N-cadherin Adhesion Research Articles

Plk2 promotes synaptic destabilization through disruption of N-cadherin adhesion complexes during homeostatic adaptation to hyperexcitation.

Synaptogenesis in the brain is highly organized and orchestrated by synaptic cellular adhesion molecules (CAMs) such as N-cadherin and amyloid precursor protein (APP) that contribute to the stabilization and structure of synapses. Although N-cadherin plays an integral role in synapse formation and synaptic plasticity, its function in synapse dismantling is not as well understood. Synapse weakening and loss are prominent features of neurodegenerative diseases, and can also be observed during homeostatic compensation to neuronal hyperexcitation. Previously, we have shown that during homeostatic synaptic plasticity, APP is a target for cleavage triggered by phosphorylation by Polo-like kinase 2 (Plk2). Here, we found that Plk2 directly phosphorylates N-cadherin, and during neuronal hyperexcitation Plk2 promotes N-cadherin proteolytic processing, degradation, and disruption of complexes with APP. We further examined the molecular mechanisms underlying N-cadherin degradation. Loss of N-cadherin adhesive function destabilizes excitatory synapses and promotes their structural dismantling as a prerequisite to eventual synapse elimination. This pathway, which may normally help to homeostatically restrain excitability, could also shed light on the dysregulated synapse loss that occurs in cognitive disorders.

EGFR Suppression Inhibits the Sphere Formation of MCF7 Cells Overexpressing EGFR.

The epidermal growth factor receptor (EGFR) is an oncogenic tyrosine kinase that is involved in tumor initiation and progression, making EGFR inhibitors and monoclonal antibodies to this receptor essential for anti-tumor therapy. We have previously shown that EGFR transgene expression in the human breast adenocarcinoma cell line MCF7 (MCF7-EGFR) stimulates the 3D spheroid-like growth. The primary focus of our present work was to investigate whether EGFR inhibition could affect the assembly of spheroids or lead to the destruction of pre-existing spheroids. We compared the effects of anti-EGFR siRNA, the anti-EGFR monoclonal antibody cetuximab, and the tyrosine kinase inhibitor AG1478 on dissociated and spheroid MCF7-EGFR cells. MCF7-EGFR cells were found to have a 2.5-fold higher sensitivity towards the cytotoxic effects of cetuximab and AG1478 compared with the parental MCF7 cell line. The suppression of EGFR mRNA with siRNA was found to reduce the sphere formation, whereas treating the pre-existing spheroids had no such effect. Treatment of dissociated spheroids with cetuximab and AG1478 was also found to inhibit the MCF7-EGFR sphere formation. We suggest that EGFR expression is important, at least, during the spheroid formation stage. The transition of a MCF7wt adherent cell culture to MCF7-EGFR spheroids was accompanied by a considerable increase in N-cadherin adhesion proteins. The level of N-cadherin decreased when MCF7-EGFR cells were treated with siRNA and cetuximab. Thus, we have demonstrated that N-cadherin is involved in the EGFR-dependent formation of MCF7-EGFR spheroids. Accordingly, MCF7-EGFR spheroids can be considered a suitable model for studying aggressive hormone-positive breast tumors.

N‐cadherin signaling stabilizes occludin tight junction via PI3K p110β signaling

The blood‐brain barrier (BBB) is a specialized microvasculature integral for brain tissue‐fluid homeostasis that is comprised of brain endothelial cells (BECs) and pericytes. Disruption of the BBB and subsequent vascular leakage of protein‐rich fluids is toxic to surrounding neurons and is an underlying risk factor in neurodegenerative disorders. BECs and pericytes form adhesions through the transmembrane protein Neural (N)‐cadherin. Although BEC‐pericyte interactions are critical for maintaining the BBB, the role of N‐cadherin adhesions in regulating the BBB remain unclear. Our previous work demonstrated that mutant mice lacking Cdh2 (N‐cadherin) in ECs or pericytes exhibited a size‐dependent increase in BBB permeability without affecting vessel or pericyte coverage. These findings raise the possibility that N‐cadherin junctions activate outside‐in signaling to strengthen the BBB. Analysis of tight junction (TJ) proteins occludin and claudins 1 and 5 in microvascular BECs of the cortex demonstrated a significant reduction in the accumulation of occludin, but not claudins, at TJs of KO mice. To delineate the signaling mechanism by which N‐cadherin adhesion‐mediated signaling regulates occludin TJs, we utilize biomimetic surfaces (Ncdh‐BioS) bearing covalently linked N‐cadherin extracellular domain to induce N‐cadherin adhesion in BEC monolayers in vitro. Consistent with our observations in mice, assembly of N‐cadherin junctions induced the accumulation of occludin and ZO1 at TJs in BEC monolayers. Depletion of N‐cadherin reversed these events, suggesting that N‐cadherin adhesion‐induced signaling assembles or stabilizes occludin TJs. Analysis of occludin‐Dendra 2 kinetics revealed decreased internalization rates of occludin from TJs in BECs grown on Ncdh‐BioS as compared to collagen, suggesting that N‐cadherin signaling stabilized occludin TJs. Furthermore, formation of N‐cadherin adhesion complexes led to activation of phosphoinositide 3‐kinase (PI3K) signaling as evidenced by the spatial redistribution of Akt to the plasma membrane as well as Akt phosphorylation. Pharmacological inhibition of class I PI3K with Copanlisib or depletion of PI3K p110β, but not of other PI3K catalytic isoforms, abolished Akt activation and increased the rate of occludin internalization from TJs in BECs grown on Ncdh‐BioS. Cumulatively, these data demonstrates that N‐cadherin outside‐in signaling strengthens the BBB by stabilizing occludin TJs through PI3K p110β signaling.

Epithelial-Mesenchymal Transition Proteins in Neuroendocrine Neoplasms: Differential Immunohistochemical Expression in Different Sites and Correlation with Clinico-Pathological Features

The first step leading to metastasis, or for the acquisition of local invasiveness, involves changes in the phenotype of neoplastic cells in the primary tumor. The epithelial–mesenchymal transition (EMT) is a process that determines the acquisition of a form and a transcriptional program that are characteristic of mesenchymal cells, in epithelial cells. The factors involved in this process are E-cadherin and N-cadherin adhesion proteins and some transcription factors such as Slug and Twist. EMT is a site-specific mechanism that is also active in embryogenesis—embryonic cells are affected if invested in certain points, probably due to the signals emanating from the cells or groups of surrounding cells. It is known that neuroendocrine neoplasms have a biological behavior that differs in grading, staging, and site. The aim of our study was to investigate the immunohistochemical expression of EMT factors (Twist, Slug, and E-cadherin) in the neuroendocrine neoplasms of the gastrointestinal tract, the pancreas, and lungs, in 65 cases retrieved from the archives of the Department of Pathology, of three hospitals. The immunoscores were compared in each site and correlated with the clinico-pathological parameters. Statistical evaluation revealed an association between the higher Twist immunoscore and higher grading (p value < 0.0001) and staging (p value = 0.0055). Slug was detected only in pancreatic cases where its reduced expression was associated with a higher grading (p value = 0.0033). This data could be of diagnostic utility in the case of metastases from neuroendocrine neoplasm, to define the site of the primitive tumor when the traditional immunohistochemical panel is not sufficient. In summary, our results indicated, first that the EMT is also an active process in neuroendocrine neoplasms. To the best of our knowledge, this was the first study that evaluated the expression of EMT factors in neuroendocrine neoplasms of different districts.

Enhanced cell-cell contact stability and decreased N-cadherin-mediated migration upon fibroblast growth factor receptor-N-cadherin cross talk.

N-cadherin adhesion has been reported to enhance cancer and neuronal cell migration either by mediating actomyosin-based force transduction or initiating fibroblast growth factor receptor (FGFR)-dependent biochemical signalling. Here we show that FGFR1 reduces N-cadherin-mediated cell migration. Both proteins are co-stabilised at cell-cell contacts through direct interaction. As a consequence, cell adhesion is strengthened, limiting the migration of cells on N-cadherin. Both the inhibition of migration and the stabilisation of cell adhesions require the FGFR activity stimulated by N-cadherin engagement. FGFR1 stabilises N-cadherin at the cell membrane through a pathway involving Src and p120. Moreover, FGFR1 stimulates the anchoring of N-cadherin to actin. We found that the migratory behaviour of cells depends on an optimum balance between FGFR-regulated N-cadherin adhesion and actin dynamics. Based on these findings we propose a positive feed-back loop between N-cadherin and FGFR at adhesion sites limiting N-cadherin-based single-cell migration.

N-cadherin signaling via Trio assembles adherens junctions to restrict endothelial permeability.

Vascular endothelial (VE)-cadherin forms homotypic adherens junctions (AJs) in the endothelium, whereas N-cadherin forms heterotypic adhesion between endothelial cells and surrounding vascular smooth muscle cells and pericytes. Here we addressed the question whether both cadherin adhesion complexes communicate through intracellular signaling and contribute to the integrity of the endothelial barrier. We demonstrated that deletion of N-cadherin (Cdh2) in either endothelial cells or pericytes increases junctional endothelial permeability in lung and brain secondary to reduced accumulation of VE-cadherin at AJs. N-cadherin functions by increasing the rate of VE-cadherin recruitment to AJs and induces the assembly of VE-cadherin junctions. We identified the dual Rac1/RhoA Rho guanine nucleotide exchange factor (GEF) Trio as a critical component of the N-cadherin adhesion complex, which activates both Rac1 and RhoA signaling pathways at AJs. Trio GEF1-mediated Rac1 activation induces the recruitment of VE-cadherin to AJs, whereas Trio GEF2-mediated RhoA activation increases intracellular tension and reinforces Rac1 activation to promote assembly of VE-cadherin junctions and thereby establish the characteristic restrictive endothelial barrier.

Abstract 340: Cardiomyocyte-Derived Mechanical Signals Function in a Paracrine Manner to Regulate Tissue Stiffness and Myocardial Proliferation

Cells sense and transduce mechanical signals through cell-cell adhesions and cell-extracellular matrix adhesions. After birth, contractile force generation and tissue stiffness increase as cardiac output increases to support the needs of the newborn organism. During this postnatal period, the N-cadherin adhesion complex assembles at the intercalated disc (ID) as integrin-fibronectin (FN) adhesions decrease, accompanying the switch from hyperplastic to hypertrophic growth. However, little is known regarding the reciprocity between integrin and N-cadherin adhesions in the regulation of cell cycle withdrawal that occurs in cardiomyocytes after birth. α-catenins function as mechanosensors and transduce the intercellular force from N-cadherin to the actin cytoskeleton. Cardiac-specific αE- and αT-catenins double knockout (DKO) mice exhibit sustained cardiomyocyte proliferation beyond the first week of life. To investigate the role of cell-matrix interactions in DKO hearts, the spatial distribution of integrin-FN complexes was examined at postnatal day (P) 4, P7, P14, and P60. DKO hearts exhibited aberrant N-cadherin localization accompanied by altered distribution of α5/β1 integrin, the primary FN receptor. Normally found at the lateral membrane, α5 and β1 accumulated at the ID while N-cadherin was reduced at the termini. FN matrix assembly, as monitored by immunofluorescent staining and its insolubility in the detergent deoxycholate, was increased in DKO hearts. Meanwhile, focal adhesion kinase (FAK) was activated in early postnatal DKO hearts. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated proliferation was dependent on FAK activity. Finally, treatment of DKO pups with the lysyl oxidase inhibitor, β-aminopropionitrile (BAPN), was sufficient to reduce cardiomyocyte proliferation to wild-type levels suggesting that matrix assembly and tissue stiffness feedback to promote cardiomyocyte proliferation in DKO hearts. This data demonstrates that α-catenins regulate the balance between cell-cell and cell-matrix adhesions, which, in turn, controls cardiomyocyte proliferation, thus providing a molecular explanation for loss of regenerative potential shortly after birth.

N‐Cadherin Juxtacrine Signaling Maintains Blood Brain Barrier and Cognitive Function

The blood brain barrier (BBB) consists of a specialized microvasculature with highly restricted permeability to both low and high molecular weight proteins. The BBB plays a critical role in maintaining tissue‐fluid homeostasis and preventing blood‐to‐brain paracellular diffusion of plasma components that can cause neuronal dysfunction. The BBB is composed of brain endothelial cells (BEC), which physically interact with pericytes at the basement membrane. BECs and pericytes express N‐cadherin, which forms homophilic adhesions to establish cross‐communications between these cell types. The role of N‐cadherin in the BBB is not well understood. We have demonstrated that an endothelial specific deletion of N‐cadherin gene (Cdh2) in mice resulted in increased permeability of the BBB in a size dependent manner, suggesting that N‐cadherin juxtacrine signaling in endothelial cells restricts BBB permeability. These changes in the BBB permeability were not associated with pericyte loss since we observed an increase in capillary coverage by pericytes in the cerebral cortex in these mice. To gain a holistic picture of N‐cadherin juxtacrine signaling in maintenance of the BBB, we utilized N‐cadherin biomimetic surfaces to induce signaling in a cell culture model. We found that Trio, a Rho guanine nucleotide exchange factor consisting of two GEFs, is recruited to N‐cadherin adhesions to induce Rac1 signaling through activation of GEF1. Genetic studies in humans provide a causal link between a deficiency of Trio GEF1 activity and neurodevelopmental diseases such as intellectual disability, suggesting that increased vascular leakage and associated neurotoxicity impairs cognitive function. Therefore, we tested whether N‐cadherin deficient mice exhibited cognitive deficit. Using the Morris water navigation task, we demonstrated that loss of N‐cadherin in endothelial cells results in a deficit in spatial learning and memory. This difference to control mice became indistinguishable with age, suggesting that deficiency of N‐cadherin juxtacrine signaling may be an underlying cause for cognitive dysfunction in the elderly population. Hence, mice lacking Cdh2 gene in endothelial cells may serve as an animal model to study vascular leakage and cognitive function in aging.Support or Funding InformationNIH T32 HL027829, NIH R01 HL103922This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hyperosmolarity induces notochordal cell differentiation with aquaporin3 upregulation and reduced N-cadherin expression.

The nucleus pulposus (NP) of intervertebral discs (IVD) undergoes dramatic changes with aging including loss of its gelatinous structure and large, vacuolated notochordal cells (NCs) in favor of a matrix-rich structure populated by small NP cells (sNPCs). NP maturation also involves a loading-pattern shift from pressurization to matrix deformations, and these events are thought to predispose to degeneration. Little is known of the triggering events and cellular alterations involved with NP maturation, which remains a fundamental open spinal mechanobiology question. A mouse IVD organ culture model was used to test the hypotheses that hyperosmotic overloading will induce NP maturation with transition of NCs to sNPCs while also increasing matrix accumulation and altering osmoregulatory and mechanotransductive proteins. Results indicated that static hyperosmolarity, as might occur during growth, caused maturation of NCs to sNPCs and involved a cellular differentiation process since known NC markers (cytokeratin-8, -19, and sonic hedgehog) persisted without increased cell apoptosis. Osmosensitive channels Aquaporin 3 (Aqp3) and transient receptor potential vanilloid-4 (TRPV4) expression were both modified with altered osmolarity, but increased Aqp3 with hyperosmolarity was associated with NC to sNPC differentiation. NC to sNPC differentiation was accompanied by a shift in cellular mechanotransduction proteins with decreased N-cadherin adhesions and increased Connexin 43 connexons. We conclude that hyperosmotic overloading can promote NC differentiation into sNPCs. This study identified osmolarity as a triggering mechanism for notochordal cell differentiation with associated shifts in osmoregulatory and mechanotransductive proteins that are likely to play important roles in intervertebral disc aging. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:788-798, 2018.

Abstract 80: Interplay Between Cell-cell and Cell-extracellular Matrix Forces Regulate Myocardial Proliferation

Changes in expression and distribution of integrin, fibronectin (FN), and N-cadherin in the postnatal heart accompany the switch from hyperplastic to hypertrophic growth. As FN decreases after birth, N-cadherin/catenin complex accumulates at the cell termini creating a specialized type of cell-cell contact called the intercalated disc (ID). Integrin-FN interactions promote cardiomyocyte and cardiac progenitor cell proliferation. However, little is known regarding the reciprocity between integrin and N-cadherin adhesions in the regulation of myocardial proliferation. Alpha-catenins function as mechanosensors and transduce the intercellular force from N-cadherin to the actin cytoskeleton. To investigate mechanotransduction in the heart, we generated cardiac-specific αE- and αT-catenins double knockout (DKO) mice. The relationship between N-cadherin, integrin, and FN was examined at postnatal day (P) 4, P7, P14, and P60. DKO hearts exhibited aberrant N-cadherin expression accompanied by increased expression of α5/β1 integrin, the primary receptor for FN. Normally found at the lateral membrane, α5 and FN accumulated at the ID along with N-cadherin in DKO hearts. FN-integrin binding leads to the formation of FN fibrils that are initially soluble in the detergent deoxycholate (DOC) but are gradually converted into a stable, DOC-insoluble form that comprises the mature matrix. Both α5 and FN were increased in the DOC-insoluble fraction consistent with enhanced matrix assembly in DKO hearts. Activation of focal adhesion kinase (FAK) and p130CAS were observed in the DKO hearts consistent with increased cell-extracellular matrix interactions. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated Yap nuclear accumulation was dependent on FAK activity. These data demonstrate that α-catenins regulate the balance between cell-cell and cell-matrix adhesions, which, in turn, controls Yap subcellular localization, thus providing a molecular explanation for loss of regenerative potential in the adult heart.

Abstract 429: The Effects of Fluvastatin and Methyl-β-Cyclodextrin-Mediated Cholesterol Depletion on the Biomechanics of Vascular Smooth Muscle Cells in Atherosclerosis

Atherosclerosis is a leading cause of death worldwide. Phenotypic shifting, alteration in cell adhesion, and migration toward inflammatory site of blood vessel wall are all critical contributions of vascular smooth muscle cells (VSMC) to the progression of atherosclerosis. Knowing that cholesterol is a main participant of fatty deposition in atherosclerotic lesions, cholesterol’s role in orchestrating cell migration, mechanics and spreading has grown more apparent. Growing evidences suggests that cholesterol loaded into VSMC in atherosclerosis is much larger than previously known, and about 40% of the total foam cells in the atherosclerotic plaque were VSMC-derived. Emerging studies have increasingly categorized cholesterol as major player in regulating VSMC stiffness and N-Cadherin mediated cell-cell adhesions. Modulating their activity or expression may block VSMC migration during the progression of atherosclerosis. In this study, the effects of a 3-hydroxy-3-methylglutaryl-coenzyme-A reductase inhibitor, fluvastatin and Methyl-β-Cyclodextrin-Mediated (MβCD) cholesterol depletion on VSMCs N-cadherin adhesion and cellular stiffness were addressed. VSMCs cholesterol quantification and lactate dehydrogenase assays demonstrated significant reduction in total cellular cholesterol content by approx. 67% for fluvastatin and 40% for MβCD. The atomic force microscope (AFM) was used to investigate real time adhesion and stiffness of living VSMCs. A proprietary software package written in Matlab was used to estimate Young’s modulus of the cell cortex. Cell adhesion was measured for 50-70% confluent cells with N-Cadherin coated stylus AFM probes on an AFM mounted on an inverted microscope. Our results suggested that fluvastatin and MβCD mediated cholesterol depletion increased N-cadherin mediated cell adhesion force by approx. 27% and 17% respectively. In addition, fluvastatin and MβCD respectively reduced VSMCs stiffness by approx. 24% and 29 % compared to control VSMCs. Altogether, the knowledge that we obtained in this project may lead to a novel therapeutic strategy that could potentially control and block VSMC migration and prevent atherosclerotic plaque formation by deciphering and regulating N-cadherin mediated adhesion

μ2-Dependent endocytosis of N-cadherin is regulated by β-catenin to facilitate neurite outgrowth.

Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N-cadherin, a calcium-dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N-cadherin internalizes through clathrin-mediated endocytosis (CME). Two tyrosine-based motifs in the cytoplasmic domain of N-cadherin recognized by the μ2 subunit of the AP-2 adaptor complex are responsible for CME of N-cadherin. Moreover, β-catenin, a core component of the N-cadherin adhesion complex, inhibits N-cadherin endocytosis by masking the 2 tyrosine-based motifs. Removal of β-catenin facilitates μ2 binding to N-cadherin, thereby increasing clathrin-mediated N-cadherin endocytosis and neurite outgrowth without affecting the steady-state level of surface N-cadherin. These results identify and characterize the mechanism controlling N-cadherin endocytosis through β-catenin-regulated μ2 binding to modulate neurite outgrowth.

N‐Cadherin, a novel and rapidly remodelling site involved in vasoregulation of small cerebral arteries

N-cadherin formed punctate adherens junctions (AJ) along the borders between vascular smooth muscle cells (VSMCs) in the pressurized rat superior cerebellar artery. The formation of N-cadherin AJs in the vessel wall depends on the intraluminal pressure and was responsive to treatment with phenylephrine (PE) (10-5 m) and ACh (10-5 m). N-cadherin-coated beads were able to induce clustering of N-cadherin-enhanced green fluorescent protein (EGFP) on the plasma membrane of isolated VSMCs, whereas treatment with PE (10-5 m) or sodium nitroprusside (10-5 m) induced a significant increase or decrease in the N-cadherin-EGFP clustering, respectively. Application of pulling force (∼1nN) to the N-cadherin-coated beads via an atomic force microscope induced a localized mechanical response from the VSMCs that opposed the pulling. N-cadherin is the major cell-cell adhesion molecule in vascular smooth muscle cells (VSMCs). We tested the hypothesis that N-cadherin is part of a novel mechanosensory mechanism in VSMCs and plays an active role in both the arteriolar myogenic response and during changes in vascular tone induced by vasomotor agonists. Intact and pressurized rat superior cerebellar arteries were labelled for confocal immunofluorescence imaging. N-cadherin formed punctate adherens junctions (AJ) along the borders between VSMCs. When the lumen pressure was raised from 50 to 90mmHg, both the density and the average size of N-cadherin AJs increased significantly. Similarly, arteriolar constriction with phenylephrine (PE) (10-5 m) induced a significant increase of N-cadherin AJ density at 50mmHg, whereas vasodilatation induced by ACh (10-5 m) was accompanied by a significant decrease in density and size of N-cadherin AJs. An atomic force microscope (AFM) was employed to further examine the mechano-responsive properties of N-cadherin adhesion sites in isolated VSMCs. AFM probes with an attached N-cadherin-coated microbead (5μm) induced a progressive clustering of N-cadherin-enhanced green fluorescent protein (EGFP) on the VSMC surface. Application of pulling force (∼1nN) to the N-cadherin-coated-beads with the AFM induced a localized mechanical response from the VSMCs that opposed the pulling. Treatment with PE (10-5 m) or sodium nitroprusside (10-5 m) induced a significant increase or decrease of the N-cadherin-EGFP clustering, respectively. These observations provide compelling evidence that N-cadherin AJs are sensitive to pressure and vasomotor agonists in VSMCs and support a functional role of N-cadherin AJs in vasomotor regulation.

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines.

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin-coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain.

N-cadherin negatively regulates collective Drosophila glial migration through actin cytoskeleton remodeling.

Cell migration is an essential and highly regulated process. During development, glia cells and neurons migrate over long distances - in most cases collectively - to reach their final destination and build the sophisticated architecture of the nervous system, the most complex tissue of the body. Collective migration is highly stereotyped and efficient, defects in the process leading to severe human diseases that include mental retardation. This dynamic process entails extensive cell communication and coordination, hence, the real challenge is to analyze it in the entire organism and at cellular resolution. We here investigate the impact of the N-cadherin adhesion molecule on collective glial migration, by using the Drosophila developing wing and cell-type specific manipulation of gene expression. We show that N-cadherin timely accumulates in glial cells and that its levels affect migration efficiency. N-cadherin works as a molecular brake in a dosage-dependent manner, by negatively controlling actin nucleation and cytoskeleton remodeling through α/β catenins. This is the first in vivo evidence for N-cadherin negatively and cell autonomously controlling collective migration.

Membrane-type 1 matrix metalloproteinase regulates fibronectin assembly and N-cadherin adhesion

Fibronectin matrix formation requires the increased cytoskeletal tension generated by cadherin adhesions, and is suppressed by membrane-type 1 matrix metalloproteinase (MT1-MMP). In a co-culture of Rat1 fibroblasts and MT1-MMP-silenced HT1080 cells, fibronectin fibrils extended from Rat1 to cell–matrix adhesions in HT1080 cells, and N-cadherin adhesions were formed between Rat1 and HT1080 cells. In control HT1080 cells contacting with Rat1 fibroblasts, cell–matrix adhesions were formed in the side away from Rat1 fibroblasts, and fibronectin assembly and N-cadherin adhesions were not formed. The role of N-cadherin adhesions in fibronectin matrix formation was studied using MT1-MMP-silenced HT1080 cells. MT1-MMP knockdown promoted fibronectin matrix assembly and N-cadherin adhesions in HT1080 cells, which was abrogated by double knockdown with either integrin β1 or fibronectin. Conversely, inhibition of N-cadherin adhesions by its knockdown or treatment with its neutralizing antibody suppressed fibronectin matrix formation in MT1-MMP-silenced cells. These results demonstrate that fibronectin assembly initiated by MT1-MMP knockdown results in increase of N-cadherin adhesions, which are prerequisite for further fibronectin matrix formation.

A Slipping Clutch in Neuronal Growth Cones Revealed by Transient Single Molecule Interactions between Flowing Actin and N-Cadherin Adhesions

The molecular clutch model proposes that the actin motile machinery generated inside cells is coupled to adhesion at the cell membrane, thus generating forces on the substrate and allowing cells to move forward. Many reports have provided evidence for clutch-like behaviors in different cell types and adhesive systems, including integrins and cadherins (Giannone et al., Trends Cell Biol 2009). However, the exact mechanisms underlying the dynamic molecular coupling between the actin retrograde flow and adhesion proteins remain elusive. We previously inferred using optical tweezers that a molecular clutch between the actin flow and N-cadherin adhesions could drive growth cone migration (Bard et al., J Neurosci 2008), but did not achieve a direct visualization of the engagement process. Here, to trigger N-cadherin homophilic adhesions at specific locations, we cultured primary neurons on micropatterned substrates comprising arrays of dots coated with purified N-cadherin, surrounded by a cytophobic background. We then tracked the trajectories of single adhesion and cytoskeletal molecules fused with photo-convertible fluorescent proteins (mEOS2), at the ventral surface of growth cones. N-cadherin and α-catenin were immobilized, while the actin retrograde flow was significantly reduced at N-cadherin coated micro-patterns, compared to non-adhesive regions. Normal actin flow on micro-patterns was restored by expression of a dominant negative N-cadherin construct inhibiting the coupling between endogenous N-cadherin and actin, demonstrating specificity of the process. Strikingly, individual actin trajectories exhibited pauses of the order of seconds selectively on N-cadherin-coated micro-patterns. Thus, at the individual molecular level, clutch engagement is characterized by transient interactions between flowing actin filaments and the immobilized N-cadherin/catenin complex. To our knowledge, this study represents the first direct demonstration of the intrinsic molecular coupling underlying the clutch process in growth cone locomotion.

AKT activation by N-cadherin regulates beta-catenin signaling and neuronal differentiation during cortical development

BackgroundDuring cerebral cortical development, neural precursor-precursor interactions in the ventricular zone neurogenic niche coordinate signaling pathways that regulate proliferation and differentiation. Previous studies with shRNA knockdown approaches indicated that N-cadherin adhesion between cortical precursors regulates β-catenin signaling, but the underlying mechanisms remained poorly understood.ResultsHere, with conditional knockout approaches, we find further supporting evidence that N-cadherin maintains β-catenin signaling during cortical development. Using shRNA to N-cadherin and dominant negative N-cadherin overexpression in cell culture, we find that N-cadherin regulates Wnt-stimulated β-catenin signaling in a cell-autonomous fashion. Knockdown or inhibition of N-cadherin with function-blocking antibodies leads to reduced activation of the Wnt co-receptor LRP6. We also find that N-cadherin regulates β-catenin via AKT, as reduction of N-cadherin causes decreased AKT activation and reduced phosphorylation of AKT targets GSK3β and β-catenin. Inhibition of AKT signaling in neural precursors in vivo leads to reduced β-catenin-dependent transcriptional activation, increased migration from the ventricular zone, premature neuronal differentiation, and increased apoptotic cell death.ConclusionsThese results show that N-cadherin regulates β-catenin signaling through both Wnt and AKT, and suggest a previously unrecognized role for AKT in neuronal differentiation and cell survival during cortical development.

Anticancer efficacy of the combination of low-dose cisplatin and trichostatin A or 5-aza-2’-deoxycytidine in ovarian cancer cells.

e15563 Background: We investigated the combination of low dose cisplatin with histone deacetylase inhibitor trichostatin A and methyl transferase inhibitor 5-aza-2’-deoxycytidine to determine if these agents could improve the sensitivity of ovarian cancer cell lines to cisplatin. The goal was to improve efficacy and decrease the toxicity of conventional chemotherapy in human ovarian cancer. Methods: Two chemoresistant ovarian cancer cell lines HEY, SKOV3 and one immortalized cell line IOSE were were treated with increasing doses of each drug alone and in combination to determine optimal cytotoxicity at the lowest dose. Markers of adhesion, metastasis, proliferation, self renewal, and measures of epigenetic regulation were measured. Spheroids, treated with single and combination drugs, were measured for cell viability. SCID mice were injected with pretreated cells and with untreated cells and then treated sequentially. Results: TSA or 5-aza-2’-deoxycytidine combined with Cisplatin had a marked reduction in E-cadherin and N-cadherin (adhesion and migration), proliferation protein Akt and Akt-1, self-renewal markers Oct4/Nanog/Sox2, and the drug transporter ABCG2. DNA methylase, DNMT3a/3b, histone deacetylase HDAC1/2 and histone demethylase LSD1, measures of epigenetic regulation, were suppressed with combined treatment. Histone methylation markers H3K4 and H3K9 were regulated differentially, with the trimethyled-H3K9 significantly suppressed, suggesting bivalent chromatin regulation. Cisplatin/TSA suppressed spheroid formation, growth, and viability. Cisplatin/TSA suppressed tumorigenesis in SCID mice when cells were pretreated. Animals treated after tumor formation with epigenetic modifiers followed by cisplatin achieved significant tumor regression, suggesting that both approaches enhance anticancer effects. Conclusions: Our research suggests the combination of low dose cisplatin with epigenetic modifiers administered together or sequentially may be promising treatments for ovarian cancer with reduced toxicity and may reverse platinum resistance.

RET Modulates Cell Adhesion via Its Cleavage by Caspase in Sympathetic Neurons

RET is a tyrosine kinase receptor involved in numerous cellular mechanisms including proliferation, neuronal navigation, migration, and differentiation upon binding with glial cell derived neurotrophic factor family ligands. RET is an atypical tyrosine kinase receptor containing four cadherin domains in its extracellular part. Furthermore, it has been shown to act as a dependence receptor. Such a receptor is active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. However, different data suggest that RET is not always associated with the cell death/survival balance but rather provides positional information. We demonstrate here that caspase cleavage of RET is involved in the regulation of adhesion in sympathetic neurons. The cleavage of RET generates an N-terminal truncated fragment that functions as a cadherin accessory protein, modifying cadherin environment and potentiating cadherin-mediated cell aggregation. Thus, the caspase cleavage of RET generates two RET fragments: one intracellular domain that can trigger cell death in apoptotic permissive settings, and one membrane-anchored ectodomain with cadherin accessory activity. We propose that this latter function may notably be important for the adequate development of the superior cervical ganglion.