Regulation Of Cell Adhesion Research Articles

Comprehensive Analysis of the Expression of TGF-β Signaling Regulators and Prognosis in Human Esophageal Cancer.

More and more evidences show that TGF-β has a crucial role in tumor initiation and development. However, the mechanism of the TGF-β signal regulator in esophageal cancer (EC) is still unclear. Here, we use a variety of bioinformatics methods to analyze the expression and survival data of TGF-β signal regulators in patients with EC. We extracted the expression of the S-TGF-β signal regulator from The Cancer Genome Atlas (TCGA). The cBioPortal database was used to assess the frequency of genetic variation. The TGF-β signal regulator is expressed in EC and normal tissues. The objective is to use the Kaplan-Meier plotter database to investigate the prognostic value of TGF-β signal regulators in cancer patients. The DAVID and clusterProfiler software package were used for functional enrichment analysis. We found that patients with TGF-β signaling mutations have shorter overall survival, disease-free survival, disease-specific survival, platinum overall survival, and platinum-free progression survival. We found that compared with the noncancerous tissues of patients with EC, ZFYVE9, BMPR1B, TGFB3, TGFBRAP1, ACVRL1, TGFBR2, SMAD4, SMAD7, ACVR2A, BMPR1, and SMAD9 were significantly downregulated in tumor tissues, while ACVR1 and Smad1 were significantly upregulated in tumor samples. Univariate survival analysis showed that ACVR1, TGFBR3, TGFBRAP1, BMPR1A, SMAD4, and TGFBR2 were positively correlated with overall survival (OS) prolongation. In addition, TGF-β signal transduction regulators could be divided into two classes. Subclass 1 was involved in regulating cell adhesion, PI3K-Akt signaling, and Rap1 signaling. Subclass 2 was related to regulating angiogenesis and PI3K signaling. In short, all members of TGF-β signal regulators can be used as biomarkers to predict the prognosis of patients with EC.

Microtubules tune mechanosensitive cell responses.

Mechanotransduction is a process by which cells sense the mechanical properties of their surrounding environment and adapt accordingly to perform cellular functions such as adhesion, migration and differentiation. Integrin-mediated focal adhesions are major sites of mechanotransduction and their connection with the actomyosin network is crucial for mechanosensing as well as for the generation and transmission of forces onto the substrate. Despite having emerged as major regulators of cell adhesion and migration, the contribution of microtubules to mechanotransduction still remains elusive. Here, we show that talin- and actomyosin-dependent mechanosensing of substrate rigidity controls microtubule acetylation (a tubulin post-translational modification) by promoting the recruitment of α-tubulin acetyltransferase 1 (αTAT1) to focal adhesions. Microtubule acetylation tunes the mechanosensitivity of focal adhesions and Yes-associated protein (YAP) translocation. Microtubule acetylation, in turn, promotes the release of the guanine nucleotide exchange factor GEF-H1 from microtubules to activate RhoA, actomyosin contractility and traction forces. Our results reveal a fundamental crosstalk between microtubules and actin in mechanotransduction that contributes to mechanosensitive cell adhesion and migration.

Poly (ADP-Ribose) Polymerase 1 Regulates Cajal-Retzius Cell Development and Neural Precursor Cell Adhesion.

Poly (ADP-ribose) polymerase 1 (PARP1) is a ubiquitously expressed enzyme that regulates DNA damage repair, cell death, inflammation, and transcription. PARP1 functions by adding ADP-ribose polymers (PAR) to proteins including itself, using NAD+ as a donor. This post-translational modification known as PARylation results in changes in the activity of PARP1 and its substrate proteins and has been linked to the pathogenesis of various neurological diseases. PARP1 KO mice display schizophrenia-like behaviors, have impaired memory formation, and have defects in neuronal proliferation and survival, while mutations in genes that affect PARylation have been associated with intellectual disability, psychosis, neurodegeneration, and stroke in humans. Yet, the roles of PARP1 in brain development have not been extensively studied. We now find that loss of PARP1 leads to defects in brain development and increased neuronal density at birth. We further demonstrate that PARP1 loss increases the expression levels of genes associated with neuronal migration and adhesion in the E15.5 cerebral cortex, including Reln. This correlates with an increased number of Cajal–Retzius (CR) cells in vivo and in cultures of embryonic neural progenitor cells (NPCs) derived from the PARP1 KO cortex. Furthermore, PARP1 loss leads to increased NPC adhesion to N-cadherin, like that induced by experimental exposure to Reelin. Taken together, these results uncover a novel role for PARP1 in brain development, i.e., regulation of CR cells, neuronal density, and cell adhesion.

Grhl3 promotes retention of epidermal cells under endocytic stress to maintain epidermal architecture in zebrafish.

Epithelia such as epidermis cover large surfaces and are crucial for survival. Maintenance of tissue homeostasis by balancing cell proliferation, cell size, and cell extrusion ensures epidermal integrity. Although the mechanisms of cell extrusion are better understood, how epithelial cells that round up under developmental or perturbed genetic conditions are reintegrated in the epithelium to maintain homeostasis remains unclear. Here, we performed live imaging in zebrafish embryos to show that epidermal cells that round up due to membrane homeostasis defects in the absence of goosepimples/myosinVb (myoVb) function, are reintegrated into the epithelium. Transcriptome analysis and genetic interaction studies suggest that the transcription factor Grainyhead-like 3 (Grhl3) induces the retention of rounded cells by regulating E-cadherin levels. Moreover, Grhl3 facilitates the survival of MyoVb deficient embryos by regulating cell adhesion, cell retention, and epidermal architecture. Our analyses have unraveled a mechanism of retention of rounded cells and its importance in epithelial homeostasis.

Criticality in Cell Adhesion

We illuminate the many-body effects underlying the structure, formation, and dissolution of cellular adhesion domains in the presence and absence of forces. We consider mixed Glauber-Kawasaki dynamics of a two-dimensional model of nearest-neighbor interacting adhesion bonds with intrinsic binding-affinity under the action of a shared pulling or pushing force. We consider adhesion bonds that are immobile due to being anchored to the underlying cytoskeleton as well as adhesion molecules that are transiently diffusing. Highly accurate analytical results are obtained on the pair-correlation level of the Bethe-Guggenheim approximation for the complete thermodynamics and kinetics of adhesion clusters of any size, including the thermodynamic limit. A new kind of dynamical phase transition is uncovered -- the mean formation and dissolution times per adhesion bond change discontinuously with respect to the bond-coupling parameter. At the respective critical points cluster formation and dissolution are fastest, while the statistically dominant transition path undergoes a qualitative change -- the entropic barrier to complete binding/unbinding is rate-limiting below, and the phase transition between dense and dilute phases above the dynamical critical point. In the context of the Ising model the dynamical phase transition reflects a first-order discontinuity in the magnetization-reversal time. Our results provide a potential explanation for the mechanical regulation of cell adhesion, and suggest that the quasi-static and kinetic response to changes in the membrane stiffness or applied forces is largest near the statical and dynamical critical point, respectively.

Guiding cell adhesion and motility by modulating cross-linking and topographic properties of microgel arrays.

Biomaterial-driven modulation of cell adhesion and migration is a challenging aspect of tissue engineering. Here, we investigated the impact of surface-bound microgel arrays with variable geometry and adjustable cross-linking properties on cell adhesion and migration. We show that cell migration is inversely correlated with microgel array spacing, whereas directionality increases as array spacing increases. Focal adhesion dynamics is also modulated by microgel topography resulting in less dynamic focal adhesions on surface-bound microgels. Microgels also modulate the motility and adhesion of Sertoli cells used as a model for cell migration and adhesion. Both focal adhesion dynamics and speed are reduced on microgels. Interestingly, Gas2L1, a component of the cytoskeleton that mediates the interaction between microtubules and microfilaments, is dispensable for the regulation of cell adhesion and migration on microgels. Finally, increasing microgel cross-linking causes a clear reduction of focal adhesion turnover in Sertoli cells. These findings not only show that spacing and rigidity of surface-grafted microgels arrays can be effectively used to modulate cell adhesion and motility of diverse cellular systems, but they also form the basis for future developments in the fields of medicine and tissue engineering.

Siglec-15 promotes progression of clear renal cell carcinoma.

Siglec-15 promotes progression of clear renal cell carcinoma.

243 PROGNOSTIC IMPACT OF CXCR7 AND CXCL12 EXPRESSION IN PATIENTS WITH ESOPHAGEAL ADENOCARCINOMA

Abstract Chemokines are major regulators of cell trafficking and adhesion. The chemokine CXCL12 and its receptors, CXCR4 and CXCR7, have recently been reported as biomarkers in various cancers, including esophageal squamous cell carcinoma; however, there are few studies of these chemokines in esophageal adenocarcinoma (EAC). In this study, we investigated the relationship between expression of CXCL12, CXCR4 and CXCR7, and prognosis in patients with EAC. Methods This study examined 55 patients with EAC who were treated in Toronto General Hospital from 2001 to 2010. Tissue microarray immunohistochemistry was used to evaluate the expression of CXCL12, CXCR4 and CXCR7. Evaluation of immunohistochemistry was performed by a pathologist without knowledge of patients’ information and scored based on a semiquantitative scoring system. The average score from multiple cancer tissues on the microarray was utilized and patients were divided into high or low expression groups using the median score as a cutoff point. These results were compared with the patients’ clinicopathological features and survival. Results The score of CXCR7 was positively correlated with that of CXCL12 (r = 0.3154). High CXCR7 expression was significantly associated with lymphatic invasion (present vs absent, P = 0.005), higher number of lymph node metastases (pN0–1 vs pN2–3, P = 0.0014) and TNM stage (Stage I-II vs III-IV, P = 0.0168). Patients with high CXCR7 (n = 23) expression was associated with worse overall (OS) and disease-free survival (DFS) (P = 0.0221, 0.0090, respectively), and patients with high CXCL12 (n = 24) tended to have worse OS and DFS (P = 0.1091, 0.1477, respectively). High expression of both CXCR7 and CXCL12 was an independent prognostic factor for DFS on multivariable analysis (HR0.3, 95%CI: 0.1–0.8, P = 0.0115). Conclusion High CXCR7 expression was associated with poor prognosis in patients with EAC, and high expression of CXCR7 and its ligand CXCL12, had a stronger association on prognosis. Further study of this potential biomarker using whole tissue samples and larger sample size is warranted.

Lamin A/C Mechanosensor Drives Tumor Cell Aggressiveness and Adhesion on Substrates With Tissue-Specific Elasticity.

Besides its structural properties in the nucleoskeleton, Lamin A/C is a mechanosensor protein involved in perceiving the elasticity of the extracellular matrix. In this study we provide evidence about Lamin A/C-mediated regulation of osteosarcoma cell adhesion and spreading on substrates with tissue-specific elasticities. Our working hypothesis is based on the observation that low-aggressive and bone-resident SaOS-2 osteosarcoma cells express high level of Lamin A/C in comparison to highly metastatic, preferentially to the lung, osteosarcoma 143B cells, thereby suggesting a role for Lamin A/C in tumor cell tropism. Specifically, LMNA gene over-expression in 143B cells induced a reduction in tumor cell aggressiveness in comparison to parental cells, with decreased proliferation rate and reduced migration capability. Furthermore, LMNA reintegration into 143B cells changed the adhesion properties of tumor cells, from a preferential tropism toward the 1.5 kPa PDMS substrate (resembling normal lung parenchyma) to the 28 kPa (resembling pre-mineralized bone osteoid matrix). Our study suggests that Lamin A/C expression could be involved in the organ tropism of tumor cells, thereby providing a rationale for further studies focused on the definition of cancer mechanism of metastatization.

The cell adhesion molecule TMIGD1 binds to moesin and regulates tubulin acetylation and cell migration

BackgroundThe cell adhesion molecule transmembrane and immunoglobulin (Ig) domain containing1 (TMIGD1) is a novel tumor suppressor that plays important roles in regulating cell–cell adhesion, cell proliferation and cell cycle. However, the mechanisms of TMIGD1 signaling are not yet fully elucidated.ResultsTMIGD1 binds to the ERM family proteins moesin and ezrin, and an evolutionarily conserved RRKK motif on the carboxyl terminus of TMIGD1 mediates the interaction of TMIGD1 with the N-terminal ERM domains of moesin and ezrin. TMIGD1 governs the apical localization of moesin and ezrin, as the loss of TMIGD1 in mice altered apical localization of moesin and ezrin in epithelial cells. In cell culture, TMIGD1 inhibited moesin-induced filopodia-like protrusions and cell migration. More importantly, TMIGD1 stimulated the Lysine (K40) acetylation of α-tubulin and promoted mitotic spindle organization and CRISPR/Cas9-mediated knockout of moesin impaired the TMIGD1-mediated acetylation of α-tubulin and filamentous (F)-actin organization.ConclusionsTMIGD1 binds to moesin and ezrin, and regulates their cellular localization. Moesin plays critical roles in TMIGD1-dependent acetylation of α-tubulin, mitotic spindle organization and cell migration. Our findings offer a molecular framework for understanding the complex functional interplay between TMIGD1 and the ERM family proteins in the regulation of cell adhesion and mitotic spindle assembly, and have wide-ranging implications in physiological and pathological processes such as cancer progression.

Carbon Nanofibers versus Silver Nanoparticles: Time-Dependent Cytotoxicity, Proliferation, and Gene Expression.

Carbon nanofibers (CNFs) are one-dimensional nanomaterials with excellent physical and broad-spectrum antimicrobial properties characterized by a low risk of antimicrobial resistance. Silver nanoparticles (AgNPs) are antimicrobial metallic nanomaterials already used in a broad range of industrial applications. In the present study these two nanomaterials were characterized by Raman spectroscopy, transmission electron microscopy, zeta potential, and dynamic light scattering, and their biological properties were compared in terms of cytotoxicity, proliferation, and gene expression in human keratinocyte HaCaT cells. The results showed that both AgNPs and CNFs present similar time-dependent cytotoxicity (EC50 of 608.1 µg/mL for CNFs and 581.9 µg/mL for AgNPs at 24 h) and similar proliferative HaCaT cell activity. However, both nanomaterials showed very different results in the expression of thirteen genes (superoxide dismutase 1 (SOD1), catalase (CAT), matrix metallopeptidase 1 (MMP1), transforming growth factor beta 1 (TGFB1), glutathione peroxidase 1 (GPX1), fibronectin 1 (FN1), hyaluronan synthase 2 (HAS2), laminin subunit beta 1 (LAMB1), lumican (LUM), cadherin 1 CDH1, collagen type IV alpha (COL4A1), fibrillin (FBN), and versican (VCAN)) treated with the lowest non-cytotoxic concentrations in the HaCaT cells after 24 h. The AgNPs were capable of up-regulating only two genes (SOD1 and MMP1) while the CNFs were very effective in up-regulating eight genes (FN1, MMP1, CAT, CDH1, COL4A1, FBN, GPX1, and TGFB1) involved in the defense mechanisms against oxidative stress and maintaining and repairing tissues by regulating cell adhesion, migration, proliferation, differentiation, growth, morphogenesis, and tissue development. These results demonstrate CNF nanomaterials’ unique great potential in biomedical applications such as tissue engineering and wound healing.

Biocompatibility pathways and mechanisms for bioactive materials: The bioactivity zone

This essay analyzes the scientific evidence that forms the basis of bioactive materials, covering the fundamental understanding of bioactivity phenomena and correlation with the mechanisms of biocompatibility of biomaterials. This is a detailed assessment of performance in areas such as bone-induction, cell adhesion, immunomodulation, thrombogenicity and antimicrobial behavior. Bioactivity is the modulation of biological activity by characteristics of the interfacial region that incorporates the material surface and the immediate local host tissue. Although the term ‘bioactive material’ is widely used and has a well understood general meaning, it would be useful now to concentrate on this interfacial region, considered as ‘the bioactivity zone’. Bioactivity phenomena are either due to topographical/micromechanical characteristics, or to biologically active species that are presented in the bioactivity zone. Examples of topographical/micromechanical effects are the modulation of the osteoblast – osteoclast balance, nanotopographical regulation of cell adhesion, and bactericidal nanostructures. Regulation of bioactivity by biologically active species include their influence, especially of metal ions, on signaling pathways in bone formation, the role of cell adhesion molecules and bioactive peptides in cell attachment, macrophage polarization by immunoregulatory molecules and antimicrobial peptides. While much experimental data exists to demonstrate the potential of such phenomena, there are considerable barriers to their effective clinical translation. This essay shows that there is solid scientific evidence of the existence of bioactivity mechanisms that are associated with some types of biomaterials, especially when the material is modified in a manner designed to specifically induce that activity.

Tissue-Specific Regulation of HNK-1 Biosynthesis by Bisecting GlcNAc.

Human natural killer—1 (HNK-1) is a sulfated glyco-epitope regulating cell adhesion and synaptic functions. HNK-1 and its non-sulfated forms, which are specifically expressed in the brain and the kidney, respectively, are distinctly biosynthesized by two homologous glycosyltransferases: GlcAT-P in the brain and GlcAT-S in the kidney. However, it is largely unclear how the activity of these isozymes is regulated in vivo. We recently found that bisecting GlcNAc, a branching sugar in N-glycan, suppresses both GlcAT-P activity and HNK-1 expression in the brain. Here, we observed that the expression of non-sulfated HNK-1 in the kidney is unexpectedly unaltered in mutant mice lacking bisecting GlcNAc. This suggests that the biosynthesis of HNK-1 in the brain and the kidney are differentially regulated by bisecting GlcNAc. Mechanistically, in vitro activity assays demonstrated that bisecting GlcNAc inhibits the activity of GlcAT-P but not that of GlcAT-S. Furthermore, molecular dynamics simulation showed that GlcAT-P binds poorly to bisected N-glycan substrates, whereas GlcAT-S binds similarly to bisected and non-bisected N-glycans. These findings revealed the difference of the highly homologous isozymes for HNK-1 synthesis, highlighting the novel mechanism of the tissue-specific regulation of HNK-1 synthesis by bisecting GlcNAc.

A Pyroptosis-Related Gene Signature for Predicting Survival in Glioblastoma.

BackgroundIn this study, a prognostic model based on pyroptosis-related genes was established to predict overall survival (OS) in patients with glioblastoma (GBM).MethodsThe gene expression data and clinical information of GBM patients were obtained from The Cancer Genome Atlas (TCGA), and bioinformatics analysis of differentially expressed genes was performed. LASSO Cox regression model was used to construct a three-pyroptosis-related gene signature, and validation was performed using an experimental cohort.ResultsA total of three pyroptosis-related genes (CASP4, CASP9, and NOD2) were used to construct a survival prognostic model, and experimental validation was performed using an experimental cohort. Receiver operating characteristic (ROC) analysis was performed, and the area under the ROC curves (AUC) was 0.921, 0.840, and 0.905 at 1, 3, and 5 years, respectively. Functional analysis revealed that T-cell activation, regulation of T-cell activation, leukocyte cell-cell adhesion, and positive regulation of cell adhesion among other immune-related functions were enriched, and immune-related processes were different between the two risk groups.ConclusionIn this study, a novel prognostic model based on three pyroptosis-related genes is constructed and used to predict the prognosis of GBM patients. The model can accurately and conveniently predict the 1-, 3-, and 5-year OS of GBM patients.

PI3K-AKT, JAK2-STAT3 pathways and cell\u2013cell contact regulate maspin subcellular localization

BackgroundMaspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved in maspin nuclear localization and explored a possible role of cell–cell contact in this process.MethodsMCF-10A cells were treated with pharmacological inhibitors against EGFR downstream pathways followed by EGF treatment. Maspin subcellular localization was determined by immunofluorescence. Proteomic and interactome analyses were conducted to identify maspin-binding proteins in EGF-treated cells only. To investigate the role of cell–cell contact these cells were either treated with chelating agents or plated on different cell densities. Maspin and E-cadherin subcellular localization was determined by immunofluorescence.ResultsWe found that PI3K-Akt and JAK2-STAT3, but not MAP kinase pathway, regulate EGF-induced maspin nuclear accumulation in MCF-10A cells. We observed that maspin is predominantly nuclear in sparse cell culture, but it is redistributed to the cytoplasm in confluent cells even in the presence of EGF. Proteomic and interactome results suggest a role of maspin on post-transcriptional and translation regulation, protein folding and cell–cell adhesion.ConclusionsMaspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-Akt and JAK2-STAT3 pathways) and cell–cell contact.DeaMyif71VSQHA76EZpJtKVideo

Identification of miRNA-target gene regulatory networks in liver fibrosis based on bioinformatics analysis.

BackgroundLiver cirrhosis is one of the leading causes of death worldwide. MicroRNAs (miRNAs) can regulate liver fibrosis, but the underlying mechanisms are not fully understood, and the interactions between miRNAs and mRNAs are not clearly elucidated.MethodsmiRNA and mRNA expression arrays of cirrhotic samples and control samples were acquired from the Gene Expression Omnibus database. miRNA-mRNA integrated analysis, functional enrichment analysis and protein-protein interaction (PPI) network construction were performed to identify differentially expressed miRNAs (DEMs) and mRNAs (DEGs), miRNA-mRNA interaction networks, enriched pathways and hub genes. Finally, the results were validated with in vitro cell models.ResultsBy bioinformatics analysis, we identified 13 DEMs between cirrhotic samples and control samples. Among these DEMs, six upregulated (hsa-miR-146b-5p, hsa-miR-150-5p, hsa-miR-224-3p, hsa-miR-3135b, hsa-miR-3195, and hsa-miR-4725-3p) and seven downregulated (hsa-miR-1234-3p, hsa-miR-30b-3p, hsa-miR-3162-3p, hsa-miR-548aj-3p, hsa-miR-548x-3p, hsa-miR-548z, and hsa-miR-890) miRNAs were further validated in activated LX2 cells. miRNA-mRNA interaction networks revealed a total of 361 miRNA-mRNA pairs between 13 miRNAs and 245 corresponding target genes. Moreover, PPI network analysis revealed the top 20 hub genes, including COL1A1, FBN1 and TIMP3, which were involved in extracellular matrix (ECM) organization; CCL5, CXCL9, CXCL12, LCK and CD24, which participated in the immune response; and CDH1, PECAM1, SELL and CAV1, which regulated cell adhesion. Functional enrichment analysis of all DEGs as well as hub genes showed similar results, as ECM-associated pathways, cell surface interaction and adhesion, and immune response were significantly enriched in both analyses.ConclusionsWe identified 13 differentially expressed miRNAs as potential biomarkers of liver cirrhosis. Moreover, we identified 361 regulatory pairs of miRNA-mRNA and 20 hub genes in liver cirrhosis, most of which were involved in collagen and ECM components, immune response, and cell adhesion. These results would provide novel mechanistic insights into the pathogenesis of liver cirrhosis and identify candidate targets for its treatment.

P73 as a Tissue Architect.

The TP73 gene belongs to the p53 family comprised by p53, p63, and p73. In response to physiological and pathological signals these transcription factors regulate multiple molecular pathways which merge in an ensemble of interconnected networks, in which the control of cell proliferation and cell death occupies a prominent position. However, the complex phenotype of the Trp73 deficient mice has revealed that the biological relevance of this gene does not exclusively rely on its growth suppression effects, but it is also intertwined with other fundamental roles governing different aspects of tissue physiology. p73 function is essential for the organization and homeostasis of different complex microenvironments, like the neurogenic niche, which supports the neural progenitor cells and the ependyma, the male and female reproductive organs, the respiratory epithelium or the vascular network. We propose that all these, apparently unrelated, developmental roles, have a common denominator: p73 function as a tissue architect. Tissue architecture is defined by the nature and the integrity of its cellular and extracellular compartments, and it is based on proper adhesive cell-cell and cell-extracellular matrix interactions as well as the establishment of cellular polarity. In this work, we will review the current understanding of p73 role as a neurogenic niche architect through the regulation of cell adhesion, cytoskeleton dynamics and Planar Cell Polarity, and give a general overview of TAp73 as a hub modulator of these functions, whose alteration could impinge in many of the Trp73–/– phenotypes.

AUY922 induces retinal toxicity through attenuating TRPM1

BackgroundOcular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed.MethodsThe impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)‑based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches.ResultsHere, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922.ConclusionsOur study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.

Bcar1/p130Cas is essential for ventricular development and neural crest cell remodelling of the cardiac outflow tract.

AimsThe adapter protein p130Cas, encoded by the Bcar1 gene, is a key regulator of cell movement, adhesion, and cell cycle control in diverse cell types. Bcar1 constitutive knockout mice are embryonic lethal by embryonic days (E) 11.5–12.5, but the role of Bcar1 in embryonic development remains unclear. Here, we investigated the role of Bcar1 specifically in cardiovascular development and defined the cellular and molecular mechanisms disrupted following targeted Bcar1 deletions.Methods and resultsWe crossed Bcar1 floxed mice with Cre transgenic lines allowing for cell-specific knockout either in smooth muscle and early cardiac tissues (SM22-Cre), mature smooth muscle cells (smMHC-Cre), endothelial cells (Tie2-Cre), second heart field cells (Mef2c-Cre), or neural crest cells (NCC) (Pax3-Cre) and characterized these conditional knock outs using a combination of histological and molecular biology techniques. Conditional knockout of Bcar1 in SM22-expressing smooth muscle cells and cardiac tissues (Bcar1SM22KO) was embryonically lethal from E14.5–15.5 due to severe cardiovascular defects, including abnormal ventricular development and failure of outflow tract (OFT) septation leading to a single outflow vessel reminiscent of persistent truncus arteriosus. SM22-restricted loss of Bcar1 was associated with failure of OFT cushion cells to undergo differentiation to septal mesenchymal cells positive for SMC-specific α-actin, and disrupted expression of proteins and transcription factors involved in epithelial-to-mesenchymal transformation (EMT). Furthermore, knockout of Bcar1 specifically in NCC (Bcar1PAX3KO) recapitulated part of the OFT septation and aortic sac defects seen in the Bcar1SM22KO mutants, indicating a cell-specific requirement for Bcar1 in NCC essential for OFT septation. In contrast, conditional knockouts of Bcar1 in differentiated smooth muscle, endothelial cells, and second heart field cells survived to term and were phenotypically normal at birth and postnatally.ConclusionOur work reveals a cell-specific requirement for Bcar1 in NCC, early myogenic and cardiac cells, essential for OFT septation, myocardialization and EMT/cell cycle regulation and differentiation to myogenic lineages.

Mechanosensation and Mechanotransduction in Natural Killer Cells

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where β actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.