Functional domains of alpha-catenin required for the strong state of cadherin-based cell adhesion

The interaction of cadherin-catenin complex with the actin-based cytoskeleton through alpha-catenin is indispensable for cadherin-based cell adhesion activity. We reported previously that E-cadherin-alpha-catenin fusion molecules showed cell adhesion and cytoskeleton binding activities when expressed in nonepithelial L cells. Here, we constructed deletion mutants of E-cadherin-alpha-catenin fusion molecules lacking various domains of alpha-catenin and introduced them into L cells. Detailed analysis identified three distinct functional domains of alpha-catenin: a vinculin/alpha-actinin-binding domain, a ZO-1-binding domain, and an adhesion-modulation domain. Furthermore, cell dissociation assay revealed that the fusion molecules containing the ZO-1-binding domain in addition to the adhesion-modulation domain conferred the strong state of cell adhesion activity on transfectants, although those lacking the ZO-1-binding domain conferred only the weak state. The disorganization of actin-based cytoskeleton by cytochalasin D treatment shifted the cadherin-based cell adhesion from the strong to the weak state. In the epithelial cells, where alpha-catenin was not precisely colocalized with ZO-1, the ZO-1-binding domain did not completely support the strong state of cell adhesion activity. Our studies showed that the interaction of alpha-catenin with the actin-based cytoskeleton through the ZO-1-binding domain is required for the strong state of E-cadherin-based cell adhesion activity.     

Expression of Ep-CAM shifts the state of cadherin-mediated adhesions from strong to weak

Various adhesion molecules play an important role in defining cell fate and maintaining tissue integrity. Therefore, cross-signaling between adhesion receptors should be a common phenomenon to support the orchestrated changes of cells' connections to the substrate and to the neighboring cells during tissue remodeling. Recently, we have demonstrated that the epithelial cell adhesion molecule Ep-CAM negatively modulates cadherin-mediated adhesions in direct relation to its expression levels. Here, we used E-cadherin/alpha-catenin chimera constructs to define the site of Ep-CAM's negative effect on cadherin-mediated adhesions. Murine L-cells transfected with either E-cadherin/alpha-catenin fusion protein, or E-cadherin fused to the carboxy-terminal half of alpha-catenin, were subsequently supertransfected with an inducible Ep-CAM construct. Introduction of Ep-CAM altered the cell's morphology, weakened the strength of cell-cell interactions, and decreased the cytoskeleton-bound fraction of the cadherin/catenin chimeras in both cell models. Furthermore, expression of Ep-CAM induced restructuring of F-actin, with changes in thickness and orientation of the actin filaments. The results showed that Ep-CAM affects E-cadherin-mediated adhesions without involvement of beta-catenin by disrupting the link between alpha-catenin and F-actin. The latter is likely achieved through remodeling of the actin cytoskeleton by Ep-CAM, possibly through pp120.     

Focal adhesion kinase is not required for integrin function or viability in Drosophila

The mammalian focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases has been implicated in controlling a multitude of cellular responses to the engagement of cell-surface integrins and G-protein-coupled receptors. The high level of sequence conservation between the mammalian proteins and the Drosophila homologue of FAK, Fak56, suggested that it would have similar functions. However, we show here that Drosophila Fak56 is not essential for integrin functions in adhesion, migration or signaling in vivo. Furthermore, animals lacking Fak56 are viable and fertile, demonstrating that Fak56 is not essential for other developmental or physiological functions. Despite this, overexpressed Fak56 is a potent inhibitor of integrins binding to the extracellular matrix, suggesting that Fak56 may play a subtle role in the negative regulation of integrin adhesion.     

Receptor tyrosine kinase signaling required for integrin alpha v beta 5- directed cell motility but not adhesion on vitronectin

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.     

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.     

Focal adhesion kinase is required for synovial fibroblast invasion,but not murine inflammatory arthritis

Introduction

Synovial fibroblasts invade cartilage and bone, leading to joint destruction in rheumatoid arthritis. However, the mechanisms that regulate synovial fibroblast invasion are not well understood. Focal adhesion kinase (FAK) has been implicated in cellular invasion in several cell types, and FAK inhibitors are in clinical trials for cancer treatment. Little is known about the role of FAK in inflammatory arthritis, but, given its expression in synovial tissue, its known role in invasion in other cells and the potential clinical availability of FAK inhibitors, it is important to determine if FAK contributes to synovial fibroblast invasion and inflammatory arthritis.

Methods

After treatment with FAK inhibitors, invasiveness of human rheumatoid synovial fibroblasts was determined with Matrigel invasion chambers. Migration and focal matrix degradation, two components of cellular invasion, were assessed in FAK-inhibited rheumatoid synovial fibroblasts by transwell assay and microscopic examination of fluorescent gelatin degradation, respectively. Using mice with tumor necrosis factor α (TNFα)–induced arthritis in which fak could be inducibly deleted, invasion and migration by FAK-deficient murine arthritic synovial fibroblasts were determined as described above and arthritis was clinically and pathologically scored in FAK-deficient mice.

Results

Inhibition of FAK in human rheumatoid synovial fibroblasts impaired cellular invasion and migration. Focal matrix degradation occurred both centrally and at focal adhesions, the latter being a novel site for matrix degradation in synovial fibroblasts, but degradation was unaltered with FAK inhibitors. Loss of FAK reduced invasion in murine arthritic synovial fibroblasts, but not migration or TNFα-induced arthritis severity and joint erosions.

Conclusions

FAK inhibitors reduce synovial fibroblast invasion and migration, but synovial fibroblast migration and TNFα-induced arthritis do not rely on FAK itself. Thus, inhibition of FAK alone is unlikely to be sufficient to treat inflammatory arthritis, but current drugs that inhibit FAK may inhibit multiple factors, which could increase their efficacy in rheumatoid arthritis.     

Choline kinase beta is required for normal endochondral bone formation

Background

Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb^−/− mice display neonatal forelimb bone deformations.

Methods

To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb^−/− mice.

Results

The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb^−/− mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb^−/− mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb^−/− mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb^−/− mice contained fewer osteoclasts along the cartilage/bone interface.

Conclusions

Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice.

General Significance

Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.     

p85 beta-PIX is required for cell motility through phosphorylations of focal adhesion kinase and p38 MAP kinase

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.     

NF-kappaB, but not p38 MAP kinase, is required for TNF-alpha-induced expression of cell adhesion molecules in endothelial cells

In response to inflammation stimuli, tumor necrosis factor-alpha (TNF-alpha) induces expression of cell adhesion molecules (CAMs) in endothelial cells (ECs). Studies have suggested that the nuclear factor-kappaB (NF-kappaB) and the p38 MAP kinase (p38) signaling pathways play central roles in this process, but conflicting results have been reported. The objective of this study is to determine the relative contributions of the two pathways to the effect of TNF-alpha. Our initial data indicated that blockade of p38 activity by chemical inhibitor SB203580 (SB) at 10 microM moderately inhibited TNF-alpha-induced expression of three types of CAMs; ICAM-1, VCAM-1 and E-selectin, indicating that p38 may be involved in the process. However, subsequent analysis revealed that neither 1 microM SB that could completely inhibit p38 nor specific knockdown of p38alpha and p38beta with small interference RNA (siRNA) had an apparent effect, indicating that p38 activity is not essential for TNF-alpha-induced CAMs. The most definitive evidence to support this conclusion was from the experiments using cells differentiated from p38alpha knockout embryonic stem cells. We could show that deletion of p38alpha gene did not affect TNF-alpha-induced ICAM-1 and VCAM-1 expression when compared with wild-type cells. We further demonstrated that inhibition of NF-kappaB completely blocked TNF-alpha-induced expression of ICAM-1, VCAM-1 and E-selectin. Taken together, our results clearly demonstrate that NF-kappaB, but not p38, is critical for TNF-alpha-induced CAM expression. The inhibition of SB at 10 microM on TNF-alpha-induced ICAM-1, VCAM-1 and E-selectin is likely due to the nonspecific effect of SB.     

Protein kinase C beta is required for human monocyte chemotaxis to MCP-1

Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused approximately 80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKCalpha and PKCbeta. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKCbeta-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKCbeta-sense ODN and PKCalpha-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR2, indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKCbeta is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKCbeta isoform in this important process.     

SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.     

DNA-Dependent protein kinase is not required for efficient lentivirus integration

How DNA is repaired after retrovirus integration is not well understood. DNA-dependent protein kinase (DNA-PK) is known to play a central role in the repair of double-stranded DNA breaks. Recently, a role for DNA-PK in retroviral DNA integration has been proposed (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Reduced transduction efficiency and increased cell death by apoptosis were observed upon retrovirus infection of cultured scid cells. We have used a human immunodeficiency virus (HIV) type 1 (HIV-1)-derived lentivirus vector system to further investigate the role of DNA-PK during integration. We measured lentivirus transduction of scid mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells. These cells are deficient in the catalytic subunit of DNA-PK and in Ku, the DNA-binding subunit of DNA-PK, respectively. At low vector titers, efficient and stable lentivirus transduction was obtained, excluding an essential role for DNA-PK in lentivirus integration. Likewise, the efficiency of transduction of HIV-derived vectors in scid mouse brain was as efficient as that in control mice, without evidence of apoptosis. We observed increased cell death in scid MEF and xrs-5 or xrs-6 cells, but only after transduction with high vector titers (multiplicity of infection [MOI], >1 transducing unit [TU]/cell) and subsequent passage of the transduced cells. At an MOI of <1 TU/cell, however, transduction efficiency was even higher in DNA-PK-deficient cells than in control cells. Taken together, the data suggest a protective role of DNA-PK against cellular toxicity induced by high levels of retrovirus integrase or integration. Another candidate cellular enzyme that has been claimed to play an important role during retrovirus integration is poly(ADP-ribose) polymerase (PARP). However, no inhibition of lentivirus vector-mediated transduction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, was observed. In conclusion, DNA-PK and PARP are not essential for lentivirus integration.     

Src SH2 arginine 175 is required for cell motility: specific focal adhesion kinase targeting and focal adhesion assembly function

Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.     

TLR4 is required for the obesity-induced pancreatic beta cell dysfunction

Obesity is an important inducing factor for type 2 diabetes. However, the mechanism underlying high-fat-（HF） diet- induced obesity in pancreatic beta cell dysfunction is still unclear. Toll-like receptor-4 （TLR4） is a key mediator of innate immunity. To investigate the effects of TLR4 in obesity-induced pancreatic beta cell dysfunction, we used male diabetic （db/db）, obese （ob/ob） mice, TLR4-wild type （WT）, and TLR4-knockout mice that were fed with normal diet or HF diet for 24 weeks. Immunostaining of TLR4 and TLR4 mRNA level in pancreatic islet were assessed. The results from biological characteristics, glucose tolerance test, insulin tolerance test, and insulin release test showed that the function of pancreatic islet was impaired in HF-fed TLR4 WT mice, but was protected in HF-fed TLR4 deficient （TLR4-/-） mice. By electron microscope detection, we observed that beta cell insulin secretory vesicles increased in HF-fed TLR4 WT mice. Ultrastructure of beta cell in HF-fed TLR4-/- mice was similar to that in normal chow diet-fed TLR4 WT mice. Then, glucose-stimulated insulin secretion assay by using primary pancreatic islet showed that the secretion function of pancreatic islet in HF-fed TLR4-/- mice was better than that in HF-fed TLR4 WT mice. Furthermore, in HF-fed TLR4-/- mice, the mRNA levels of IL-6, TNF-α, and MCP-1 genes in pancreatic islet were sig- nificantly lower than those in HF-fed TLR4 WT mice. Consistent with the change in gene expression, HF-fed TLR4 WT mice but not HF-fed TLR4-/- mice exhibited macro- phage invasion in pancreatic island. Taken together, our data indicated that HF diet-induced obesity can stimulate the up-regulation of TLR4 locating on the surface of pancreatic beta cell, and subsequently lead to the recruitment of macro- phage into pancreatic islet, which finally results in pancreatic beta cell dysfunction. This process is a possible mechanism involved in obesity-induced pancreatic beta cell dysfunction.     

The Rac exchange factor Tiam1 is required for the establishment and maintenance of cadherin-based adhesions

Rho family proteins are essential for the formation of adherens junctions, which are required for the maintenance of epithelial integrity. Activated Rac and the Rac exchange factor Tiam1 have been shown to promote the formation of adherens junctions and the accompanying induction of an epithelioid phenotype in a number of cell lines. Here we show that Madin-Darby canine kidney II cells in which Tiam1 was down-regulated using short interfering RNA disassembled their cadherin-based adhesions and acquired a flattened, migratory, and mesenchymal morphology. In addition, the expression of E1A in mesenchymal V12Ras-transformed Madin-Darby canine kidney II cells led simultaneously to the up-regulation of the Tiam1 protein, the activation of Rac, the formation of cadherin-based adhesions, and reversion to an epithelial phenotype. This finding suggests that E1A induces an epithelial morphology through the up-regulation of Tiam1 and, thereby, the activation of Rac and the formation of cadherin-based adhesions. Indeed, we found that E1A is able to induce an epithelial-like morphology accompanied by the formation of cadherin-based adhesions only in wild-type but not in Tiam1-deficient primary mouse embryonic fibroblasts. These studies indicate that the Rac activator Tiam1 is essential for the formation as well as the maintenance of cadherin-based adhesions.     

A novel ubiquitin-like domain in IkappaB kinase beta is required for functional activity of the kinase

Activation of NF-kappaB requires two highly related kinases named IKKalpha and IKKbeta that share identity in the nature and positioning of their structural domains. Despite their similarity, the kinases are functionally divergent, and we therefore sought to identify any structural features specific for IKKalpha or IKKbeta. We performed bioinformatics analysis, and we identified a region resembling a ubiquitin-like domain (UBL) that exists only in IKKbeta and that we named the UBL-like domain (ULD). Deletion of the ULD rendered IKKbeta catalytically inactive and unable to induce NF-kappaB activity, and overexpression of only the ULD dose-dependently inhibited tumor necrosis factor-alpha-induced NF-kappaB activity. The ULD could not be functionally replaced within IKKbeta by ubiquitin or the corresponding region of IKKalpha, whereas deletion of the equivalent section of IKKalpha did not affect its catalytic activity against IkappaBalpha or its activation by NF-kappaB-inducing kinase. We identified five residues conserved among the larger family of UBL-containing proteins and IKKbeta, and alanine scanning revealed that the leucine at position 353 (Leu(353)) is absolutely critical for IKKbeta-induced NF-kappaB activation. Most intriguingly, the L353A mutant was catalytically active but, unlike wild-type IKKbeta, formed a stable complex with the NF-kappaB p65 subunit. Our findings therefore establish the ULD as a critical functional domain specific for IKKbeta that might play a role in dissociating IKKbeta from p65.     

IKK beta is required for peripheral B cell survival and proliferation

NF-kappaB activity in mammalian cells is regulated through the IkappaB kinase (IKK) complex, consisting of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma). Targeted deletion of Ikkbeta results in early embryonic lethality, thus complicating the examination of IKKbeta function in adult tissues. Here we describe the role of IKKbeta in B lymphocytes made possible by generation of a mouse strain that expresses a conditional Ikkbeta allele. We find that the loss of IKKbeta results in a dramatic reduction in all peripheral B cell subsets due to associated defects in cell survival. IKKbeta-deficient B cells are also impaired in mitogenic responses to LPS, anti-CD40, and anti-IgM, indicating a general defect in the ability to activate the canonical NF-kappaB signaling pathway. These findings are consistent with a failure to mount effective Ab responses to T cell-dependent and independent Ags. Thus, IKKbeta provides a requisite role in B cell activation and maintenance and thus is a key determinant of humoral immunity.     

Protein kinase C is required for responses to T cell receptor ligands but not to interleukin-2 in T cells

We have tested the role of protein kinase C in mRNA expression and T cell proliferation mediated through the T cell receptor and through the interleukin-2 (IL-2) receptor. Chronic treatment of a mouse T cell clone with phorbol esters caused a complete loss of protein kinase C activity and a concomitant loss of proliferation to T cell receptor ligands (antigen, lectins, antireceptor antibodies). In contrast, kinase C-depleted T cells retained the ability to proliferate to IL-2. Loss of the T cell receptor response was not due to decreased cell surface expression of receptor or impairment of early receptor function (phosphatidylinositol turnover, calcium mobilization). Kinase C-depleted T cells showed no induction of mRNAs for activation-associated genes on exposure to the T cell receptor ligand Concanavalin A; expression of a subset of the same mRNAs in response to IL-2 was unaffected. We conclude that kinase C is required for mRNA expression and subsequent proliferation mediated through the T cell receptor pathway but is not involved in mRNA expression and proliferation in response to IL-2.     

EphA4 is required for cell adhesion and rhombomere-boundary formation in the zebrafish

The formation of boundaries between or within tissues is a fundamental aspect of animal development. In the developing vertebrate hindbrain, boundaries separate molecularly and neuroanatomically distinct segments called rhombomeres. Transplantation studies have suggested that rhombomere boundaries form by the local sorting out of cells with different segmental identities. This sorting-out process has been shown to involve repulsive interactions between cells expressing an Eph receptor tyrosine kinase, EphA4, and cells expressing its ephrinB ligands. Although a model for rhombomere-boundary formation based on repulsive Eph-ephrin signaling is well established in the literature, the predictions of this model have not been tested in loss-of-function experiments. Here, we eliminate EphA4 and ephrinB2a proteins in zebrafish with antisense morpholinos (MO) and find that rhombomere boundaries are disrupted in EphA4MO embryos, consistent with a requirement for Eph-ephrin signaling in boundary formation. However, in mosaic embryos, we observe that EphA4MO cells and EphA4-expressing cells sort from one another, an observation that is not predicted by the Eph-ephrin repulsion model but instead suggests that EphA4 promotes cell adhesion within the rhombomeres in which it is expressed. Differential cell adhesion is known to be an effective mechanism for cell sorting. We therefore propose that the well-known EphA4-dependent repulsion between rhombomeres operates in parallel with the EphA4-dependent adhesion within rhombomeres described here to drive the cell sorting that underlies rhombomere-boundary formation.