Presence Of Cell-cell Contact Research Articles

Dictyostelium cells migrate similarly on surfaces of varying chemical composition.

During cell migration, cell-substrate binding is required for pseudopod anchoring to move the cell forward, yet the interactions with the substrate must be sufficiently weak to allow parts of the cell to de-adhere in a controlled manner during typical protrusion/retraction cycles. Mammalian cells actively control cell-substrate binding and respond to extracellular conditions with localized integrin-containing focal adhesions mediating mechanotransduction. We asked whether mechanotransduction also occurs during non-integrin mediated migration by examining the motion of the social amoeba Dictyostelium discoideum, which is thought to bind non-specifically to surfaces. We discovered that Dictyostelium cells are able to regulate forces generated by the actomyosin cortex to maintain optimal cell-surface contact area and adhesion on surfaces of various chemical composition and that individual cells migrate with similar speed and contact area on the different surfaces. In contrast, during collective migration, as observed in wound healing and metastasis, the balance between surface forces and protrusive forces is altered. We found that Dictyostelium collective migration dynamics are strongly affected when cells are plated on different surfaces. These results suggest that the presence of cell-cell contacts, which appear as Dictyostelium cells enter development, alter the mechanism cells use to migrate on surfaces of varying composition.

Cell density regulates in vitro activation of heart valve interstitial cells

Valve interstitial cells, the most prominent cell type in the heart valve, are activated and express α-smooth muscle actin in valve repair and in diseased valves. We hypothesize that cell density, time in culture, and the establishment of cell-cell contacts may be involved in regulating valve interstitial cell activation in vitro. To study cell density, valve interstitial cells were plated at passages 3 to 5, at a density of 17,000 cells/22 × 22 mm(2) coverslip, and grown for 1, 2, 4, 7, and 10 days. Valve interstitial cells were stained for α-smooth muscle actin and viewed under confocal microscopy to characterize the intensity of staining. To study time in culture, valve interstitial cells were plated at a 10-fold higher density to achieve similar growth densities over a shorter time period compared with valve interstitial cells plated at low density. α-Smooth muscle actin staining was compared at the same time points between those plated at high and low densities. To confirm valve interstitial cell activation as indicated by α-smooth muscle actin staining, valve interstitial cells were stained for cofilin at days 2, 5, 8, and 14 days postplating. To study the association of transforming growth factor β with valve interstitial cell activation with respect to cell density, valve interstitial cells were stained for α-smooth muscle actin and transforming growth factor β at 2, 4, 6, and 8 days postplating. To study the activation of the transforming growth factor β signaling pathway, valve interstitial cells were stained for pSmad2/3 at days 2, 4, 6, 8, 10, and 12 days postplating. To study cell contacts and activation, subconfluent and confluent cultures of valve interstitial cells were stained for β-catenin, N-cadherin, and α-smooth muscle actin. Also, whole-cell lysates of subconfluent and confluent valve interstitial cell cultures were probed by Western blot analysis for phospho-β-catenin at Ser33/37/Thr41, which is the form of β-catenin targeted for proteosomal degradation. The percentage valve interstitial cells with high-intensity α-smooth muscle actin staining decreases significantly between days 1 and 4, and at confluency, most cells show absent or low-intensity staining, regardless of time in culture. Similar results are obtained with cofilin staining. Transforming growth factor β and nuclear pSmad2/3 staining in valve interstitial cells decreases concurrently with valve interstitial cell activation as cell density increases. Examining β-catenin and N-cadherin staining, single valve interstitial cells show no cell-cell contact with strong cytoplasmic staining, with some showing nuclear staining of β-catenin, while confluent monolayers show strong staining of fully established cell-cell contacts, weak cytoplasmic staining, and absent nuclear staining. The presence of cell-cell contacts is associated with a decreased α-smooth muscle actin. The level of phospho-β-catenin at Ser33/37/Thr41 is lower in confluent cultures compared with low-density subconfluent valve interstitial cell cultures. Cell-cell contacts may inhibit valve interstitial cell activation, while absence of cell-cell contacts may contribute to activation.

Angiopoietin-1/Tie2 Signal Augments Basal Notch Signal Controlling Vascular Quiescence by Inducing Delta-Like 4 Expression through AKT-mediated Activation of β-Catenin

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group previously showed that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts. We further demonstrated that the former up-regulates Notch ligand delta-like 4 (Dll4) only in the presence of cell-cell contacts. Because Dll4/Notch signal restricts sprouting angiogenesis and promotes vascular stabilization, we investigated the mechanism of how the Ang1/Tie2 signal induces Dll4 expression to clarify the role of the Dll4/Notch signal in Ang1/Tie2 signal-mediated vascular quiescence. Under confluent endothelial cells, the basal Notch signal was observed. Ang1, moreover, induced Dll4 expression and production of the Notch intracellular domain (NICD). Ang1 stimulated transcriptional activity of β-catenin through phosphoinositide 3-kinase (PI3K)/AKT-mediated phosphorylation of glycogen synthase kinase 3β (GSK3β). Correspondingly, the GSK3β inhibitor up-regulated Dll4, whereas depletion of β-catenin by siRNA blocked Ang1-induced Dll4 expression, indicating the indispensability of β-catenin in Ang1-mediated up-regulation of Dll4. In addition, Dll4 expression by the GSK3β inhibitor was only observed in confluent cells, and was impeded by DAPT, a γ-secretase inhibitor, implying requirement of the Notch signal in β-catenin-dependent Dll4 expression. Consistently, we found that either Ang1 or NICD up-regulates Dll4 through the RBP-J binding site within intron 3 of the DLL4 gene and that β-catenin forms a complex with NICD/RBP-J to enhance Dll4 expression. Ang1 induced the deposition of extracellular matrix that is preferable for basement membrane formation through Dll4/Notch signaling. Collectively, the Ang1/Tie2 signal potentiates basal Notch signal controlling vascular quiescence by up-regulating Dll4 through AKT-mediated activation of β-catenin.

Engineered 3D environments to elucidate the effect of environmental parameters on drug response in cancer

Traditional in vitro models used for the development of anti-cancer drugs are based on the monolayer culture of cells, which has a limited predictivity of in vivo efficacy. A number of cell culture platforms have been developed in recent years to improve predictivity and further to elucidate the mechanisms governing the differing responses observed in vitro versus in vivo. One detrimental aspect of current in vitro models is their inability to decouple the effect of different extrinsic factors on the responsiveness of the cells to drug treatment. Here, we have used an engineered poly(dimethylsiloxane) (PDMS) microwell array as a reductionist approach to study the effect of environmental parameters, independently of each other. It is observed for MCF-7 breast cancer cells, that culture within the three-dimensional (3D) environment of the microwells alone had an effect on the response to Taxol and results in a reduction of cell death in comparison to cells cultured on flat substrates. Additionally the microwells allowed the response of single versus multicell clusters to be differentiated. It was found that the formation of cell-cell contacts alters the drug response, depending on the type of adhesive protein present. Thus, with this microwell platform it is revealed that the presence of cell-cell contacts in addition to the dimensionality and the matrix composition of the environment are important mediators of altered drug responses. In conclusion the microwell array can not only serve as a platform to reveal which parameters of the extracellular environment affect drug response but further the interdependence of these parameters.

Angiopoietin-1 Induces Krüppel-like Factor 2 Expression through a Phosphoinositide 3-Kinase/AKT-dependent Activation of Myocyte Enhancer Factor 2

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group have recently shown that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts and further demonstrated that the former selectively induces expression of Krüppel-like factor 2 (KLF2), a transcription factor involved in vascular quiescence. Here, we investigated the mechanism of how Ang1/Tie2 signal induces KLF2 expression to clarify the role of KLF2 in Ang1/Tie2 signal-mediated vascular quiescence. Ang1 stimulated KLF2 promoter-driven reporter gene expression in endothelial cells, whereas it failed when a myocyte enhancer factor 2 (MEF2)-binding site of KLF2 promoter was mutated. Depletion of MEF2 by siRNAs abolished Ang1-induced KLF2 expression, indicating the requirement of MEF2 in KLF2 induction by Ang1. Constitutive active phosphoinositide 3-kinase (PI3K) and AKT increased the MEF2-dependent reporter gene expression by enhancing its transcriptional activity and stimulated the KLF2 promoter activity cooperatively with MEF2. Consistently, inhibition of either PI3K or AKT and depletion of AKT abrogated Ang1-induced KLF2 expression. In addition, we confirmed the dispensability of extracellular signal-regulated kinase 5 (ERK5) for Ang1-induced KLF2 expression. Furthermore, depletion of KLF2 resulted in the loss of the inhibitory effect of Ang1 on vascular endothelial growth factor (VEGF)-mediated expression of vascular cell adhesion molecule-1 in endothelial cells and VEGF-mediated monocyte adhesion to endothelial cells. Collectively, these findings indicate that Ang1/Tie2 signal stimulates transcriptional activity of MEF2 through a PI3K/AKT pathway to induce KLF2 expression, which may counteract VEGF-mediated inflammatory responses.

Differential function of Tie2 at cell–cell contacts and cell–substratum contacts regulated by angiopoietin-1

Tie2 belongs to the receptor tyrosine kinase family and functions as a receptor for Angiopoietin-1 (Ang1). Gene-targeting analyses of either Ang1 or Tie2 in mice reveal a critical role of Ang1-Tie2 signalling in developmental vascular formation. It remains elusive how the Tie2 signalling pathway plays distinct roles in both vascular quiescence and angiogenesis. We demonstrate here that Ang1 bridges Tie2 at cell-cell contacts, resulting in trans-association of Tie2 in the presence of cell-cell contacts. In clear contrast, in isolated cells, extracellular matrix-bound Ang1 locates Tie2 at cell-substratum contacts. Furthermore, Tie2 activated at cell-cell or cell-substratum contacts leads to preferential activation of Akt and Erk, respectively. Microarray analyses and real-time PCR validation clearly show the differential gene expression profile in vascular endothelial cells upon Ang1 stimulation in the presence or absence of cell-cell contacts, implying downstream signalling is dependent upon the spatial localization of Tie2.

Major role for active extension in the formation of processes by ras-transformed fibroblasts.

Expression of constitutively active Ras protein in fibroblasts results in enhanced cell motility, invasion competence and morphological changes including the formation of elongate cellular processes. These processes have been shown to resemble retraction tails formed passively behind nontransformed cells by movement relative to sites of cell-substrate attachment. However, analysis presented here reveals that active extension mechanisms also play a role in the formation of these processes. Extension of distal process ends occurs at 0.42+/-0.44 microm/min in ras-transformed fibroblasts and accounts for 63.6+/-27.5% of observed process lengths. Active process extension by ras-transformed fibroblasts also persists in the presence of cell-cell contacts. Studies conducted using actin or microtubule antagonists, and correlation of process behavior followed by fixation and immunostaining reveal that process extension requires intact actin and microtubule networks. Other analyses reveal that active extension plays a significantly smaller role in the formation of processes by non-transformed control fibroblasts. These observations demonstrate that constitutively active Ras enhances process extension in fibroblasts and is a causal factor in process extension by fibroblasts in the presence of cell-cell contacts. Moreover, these studies demonstrate that process extension by ras-transformed fibroblasts is accomplished through mechanisms similar to those thought to drive active extension of processes by other cell types including neurons. These findings suggest that extension of cellular processes could play an important role in the metastatic behavior of ras-transformed fibroblasts as well as the response of untransformed fibroblasts to receptor mediated signal transduction events.

CD45-mediated signals can trigger shedding of lymphocyte L-selectin.

The adhesion molecule L-selectin is proteolytically cleaved from the surface of lymphocytes and neutrophils within minutes after stimulation by phorbol ester or calcium ionophores. In contrast to neutrophils, soluble factors have not been shown to induce down-regulation of L-selectin on lymphocytes. We therefore examined whether signals generated by interaction with cell surface receptors could deliver physiological stimuli inducing this regulatory mechanism. While cross-linking of several adhesion molecules (CD2, CD44, alpha 4-integrin, LFA-1) by antibody did not result in a significant reduction of the expression of L-selectin, antibodies against CD45 and Thy-1.2, both involved in the regulation of lymphocyte activation, induced loss of cell surface L-selectin within minutes, even at 4 degrees C, by shedding into the supernatant. Cross-linking of these molecules was shown to be essential, but Fc interactions or adherent cells were not required. A similar response, albeit less effective, was found after cross-linking of CD3. Interestingly, initiation of shedding only occurred in the presence of cell-cell contact, pointing to a second, as yet unknown, signal required. Loss of L-selectin induced by CD45 cross-linking is followed by a rapid re-expression of the molecule upon incubation at 37 degrees C. This reaction is also dependent on specific triggering signals as rapid re-expression was not observed after removal of L-selectin by trypsin. The data indicate that the protein phosphatase CD45 as well as the TCR complex itself in combination with a further, as yet unknown, cell-cell contact-dependent stimulus have a regulatory role in the dynamic control of L-selectin expression in lymphocytes.

Desmosome assembly in MDCK epithelial cells does not require the presence of functional microtubules.

Desmosomes, complex multisubunit structures that assemble at sites of cell-cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645-655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin-Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post-translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell-cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstrated an apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell-cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell-cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell-cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules.

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis.

The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. II. Morphological analysis.

Biochemical analysis of the kinetics of assembly of two cytoplasmic plaque proteins of the desmosome, desmoplakins I (250,000 Mr) and II (215,000 Mr), in Madin-Darby canine kidney (MDCK) epithelial cells, demonstrated that these proteins exist in a soluble and insoluble pool, as defined by their extract ability in a Triton X-100 high salt buffer (CSK buffer). Upon cell-cell contact, there is a rapid increase in the capacity of the insoluble pool at the expense of the soluble pool; subsequently, the insoluble pool is stabilized, while proteins remaining in the soluble pool continue to be degraded rapidly (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685). In this paper, we have sought to determine the spatial distribution of the soluble and insoluble pools of desmoplakins I and II, and their organization in the absence and presence of cell-cell contact by using differential extraction procedures and indirect immunofluorescence microscopy. In the absence of cell-cell contact, two morphologically and spatially distinct patterns of staining of desmoplakins I and II were observed: a pattern of discrete spots in the cytoplasm and perinuclear region, which is insoluble in CSK buffer; and a pattern of diffuse perinuclear staining, which is soluble in CSK buffer, but which is preserved when cells are fixed in 100% methanol at -20 degrees C. Upon cell-cell contact, in the absence or presence of protein synthesis, the punctate staining pattern of desmoplakins I and II is cleared rapidly and efficiently from the cytoplasm to the plasma membrane in areas of cell-cell contact (less than 180 min). The distribution of the diffuse perinuclear staining pattern remains relatively unchanged and becomes the principal form of desmoplakins I and II in the cytoplasm 180 min after induction of cell-cell contact. Thereafter, the relative intensity of staining of the diffuse pattern gradually diminishes and is completely absent 2-3 d after induction of cell-cell contact. Significantly, double immunofluorescence shows that during desmosome assembly on the plasma membrane both staining patterns coincide with a subpopulation of cytokeratin intermediate filaments. Taken together with the preceding biochemical analysis, we suggest that the assembly of desmoplakins I and II in MDCK epithelial cells is regulated at three discrete stages during the formation of desmosomes.

Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells.

During growth of Madin-Darby canine kidney (MDCK) epithelial cells, there is a dramatic change in the stability, biophysical properties, and distribution of the membrane skeleton (fodrin) which coincides temporally and spatially with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral domain of the plasma membrane. These changes occur maximally upon the formation of a continuous monolayer of cells, indicating that extensive cell-cell contact may play an important role in the organization of polarized MDCK cells (Nelson, W. J., and P. J. Veshnock, 1986, J. Cell Biol., 103:1751-1766). To directly analyze the role of cell-cell contact in these events, we have used an assay in which the organization of fodrin and membrane proteins is analyzed in confluent monolayers of MDCK cells in the absence or presence of cell-cell contact by adjusting the concentration Ca++ in the growth medium. Our results on the stability and solubility properties of fodrin reported here show directly that there is a positive correlation between cell-cell contact and increased stability and insolubility of fodrin. Furthermore, we show that fodrin can be recruited from an unstable pool of protein to a stable pool during induction of cell-cell contact; significantly, the stabilization of fodrin is not affected by the addition of cyclohexamide, indicating that proteins normally synthesized during the induction of cell-cell contact are not required. Together these results indicate that cell-cell contact may play an important role in the development of polarity in MDCK cells by initiating the formation of a stable, insoluble matrix of fodrin with preexisting (membrane) proteins at the cell periphery. This matrix may function subsequently to trap proteins targeted to the membrane, resulting in the maintenance of membrane domains.

Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type

When adult rat hepatocytes were co-cultured with another liver epithelial cell type in a medium supplemented or not with fetal calf serum (FCS), it was found that 1. 1. They survived for more than 2 months 2. 2. Albumin secretion levels remained high over the whole culture period 3. 3. Decreased secretion might be reversed 4. 4. This protein secretion activity appeared to be dependent upon both the presence of cell-cell contacts and the production of an extracellular material. The results demonstrate for the first time long-term stabilization and reversibility of a specific function (albumin secretion) at high levels by adult hepatoeytes cultured in serum-free medium and suggest that both the presence of other liver cell type(s) and the production of an extracellular matrix are needed for the maintenance of specific functions in cultured hepatocytes.