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Development (Cambridge, England) Jun 2024Visualization of protein dynamics is a crucial step in understanding cellular processes. Chromobodies, fluorescently labelled single-domain antibodies, have emerged as...
Visualization of protein dynamics is a crucial step in understanding cellular processes. Chromobodies, fluorescently labelled single-domain antibodies, have emerged as versatile probes for live cell imaging of endogenous proteins. However, how these chromobodies behave in vivo and how accurately they monitor tissue changes remain poorly explored. Here, we generated an endothelial-specific β-catenin chromobody-derived probe and analyzed its expression pattern during cardiovascular development in zebrafish. Using high-resolution confocal imaging, we show that the chromobody signal correlates with the localization of β-catenin in the nucleus and at cell-cell junctions, and thereby can be used to assess endothelial maturation. Loss of Cadherin 5 strongly affects the localization of the chromobody at the cell membrane, confirming the cadherin-based adherens junction role of β-catenin. Furthermore, using a genetic model to block blood flow, we observed that cell junctions are compromised in most endothelial cells but not in the endocardium, highlighting the heterogeneous response of the endothelium to the lack of blood flow. Overall, our data further expand the use of chromobodies for in vivo applications and illustrate their potential to monitor tissue morphogenesis at high resolution.
Topics: Animals; Zebrafish; beta Catenin; Cadherins; Morphogenesis; Zebrafish Proteins; Adherens Junctions; Endothelial Cells; Endothelium, Vascular; Antigens, CD
PubMed: 38847494
DOI: 10.1242/dev.202122 -
Journal of Translational Medicine Jun 2024Inflammation and endothelial barrier dysfunction are the major pathophysiological changes in acute respiratory distress syndrome (ARDS). Sphingosine-1-phosphate receptor...
BACKGROUND
Inflammation and endothelial barrier dysfunction are the major pathophysiological changes in acute respiratory distress syndrome (ARDS). Sphingosine-1-phosphate receptor 3 (S1PR3), a G protein-coupled receptor, has been found to mediate inflammation and endothelial cell (EC) integrity. However, the function of S1PR3 in ARDS has not been fully elucidated.
METHODS
We used a murine lipopolysaccharide (LPS)-induced ARDS model and an LPS- stimulated ECs model to investigate the role of S1PR3 in anti-inflammatory effects and endothelial barrier protection during ARDS.
RESULTS
We found that S1PR3 expression was increased in the lung tissues of mice with LPS-induced ARDS. TY-52156, a selective S1PR3 inhibitor, effectively attenuated LPS-induced inflammation by suppressing the expression of proinflammatory cytokines and restored the endothelial barrier by repairing adherens junctions and reducing vascular leakage. S1PR3 inhibition was achieved by an adeno-associated virus in vivo and a small interfering RNA in vitro. Both the in vivo and in vitro studies demonstrated that pharmacological or genetic inhibition of S1PR3 protected against ARDS by inhibiting the NF-κB pathway and improving mitochondrial oxidative phosphorylation.
CONCLUSIONS
S1PR3 inhibition protects against LPS-induced ARDS via suppression of pulmonary inflammation and promotion of the endothelial barrier by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation, indicating that S1PR3 is a potential therapeutic target for ARDS.
Topics: Animals; Humans; Male; Mice; Cytokines; Endothelial Cells; Inflammation; Lipopolysaccharides; Lung; Mice, Inbred C57BL; Mitochondria; NF-kappa B; Oxidative Phosphorylation; Protective Agents; Receptors, Lysosphingolipid; Respiratory Distress Syndrome; Sphingosine-1-Phosphate Receptors
PubMed: 38840216
DOI: 10.1186/s12967-024-05220-9 -
European Journal of Cell Biology Jun 2024Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and...
Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and transducer by recruiting vinculin and transducing signals that influence cell behaviors. α-catenin/vinculin complex-mediated mechanotransduction regulates multiple pathways, such as Hippo pathway. However, their associations with the α-catenin-based tension sensors at cell junctions are still not fully addressed. Here, we uncovered the TRIP6/LATS1 complex co-localizes with α-catenin/vinculin at both bicellular junctions (BCJs) and tricellular junctions (TCJs). The localization of TRIP6/LATS1 complex to both TCJs and BCJs requires ROCK1 and α-catenin. Treatment by cytochalasin B, Y-27632 and blebbistatin all impaired the BCJ and TCJ junctional localization of TRIP6/LATS1, indicating that the junctional localization of TRIP6/LATS1 is mechanosensitive. The α-catenin/vinculin/TRIP6/LATS1 complex strongly localized to TCJs and exhibited a discontinuous button-like pattern on BCJs. Additionally, we developed and validated an α-catenin/vinculin BiFC-based mechanosensor that co-localizes with TRIP6/LATS1 at BCJs and TCJs. The mechanosensor exhibited a discontinuous distribution and motile signals at BCJs. Overall, our study revealed that TRIP6 and LATS1 are novel compositions of the tension sensor, together with the core complex of α-catenin/vinculin, at both the BCJs and TCJs.
Topics: alpha Catenin; Humans; Protein Serine-Threonine Kinases; Vinculin; Mechanotransduction, Cellular; Adaptor Proteins, Signal Transducing; Intercellular Junctions; HEK293 Cells; rho-Associated Kinases; Transcription Factors
PubMed: 38805800
DOI: 10.1016/j.ejcb.2024.151426 -
Cell Communication and Signaling : CCS May 2024The Crumbs protein (CRB) family plays a crucial role in maintaining the apical-basal polarity and integrity of embryonic epithelia. The family comprises different... (Review)
Review
The Crumbs protein (CRB) family plays a crucial role in maintaining the apical-basal polarity and integrity of embryonic epithelia. The family comprises different isoforms in different animals and possesses diverse structural, localization, and functional characteristics. Mutations in the human CRB1 or CRB2 gene may lead to a broad spectrum of retinal dystrophies. Various CRB-associated experimental models have recently provided mechanistic insights into human CRB-associated retinopathies. The knowledge obtained from these models corroborates the importance of CRB in retinal development and maintenance. Therefore, complete elucidation of these models can provide excellent therapeutic prospects for human CRB-associated retinopathies. In this review, we summarize the current animal models and human-derived models of different CRB family members and describe the main characteristics of their retinal phenotypes.
Topics: Humans; Animals; Membrane Proteins; Retinal Diseases; Retina; Eye Proteins; Disease Models, Animal; Nerve Tissue Proteins; Mutation
PubMed: 38802833
DOI: 10.1186/s12964-024-01673-z -
Scientific Reports May 2024Hypoxia-inducible factors (HIF) 1 and 2 regulate similar but distinct sets of target genes. Although HIFs are best known for their roles in mediating the hypoxia...
Hypoxia-inducible factors (HIF) 1 and 2 regulate similar but distinct sets of target genes. Although HIFs are best known for their roles in mediating the hypoxia response accumulating evidence suggests that under certain conditions HIFs, particularly HIF2, may function also under normoxic conditions. Here we report that HIF2α functions under normoxic conditions in kidney epithelial cells to regulate formation of adherens junctions. HIF2α expression was required to induce Dock4/Rac1/Pak1-signaling mediating stability and compaction of E-cadherin at nascent adherens junctions. Impaired adherens junction formation in HIF2α- or Dock4-deficient cells led to aberrant cyst morphogenesis in 3D kidney epithelial cell cultures. Taken together, we show that HIF2α functions in normoxia to regulate epithelial morphogenesis.
Topics: Adherens Junctions; Signal Transduction; Cell Polarity; Animals; Basic Helix-Loop-Helix Transcription Factors; rac1 GTP-Binding Protein; GTPase-Activating Proteins; Cadherins; Mice; Humans; Epithelial Cells; p21-Activated Kinases; Cell Line
PubMed: 38802496
DOI: 10.1038/s41598-024-62955-7 -
International Journal of Molecular... May 2024Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane...
Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane (MAM) region. Sigma-1R is abundant in the brain and is involved in several physiological processes as well as in various disease states. The role of Sigma-1R at the blood-brain barrier (BBB) is incompletely characterized. In this study, the effect of Sigma-1R activation was investigated in vitro on rat brain microvascular endothelial cells (RBMVEC), an important component of the blood-brain barrier (BBB), and in vivo on BBB permeability in rats. The Sigma-1R agonist PRE-084 produced a dose-dependent increase in mitochondrial calcium, and mitochondrial and cytosolic reactive oxygen species (ROS) in RBMVEC. PRE-084 decreased the electrical resistance of the RBMVEC monolayer, measured with the electric cell-substrate impedance sensing (ECIS) method, indicating barrier disruption. These effects were reduced by pretreatment with Sigma-1R antagonists, BD 1047 and NE 100. In vivo assessment of BBB permeability in rats indicates that PRE-084 produced a dose-dependent increase in brain extravasation of Evans Blue and sodium fluorescein brain; the effect was reduced by the Sigma-1R antagonists. Immunocytochemistry studies indicate that PRE-084 produced a disruption of tight and adherens junctions and actin cytoskeleton. The brain microcirculation was directly visualized in vivo in the prefrontal cortex of awake rats with a miniature integrated fluorescence microscope (aka, miniscope; Doric Lenses Inc.). Miniscope studies indicate that PRE-084 increased sodium fluorescein extravasation in vivo. Taken together, these results indicate that Sigma-1R activation promoted oxidative stress and increased BBB permeability.
Topics: Animals; Receptors, sigma; Blood-Brain Barrier; Sigma-1 Receptor; Rats; Reactive Oxygen Species; Endothelial Cells; Male; Mitochondria; Calcium; Morpholines; Brain; Cells, Cultured
PubMed: 38791182
DOI: 10.3390/ijms25105147 -
PLoS Neglected Tropical Diseases May 2024Cryptosporidium spp. cause watery diarrhea in humans and animals, especially in infants and neonates. They parasitize the apical surface of the epithelial cells in the...
BACKGROUND
Cryptosporidium spp. cause watery diarrhea in humans and animals, especially in infants and neonates. They parasitize the apical surface of the epithelial cells in the intestinal lumen. However, the pathogenesis of Cryptosporidium-induced diarrhea is not fully understood yet.
METHODOLOGY/PRINCIPAL FINDINGS
In this study, we infected C57BL/6j neonatal mice with C. parvum IIa and IId subtypes, and examined oocyst burden, pathological changes, and intestinal epithelial permeability during the infection. In addition, transcriptomic analyses were used to study the mechanism of diarrhea induced by the C. parvum IId subtype. The neonatal mice were sensitive to both C. parvum IIa and IId infection, but the IId subtype caused a wide oocyst shedding window and maintained the high oocyst burden in the mice compared with the IIa subtype. In addition, the mice infected with C. parvum IId resulted in severe intestinal damage at the peak of infection, leading to increased permeability of the epithelial barrier. The KEGG, GO and GSEA analyses revealed that the downregulation of adherens junction and cell junction molecules at 11 dpi. Meanwhile, E-cadherin, which is associated with adherens junction, was reduced at the protein level in mouse ileum at peak and late infection.
CONCLUSIONS/SIGNIFICANCE
C. parvum IId infection causes more severe pathological damage than C. parvum IIa infection in neonatal mice. Furthermore, the impairment of the epithelial barrier during C. parvum IId infection results from the downregulation of intestinal junction proteins.
Topics: Animals; Cryptosporidium parvum; Cryptosporidiosis; Animals, Newborn; Mice; Mice, Inbred C57BL; Intestinal Mucosa; Down-Regulation; Cadherins; Diarrhea; Epithelial Cells; Female; Oocysts; Ileum; Disease Models, Animal
PubMed: 38787872
DOI: 10.1371/journal.pntd.0012212 -
Frontiers in Molecular Neuroscience 2024Functions of the cerebellar cortex, from motor learning to emotion and cognition, depend on the appropriate molecular composition at diverse synapse types. Glutamate...
Functions of the cerebellar cortex, from motor learning to emotion and cognition, depend on the appropriate molecular composition at diverse synapse types. Glutamate receptor distributions have been partially mapped using immunogold electron microscopy. However, information is lacking on the distribution of many other components, such as Shank2, a postsynaptic scaffolding protein whose cerebellar dysfunction is associated with autism spectrum disorders. Here, we used an adapted Magnified Analysis of the Proteome, an expansion microscopy approach, to map multiple glutamate receptors, scaffolding and signaling proteins at single synapse resolution in the cerebellar cortex. Multiple distinct synapse-selective distribution patterns were observed. For example, AMPA receptors were most concentrated at synapses on molecular layer interneurons and at climbing fiber synapses, Shank1 was most concentrated at parallel fiber synapses on Purkinje cells, and Shank2 at both climbing fiber and parallel fiber synapses on Purkinje cells but little on molecular layer interneurons. Our results are consistent with gene expression data but also reveal input-selective targeting within Purkinje cells. In specialized glomerular structures of the granule cell layer, AMPA receptors as well as most other synaptic components preferentially targeted to synapses. However, NMDA receptors and the synaptic GTPase activating protein SynGAP preferentially targeted to extrasynaptic sites. Thus, glomeruli may be considered integrative signaling units through which mossy fibers differentially activate synaptic AMPA and extrasynaptic NMDA receptor complexes. Furthermore, we observed NMDA receptors and SynGAP at adherens junctions, suggesting a role in structural plasticity of glomeruli. Altogether, these data contribute to mapping the cerebellar 'synaptome'.
PubMed: 38783902
DOI: 10.3389/fnmol.2024.1381534 -
International Journal of General... 2024Evidence has indicated that is involved in regulating adherens junction. However, the distinct effect of its aberrant expression on epithelial ovarian cancer (EOC)...
PURPOSE
Evidence has indicated that is involved in regulating adherens junction. However, the distinct effect of its aberrant expression on epithelial ovarian cancer (EOC) awaits clarification.
METHODS
In this study, public databases (Gene Expression Omnibus, The Cancer Genome Atlas, and The Genotype-Tissue Expression), online analysis tools (Kaplan-Meier plotter and TIMER), and data analysis methods (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and the CIBERSORT algorithm) were fully utilized to analyze the differential expression, diagnostic efficiency, prognostic significance, potential function, and correlation with immune infiltration of PDZD11. The differential expression of was tested by immunohistochemistry in EOC tissues (78 cases) and control tissues (37 cases).
RESULTS
Our results indicate that was remarkably overexpressed in EOC, which was associated with advanced cancer stages, no lymphatic metastasis status, and poor prognosis. Moreover, played a role in cell adhesion, cell proliferation, and immune responses. Also, was significantly related to the abundances of infiltrating immune cells in EOC, including neutrophils, macrophages, dendritic cells, CD8+ T cells, and CD4+ T cells, and its expression was positively co-expressed with well-known immune checkpoints, including TIGIT, TIM3, LAG3, CTLA4, and PD-1.
CONCLUSION
These results suggest that could be a potential diagnostic and prognostic biomarker associated with immune infiltration in EOC, and our findings might help elucidate the function of in carcinogenesis.
PubMed: 38766598
DOI: 10.2147/IJGM.S459418 -
Parasite (Paris, France) 2024Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host...
Quantitative phosphoproteomic analysis of chicken DF-1 cells infected with Eimeria tenella, using tandem mass tag (TMT) and parallel reaction monitoring (PRM) mass spectrometry.
Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host cell-E. tenella interactions. However, no comprehensive phosphoproteomic analyses of host cells at various phases of E. tenella infection have been published. In this study, quantitative phosphoproteomic analysis of chicken embryo DF-1 fibroblasts that were uninfected (UI) or infected with E. tenella for 6 h (PI6, the early invasion phase) or 36 h (PI36, the trophozoite development phase) was conducted. A total of 10,122 phosphopeptides matched to 3,398 host cell phosphoproteins were identified and 13,437 phosphorylation sites were identified. Of these, 491, 1,253, and 275 differentially expressed phosphorylated proteins were identified in the PI6/UI, PI36/UI, and PI36/PI6 comparisons, respectively. KEGG pathway enrichment analysis showed that E. tenella modulated host cell processes through phosphorylation, including focal adhesion, regulation of the actin cytoskeleton, and FoxO signaling to support its early invasion phase, and modulating adherens junctions and the ErbB signaling pathway to favor its trophozoite development. These results enrich the data on the interaction between E. tenella and host cells and facilitate a better understanding of the molecular mechanisms underlying host-parasite relationships.
Topics: Animals; Eimeria tenella; Chickens; Proteomics; Phosphoproteins; Tandem Mass Spectrometry; Phosphorylation; Fibroblasts; Cell Line; Poultry Diseases; Host-Parasite Interactions; Coccidiosis; Chick Embryo; Signal Transduction
PubMed: 38759153
DOI: 10.1051/parasite/2024027