41 research outputs found
Protein Tyrosine Phosphatase PTP1B is Involved in Hippocampal Synapse Formation and Learning
ER-bound PTP1B is expressed in hippocampal neurons, and accumulates among neurite contacts. PTP1B dephosphorylates ß-catenin in N-cadherin complexes ensuring cell-cell adhesion. Here we show that endogenous PTP1B, as well as expressed GFP-PTP1B, are present in dendritic spines of hippocampal neurons in culture. GFP-PTP1B overexpression does not affect filopodial density or length. In contrast, impairment of PTP1B function or genetic PTP1B-deficiency leads to increased filopodia-like dendritic spines and a reduction in mushroom-like spines, while spine density is unaffected. These morphological alterations are accompanied by a disorganization of pre- and post-synapses, as judged by decreased clustering of synapsin-1 and PSD-95, and suggest a dynamic synaptic phenotype. Notably, levels of ß-catenin-Tyr-654 phosphorylation increased ∼5-fold in the hippocampus of adult PTP1B−/− (KO) mice compared to wild type (WT) mice and this was accompanied by a reduction in the amount of ß-catenin associated with N-cadherin. To determine whether PTP1B-deficiency alters learning and memory, we generated mice lacking PTP1B in the hippocampus and cortex (PTP1Bfl/fl–Emx1-Cre). PTP1Bfl/fl–Emx1-Cre mice displayed improved performance in the Barnes maze (decreased time to find and enter target hole), utilized a more efficient strategy (cued), and had better recall compared to WT controls. Our results implicate PTP1B in structural plasticity within the hippocampus, likely through modulation of N-cadherin function by ensuring dephosphorylation of ß-catenin on Tyr-654. Disruption of hippocampal PTP1B function or expression leads to elongation of dendritic filopodia and improved learning and memory, demonstrating an exciting novel role for this phosphatase
ER-Bound Protein Tyrosine Phosphatase PTP1B Interacts with Src at the Plasma Membrane/Substrate Interface
PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research
Development and validation of a population-based prediction scale for osteoporotic fracture in the region of Valencia, Spain: the ESOSVAL-R study
<p>Abstract</p> <p>Background</p> <p>Today, while there are effective drugs that reduce the risk of osteoporotic fracture, yet there are no broadly accepted criteria that can be used to estimate risks and decide who should receive treatment. One of the actual priorities of clinical research is to develop a set of simple and readily-available clinical data that can be used in routine clinical practice to identify patients at high risk of bone fracture, and to establish thresholds for therapeutic interventions. Such a tool would have high impact on healthcare policies. The main objective of the ESOSVAL-R is to develop a risk prediction scale of osteoporotic fracture in adult population using data from the Region of Valencia, Spain.</p> <p>Methods/Design</p> <p><it>Study design</it>: An observational, longitudinal, prospective cohort study, undertaken in the Region of Valencia, with an initial follow-up period of five years; <it>Subjects</it>: 14,500 men and women over the age of 50, residing in the Region and receiving healthcare from centers where the ABUCASIS electronic clinical records system is implanted; <it>Sources of data</it>: The ABUCASIS electronic clinical record system, complemented with hospital morbidity registers, hospital Accidents & Emergency records and the Regional Ministry of Health's mortality register; <it>Measurement of results</it>: Incident osteoporotic fracture (in the hip and/or major osteoporotic fracture) during the study's follow-up period. Independent variables include clinical data and complementary examinations; <it>Analysis</it>: 1) Descriptive analysis of the cohorts' baseline data; 2) Upon completion of the follow-up period, analysis of the strength of association between the risk factors and the incidence of osteoporotic fracture using Cox's proportional hazards model; 3) Development and validation of a model to predict risk of osteoporotic fracture; the validated model will serve to develop a simplified scale that can be used during routine clinical visits.</p> <p>Discussion</p> <p>The ESOSVAL-R study will establish a prediction scale for osteoporotic fracture in Spanish adult population. This scale not only will constitute a useful prognostic tool, but also it will allow identifying intervention thresholds to support treatment decision-making in the Valencia setting, based mainly on the information registered in the electronic clinical records.</p
Moving beyond neurons: the role of cell type-specific gene regulation in Parkinson's disease heritability
Parkinson’s disease (PD), with its characteristic loss of nigrostriatal dopaminergic neurons and deposition of α-synuclein in neurons, is often considered a neuronal disorder. However, in recent years substantial evidence has emerged to implicate glial cell types, such as astrocytes and microglia. In this study, we used stratified LD score regression and expression-weighted cell-type enrichment together with several brain-related and cell-type-specific genomic annotations to connect human genomic PD findings to specific brain cell types. We found that PD heritability attributable to common variation does not enrich in global and regional brain annotations or brain-related cell-type-specific annotations. Likewise, we found no enrichment of PD susceptibility genes in brain-related cell types. In contrast, we demonstrated a significant enrichment of PD heritability in a curated lysosomal gene set highly expressed in astrocytic, microglial, and oligodendrocyte subtypes, and in LoF-intolerant genes, which were found highly expressed in almost all tested cellular subtypes. Our results suggest that PD risk loci do not lie in specific cell types or individual brain regions, but rather in global cellular processes detectable across several cell types
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PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility
ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration
P120-Catenin Regulates Early Trafficking Stages of the N-Cadherin Precursor Complex.
It is well established that binding of p120 catenin to the cytoplasmic domain of surface cadherin prevents cadherin endocytosis and degradation, contributing to cell-cell adhesion. In the present work we show that p120 catenin bound to the N-cadherin precursor, contributes to its anterograde movement from the endoplasmic reticulum (ER) to the Golgi complex. In HeLa cells, depletion of p120 expression, or blocking its binding to N-cadherin, increased the accumulation of the precursor in the ER, while it decreased the localization of mature N-cadherin at intercellular junctions. Reconstitution experiments in p120-deficient SW48 cells with all three major isoforms of p120 (1, 3 and 4) had similar capacity to promote the processing of the N-cadherin precursor to the mature form, and its localization at cell-cell junctions. P120 catenin and protein tyrosine phosphatase PTP1B facilitated the recruitment of the N-ethylmaleimide sensitive factor (NSF), an ATPase involved in vesicular trafficking, to the N-cadherin precursor complex. Dominant negative NSF E329Q impaired N-cadherin trafficking, maturation and localization at cell-cell junctions. Our results uncover a new role for p120 catenin bound to the N-cadherin precursor ensuring its trafficking through the biosynthetic pathway towards the cell surface
Optical fiber sensors based on Layer-by-Layer nanostructured films
AbstractDifferent optical fiber sensors based on Layer-by-Layer nanostructured coatings are shown. Due to the precise control on the nanometer scale that this technique provides it is possible to optimize the response of the optical fiber sensors and also the fabrication of sensors based on sensing phenomena which are possible to observe only with nanocoatings. Sensors based on nanoFabry-Perots, microgratings, tapered ends, biconically tapered fibers, long period gratings or photonic crystal fiber have made possible the monitoring of temperature, humidity, pH, gases, volatile organic compounds, H2O2, copper or glucose. The possibility of incorporating proteins, enzymes or antibodies makes this technique especially useful for the fabrication of biosensors for biological recognition
Expression and distribution of BiFC constructs.
<p>(A) Diagram of fusion proteins. YN (residues 1–154) and YC (residues 155–238) fragments of enhanced YFP were fused to the N-terminus of wild type (WT) and substrate trap mutant D181A (DA) PTP1B. The same fragments were fused at the C-terminus of mouse Src and Fyn. The amino acids of the linker region are indicated in italics. (B) Constructs were transiently expressed in CHO-K1 cells and probed in Western blots with anti-PTP1B (left panel), anti-Src (middle panel), and anti-Fyn (right panel) antibodies. YC and YN fragments of YFP add ∼10 and 18 kDa, respectively, to the partner fused proteins (PTP1B, Src and Fyn). Arrowheads indicate the migration of the endogenous proteins. Anti-PTP1B does not recognize the endogenous CHO-K1 protein; thus, a cell extract from PTP1B knockout cells reconstituted with human PTP1B was probed with this antibody and shown in the lane marked as PTP1BWT. Subcellular distribution of constructs used for BiFC was assessed by fluorescence microscopy. CHO-K1 cells expressing YC-PTP1BWT (C, C′, D), YC-PTP1BDA (E, E′, F), and SYF cells expressing Fyn-YN (G, H), and Src-YN (I, J) were immunolabeled with anti-PTP1B (C, Ć, E, É), anti-Fyn (G) and anti-Src (I) followed by secondary antibodies conjugated with Alexa Fluor 568 nm. Images on the red channel show that YC-PTP1BWT (C, Ć) and YC-PTP1BDA (E, É) localize in the ER, as expected (Ćand É are magnifications of regions within boxes in C and E, respectively). In addition, YCPTP1BDA accumulates in small puncta (arrows in É). Fyn-YN (G) and Src-YN (I) are enriched at the cell margin and in a perinuclear compartment. All constructs display background fluorescence at the green channel in which BiFC is analyzed (D, F, H, J). (K-N) Starved SYF cells expressing Src-YN were plated for 30 min in the absence (K, L) and in the presence (M, N) of serum. Note that Src-YN localizes in a perinuclear compartment in the absence of serum (L) and redistributes to peripheral, radial focal adhesions in the presence of serum (N), as expected. (K, M) Surface reflectance interference contrast images showing the membrane in contact with the substrate. Dashed lines indicate the perimeter of cells. Scale bar, 40 µm.</p
Schematic view of BiFC results.
<p>ER membranes (green) are positioned close to the plasma membrane (brown) by microtubules (black). PTP1B anchored to the cytosolic surface of the ER membrane (green pins) can interact with Src (red pins) associated to the cytosolic side of the plasma membrane. The substrate trap mutant PTP1BDA enhances the interaction leading to the visualization of large puncta (inset, and green beads on the ER in the upper/lower view). TIRF microscopy reveals BiFC puncta at the ventral membrane and SRIC shows that some of BiFC puncta occur in close contact with the substrate (SRIC dark spots).</p