Sparse Cultures Research Articles

Abstract 327: NADPH Oxidase-dependent Signaling Involved In Endothelial Cell Survival And Proliferation: Role Of Nox2 And Nox4

Objective: Endothelial cells (EC) produce a variety of reactive oxygen species (ROS) which act as mediators of signaling for proliferation, migration, differentiation and cell death. The major source of ROS in EC at rest is the NADPH oxidase enzyme complex. We aimed to determine the roles of the Nox2 and Nox4 catalytic subunits of NADPH oxidase in proliferation and survival of human microvascular endothelial cells (HMEC-1). Methods: Intracellular superoxide was measured by dihydroethidium fluorescence and lucigenin chemiluminescence, and total ROS by dichlorodihydrofluorescein-diacetate assay. Nox4 and Nox2 protein expression was measured by Western blot, and cell proliferation was quantitated by trypan blue exclusion and a tetrazolium-based MTS assay. EC survival was measured by caspase3/7 activity. Results: The expression of Nox4 (but not Nox2) protein, superoxide and total ROS, were all significantly higher (P < 0.05, N = 4) under proliferative conditions (sparse culture or serum addition) than in quiescent (dense culture or serum-starved) cells. Suppression of NADPH oxidase-derived ROS with specific siRNA significantly reduced cell proliferation as shown below. Under proliferative conditions phosphorylation of ERK and Akt were increased. Suppression of Nox4-derived ROS production reduced the phosphorylation of both ERK and Akt, whereas suppression of Nox2 reduced only Akt phosphorylation. Further, suppression of Nox2, but not Nox4, significantly activated caspase3/7 (P < 0.05, N = 4). Thus Nox4-derived ROS act to promote proliferation of EC via ERK and Akt signaling, whereas Nox2 prevents apoptotic signaling via Akt and caspase3/7. Conclusion: Nox isoform-derived ROS act via distinct signaling pathways to promote EC proliferation and survival. Selective modulation of Nox-type NADPH oxidase could be a useful approach for blocking angiogenesis or for therapeutic angiogenesis in chronic ischemia or tissue engineering.

Cellular processes underlying maturation of P19 neurons: Changes in protein folding regimen and cytoskeleton organization

Embryonal carcinoma P19 cells provide an ideal model to study molecular programs along differentiation. Upon induction by retinoic acid (RA), the cells undergo a program of differentiation that generates functioning neurons within 60 h. RA induced cells that were plated as sparse (1000 cells/mm(2)) or dense (4000 cells/mm(2)) cultures showed a marked difference in the culture morphology with the dense cultures exhibiting rapid maturation and accelerated neurite outgrowth. The protein expression levels of the sparse and dense cultures were compared 48 h following RA. Cell extracts were separated by 1-DE and 2-DE and differential expression (>four-fold) proteins were identified by MS. Here, we focus on 20 proteins associated with cytoskeletal regulation and stress-dependent protein refolding. The first group includes drebrin, cofilin, alpha-internexin, vimentin, and nestin. Among the proteins in the second group are subunits of the TCP-1, and several chaperones of the Hsp70 and Hsp90 families. We show that coordinated remodeling of the cytoskeleton and modulations in chaperone activity underlie the change in neurite extension rate. Furthermore, a proteomics-based analysis applied on P19 neurons demonstrated pathways underlying neuronal outgrowth, suggesting that a malfunction of such pathways leads to neuropathological conditions.

Involvement of protein kinases in self‐organization of the rhythm of protein synthesis by direct cell—cell communication

Primary cultures of rat hepatocytes grown on slides were studied in serum-free medium. Ultradian protein synthesis rhythm was used as a marker of synchronization of individual oscillations, resulting in the formation of a common rhythm of the cell population, i.e. cell-cell self-organization. Dense synchronous and sparse non-synchronous cultures were used to estimate effect of protein kinase activity on the kinetics of protein synthesis. Treatment of dense cultures with the inhibitors H7 (40 microM) or H8 (25 microM) resulted in a loss of the protein synthesis rhythm, a suppression of the cell-cell self-organization. Stimulation of protein kinase activity with either 0.5 or 1.0 microM phorbol 12-miristate-13-acetate (PMA) or 10 microM forskolin caused the appearance of the synthetic rhythm in non-synchronous sparse cultures under otherwise normal conditions. Inhibition of protein kinase activity with H7 resulted in signal factors, such as gangliosides and phenylephrine, failing to initiate this rhythm in sparse cultures. Activation of protein kinase activity with PMA shifted the phase pattern of the protein synthesis rhythm. Thus, according to our previous and the new data, protein kinase activity and consequently protein phosphorylation is the crucial step of sequence of processes resulting in synchronization during self-organization of cells in producing a common rhythm in the population. The general pathway can be presented as follows: signaling of gangliosides or other calcium agonists-->efflux of calcium ion from intracellular stores, with elevation of calcium concentration in the cytoplasm-->activation of protein kinases-->protein phosphorylation-->synchronization of individual oscillations in protein synthesis rates-->induction of a common rhythm throughout this population. The data have been discussed concerning similarity of the direct cell-cell communication and the cell self-organization in cultures and in organism.

Confluence-dependent resistance to doxorubicin in human MDA-MB-231 breast carcinoma cells requires hypoxia-inducible factor-1 activity

Compared with tumour cells cultured as sparse monolayers, tumour cells cultured as confluent monolayers exhibit high levels of resistance to anti-cancer agents. This phenomenon is called confluence-dependent resistance (CDR). We determined the contribution of hypoxia-inducible factor-1 (HIF-1), a key transcriptional regulator of cellular adaptations to hypoxia, to the development of CDR in MDA-MB-231 breast cancer cells. Clonogenic assays revealed a density-dependent increase in resistance to doxorubicin. Cell density also correlated with increased staining for reductively activated pimonidazole (a marker for hypoxia), as well as with increased levels of the HIF-1α subunit and HIF transcriptional activity as determined by immunocytochemistry, Western blot, and luciferase reporter assays. Importantly, inhibition of HIF-1α expression with siRNA significantly attenuated CDR. Similarly, shaking of cultures attenuated CDR, pimonidazole immunostaining, HIF-1α accumulation, and HIF-1 transcriptional activity. Having established a link between HIF-1 and CDR, we used HIF-1α and HIF-1 transcriptional activity as markers to investigate the role of additional factors in the regulation of CDR. Confluence-dependent increases in HIF-1α accumulation and HIF-1 transcriptional activity were observed even in cells cultured at 0.1% O 2 as well as in sparse cultures incubated with conditioned medium from confluent monolayers. Serum deprivation in both sparse and confluent cultures also resulted in HIF-1α accumulation. These results reveal that, although pericellular hypoxia may be a major contributor to HIF-1 activity, changes in the levels of soluble factors may also play a role. This study demonstrates that HIF-1 is required for CDR.

Mechanism of direct cell interactions. Self-organization of protein synthesis rhythm

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 microM or forskolin at 10 microM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 microM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 microM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists-->changes in the level of calcium ions in cytoplasm-->increased protein kinase activities-->protein phosphorylation-->modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.

NMR-based metabolic profiling of human hepatoma cells in relation to cell growth by culture media analysis

Metabolic profiling is a metabolomic approach that allows the characterization of metabolic phenotypes under specific set of conditions. In the present paper we investigated the metabolism of sparse and high density cultures in relation to different cell growth phases. Changes in the metabolome were evaluated by using 1H-NMR spectroscopy, correlation map and Multivariate Data Analysis on the net balances of metabolites in the medium. This approach allowed us to identify two different metabolic profiles in relation to the cell growth phases in subconfluence and confluence cultures. The results have been interpreted on the basis of patterns of correlations obtained in the two physiological cell states. Cells almost arrested in G0/G1 phase by contact dependent growth inhibition underwent changes in the channeling of amino acids utilization from synthetic to energetic purpose and in anaplerosis/cataplerosis regulation of the TCA cycle.

Stuck (and Inhibited) at the Surface

Vascular endothelial cadherin (VEC) inhibits the signaling of the vascular endothelial growth factor receptor (VEGFR), which may contribute to contact-mediated growth inhibition, a phenomenon that is lost in cancerous cells. Lampugnani et al. show that VEC sequesters VEGFR at the plasma membrane, preventing internalization. When sequestered at the plasma membrane at the site of cell-cell junctions, VEC promotes the dephosphorylation of VEGFR through the recruitment of the phosphatase DEP-1 to the complex. The authors studied VEGFR endocytosis and signaling in human umbilical vein endothelial cells (HUVECs) and VEC-null or VEC-positive cultured cell lines. In HUVECs, VEGF-stimulated internalization of VEGFR was decreased in confluent cultures compared with that observed in sparse cultures. A similar lower amount of internalization was observed in VEC-positive cultured cells compared with that in VEC-null cells. Internalized VEGFR was active, and the VEC-null cells showed greater abundance of tyrosine-phosphorylated VEGFR and activated phospholipase C-γ on internal membranes than did the VEC-positive cells. Internalization was clathrin mediated, and inhibition of clathrin-mediated endocytosis in the VEC-null cells returned VEGFR signaling--measured as tyrosine phosphorylation of mitogen-activated protein kinases (MAPKs) p44 and p42--to levels similar to those in VEC-positive cells. VEC was not endocytosed with VEGFR, suggesting that VEGFR activity was inhibited at the plasma membrane. VEC-positive cells in which DEP-1 was knocked down with silencing RNA showed enhanced VEGFR internalization and VEGFR phosphorylation and MAPK activation. Thus, VEC appears to stabilize VEGFR at the cell surface by stimulating VEGFR dephosphorylation at sites of cell-cell contact through the recruitment of DEP-1. Signaling by VEGFR after internalization appears to be an important mechanism of signal transduction by this growth factor receptor, and blocking this internalization is one mechanism by which activity can be controlled. M. G. Lampugnani, F. Orsenigo, M. C. Gagliani, C. Tacchetti, E. Dejana, Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J. Cell Biol. 174 , 593-604 (2006). [Abstract] [Full Text]

The tight junction protein ZO-2 has several functional nuclear export signals

The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser 369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein.

Development of multidrug resistance due to multiple factors including P-glycoprotein overexpression underK-selection afterMYC andHRAS oncogene activation

Multistep tumorigenesis is a form of microevolution consisting of mutation and selection. To clarify the role of selection modalities in tumor development, we examined two alternative evolutionary conditions, r-selection in sparse culture, which allows cells to proliferate rapidly, and K-selection in confluent culture, in which overcrowding constrains cell proliferation. Using MYC- and EJ-RAS-transformed rat embryo fibroblasts, we found that K-selected cells acquired and stably maintained multidrug resistance (MDR) to DOX, VCR, MTX and Ara-C. Then, we examined the involvement of a number of factors potentially causal of the development of MDR, that is, ploidy, Tp53 mutation, doubling time and the expression levels of genes related to drug resistance. Although ploidy status and Tp53 mutations did not correlate with MDR, we found that Abcb1/Mdr1, encoding P-glycoprotein (Pgp), was significantly upregulated after K-selection. Cyclosporin A, a competitive inhibitor of Pgp, increased the intracellular accumulation of DOX and reduced the resistance to it. Indeed, the population of Pgp-transfected cells significantly expanded under K-, but not under r-selection. In addition to Pgp upregulation, altered expression of other genes such as Cda/cytidine deaminase and Slc29a1/equilibrative nucleoside transporter 1 and prolonged doubling times were associated with MDR. This system reproduces events associated with MDR in vivo and would be useful for analysis of MDR development.

Hepatocyte Growth Factor/Scatter Factor and MET Are Involved in Arterial Repair and Atherogenesis

Several studies have shown that in the arterial wall hepatocyte growth factor/scatter factor (HGF/SF) is expressed by smooth muscle cells (SMCs) but acts on endothelial cells, not SMCs. Other studies, however, have indicated that SMCs can respond to HGF/SF. We have reinvestigated expression and activity of HGF/SF and its receptor MET in arterial SMC and endothelial cell cultures and in whole arteries after superficial or deep injury or atherogenesis. High-density cultures of SMCs produced HGF/SF but did not express MET, whereas SMCs, at the leading edge of injured cultures, expressed both ligand and receptor and showed a dramatic motility and growth response to HGF/SF. In line with these results, HGF/SF and MET expression was undetectable in the media of uninjured carotid arteries but was induced after deep arterial injury in areas of SMC migration in the neointima. Strong MET expression was also observed in the SMCs of the atherosclerotic lesions of homozygous apoE(-/-) mice, whereas HGF/SF was expressed by macrophage-derived foam cells. These results demonstrate that MET is induced in migrating and proliferating SMCs and that HGF/SF and MET are key mediators of the SMC response in atherogenesis.

Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells.

BackgroundCCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro.ResultsIn cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-β. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis.ConclusionThese results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament.

Factors Modulating p63 Expression in Cultured Limbal Epithelial Cells

The expression pattern of p63, a homologue of the transcription factor p53, and whether it can be used as a corneal epithelial stem cell specific marker remain controversial. We investigated the p63 expression pattern in cultured limbal epithelial cells at different time points in culture, in sparse and confluent cultures, after growth factor starvation, and in single-cell-derived colonies. Harvested limbal epithelial cells were plated at 2.5 (sparse) or 5 (dense) x 10 cells/cm and evaluated for p63 expression at day 1, day 4, day 7, after starvation for 72 hours, or in colonies derived from single cells. Expression of corneal lineage specific differentiation marker keratin 3 (K3) was correlated with p63 expression. Results were compared by 1-way ANOVA. More than 85% (85%-90%) of cells expressed p63 on day 1 regardless of cell plating density. On day 4, sparsely plated cultures were subconfluent and demonstrated high p63 expression (87.4%), whereas densely plated cells were confluent and had markedly reduced p63 expression (16.9%). Starvation of subconfluent cultures arrested cell division but did not decrease p63 expression. High-p63-expressing cultures expressed low levels of K3, and this trend was reversed in confluent cultures. Most cells in all colonies derived from single cells expressed p63. The majority of corneal limbal epithelial cells express p63 in colonies derived from single cells and in subconfluent cultures regardless of time in culture or continuance of cell division. This suggests that p63 expression in culture cannot be used as a marker for stem cells. Significantly reduced number of cells express p63 in confluent cultures, associated with increased cell-cell contact. It is notable that these cells continue to express p63 amid areas of increased cell-cell contact several days after cultures have attained full confluency. This may represent a unique subpopulation of cells that retain proliferative potential in a confluent culture and may be analogous to a subpopulation of stem cells present in vivo.

Biphasic calcium response of platelet‐derived growth factor stimulated glioblastoma cells is a function of cell confluence

Previous reports have linked the spiking or two-phased character of calcium transients evoked by platelet-derived growth factor (PDGF) to the position of cells in the cell cycle without regard to cell-cell contact and communication. Because cell confluence can regulate growth factor receptor expression and dephosphorylation, we investigated the effect of cell culture confluence and cell cycle on calcium responses of PDGF-BB-stimulated A172 glioblastoma cells. Digital imaging cytometry was used to correlate the peak and duration of calcium response with bromodeoxyuridine positivity and DNA content and with culture confluence on a cell-by-cell basis. In serum-starved cultures, complete two-phase calcium signals and shorter, lower spikes occurred independent of cell cycle phase. However, the confluence of cell culture seemed essential for inducing a complete response because cells in sparse cultures exhibited mostly short spikes with lower peaks or no transients at all. Because cell confluence, by virtue of cell-cell contacts, is assumed to be an important regulator of proliferation, one is tempted to speculate that in transformed cells the ability to produce stronger growth signals upon reaching confluence and facing contact inhibition could provide a proliferative advantage.

Methylation of the CA9 promoter can modulate expression of the tumor-associated carbonic anhydrase IX in dense carcinoma cell lines

CA IX is a tumor-associated transmembrane isoform of the carbonic anhydrase with a high enzyme activity and a functional involvement in the pH regulation and cell adhesion. Expression of CA9 gene in tumor cells is principally regulated by the high cell density and the hypoxia-related VHL-HIF pathway. In renal cell carcinomas with VHL inactivation, CA9 transcription is further controlled by site-specific promoter methylation. Here we explored a possible role of methylation in the non-RCC cell lines represented mainly by HeLa cervical carcinoma cells. Using metabisulfite sequencing and treatment with the methylation inhibitor 5-aza-2'-deoxycytidine we showed that the methylation of a single CpG site at -74 position with respect to the transcription start can down-modulate the expression of CA9 in cells cultivated at high density, but not in cells grown in sparse culture nor in cells exposed to hypoxia. Methylation appears to act in tumor cells expressing intermediate levels of CA IX protein, but not in cell lines expressing high CA IX levels. Our results indicate that promoter methylation is not crucial for the control of CA9 gene expression in the non-RCC cell lines but could represent an accessory mechanism restricting its expression in highly dense carcinoma cell cultures.

L1, a novel target of β-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers

Aberrant β-catenin-TCF target gene activation plays a key role in colorectal cancer, both in the initiation stage and during invasion and metastasis. We identified the neuronal cell adhesion molecule L1, as a target gene of β-catenin-TCF signaling in colorectal cancer cells. L1 expression was high in sparse cultures and coregulated with ADAM10, a metalloprotease involved in cleaving and shedding L1's extracellular domain. L1 expression conferred increased cell motility, growth in low serum, transformation and tumorigenesis, whereas its suppression in colon cancer cells decreased motility. L1 was exclusively localized in the invasive front of human colorectal tumors together with ADAM10. The transmembrane localization and shedding of L1 by metalloproteases could be useful for detection and as target for colon cancer therapy.

Transcriptional control of cell density dependent regulation of matrix metalloproteinase and TIMP expression in breast cancer cell lines

Our recent studies on breast carcinoma cell lines with differing tumorigenicity/invasiveness (MCF-7<MDA-MB-468<MDAMB-231<MDA-MB-435) had shown significantly decreasing expression levels of MMPs-1,-2,-3,-8,-9,-10,-11 and -13 with increasing cell density while the levels of TIMP-1 and -2 increased. This correlated well with a lower invasiveness of confluent cells. In the present study, we extend our in vitro studies on three-dimensional cultures of breast cancer cell lines MCF-7 and MDAMB-435 and the transcriptional control of MMP and TIMP-expression in two-dimensional cultures of MDA-MB-231 and -435 cells. The tumor spheroid model showed that MMP expression and proteolytic activity were considerably higher in loosely structured tumor groups as compared to densely growing "compact" cell complexes. These data suggested that cell density regulates MMP and TIMP transcription and therefore, we tested whether AP-1, NF kappa B and CRE are involved in this process. Gene silencing of c-jun in sparse cultures had an inhibitory effect on MMP-3, -9 and -13 expression, on proteolytic activity as well as on the invasive potential of the cells, thus confirming a role for AP-1.TIMP-1, and -2 expression was up-regulated as compared to control cells. Consistent with this, overexpression of c-jun and c-fos in confluent breast cancer cell lines leads to up-regulation of MMP expression, proteolytic activity and invasion as well as down-regulation of TIMP-1. In summary, we provide evidence that cell density influences the invasive potential of tumor cells via regulation of MMPs and TIMPs by AP-1, NF kappa B and CRE transcription factors. Overexpression of MMPs in sparse cultures could help explain early dissemination of potentially metastatic cells.

The role of extracellular signal-regulated protein kinase in transcriptional regulation of the hypoxia marker carbonic anhydrase IX

In the present study, we investigated the role of the extracellular signal-regulated protein kinase (ERK) in regulation of the hypoxia marker, carbonic anhydrase IX (CAIX). U0126, a specific inhibitor of MEK1/2, downregulated CAIX expression induced by true hypoxia and cell density. CA9 promoter activity was similarly affected. Mapping of the U0126 effect revealed that both critical elements within the CA9 promoter, the hypoxia response element and the juxtaposed SP1-binding PR1, were inhibited. This confirmed that ERK signaling modulates CA9 promoter activity via its effects on hypoxia inducible factor-1 (HIF-1) and SP1. Further analysis of the U0126 effect on HIF-1-dependent transcription in MCF-7 cells identified p300, a transcriptional co-activator of HIF-1, as the target of ERK. Constitutively increased ERK activity in isogenic fibrosarcoma cell lines did not cause increased cell density-dependent CAIX expression/CA9 promoter activity. In HeLa cells, an inverse correlation between cell density-induced CAIX expression and ERK activation was observed: sparse cultures did not express CAIX and displayed high ERK activation, whereas CAIX expression in dense cultures was associated with low ERK activation. Collectively, our data do not support any quantitative relationship between ERK activation and CAIX expression. Thus, although ERK signaling is required for optimal CAIX expression, our data are consistent with a model in which constitutive basal ERK activity plays an auxiliary role in CA9 promoter transactivation by modulating activity of the transcription factor SP1 and the transcriptional co-activator p300.

Functional Fixedness in a Technologically Sparse Culture

Problem solving can be inefficient when the solution requires subjects to generate an atypical function for an object and the object's typical function has been primed. Subjects become "fixed" on the design function of the object, and problem solving suffers relative to control conditions in which the object's function is not demonstrated. In the current study, such functional fixedness was demonstrated in a sample of adolescents (mean age of 16 years) among the Shuar of Ecuadorian Amazonia, whose technologically sparse culture provides limited access to large numbers of artifacts with highly specialized functions. This result suggests that design function may universally be the core property of artifact concepts in human semantic memory.

Association of ARVCF with zonula occludens (ZO)-1 and ZO-2: binding to PDZ-domain proteins and cell-cell adhesion regulate plasma membrane and nuclear localization of ARVCF.

ARVCF, an armadillo-repeat protein of the p120(ctn) family, associates with classical cadherins and is present in adherens junctions, but its function is poorly understood. Here, we show that ARVCF interacts via a C-terminal PDZ-binding motif with zonula occludens (ZO)-1 and ZO-2. ARVCF and ZO-1 partially colocalize in the vicinity of the apical adhesion complex in polarized epithelial Madin-Darby canine kidney cells. ARVCF, ZO-1, and E-cadherin form a complex and are recruited to sites of initial cell-cell contact in sparse cell cultures. E-cadherin binding and plasma membrane localization of ARVCF require the PDZ-binding motif. Disruption of cell-cell adhesion releases ARVCF from the plasma membrane and an increased fraction of the protein localizes to the nucleus. Nuclear localization of ARVCF also requires the PDZ-binding motif and can be mediated by the PDZ domains of ZO-2. Thus, the interaction of ARVCF with distinct PDZ-domain proteins determines its subcellular localization. Interactions with ZO-1 and ZO-2, in particular, may mediate recruitment of ARVCF to the plasma membrane and the nucleus, respectively, possibly in response to cell-cell adhesion cues.

Cell Growth Regulation through GM3-enriched Microdomain (Glycosynapse) in Human Lung Embryonal Fibroblast WI38 and Its Oncogenic Transformant VA13

Cell growth control mechanisms were studied based on organization of components in glycosphingolipid-enriched microdomain (GEM) in WI38 cells versus their oncogenic transformant VA13 cells. Levels of fibroblast growth factor receptor (FGFR) and cSrc were 4 times and 2-3 times higher, respectively, in VA13 than in WI38 GEM, whereas the level of tetraspanin CD9/CD81 was 3-5 times higher in WI38 than in VA13 GEM. Csk, the physiological inhibitor of cSrc, was present in WI38 but not in VA13 GEM. Functional association of GEM components in control of cell growth in WI38 is indicated by several lines of evidence. (i) Confluent, growth-inhibited WI38 showed a lower degree of FGF-induced MAPK activation than actively growing cells in sparse culture. (ii) The level of inactive cSrc (with Tyr-527 phosphate) was higher in confluent cells than in actively growing cells. Both processes i and ii were inhibited by GM3 since they were enhanced by GM3 depletion with d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4). (iii) The high level of inactive cSrc associated with growth-inhibited cells was caused by coexisting Csk in WI38 GEM. (iv) Interaction of GM3 with FGFR was demonstrated by binding of GM3 to FGFR in the GEM fraction, as probed with GM3-coated beads, and by confocal microscopy. In contrast to WI38, both cSrc and MAPK in VA13 were strongly activated regardless of FGF stimulation or GM3 depletion by P4. Continuous, constitutive activation of both cSrc and MAPK was due to (i) a much higher level of cSrc and FGFR in VA13 than in WI38 GEM, (ii) their close association/interaction in VA13 GEM as indicated by clear coimmunoprecipitation between cSrc and FGFR, and (iii) the absence of Csk in VA13 GEM, making GEM incapable of inhibiting cSrc activation.