Guidance of cortical radial migration by gradient of diffusible factors

During the development of cerebral cortex, newborn pyramidal neurons originated from the ventricle wall migrate outwardly to the superficial layer of cortex under the guidance of radial glial filaments. Whether this radial migration of young neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the ventricular zone is entirely unknown, a potential guidance mechanism that has long been overlooked. Our recent study showed that a guidance molecule semaphorin-3A, which is expressed in descending gradient across cortical layers, may serve as a chemoattractive guidance signal for radial migration of newborn cortical neurons toward upper layers. We hypothesize the existence of four groups of extracellular factors that can guide the radial migration of young neurons: (1) attractive factors expressing in superficial layers of cortex, (2) repulsive factors enriched in the ventricular zone, (3) pro-migratory factors uniformly expressed in all cortical layers and (4) stop signals locally expressed in the outmost layer of cortex.Key words: radial migration, cortex, guidance, semaphorin, diffusible factors, growth coneThe mammalian cerebral cortex has the typical laminar structure, the formation of which is essential for neurons in each cortical layer to establish the specific input and output connections with other brain regions. The development of the cortical laminar structure is known to involve the well-coordinated radial migration of newborn pyramidal neurons during development.¹ After young neurons are generated from the ventricular zone (VZ) and subventricular zone (SVZ), they leave their birthplace and migrate along radial glial filaments toward the surface of cortical plate (CP), crossing existing cortical layers composed of earlier born neurons and eventually settling down beneath the marginal zone (MZ, layer I).^1–3 It is generally accepted that the adhesion between neurons and radial glial filaments provides the directionality for these young neurons, and the targeting of neurons to specific lamina was controlled by the selective detachment of migrating neurons from radial glial fibers upon reaching the designated cortical layer.^2,3 However, we believe that the radial glial fibers can only serve as the adhesive scaffold for migrating neurons and constrain their migration in the radial dimension; it remains an open question regarding the nature of the signals that cause newborn neurons to migrate consistently outward along the fiber rather than inward. Whether the radial migration of cortical neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the VZ is entirely unknown, a potential guidance mechanism that has long been overlooked.Recently we found that the radial migration of layer II/III cortical neurons during development is guided by an extracellular guidance molecule semaphorin-3A (Sema3A).⁴ We observed that Sema3A is expressed in a descending gradient across the cortical layers, whereas its receptor neuropilin-1 (NP1) is expressed at a high level in migrating neurons. By in utero electroporation, we were able to monitor the migration of a subpopulation of cortical neurons in their native environment and examine the effect of perturbing Sema3A signaling. We found that downregulation or conditional knockout of NP1 in young neurons impeded their radial migration with severe misorientation of affected neurons during their migration without altering their cell fate. Studies in cultured cortical slices further showed the requirement of the endogenous gradient of Sema3A for the proper migration of newborn neurons. Results from transwell chemotaxis assays in dissociated culture of newborn cortical neurons also supported the notion that Sema3A attracts the migration of these neurons through the receptor NP1. Thus, Sema3A may serve as a chemoattractive guidance signal for the radial migration of newborn cortical neurons toward upper layers. This is the first demonstration that radial migration of cortical neurons is guided by gradient of extracellular guidance factors. This study also suggests that guidance factors may guide the radial migration by their actions on the growth cone of the leading process of migrating neurons, via mechanisms similar to that found for their actions on axon guidance and dendritic orientation, followed by long-range cytoplasmic signaling that coordinates the forward motility of the entire neuron.⁵In this study, we have only observed an attractive effect of Sema3A in the radial migration of the layer II/III cortical neurons. However, to form the highly ordered laminar structure of the cortex, the entire process of neuronal migration is likely to depend on coordinated actions of multiple factors in the developing cortex, including other semaphorin family members and other guidance molecules, e.g., slits⁶ and ephrins,⁷ which are also expressed in the CP. We hypothesize that four groups of extracellular factors orchestrate to promote the proper radial migration and cortical lamination: (1) factors that are expressed in superficial layers of cortex and in a descending gradient, like Sema3A, may attract the upward migration of newborn neurons (attractive factors), (2) factors enriched in the VZ may exert repulsive action and help to “push” newborn neurons out of their birthplace (repulsive factors), (3) those factors widely expressed in all cortical layers may promote the motility of migrating neurons (pro-migratory factors) and (4) Some repulsive cues may be locally expressed in the superficial layer of cortex to prevent the over migration of neurons when they have arrived at the outmost layer (stop signal). Under the guidance of these four groups of factors, newborn neurons migrate all the way from VZ to the outmost layer of CP and then settle down. One of our recent tasks is to try to identify these four groups of factors.If the radial migration and cortical lamination are guided by diffusible factors, why is radial glial system necessary for this migration process? In other words, why earlier-born neurons in different layers cannot provide the supportive adhesion to young neurons during their radial migration? A potential explanation is that neurons in cortex undergo maturation after terminating their migration, accompanying with changes in their expression profiles of adhesion ligands, and become less and less supportive to the neuronal migration. In contrast, as a kind of cortical progenitor cells, radial glial cells maintain a relatively ‘young’ state and continue to express supportive adhesion ligands over a very long developmental stage. Thus, only the radial glial filament is capable of providing a bridge for newborn neurons to migrate over a very long distance across the non-permissive cell layers. In summary, we believe that during the cortical radial migration, signals from diffusible factors override the adhesive signal from radial glial fibers to promote the appropriate migration and placement of newborn neurons.? Open in a separate windowFigure 1A schematic diagram for the guidance of cortical radial migration by diffusible factors. (A) A model for the distribution of four groups of guidance factors in developing cortex. Radial glial filaments are shown in red, young neurons are in green. There may exist a descending gradient of attractive factors in upper cortical layers (yellow) and an ascending gradient of repulsive factors (blue) near the ventricular zone (VZ). Stop signals (purple) may come from the surface of cortex, and pro-migratory factors (dots) may be widely distributed. (B) Representative image of EGFP-labeled neurons migrating along radial glial filaments in the cortical tissue of E20 mouse. Sections were counterstained with DAPI (Red). Scale bar, 100 µm.     

Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of alpha3 chain

The basement membrane protein laminin-5 promotes cell adhesion and migration. The carboxyl-terminal G3 domain in the alpha3 chain is essential for the unique activity of laminin-5. To investigate the function of the G3 domain, we prepared various recombinant laminin-5 forms with a partially deleted or mutated G3 domain. The deletion of the carboxyl-terminal 28 amino acids (region III) markedly decreased the cell adhesion activity with a slight loss of the cell motility activity toward BRL and EJ-1 cells. This change was attributed to the loss of Lys-Arg-Asp sequence. Further deletion of 83 amino acids (region II) led to almost complete loss of the cell motility activity. All charged amino acid residues tested in this region were not responsible for the activity loss. These results suggest that the G3 domain contains two distinct regions that differently regulate cell adhesion and migration. Analysis of laminin-5 receptors showed that integrins alpha3beta1, alpha6beta1, and alpha6beta4 had different but synergistic effects on cell adhesion and migration on laminin-5. However, the structural change of the G3 domain appeared not to change integrin specificity. The present study demonstrates that the G3 domain in laminin-5 plays a central role to produce different biological effects on cells.     

Crystal structure of the ligand binding domain of netrin G2

Netrin G proteins represent a small family of synaptic cell adhesion molecules related to netrins and to the polymerization domains of laminins. Two netrin G proteins are encoded in vertebrate genomes, netrins G1 and G2, which are known to bind the leucine-rich repeat proteins netrin G ligand (NGL)-1 and NGL-2, respectively. Netrin G proteins share a common multi-domain architecture comprising a laminin N-terminal (LN) domain followed by three laminin epidermal growth factor-like (LE) domains and a C′ region containing a glycosylphosphatidylinositol anchor. Here, we use deletion analysis to show that the LN domain region of netrin Gs contains the binding site for NGLs to which they bind with 1:1 stoichiometry and sub-micromolar affinity. Netrin Gs are alternatively spliced in their LE domain regions, but the binding region, the LN domain, is identical in all splice forms. We determined the crystal structure for a fragment comprising the LN domain and domain LE1 of netrin G2 by sulfur single-wavelength anomalous diffraction phasing and refined it to 1.8 Å resolution. The structure reveals an overall architecture similar to that of laminin α chain LN domains but includes significant differences including a Ca²⁺ binding site in the LN domain. These results reveal the minimal binding unit for interaction of netrin Gs with NGLs, define structural features specific to netrin Gs, and suggest that netrin G alternative splicing is not involved in NGL recognition.     

Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9′10), (ii) an Arg to Ala synergy site mutation (FnIII9^R→^A10), (iii) a two-Gly (FnIII9^2G10) insertion, and (iv) a four-Gly (FNIII9^4G10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9′10 and FnIII9^2G10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9′10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9′10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn.     

Stimulation of endothelial cell migration in culture by ladsin,a laminin-5-like cell adhesion protein

Summary Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin α3β1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.     

Integrins: versatile receptors controlling melanocyte adhesion, migration and proliferation

From the onset of melanocyte specification from the neural crest, throughout their migration during embryogenesis and until they reside in their niche in the basal keratinocyte layer, melanocytes interact in dynamic ways with the extracellular environment of the growing embryo. To recognize and to adhere to their environment, melanocytes depend on heterodimeric cell surface receptors of the family of integrins. In addition to the control of adhesive interactions between melanocytes and the extracellular matrix scaffold secreted by fibroblasts and keratinocytes, the integrin receptors allow cells also to sense the mechanical condition of the extracellular environment, responding by intracellular signaling, triggering cell survival, proliferation or migration events. In this review, we summarize the recently emerged concepts that explain integrin-dependent adhesion and how this adhesion system interfaces with integrin-dependent signaling events. The gained information will help to understand melanocyte behavior in pathological situations such as melanoma growth and metastasis formation.     

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells

Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs.     

Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation

Pancreatic cancer is characterized by aggressive local invasion and early metastasis formation. Active migration of the pancreatic cancer cells is essential for these processes. We have shown previously that the pancreatic cancer cells lines CFPAC1 and IMIM-PC2 show high migratory activity, and we have investigated herein the reason for this observation. Cell migration was assessed using a three-dimensional, collagen-based assay and computer-assisted cell tracking. The expression of receptor tyrosine kinases was determined by flow-cytometry and cytokine release was measured by an enzyme-linked immunoassay. Receptor function was blocked by antibodies or pharmacological enzyme inhibitors. Both cells lines express the epidermal growth factor receptor (EGFR) as well as its family-member ErbB2 and the platelet-derived growth factor receptor (PDGFR)α, whereas only weak expression was detected for ErbB3 and no expression of PDGFRβ. Pharmacological inhibition of the EGFR or ErbB2 significantly reduced the migratory activity in both cell lines, as did an anti-EGFR antibody. Interestingly, combination of the latter with an anti-PDGFR antibody led to an even more pronounced reduction. Both cell lines release detectable amounts of EGF. Thus, the high migratory activity of the investigated pancreatic cancer cell lines is due to autocrine EGFR activation and possibly of other receptor tyrosine kinases.     

Migration of connective tissue-derived cells is mediated by ultra-low concentration gradient fields of EGF

The directed migration of cells towards chemical stimuli incorporates simultaneous changes in both the concentration of a chemotactic agent and its concentration gradient, each of which may influence cell migratory response. In this study, we utilized a microfluidic system to examine the interactions between epidermal growth factor (EGF) concentration and EGF gradient in stimulating the chemotaxis of connective tissue-derived fibroblast cells. Cells seeded within microfluidic devices were exposed to concentration gradients established by EGF concentrations that matched or exceeded those required for maximum chemotactic responses seen in transfilter migration assays. The migration of individual cells within the device was measured optically after steady-state gradients had been experimentally established. Results illustrate that motility was maximal at EGF concentration gradients between .01- and 0.1-ng/(mL.mm) for all concentrations used. In contrast, the number of motile cells continually increased with increasing gradient steepness for all concentrations examined. Microfluidics-based experiments exposed cells to minute changes in EGF concentration and gradient that were in line with the acute EGFR phosphorylation measured. Correlation of experimental data with established mathematical models illustrated that the fibroblasts studied exhibit an unreported chemosensitivity to minute changes in EGF concentration, similar to that reported for highly motile cells, such as macrophages. Our results demonstrate that shallow chemotactic gradients, while previously unexplored, are necessary to induce the rate of directed cellular migration and the number of motile cells in the connective tissue-derived cells examined.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

p53 mediates interstitial cystitis antiproliferative factor (APF)-induced growth inhibition of human urothelial cells

Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis, a urinary bladder disorder of unknown etiology that is characterized by chronic pelvic pain. The present study was directed toward uncovering a pathway through which APF signals. Treatment of human urothelial cells with native APF resulted in growth inhibition accompanied by blockade of cell cycle transit and increased p53. Reduced expression of p53 by RNA interference diminished, while ectopic expression of p53 mimicked, the effects of APF. These are the first findings implicating the network of p53 target genes in urothelial defects associated with interstitial cystitis.     

Inhibition of rat glioma cell migration and proliferation by a calix[8]arene scaffold exposing multiple GlcNAc and ureido functionalities

Beta1,4-Galactosyltransferases (beta1,4-GalTase) exposed on the cell surface are involved in cell migration. Specifically, beta1,4-GalTase V is highly expressed in glioma and promotes invasion, growth, and survival of glioma cells. A glycocalix[8]arene exposing N-acetylglucosamine (GlcNAc) residues (compound 1) inhibited rat C6 glioma cell migration as assessed in a scratch wound model. This effect was related to inhibition of focal adhesion kinase phosphorylation, measured by western blot analysis, and specifically observed in the area bordering the scratch wound. Compound 1 inhibited also C6 cell proliferation, an effect unrelated to its ability to interact with GalTase as it was mimicked by different calix[8]arene derivatives, all characterized by multivalency and ureido groups. Compound 1 did not induce apoptotic death, but caused a different distribution of C6 cells within the cell cycle. The results here reported identify compound 1 as a molecule able to exert inhibitory effects on C6 cell migration and proliferation, independently, because of distinct components in its structure.     

Mechanism of hydrogen peroxide-induced keratinocyte migration in a scratch-wound model

Recent studies have shown that low concentrations of H₂O₂ are produced endogenously by nonphagocytes after wounding. We observed that H₂O₂ at such concentrations can stimulate proliferation as well as migration of keratinocytes in a scratch-wound assay. Both wounding and H₂O₂ can induce phosphorylation of ERK1/2 via EGFR, but the activation of ERK1/2 by H₂O₂ is more sustained and can last more than 8 h. Sustained ERK1/2 activation is required for the increased proliferation and migration induced by H₂O₂. The p38 MAPK was also found to be phosphorylated upon treatment with H₂O₂ but it was not required for H₂O₂-induced migration or proliferation. Furthermore, it was observed that there is a cross talk between the ERK1/2 and the p38 pathways whereby inhibition of either pathway can lead to activation of the other. As a result, the motogenic effects of H₂O₂ were further enhanced when p38 was inhibited. Our data are consistent with the view that H₂O₂ may play an important signaling role in wound healing.     

A method for N-terminal de novo sequence analysis of proteins by matrix-assisted laser desorption/ionization mass spectrometry

A novel method for isolation and de novo sequencing of N-terminal peptides from proteins is described. The method presented here combines selective chemical tagging using succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) at the N^α-amino group of peptides after digestion by metalloendopeptidase (from Grifola frondosa) and selective capture procedures using p-phenylenediisothiocyanate resin, by which the N-terminal peptide can be isolated, whether or not it is N-terminally blocked. The isolated N-terminal peptide modified N-terminally with TMPP-Ac-OSu reagent produces a simple fragmentation pattern under tandem mass spectrometric analysis to significantly facilitate sequencing.     

Liver epithelial cell migration induced by epidermal growth factor or transforming growth factor alpha is associated with changes in the gene expression of secreted proteins

Summary Rat liver epithelial cells are induced to migrate by epidermal growth factor (EGF) or transforming growth factor alpha (TGF-α) in serum-free medium supplemented with insulin. Immunohistological staining of the migration tracks containing laminin and fibronectin has allowed a quantitative analysis of the process. The growth factor-induced migration is relatively slow, but very efficient. Between 24 and 48 h after exposure to EGF (or TGF-α), 50 to 70% of the cells have migrated away from their site of initial attachment and spreading. This delayed effect of the interaction of the receptor with its ligands is associated with changes in gene expression, but is not associated with a stimulation of cell proliferation. In serum-free medium supplemented with insulin, the cells secrete six major proteins, as revealed by SDS-polyacrylamide gel electrophoresis. The media of cultures supplemented with insulin plus EGF (or TGF-α) contain in addition two new proteins and an increased amount of fibronectin. One secreted protein is synthesized in significantly reduced amounts. The most conspicuously EGF-induced protein (EIP-1, Mr 47 000) is detected within 2 h, depends on the continued presence of the growth factor, and has not been detected as bound to the substratum. The stringent regulation of EIP-1 suggests that this gene product might participate in the modulation of the changes induced by the growth factor. The system is being used for the further analysis of the regulation of gene expression by EGF and of the migration of normal and neoplastically transformed epithelial cells. This paper is dedicated to the memory of Dr. Luis F. Leloir. A preliminary communication has been presented at the Cold Spring Harbor Meeting on Liver Gene Expression, May 1987. Editor's Statement Mitogen-stimulated gnees are an active area of study with fibroblastic systems. In this paper the approach is extended to epithelial cells and functional correlations are also made.     

Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities

Elevated levels of p130^Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130^Cas promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130^Cas protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130^Cas-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130^Cas exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130^Cas on cell biology and therefore will be the target of future studies.     

Arsenic Inhibits DNA Mismatch Repair by Promoting EGFR Expression and PCNA Phosphorylation

Both genotoxic and non-genotoxic chemicals can act as carcinogens. However, while genotoxic compounds lead directly to mutations that promote unregulated cell growth, the mechanism by which non-genotoxic carcinogens lead to cellular transformation is poorly understood. Using a model non-genotoxic carcinogen, arsenic, we show here that exposure to arsenic inhibits mismatch repair (MMR) in human cells, possibly through its ability to stimulate epidermal growth factor receptor (EGFR)-dependent tyrosine phosphorylation of proliferating cellular nuclear antigen (PCNA). HeLa cells exposed to exogenous arsenic demonstrate a dose- and time-dependent increase in the levels of EGFR and tyrosine 211-phosphorylated PCNA. Cell extracts derived from arsenic-treated HeLa cells are defective in MMR, and unphosphorylated recombinant PCNA restores normal MMR activity to these extracts. These results suggest a model in which arsenic induces expression of EGFR, which in turn phosphorylates PCNA, and phosphorylated PCNA then inhibits MMR, leading to increased susceptibility to carcinogenesis. This study suggests a putative novel mechanism of action for arsenic and other non-genotoxic carcinogens.     

Detection of bovine leukocyte adhesion deficiency by nonisotopic ligase chain reaction

A nonisotopic ligase chain reaction (LCR) assay was developed to detect the mutation (D128G; Shuster et al. (1992) PNAS 89, 9225-9) for bovine leukocyte adhesion deficiency (BLAD). Two sets of diagonally opposed discriminating LCR primers that differentiate the normal and BLAD allele were designed so that the 3′ end of each primer overlapped the D128G mutation. These discriminating primers were synthesized with a 5′ biotin and could be captured using streptavidin-coated microtitre wells. A common set of primers that abut these discriminating primers were also synthesized and 3′-tailed with digoxigenin-ddUTP. Captured LCR products were then detected using antidigoxigenin antibodies coupled to alkaline phosphatase. The assay readout was a chemiluminescent signal generated by the hydrolysis of Lumi-Phos TM 530 and the entire assay including DNA isolation can be completed within 8 h.     

Effects on the conformation of FVIIa by sTF and Ca binding: Studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5 Å for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044–1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.It is known from amidolytic activity measurements that Ca²⁺ binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca²⁺ binding. Interestingly, this Ca²⁺-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.