Cell segregation and border sharpening by Eph receptor-ephrin-mediated heterotypic repulsion.

Eph receptor and ephrin signalling has a major role in cell segregation and border formation, and may act through regulation of cell adhesion, repulsion or tension. To elucidate roles of cell repulsion and adhesion, we combined experiments in cell culture assays with quantitations of cell behaviour which are used in computer simulations. Cells expressing EphB2, or kinase-inactive EphB2 (kiEphB2), segregate and form a sharp border with ephrinB1-expressing cells, and this is disrupted by knockdown of N-cadherin. Measurements of contact inhibition of locomotion reveal that EphB2-, kiEphB2- and ephrinB1-expressing cells have strong heterotypic and weak homotypic repulsion. EphB2 cells have a transient increase in migration after heterotypic activation, which underlies a shift in the EphB2-ephrinB1 border but is not required for segregation or border sharpening. Simulations with the measured values of cell behaviour reveal that heterotypic repulsion can account for cell segregation and border sharpening, and is more efficient than decreased heterotypic adhesion. By suppressing homotypic repulsion, N-cadherin creates a sufficient difference between heterotypic and homotypic repulsion, and enables homotypic cohesion, both of which are required to sharpen borders.

T-Cadherin Expression in Melanoma Cells Stimulates Stromal Cell Recruitment and Invasion by Regulating the Expression of Chemokines, Integrins and Adhesion Molecules.

T-cadherin is a glycosyl-phosphatidylinositol (GPI) anchored member of the cadherin superfamily involved in the guidance of migrating cells. We have previously shown that in vivo T-cadherin overexpression leads to increased melanoma primary tumor growth due to the recruitment of mesenchymal stromal cells as well as the enhanced metastasis. Since tumor progression is highly dependent upon cell migration and invasion, the aim of the present study was to elucidate the mechanisms of T-cadherin participation in these processes. Herein we show that T-cadherin expression results in the increased invasive potential due to the upregulated expression of pro-oncogenic integrins, chemokines, adhesion molecules and extracellular matrix components. The detected increase in chemokine expression could be responsible for the stromal cell recruitment. At the same time our previous data demonstrated that T-cadherin expression inhibited neoangiogenesis in the primary tumors. We demonstrate molecules and reduction in pro-angiogenic factors. Thus, T-cadherin plays a dual role in melanoma growth and progression: T-cadherin expression results in anti-angiogenic effects in melanoma, however, this also stimulates transcription of genes responsible for migration and invasion of melanoma cells.

Proteomics analysis of contact-initiated eph receptor-ephrin signaling.

Large-scale biochemical analysis of cell-specific signaling can be interrogated in cocultures of Eph receptor- and ephrin-expressing cells by combining proteomics analysis with cell-specific metabolic labeling. In this chapter, we describe how to perform such large-scale analysis, including the generation of cells stably expressing the receptors and ligands of interest, optimization steps for Eph-ephrin coculture, and the proteomics analysis. As the experimental details may vary depending on the specific system that is being interrogated, the goal of the chapter is mainly to provide sufficient experimental context for experienced researchers to set up and conduct these experiments.

A mechanical model of cell segregation driven by differential adhesion.

From simulations that begin with a random mix of two cell types, we monitor progress towards segregation driven by contact-mediated linkage of model cells, which is equivalent to the cell-cell adhesion of real cells. In comparison with real cell experiments, we show that this mechanical model can account for the observed extent of segregation obtained by differential adhesion in a 2D cell culture assay of cells with differentially expressed cadherin molecules. Calibration of virtual to real time allowed us to estimate a time course for these experiments that was within 50% agreement for the simulations compared to differential adhesion of cells. In contrast, simulations of differential adhesion do not account for the rate of segregation driven by interactions between EphB2 receptor and ephrinB1 expressing cells which occurs an order of magnitude faster. The latter result suggests that mechanisms additional or alternative to differential adhesion contribute to Eph-ephrin mediated cell segregation.

Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated ß1-integrin-dependent cell migration.

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human ß1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of ß1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from ß1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

Simulation of cell movement and interaction.

A mechanical model of cell motion was developed that reproduced the behaviour of cells in 2-dimensional culture. Cell adhesion was modelled with inter-cellular cross-links that attached for different times giving a range of adhesion strength. Simulations revealed an adhesion threshold below which cell motion was almost unaffected and above which cells moved as if permanently linked. Comparing simulated cell clusters (with known connections) to calculated clusters (based only on distance) showed that the calculated clusters did not correspond well across the full size range from small to big clusters. The radial distribution function of the cells was found to be a better measure, giving a good correlation with the known cell linkage throughout the simulation run. This analysis showed that cells were best modelled with a degree of stickiness just under the critical threshold level. This allowed fluidlike motion while maintaining cohesiveness across the population.

Myosin VI and optineurin are required for polarized EGFR delivery and directed migration.

The polarized trafficking of membrane proteins into the leading edge of the cell is an integral requirement for cell migration. Myosin VI and its interacting protein optineurin have previously been shown to operate in anterograde trafficking pathways, especially for the polarized delivery of cargo to the basolateral domain in epithelial cells. Here we show that in migratory cells ablation of myosin VI or optineurin inhibits the polarized delivery of the epidermal growth factor receptor (EGFR) into the leading edge and leads to profound defects in lamellipodia formation. Depletion of either myosin VI or optineurin, however, does not impair the overall ability of cells to migrate in a random migration assay, but it dramatically reduces directed migration towards a growth factor stimulus. In summary, we identified a specific role for myosin VI and optineurin in directionally persistent cell migration, which involves the polarized delivery of vesicles containing EGFR into the leading edge of the cell.

A feedback loop mediated by degradation of an inhibitor is required to initiate neuronal differentiation.

Neuronal differentiation is regulated by proneural genes that promote neurogenesis and inhibitory mechanisms that maintain progenitors. This raises the question of how the up-regulation of proneural genes required to initiate neurogenesis occurs in the presence of such inhibition. We carried out loss and gain of gene function, an interaction screen for binding partners, and biochemical analyses to uncover the regulation, developmental role, and mechanism of action of a ubiquitination adaptor protein, Btbd6a (BTB domain containing 6a). We find that the proneural gene neurog1 up-regulates btbd6a, which in turn is required for up-regulation of neurog1. Btbd6a is an adaptor for the Cul3 ubiquitin ligase complex, and we find that it binds to the transcriptional repressor Plzf (promyelocytic leukemia zinc finger). Btbd6a promotes the relocation of Plzf from nucleus to cytoplasm and targets Plzf for ubiquitination and degradation. plzfa is expressed widely in the neural epithelium; when overexpressed, it inhibits neurogenesis, and this inhibition is reversed by btbd6a. The antagonism of endogenous plzfa by btbd6a is required for neurogenesis, since the block in neuronal differentiation caused by btbd6a knockdown is alleviated by plzfa knockdown. These findings reveal a feedback loop mediated by degradation of an inhibitor that is essential for progenitors to undergo the transition to neuronal differentiation.

Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells.

Cells have self-organizing properties that control their behavior in complex tissues. Contact between cells expressing either B-type Eph receptors or their transmembrane ephrin ligands initiates bidirectional signals that regulate cell positioning. However, simultaneously investigating how information is processed in two interacting cell types remains a challenge. We implemented a proteomic strategy to systematically determine cell-specific signaling networks underlying EphB2- and ephrin-B1-controlled cell sorting. Quantitative mass spectrometric analysis of mixed populations of EphB2- and ephrin-B1-expressing cells that were labeled with different isotopes revealed cell-specific tyrosine phosphorylation events. Functional associations between these phosphotyrosine signaling networks and cell sorting were established with small interfering RNA screening. Data-driven network modeling revealed that signaling between mixed EphB2- and ephrin-B1-expressing cells is asymmetric and that the distinct cell types use different tyrosine kinases and targets to process signals induced by cell-cell contact. We provide systems- and cell-specific network models of contact-initiated signaling between two distinct cell types.

Regulation of EphB2 activation and cell repulsion by feedback control of the MAPK pathway.

In this study, we investigated whether the ability of Eph receptor signaling to mediate cell repulsion is antagonized by fibroblast growth factor receptor (FGFR) activation that can promote cell invasion. We find that activation of FGFR1 in EphB2-expressing cells prevents segregation, repulsion, and collapse responses to ephrinB1 ligand. FGFR1 activation leads to increased phosphorylation of unstimulated EphB2, which we show is caused by down-regulation of the leukocyte common antigen-related tyrosine phosphatase receptor that dephosphorylates EphB2. In addition, FGFR1 signaling inhibits further phosphorylation of EphB2 upon stimulation with ephrinB1, and we show that this involves a requirement for the mitogen-activated protein kinase (MAPK) pathway. In the absence of activated FGFR1, EphB2 activates the MAPK pathway, which in turn promotes EphB2 activation in a positive feedback loop. However, after FGFR1 activation, the induction of Sprouty genes inhibits the MAPK pathway downstream of EphB2 and decreases cell repulsion and segregation. These findings reveal a novel feedback loop that promotes EphB2 activation and cell repulsion that is blocked by transcriptional targets of FGFR1.

T-cadherin suppresses angiogenesis in vivo by inhibiting migration of endothelial cells.

Our previous studies have revealed the abundant expression of T-cadherin--a glycosylphosphatidylinositol (GPI)-anchored member of cadherin superfamily--in endothelial and mural cells in the heart and vasculature. The upregulation of T-cadherin in vascular proliferative disorders such as atherosclerosis and restenosis suggests the involvement of T-cadherin in vascular growth and remodeling. However, the functional significance of this molecule in the vasculature remains unknown. The effect of T-cadherin on angiogenesis in vivo was evaluated using Matrigel implant model. We demonstrate that T-cadherin overexpression in L929 cells injected in Matrigel inhibits neovascularization of the plug. In vitro T-cadherin inhibits the directional migration of endothelial cells, capillary growth, and tube formation but has no effect on endothelial cell proliferation, adhesion, or apoptosis in vitro. These data suggest that T-cadherin expressed in the stroma could act as a negative guidance cue for the ingrowing blood vessels and thus could have an important potential therapeutic application.

Modes of faulting at mid-ocean ridges.

Abyssal-hill-bounding faults that pervade the oceanic crust are the most common tectonic feature on the surface of the Earth. The recognition that these faults form at plate spreading centres came with the plate tectonic revolution. Recent observations reveal a large range of fault sizes and orientations; numerical models of plate separation, dyke intrusion and faulting require at least two distinct mechanisms of fault formation at ridges to explain these observations. Plate unbending with distance from the top of an axial high reproduces the observed dip directions and offsets of faults formed at fast-spreading centres. Conversely, plate stretching, with differing amounts of constant-rate magmatic dyke intrusion, can explain the great variety of fault offset seen at slow-spreading ridges. Very-large-offset normal faults only form when about half the plate separation at a ridge is accommodated by dyke intrusion.

Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme.

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.

Diverse roles of eph receptors and ephrins in the regulation of cell migration and tissue assembly.

Eph receptor tyrosine kinases and ephrins have key roles in regulation of the migration and adhesion of cells required to form and stabilize patterns of cell organization during development. Activation of Eph receptors or ephrins can lead either to cell repulsion or to cell adhesion and invasion, and recent work has found that cells can switch between these distinct responses. This review will discuss biochemical mechanisms and developmental roles of the diverse cell responses controlled by Eph receptors and ephrins.