylation, which may in turn contribute to diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Summary: Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior. [ABSTRACT FROM AUTHOR]

The systemic capillary leak syndrome (SCLS) is a rare disorder characterized by transient episodes of hypotensive shock and anasarca thought to arise from revers-ible microvascular barrier dysfunction. Although the high prevalence of a mono-clonal gammopathy of unknown signifi-cance in SCLS suggests a pathogenic contribution of endogenous immuno-globulins, the mechanisms of vascular hyperpermeability remain obscure. Herein, we report clinical and molecular findings on 23 patients, the largest SCLS case series to date. Application of epi-sodic SCLS sera, but neither the purified immunoglobulin fraction nor sera ob-talned from patients during remission, to human microvascular endothelial cells caused vascular endothelial Cadherin in-ternalization, disruption of interendothe-lial junctions, actin stress fiber formation, and increased permeability in comple-mentary functional assays without induc-ing endothelial apoptosis. Intravenous Im-munoglobulin, one promising therapy for SCLS, mitigated the permeability effects of episodic sera. Consistent with the pres-ence of endogenous, nonimmunoglobu-lln, circulating permeability factor(s) con-strained to SCLS episodes, we found that vascular endothelial growth factor (VEGF) and anglopoietin 2 (Ang2), were elevated in episodic SCLS sera but not in remis-sion sera. Ab-based inhibition of Ang2 counteracted permeability induced by ep-isodic SCLS sera. Comparable experi-ments with anti-VEGF Ab (bevacizumab) yielded less interpretable results, prob-ably because of endothelial toxicity of VEGF withdrawal. Our results support a model of SCLS pathogenesis in which nonimmunoglobulin humoral factors such as VEGF and Ang2 contribute to transient endothelial contraction, suggesting a mo-lecular mechanism for this highly lethal disorder. (Blood. 2012;119(18):4321-4332) [ABSTRACT FROM AUTHOR]

Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin-Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood. Here, we demonstrate that nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in nephrin tyrosine phosphorylation, nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate nephrin turnover and podocyte health. [ABSTRACT FROM AUTHOR]

Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation. [ABSTRACT FROM AUTHOR]

We have analyzed the means by which the Nck family of adaptor proteins couples adhesion proteins to actin reorganization. The nephrin adhesion protein is essential for the formation of actin-based foot processes in glomerular podocytes. The clustering of nephrin induces its tyrosine phosphorylation, Nck recruitment, and sustained localized actin polymerization. Any one of three phosphorylated (p)YDXV motifs on nephrin is sufficient to recruit Nck through its Src homology 2 (SH2) domain and induce localized actin polymerization at these clusters. Similarly, Nck SH3 mutants in which only the second or third SH3 domain is functional can mediate nephrin-induced actin polymerization. However, combining such nephrin and Nck mutants attenuates actin polymerization at nephrin-Nck clusters. We propose that the multiple Nck SH2-binding motifs on nephrin and the multiple SH3 domains of Nck act cooperatively to recruit the high local concentration of effectors at sites of nephrin activation that is required to initiate and maintain actin polymerization in vivo. We also find that YDXV motifs in the Tir protein of enteropathogenic Escherichia coli and nephrin are functionally interchangeable, indicating that Tir reorganizes the actin cytoskeleton by molecular mimicry of nephrin-like signaling. Together, these data identify pYDXV/Nck signaling as a potent and portable mechanism for physiological and pathological actin regulation. [ABSTRACT FROM AUTHOR]

Modular interaction domains that recognize peptide motifs in target proteins can impart selectivity in signaling pathways. Phosphotyrosine binding (PTB) domains are components of cytoplasmic docking proteins that bind cell surface receptors through NPXY motifs. We have employed a library of human proteome-derived NXXY sequences to explore PTB domain specificity and function. SPOTS peptide arrays were used to create a comprehensive matrix of receptor motifs that were probed with a set of 10 diverse PTB domains. This approach confirmed that individual PTB domains have selective and distinct recognition properties and provided a means to explore over 2,500 potential PTB domain-NXXY interactions. The results correlated well with previously known associations between full-length proteins and predicted novel interactions, as well as consensus binding data for specific PTB domains. Using the Ret, MuSK, and ErbB2 receptor tyrosine kinases, we show that interactions of these receptors with PTB domains predicted to bind by the NXXY arrays do occur in cells. Proteome-based peptide arrays can therefore identify networks of receptor interactions with scaffold proteins that may be physiologically relevant. [ABSTRACT FROM AUTHOR]