Contact response of cells can mediate morphogenetic pattern formation

Theories of morphogenetic pattern formation have included Turing's chemical prepatterns, mechanochemical interactions, cell sorting, and other mechanisms involving guided motion or signalling of cells. Many of these theories presuppose long-range cellular communication or other controls such as chemical concentration fields. However, the possibility that direct interactions between cells can lead to order and structure has not been seriously investigated in mathematical models. In this paper we consider this possibility, with emphasis on cells that reorient and align with each other when they come into contact. We show that such contact responses can account for the formation of multicellular patterns called parallel arrays. These patterns typically occur in tissue cultures of fibroblasts, and consist of clusters of cells sharing a common axis of orientation. Using predictions of a mathematical model and computer simulations of cell motion and interactions we show that contact responses alone, in the absence of other global controls, can promote the formation of these patterns. We suggest other situations in which patterns may result from direct cellular communication. Previous theories of morphogenesis are briefly reviewed and compared with this proposed mechanism.     

Characterization of sorCS1, an alternatively spliced receptor with completely different cytoplasmic domains that mediate different trafficking in cells

We previously isolated and sequenced murine sorCS1, a type 1 receptor containing a Vps10p-domain and a leucine-rich domain. We now show that human sorCS1 has three isoforms, sorCS1a-c, with completely different cytoplasmic tails and differential expression in tissues. The b tail shows high identity with that of murine sorCS1b, whereas the a and c tails have no reported counterparts. Like the Vps10p-domain receptor family members sortilin and sorLA, sorCS1 is synthesized as a proreceptor that is converted in late Golgi compartments by furin-mediated cleavage. Mature sorCS1 bound its own propeptide with low affinity but none of the ligands previously shown to interact with sortilin and sorLA. In transfected cells, about 10% of sorCS1a was expressed on the cell surface and proved capable of rapid endocytosis in complex with specific antibody, whereas sorCS1b presented a high cell surface expression but essentially no endocytosis, and sorCS1c was intermediate. This is an unusual example of an alternatively spliced single transmembrane receptor with completely different cytoplasmic domains that mediate different trafficking in cells.     

Dextran sulfate can act as an artificial receptor to mediate a type-specific herpes simplex virus infection via glycoprotein B.

Herpes simplex virus (HSV) adsorption to host cells is mediated, at least in part, by the interaction of viral glycoproteins with cell surface glycosaminoglycans such as heparan sulfate and chondroitin sulfate. To investigate the contribution of various cell surface components in the infection pathway, we isolated a mutant cell line, sog9, which is unable to synthesize glycosaminoglycans (B. W. Banfield, Y. Leduc, L. Esford, K. Schubert, and F. Tufaro, J. Virol. 69:3290-3298, 1995). Although HSV-1 and HSV-2 infection of sog9 cells is diminished, the cells are still infected at about 0.5% efficiency, which suggests that these cells normally express at least one nonglycosaminoglycan receptor. In this report, we used sog9 cells to test whether glycosaminoglycan analogs, such as dextran sulfate (DS), could functionally substitute for cellular glycosaminoglycans to initiate HSV infection. We show that high-molecular-weight DS added either prior to or during inoculation stimulated HSV-1 but not HSV-2 infection by up to 35-fold; DS added after viral adsorption had no effect on infection efficiency. Moreover, DS stimulated HSV-1 infection at 4 degrees C, indicating that this compound impinged on an early, energy-independent step in infection. Using radiolabeled virus, we showed that HSV-1 is more efficient than HSV-2 in adsorbing to DS immobilized on microtiter wells. This raised the possibility that only HSV-1 could engage additional receptors to initiate infection in the presence of DS. To determine which viral component(s) facilitated DS stimulation, a panel of intertypic recombinants and deletion mutant viruses was investigated. These assays showed that DS stimulation of infection is mediated primarily by gB-1. Thus, this study provides direct evidence that a principal role for cell surface glycosaminoglycans in HSV infection is to provide an efficient matrix for virus adsorption. Moreover, by using DS as an alternative adsorption matrix (a trans receptor), we uncovered a functional, type-specific interaction of HSV-1 with a cell surface receptor.     

Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin

Intestinal P-glycoprotein, which is encoded by the MDR1 gene, plays an important role in the absorption and presystemic elimination of many xenobiotics. Hence, an understanding of the factors regulating its expression and function is of substantial interest. In addition to genetic factors, exposure to drugs such as rifampin can profoundly affect its expression. So far, the mechanisms by which rifampin induces MDR1 expression are poorly understood. Recent studies demonstrate that the nuclear receptor PXR (pregnane X receptor) is involved in xenobiotic induction of CYP3A4. Because CYP3A4 and MDR1 are often co-induced, we investigated whether a similar mechanism is also involved in MDR1 induction. The human colon carcinoma cell line LS174T was used as an intestinal model to study induction because in these cells the endogenous MDR1 gene is highly inducible by rifampin. The 5'-upstream region of human MDR1 was examined for the presence of potential PXR response elements. Several binding sites were identified that form a complex regulatory cluster at about -8 kilobase pairs. Only one DR4 motif within this cluster is necessary for induction by rifampin. We conclude that induction of MDR1 is mediated by a DR4 motif in the upstream enhancer at about -8 kilobase pairs, to which PXR binds.     

Dynamic larval dispersal can mediate the response of marine metapopulations to multiple climate change impacts

Climate change is having multiple impacts on marine species characterized by sedentary adult and pelagic larval phases, from increasing adult mortality to changes in larval duration and ocean currents. Recent studies have shown impacts of climate change on species persistence through direct effects on individual survival and development, but few have considered the indirect effects mediated by ocean currents and species traits such as pelagic larval duration. We used a density-dependent and stochastic metapopulation model to predict how changes in adult mortality and dynamic connectivity can affect marine metapopulation stability. We analyzed our model with connectivity data simulated from a biophysical ocean model of the northeast Pacific coast forced under current (1998–2007) and future (2068–2077) climate scenarios in combination with scenarios of increasing adult mortality and decreasing larval duration. Our results predict that changes of ocean currents and larval duration mediated by climate change interact in complex and opposing directions to shape local mortality and metapopulation connectivity with synergistic effects on regional metapopulation stability: while species with short larval duration are most sensitive to temperature-driven reduction in larval duration, the response of species with longer larval duration are mostly mediated by changes in both the mean and variance of larval connectivity driven by ocean currents. Our results emphasize the importance of considering the spatiotemporal structure of connectivity in order to predict how the multiple effects of climate change will impact marine populations.     

Mutualism can mediate competition and promote coexistence

Mutualistic interactions are not believed to promote coexistence of competitors because mutualisms produce positive feedbacks on abundances whereas coexistence requires negative feedbacks. Here we show that a mutualism between an anemonefish (Amphiprion) and its sea anemone host mediates the effect of asymmetrical competition for space between the anemonefish and another damselfish (Dascyllus) in a manner that fosters their coexistence. Amphiprion stimulates increases in host area, the shared resource, but social interactions cap the number of anemonefish to two adults per host. Space generated by the mutualism becomes differentially available to Dascyllus because the effectiveness of an anemonefish in excluding its competitor declines with increases in the area it defends. This alters Amphiprion's ratio of per capita intra‐ to interspecific effects and thus facilitates coexistence of the fishes. This mechanism may be prevalent in nature, adding another major pathway by which mutualism can enhance diversity.     

Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.     

Platelet-derived growth factor receptor can mediate tumorigenic transformation by the bovine papillomavirus E5 protein.

We showed previously that the beta receptor for platelet-derived growth factor (PDGF) is constitutively activated in fibroblasts transformed by the 44-amino-acid bovine papillomavirus type 1 (BPV) E5 protein and that the E5 protein and the PDGF receptor exist in a stable complex in E5-transformed fibroblasts. On the basis of these results, we proposed that activation of the PDGF receptor by the BPV E5 protein generates a sustained proliferative signal, resulting in fibroblast transformation. In this study, we used a gene transfer approach to provide functional evidence that the PDGF receptor can mediate transformation by the E5 protein. We show that normal mouse mammary gland (NMuMG) cells, a murine mammary epithelial cell line that does not express PDGF receptors, are not susceptible to transformation by the E5 protein. Coexpression of the PDGF beta receptor and E5 genes in these cells results in markedly increased tyrosine phosphorylation of an immature PDGF receptor species and the formation of a stable complex between the E5 protein and this immature PDGF receptor form. Importantly, introduction of the PDGF receptor gene into NMuMG cells renders them highly susceptible to E5-mediated tumorigenic transformation. In contrast, the E5 protein does not induce transformation via the endogenous epidermal growth factor receptor pathway in these cells. These results demonstrate that the PDGF receptor, a cellular protein with a well-characterized role in the positive control of cell proliferation, can mediate transformation by a DNA virus transforming protein.     

gamma -glutamyltransferase and its isoform mediate an endoplasmic reticulum stress response

Although use of multiple alternative first exons generates unique noncoding 5'-ends for gamma-glutamyltransferase (GGT) cDNAs in several species, we show here that alternative splicing events also alter coding exons in mouse GGT to produce at least four protein isoforms. GGTDelta1 introduces CAG four bases upstream of the primary ATG codon and encodes an active GGT heterodimeric ectoenzyme identical to constitutive GGT cDNA but translational efficiency is reduced 2-fold. GGTDelta2-5 deletes the last eight nucleotides of exon 2 through most of exon 5 in-frame, selectively eliminating residues 96-231 from the amphipathic N-terminal subunit, including four N-glycan consensus sites, while leaving the C-terminal hydrophilic subunit intact. GGTDelta7 introduces 22 bases from intron 7 causing a frameshift and a premature stop codon so a truncated polypeptide is encoded terminating with 14 novel residues but retaining the first 339 residues of the native GGT protein. GGTDelta8-9 deletes the terminal four nucleotides of exon 8 plus all of exon 9 and inserts 24 bases from intron 9 in-frame so the C-terminal subunit of the encoded polypeptide loses residues 401-444 but gains eight internal hydrophobic residues. In contrast to the product of GGTDelta1, those derived from GGTDelta2-5, Delta7, Delta8-9 all lack transferase activity and persist as single-chain glycoproteins retained largely in the endoplasmic reticulum as determined by immunofluorescence microscopy and constitutive endoglycosidase H sensitivity in metabolically labeled cells. The developmental-stage plus tissue-specific regulation of the alternative splicing events at GGTDelta7 and GGTDelta8-9 implies unique roles for these GGT protein isoforms. The ability of the GGTDelta1 and GGTDelta7 to mediate the induction of C/EBP homologous protein-10, CHOP-10, and immunoglobulin heavy chain binding protein, BiP, implicates a specific role for these two GGT protein isoforms in the endoplasmic reticulum stress response.     

Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non-FcgammaR-expressing cells. The data indicate that complexes formed from Ad and anti-Ad neutralizing antibodies, while compromised with respect to infection of non-FcgammaR-expressing target cells, maintain the potential to transfer genes to FcgammaR-expressing cells, with consequent expression of the transgene. The formation of Ad-immune complexes that can target viable virus to antigen-presenting cells may account for the success of Ad-based vaccines administered in the presence of low levels of neutralizing anti-Ad antibody.     

The limits of the cellular capacity to mediate an estrogen response.

While steroid response is generally restricted by the availability of steroid receptors, the theoretical limits of the response are not known. We have constructed a series of cell lines that stably express the estrogen receptor (ER) at levels up to 5,000,000 ERs per cell and employed these cells to explore the limits of the estrogen response. Several reporter genes with estrogen response elements upstream of the herpes thymidine kinase promoter showed hyperbolic saturation kinetics with increasing ER. Maximum response was 10 times that seen in cell lines with receptor titers comparable to physiological levels. Half-maximal responses required 500,000 receptors per cell, and cells with 5,000,000 ERs showed greater than 90% maximum induction. Estradiol dose-response studies indicated that the receptors are limiting below 500,000 ERs per cell, but at higher ER titers there are spare receptors. In contrast to most reporters, the widely used reporter pA2-CAT, which has 200 base pairs of Xenopus vitellogenin DNA between the response element and the promoter, showed squelching at ER levels beyond 500,000 per cell. Cell lines that expressed ER above this level activated pA2-CAT with a distorted hormone dependence, where saturating ligand concentrations were inhibitory. All reporters displayed squelching when the ER was provided by transient transfection at a level that we judge is 20,000,000 per cell by extrapolation from the behavior of stable cell lines. These findings suggest that saturation of the cellular capacity to mediate an estrogen response and ER-dependent squelching occur at receptor titers well above those encountered in nature. If current models of steroid hormone action are correct, the findings also imply that estrogen response elements are occupied to very small extents under normal conditions.     

Saliva can mediate HIV-1-specific antibody-dependent cell-mediated cytotoxicity

HIV is not usually transmitted by saliva from HIV-1-infected individuals. Antiviral substances in saliva responsible for this may include HIV-1-specific antibody-dependent cell-mediated cytotoxicity (ADCC). We evaluated saliva ADCC titers of 62 HIV-1-infected women from the Women's Interagency HIV Study (WIHS) and 55 uninfected individuals. HIV-1-infected women were less likely to have ADCC activity in saliva than in serum or cervical lavage fluid (CVL). 24% of HIV-1-positive women and a similar percentage of uninfected women had HIV-1-specific saliva ADCC activity. A significant amount of saliva ADCC activity in infected women was HIV-gp120-specific. These studies demonstrate that HIV-specific ADCC activity can be present in saliva. This activity may contribute to host defence against initial infection with HIV.     

Envelope glycoproteins of hantavirus can mediate cell-cell fusion independently

Hantaviruses (HTVs) are enveloped viruses and can induce low PH-dependent cell fusion. In this report we molecularly cloned viral glycoproteins (GPs) cDNA and nucleocapsid (NP) cDNA of two strains of Hantaan virus and one strain of Seoul virus and expressed in Vero E6 cells under control of a CMV promoter. The examinations of viral gene expressions were carried out by IFA and immune-precipitation. After treatment with low PH (PH 5.8) medium the syncytium were observed in the cells transfected with the GPs clones while in the cells transfected with the NP clones we did not find this phenomenon. Furthermore cotransfection of the NP and GPs did not enhance fusion activity. Treatment with anti-GP monoclonal antibodies could inhibit fusion activity whereas the antibodies against NP could not. These results indicated that GPs can mediate cell-cell fusion independently.     

Activin receptor-like kinase 2 can mediate atrioventricular cushion transformation

The development of enteric and sympathetic neurons from neural crest precursor cells is regulated by signals produced by the embryonic environments to which the cells migrate. Bone morphogenetic proteins (BMPs) are present in the developing embryo and act to induce neuronal differentiation and noradrenergic properties of neural crest cells. We have investigated the role of BMP2 in regulating the appearance of distinct populations of autonomic neurons from postmigratory, HNK-1-positive neural crest precursor cells. BMP2 promotes neuronal differentiation of sympathetic and enteric precursor cells isolated from E14.5 rat. The effects of BMP2 change over time, resulting in a decrease in neuron number that can be attributed to apoptotic cell death. BMP2-dependent neuron death is rescued by gut-derived factors that provide trophic support to maturing neurons, indicating that BMP2 regulates the acquisition of trophic dependence of developing peripheral neurons. In addition to regulating neuron number, BMP2 promotes both panneuronal maturation and the acquisition of an enteric phenotype, as measured by lineage-specific changes in the expression of tyrosine hydroxylase and MASH-1. While BMP2 is sufficient to induce neuronal differentiation and panneuronal development, these results suggest that additional factors in the environment must collaborate with BMP2 to promote the final noradrenergic phenotype of sympathetic neurons.