Cell Aggregation Assays Research Articles

Gene Deletion Screen for Cardiomyopathy in Adult Drosophila Identifies a New Notch Ligand

Drosophila has been recognized as a model to study human cardiac diseases. Despite these findings, and the wealth of tools that are available to the fly community, forward genetic screens for adult heart phenotypes have been rarely performed because of the difficulty in accurately measuring cardiac function in adult Drosophila. Using optical coherence tomography to obtain real-time analysis of cardiac function in awake Drosophila, we performed a genomic deficiency screen in adult flies. Based on multiple complementary approaches, we identified CG31665 as a novel gene causing dilated cardiomyopathy. CG31665, which we name weary (wry), has structural similarities to members of the Notch family. Using cell aggregation assays and gamma-secretase inhibitors we show that Wry is a novel Notch ligand that can mediate cellular adhesion with Notch expressing cells and transactivates Notch to promote signaling and nuclear transcription. Importantly, Wry lacks a DSL (Delta-Serrate-Lag) domain that is common feature to the other Drosophila Notch ligands. We further show that Notch signaling is critically important for the maintenance of normal heart function of the adult fly. In conclusion, we identify a previously unknown Notch ligand in Drosophila that when deleted causes cardiomyopathy. Our study suggests that Notch signaling components may be a therapeutic target for dilated cardiomyopathy.

Ganoderic acid T inhibits tumor invasion in vitro and in vivo through inhibition of MMP expression

The traditional Chinese medicinal mushroom, Ganoderma lucidum, has been used in Asia for several thousand years for the prevention and treatment of a variety of diseases, including cancer. In previous work, we purified ganoderic acid T (GA-T) from G. lucidum [28]. In the present study, we investigate the functions of GA-T in terms of its effects on invasion in vitro and metastasis in vivo. A trypan blue dye exclusion assay indicates that GA-T inhibits proliferation of HCT-116 cells, a human colon carcinoma cell line. Cell aggregation and adhesion assays show that GA-T promotes homotypic aggregation and simultaneously inhibits the adhesion of HCT-116 cells to the extracellular matrix (ECM) in a dose-dependent manner. Wound healing assays indicate that GA-T also inhibits the migration of HCT-116 cells in a dose-dependent manner, and it suppresses the migration of 95-D cells, a highly metastatic human lung tumor cell line, in a dose- and time-dependent manner. In addition, GA-T inhibits the nuclear translocation of nuclear factor-κB (NF-κB) and the degradation of inhibitor of κB-α (IκBα), which leads to down-regulated expression of matrix metalloproteinase-9 (MMP-9), inducible nitric oxide synthase (iNOS), and urokinase-type plasminogen activator (uPA). Animal and Lewis Lung Carcinoma (LLC) model experiments demonstrate that GA-T suppresses tumor growth and LLC metastasis and down-regulates MMP-2 and MMP-9 mRNA expression in vivo. Taken together, these results demonstrate that GA-T effectively inhibits cancer cell invasion in vitro and metastasis in vivo, and thus it may act as a potential drug for treating cancer.

Rac1 activation inhibits E-cadherin-mediated adherens junctions via binding to IQGAP1 in pancreatic carcinoma cells

BackgroundMonomeric GTPases of the Rho family control a variety of cellular functions including actin cytoskeleton organisation, cell migration and cell adhesion. Defects in these regulatory processes are involved in tumour progression and metastasis. The development of metastatic carcinoma is accompanied by deregulation of adherens junctions, which are composed of E-cadherin/β- and α-catenin complexes.ResultsHere, we show that the activity of the monomeric GTPase Rac1 contributes to inhibition of E-cadherin-mediated cell-cell adhesion in pancreatic carcinoma cells. Stable expression of constitutively active Rac1(V12) reduced the amount of E-cadherin on protein level in PANC-1 pancreatic carcinoma cells, whereas expression of dominant negative Rac1(N17) resulted in an increased amount of E-cadherin. Extraction of proteins associated with the actin cytoskeleton as well as coimmunoprecipitation analyses demonstrated markedly decreased amounts of E-cadherin/catenin complexes in Rac1(V12)-expressing cells, but increased amounts of functional E-cadherin/catenin complexes in cells expressing Rac1(N17). Cell aggregation and migration assays revealed, that cells containing less E-cadherin due to expression of Rac1(V12), exhibited reduced cell-cell adhesion and increased cell motility. The Rac/Cdc42 effector protein IQGAP1 has been implicated in regulating cell-cell adhesion. Coimmunoprecipitation studies showed a decrease in the association between IQGAP1 and β-catenin in Rac1(V12)-expressing PANC-1 cells and an association of IQGAP1 with Rac1(V12). Elevated association of IQGAP1 with the E-cadherin adhesion complex via β-catenin correlated with increased intercellular adhesion of PANC-1 cells.ConclusionThese results indicate that active Rac1 destabilises E-cadherin-mediated cell-cell adhesion in pancreatic carcinoma cells by interacting with IQGAP1 which is associated with a disassembly of E-cadherin-mediated adherens junctions. Inhibition of Rac1 activity induced increased E-cadherin-mediated cellular adhesion.

Drosophila big brain does not act as a water channel, but mediates cell adhesion

The neurogenic gene Drosophila big brain ( bib) has a high sequence homology to aquaporin-4. However, its cellular functions in Drosophila neurogenesis have remained elusive. Here we investigated cell adhesion, and the ion and water permeability of Bib. The adhesive function was examined by a cell aggregation assay using L cells. Bib-transfected L cells formed aggregated clusters, while control-L cells remained as a single cell suspension. Ion permeation was not confirmed in L cells stably expressing Bib. When expressed in COS7 cells, Bib exhibited limited water permeability. This newly found cell adhesive function of Bib may be important for Drosophila neurogenesis.

Overexpression of α-catenin increases osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells

α- and β-Catenin link cadherins to the actin-based cytoskeleton at adherens junctions and regulate cell–cell adhesion. Although roles of cadherins and canonical Wnt-/β-catenin-signaling in osteoblastic differentiation have been extensively studied, the role of α-catenin is not known. Murine embryonic mesenchymal stem cells, C3H10T1/2 cells, were transduced with retrovirus encoding α-catenin (MSCV-α-catenin-HA-GFP). In the presence of Wnt-3A conditioned medium or osteogenic medium (β-glycerol phosphate and ascorbic acid), cells overexpressing α-catenin showed enhanced osteoblastic differentiation as measured by alkaline phosphatase (ALP) staining and ALP activity assay compared to cells transduced with empty virus (MSCV-GFP). In addition, mRNA expression of osteocalcin and Runx2 was significantly increased compared to control. Cell aggregation assay revealed that α-catenin overexpression has significantly increased cell–cell aggregation. However, cellular β-catenin levels (total, cytoplasmic-nuclear ratio) and β-catenin–TCF/LEF transcriptional activity did not change by overexpression of α-catenin. Knock-down of α-catenin using siRNA decreased osteoblastic differentiation as measured by ALP assay. These results suggest that α-catenin overexpression increases osteoblastic differentiation by increasing cell–cell adhesion rather than Wnt-/β-catenin-signaling.

Biophysical Properties of Cadherin Bonds Do Not Predict Cell Sorting

Differential binding between cadherin subtypes is widely believed to mediate cell sorting during embryogenesis. However, a fundamental unanswered question is whether cell sorting is dictated by the biophysical properties of cadherin bonds, or by broader, cadherin-dependent differences in intercellular adhesion or membrane tension. This report describes atomic force microscope measurements of the strengths and dissociation rates of homophilic and heterophilic cadherin (CAD) bonds. Measurements conducted with chicken N-CAD, canine E-CAD, and Xenopus C-CAD demonstrated that all three cadherins cross-react and form multiple, intermolecular bonds. The mechanical and kinetic properties of the heterophilic bonds are similar to the homophilic interactions. The thus quantified bond parameters, together with previously reported adhesion energies were further compared with in vitro cell aggregation and sorting assays, which are thought to mimic in vivo cell sorting. Trends in quantified biophysical properties of the different cadherin bonds do not correlate with sorting outcomes. These results suggest that cell sorting in vivo and in vitro is not governed solely by biophysical differences between cadherin subtypes.

Initiation of testicular tubulogenesis is controlled by neurotrophic tyrosine receptor kinases in a three-dimensional Sertoli cell aggregation assay

The first morphological sign of testicular differentiation is the formation of testis cords. Prior to cord formation, newly specified Sertoli cells establish adhesive junctions, and condensation of somatic cells along the surface epithelium of the genital ridge occurs. Here, we show that Sertoli cell aggregation is necessary for subsequent testis cord formation, and that neurotrophic tyrosine kinase receptors (NTRKs) regulate this process. In a three-dimensional cell culture assay, immature rat Sertoli cells aggregate to form large spherical aggregates (81.36+/-7.34 microm in diameter) in a highly organized, hexagonal arrangement (376.95+/-21.93 microm average distance between spherical aggregates). Exposure to NTRK inhibitors K252a and AG879 significantly disrupted Sertoli cell aggregation in a dose-dependent manner. Sertoli cells were prevented from establishing cell-cell contacts and from forming spherical aggregates. In vitro-derived spherical aggregates were xenografted into immunodeficient nude mice to investigate their developmental potential. In controls, seminiferous tubule-like structures showing polarized single-layered Sertoli cell epithelia, basement membranes, peritubular myoid cells surrounding the tubules, and lumen were observed in histological sections. By contrast, grafts from treatment groups were devoid of tubules and only few single Sertoli cells were present in xenografts after 4 weeks. Furthermore, the grafts were significantly smaller when Sertoli cell aggregation was disrupted by K252a in vitro (20.87 vs 6.63 mg; P<0.05). We conclude from these results that NTRK-regulated Sertoli-Sertoli cell contact is essential to the period of extensive growth and remodeling that occurs during testicular tubulogenesis, and our data indicate its potential function in fetal and prepubertal testis differentiation.

Tumor-Derived Extracellular Mutations of PTPRT/PTPρ Are Defective in Cell Adhesion

Receptor protein tyrosine phosphatase T (PTPRT/PTPrho) is frequently mutated in human cancers including colon, lung, gastric, and skin cancers. More than half of the identified tumor-derived mutations are located in the extracellular part of PTPrho. However, the functional significance of those extracellular domain mutations remains to be defined. Here we report that the extracellular domain of PTPrho mediates homophilic cell-cell aggregation. This homophilic interaction is very specific because PTPrho does not interact with its closest homologue, PTPmu, in a cell aggregation assay. We further showed that all five tumor-derived mutations located in the NH(2)-terminal MAM and immunoglobulin domains impair, to varying extents, their ability to form cell aggregates, indicating that those mutations are loss-of-function mutations. Our results suggest that PTPrho may play an important role in cell-cell adhesion and that mutational inactivation of this phosphatase could promote tumor migration and metastasis.

A novel function of CD82/KAI-1 on E-cadherin-mediated homophilic cellular adhesion of cancer cells

In this study, we analyzed the effect of the metastasis suppressor CD82/KAI-1, a member of the tetraspanin superfamily, on intercellular adhesion on cancer cells. The newly established invasion assay and the cell aggregation assay revealed that CD82 strengthens E-cadherin-mediated intercellular adhesion. Interestingly, ectopic expression of CD82 stabilized E-cadherin/β-catenin complex formation. Furthermore, CD82 reduced tyrosine phosphorylation of β-catenin on HGF stimulation. Taken together, CD82 may stabilize or strengthen E-cadherin-dependent intercellular adhesion by regulating β-catenin-mediated signal transduction on cancer cells, and consequently, prevent cancer cells from seceding from the primary tumor site.

Nectin-like molecule 1 is a glycoprotein with a single N-glycosylation site at N290KS which influences its adhesion activity

Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3, from now on referred to as NECL1, is a neural tissue-specific immunoglobulin-like cell–cell adhesion molecule which has Ca 2+-independent homo- or heterophilic cell–cell adhesion activity and plays an important role in the formation of synapses, axon bundles and myelinated axons. Here we first detected the expression of NECL1 in human fetal and adult brains, and mouse brains at different developmental stages. The results indicated that two bands with molecular weights of about 62 kDa and 48 kDa were found in human fetal brain, while only one band with a molecular weight of about 48 kDa was found in human adult brain; two bands with molecular weights of about 62 kDa and 48 kDa whose expression level gradually increased were also found from mouse E16 to P14, while only one band with a molecular weight of about 48 kDa was found from P14. Bioinformatics analysis showed there were two putative N-glycosylation sites within human NECL1 at positions N25LS and N290KS and within mouse Necl1 at positions N23LS and N288KS, respectively. There was no O-glycosylation site in either human NECL1 or mouse Necl1. Based on the results of N-Glycosidase F treatment with human fetal brain tissue and lysates from transient transfection with human wild-type or glycosylation site mutant NECL1 in 293ET cells, we demonstrated that human NECL1 is an N-linked glycoprotein with a single glycosylation site at position N290KS. Cell aggregation assay further showed there was an increased adhesion activity after the glycosylation site mutation of NECL1 molecule.

COX-2 Inhibitory Activity of Cafestol and Analogs from Coffee Beans

Two kaurane diterpenes, namely cafestol (1) and kahweol (2), were isolated from hydrolyzed fraction of the fixed oil of Ethiopian Coffea arabica unroasted beans, using AgNO3-impregnated silica gel chromatography. In addition, cafestol palmitate (3) and kahweol palmitate (4), the two natural diterpene esters of C. arabica, were also synthesized. Compounds 1–4 were evaluated for anti-inflammatory activity using cyclooxygenase-2 (COX-2), cell aggregation, cell proliferation, cell adhesion, iNOS and antioxidant assays. The COX-2 inhibitory activity of cafestol (1) was found to be 20-fold more potent than kahweol (2) (IC50 value 0.25 μg/mL vs. 5.0 μg/mL), while compounds 3 and 4 were weakly active. The isolation and structure elucidation of the diterpenes 1 and 2, preparation of compounds 3 and 4, and their biological activities are presented in this paper.

Ganoderic Acid Me Inhibits Tumor Invasion Through Down-Regulating Matrix Metalloproteinases 2/9 Gene Expression

The effect of ganoderic acid Me (GA-Me), which was purified from the fermentation mycelia of the traditional Chinese medicinal mushroom Ganoderma lucidum as reported (Tang W, Gu TY, Zhong JJ. Biochem Eng J. 2006;32:205-210), on anti-invasion was investigated. Wound healing assay indicated that GA-Me inhibited cell migration of 95-D, a human highly metastatic lung tumor cell line, in dose- and time-dependent manners. Results of cell aggregation and adhesion assays showed that GA-Me promoted cell homotypic aggregation and inhibited cell adherence to extracellular matrix (ECM). In addition, GA-Me suppressed matrix metalloproteinases 2/9 (MMP2/9) gene expressions at both mRNA and protein levels in 95-D cells according to qRT-PCR and Western blotting, respectively. The results demonstrated that GA-Me effectively inhibited tumor invasion, and it might act as a new MMP2/9 inhibitor for anti-metastasis treatment of carcinoma cells.

Control of Bacterial Aggregation by Thermoresponsive Glycopolymers

Thermoresponsive polymers with pendant glucose functionality form aggregates with bacterial cells that can be assembled/disassembled reversibly through temperature cycling. The polymers are chain extended at temperatures below 40 °C and display glucose moieties to solution, whereas above this temperature the globular state of the polymer “hides” the sugar groups. The specificity and reversibility of the glucose display is demonstrated chemically via a boronic acid−alizarin coupled assay and through cell aggregation assays utilizing bacteria with and without fimH sugar recognition sites.

ADAM-15/Metargidin Mediates Homotypic Aggregation of Human T Lymphocytes and Heterotypic Interactions of T Lymphocytes with Intestinal Epithelial Cells

Intestinal epithelial cells (IEC) play an immunoregulatory role in the intestine. This role involves cell-cell interactions with intraepithelial lymphocytes that may also play a role in some enteropathies. The discovery of the RGD motif-containing Protein ADAM-15 (a disintegrin and metalloprotease-15) raises the question of its involvement in these cell-cell interactions. Cell adhesion assays were performed using the Jurkat E6.1 T cell line as a model of T lymphocytes and Caco2-BBE monolayers as a model of intestinal epithelia. Our results show that an anti-ADAM-15 ectodomain antibody inhibited the attachment of Jurkat cells on Caco2-BBE monolayers. Overexpression of ADAM-15 in Caco2-BBE cells enhanced Jurkat cell binding, and overexpression of ADAM-15 in Jurkat cells enhanced their aggregation. Mutagenesis experiments showed that both the mutation of ADAM-15 RGD domain or the deletion of its cytoplasmic tail decreased these cell-cell interactions. Moreover, wound-healing experiments showed that epithelial ADAM-15-mediated Jurkat cell adhesion to Caco2-BBE cells enhances the mechanisms of wound repair. We also found that ADAM-15-mediated aggregation of Jurkat cells increases the expression of tumor necrosis factor-alpha mRNA. These results demonstrate the following: 1) ADAM-15 is involved in heterotypic adhesion of intraepithelial lymphocytes to IEC as well as in homotypic aggregation of T cells; 2) both the RGD motif and the cytoplasmic tail of ADAM-15 are involved for these cell-cell interactions; and 3) ADAM-15-mediated cell-cell interactions are involved in mechanisms of epithelial restitution and production of pro-inflammatory mediators. Altogether these findings point to ADAM-15 as a possible therapeutic target for prevention of inappropriate T cell activation involved in some pathologies.

Characterization of the P373L E-cadherin germline missense mutation and implication for clinical management

Aim Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome caused by E-cadherin germline mutations. One-third of these mutations are of the missense type, representing a burden in genetic counselling. A new germline missense mutation (P373L) was recently identified in a HDGC Italian family. The present work aimed at addressing the disease-causative nature of the P373L mutant. Methods Assessment of the P373L mutation effect was based on cell aggregation and invasion assays. LOH analysis at the E-cadherin locus, search for somatic E-cadherin mutations and for promoter hypermethylation were performed to identify the mechanism of inactivation of the E-cadherin wild-type allele in the tumour. Results In vitro the P373L germline mutation impaired the E-cadherin functions. E-cadherin promoter hypermethylation was observed in the tumour of the P373L mutation carrier. Conclusion We conclude that the combination of clinical, in vitro and molecular genetic data is helpful for establishing an accurate analysis of HDGC-associated CDH1 germline missense mutations and subsequently for appropriate clinical management of asymptomatic mutation carriers.

Cadherin Conformations Associated with Dimerization and Adhesion

To investigate conformations of C-cadherin associated with functional activity and physiological regulation, we generated monoclonal antibodies (mAbs) that bind differentially to monomeric or dimeric forms. These mAbs recognize conformational epitopes at multiple sites along the C-cadherin ectodomain aside from the well known Trp-2-mediated dimer interface in the N-terminal EC1 domain. Group 1 mAbs, which bind monomer better than dimer and the Trp-2-mutated protein (W2A) better than wild type, recognize epitopes in EC4 or EC5. Dimerization of the W2A mutant protein via a C-terminal immunoglobulin Fc domain restored the dimeric mAb-binding properties to EC4-5 and partial homophilic binding activity but did not restore full cell adhesion activity. Group 2 and Group 3 mAbs, which bind dimer better than monomer and wild type better than W2A, recognize epitopes in EC1 and the interface between EC1 and EC2, respectively. None of the mAbs could distinguish between different physiological states of C-cadherin at the cell surface of either Xenopus embryonic cells or Colo 205 cultured cells, demonstrating that changes in dimerization do not underlie regulation of adhesion activity. On the cell surface the EC3-EC5 domains are much less accessible to mAb binding than EC1-EC2, suggesting that they are masked by the state of cadherin organization or by other molecules. Thus, the EC2-EC5 domains either reflect, or are involved in, cadherin dimerization and organization at the cell surface.

The Variable Transmembrane Domain of Drosophila N-Cadherin Regulates Adhesive Activity

Drosophila N-cadherin (CadN) is an evolutionarily conserved classic cadherin which has a large, complex extracellular domain and a catenin-binding cytoplasmic domain. The CadN locus contains three modules of alternative exons (7a/b, 13a/b, and 18a/b) and undergoes alternative splicing to generate multiple isoforms. Using quantitative transcript analyses and green fluorescent protein-based cell sorting, we found that during development CadN alternative splicing is regulated in a temporal but not cell-type-specific fashion. In particular, exon 18b is predominantly expressed during early developmental stages, while exon 18a is prevalent at the late developmental and adult stages. All CadN isoforms share the same molecular architecture but have different sequences in their extracellular and transmembrane domains, suggesting functional diversity. In vitro quantitative cell aggregation assays revealed that all CadN isoforms mediate homophilic interactions, but the isoforms encoded by exon 18b have a higher adhesive activity than those by its alternative, 18a. Domain-swapping experiments further revealed that the different sequences in the transmembrane domains of isoforms are responsible for their differential adhesive activities. CadN alternative splicing might provide a novel mechanism to fine-tune its adhesive activity at different developmental stages or to restrict the use of high-affinity 18b-type isoforms at the adult stage.

Regulated ADAM10-dependent Ectodomain Shedding of γ-Protocadherin C3 Modulates Cell-Cell Adhesion

Gamma-protocadherins (Pcdh gamma) are type I transmembrane proteins, which are most notably expressed in the nervous system. They are enriched at synapses and involved in synapse formation, specification, and maintenance. In this study, we show that Pcdh gamma C3 and Pcdh gamma B4 are specifically cleaved within their ectodomains by the disintegrin and metalloprotease ADAM10. Analysis of ADAM10-deficient fibroblasts and embryos, inhibitor studies, as well as RNA interference-mediated down-regulation demonstrated that ADAM10 is not only responsible for the constitutive but also for the regulated shedding of these proteins in fibroblasts and in neuronal cells. In contrast to N-cadherin shedding, which was activated by N-methyl-D-aspartic acid receptor activation in neuronal cells, Pcdh gamma shedding was induced by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate stimulation, suggesting differential regulation mechanisms of cadherin-mediated functions at synapses. Cell aggregation assays in the presence or absence of metalloprotease inhibitors strongly suggest that the ectodomain shedding events modulate the cell adhesion role of Pcdh gamma. The identification of ADAM10 as the protease responsible for constitutive and regulated Pcdh gamma shedding may therefore provide new insight into the regulation of Pcdh gamma functions.

ZO-1 and ZO-2 Independently Determine Where Claudins Are Polymerized in Tight-Junction Strand Formation

A fundamental question in cell and developmental biology is how epithelial cells construct the diffusion barrier allowing them to separate different body compartments. Formation of tight junction (TJ) strands, which are crucial for this barrier, involves the polymerization of claudins, TJ adhesion molecules, in temporal and spatial manners. ZO-1 and ZO-2 are major PDZ-domain-containing TJ proteins and bind directly to claudins, yet their functional roles are poorly understood. We established cultured epithelial cells (1(ko)/2(kd)) in which the expression of ZO-1/ZO-2 was suppressed by homologous recombination and RNA interference, respectively. These cells were well polarized, except for a complete lack of TJs. When exogenously expressed in 1(ko)/2(kd) cells, ZO-1 and ZO-2 were recruited to junctional areas where claudins were polymerized, but truncated ZO-1 (NZO-1) containing only domains PDZ1-3 was not. When NZO-1 was forcibly recruited to lateral membranes and dimerized, claudins were dramatically polymerized. These findings indicate that ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized.

Establishment of four pairs of canine mammary tumour cell lines derived from primary and metastatic origin and their E‐cadherin expression

Four new pairs of canine mammary carcinoma cell lines derived from both primary and metastatic lesions were established. The cells were cultured in RPMI-1640 with 10% fetal bovine serum and they showed stable growth for more than 120 passages. Using these cell lines, the expression of E-cadherin was measured by flow cytometry and the function of E-cadherin was evaluated by cell aggregation assay and results from the primary and metastatic lesions were compared statistically. E-cadherin was strongly expressed in all of the cell lines, without a notable difference between cells of primary and metastatic origin. In the cell aggregation assay, the function of E-cadherin was significantly weaker in the cells of primary origin (p < 0.05), as compared with cells of metastatic origin. The present results suggest that a reduction in E-cadherin function may be implicated in the invasive and metastatic potential of canine mammary tumour cells; however, further study will be needed to clarify E-cadherin function in the context of the metastasis of canine mammary carcinoma.