Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

The active digestion of uniparental chloroplast DNA in a single zygote of Chlamydomonas reinhardtii is revealed by using the optical tweezer

The non-Mendelian inheritance of organelle genes is a phenomenon common to almost all eukaryotes, and in the isogamous alga Chlamydomonas reinhardtii, chloroplast (cp) genes are transmitted from the mating type positive (mt+) parent. In this study, the preferential disappearance of the fluorescent cp nucleoids of the mating type negative (mt−) parent was observed in living young zygotes. To study the change in cpDNA molecules during the preferential disappearance, the cpDNA of mt+ or mt− origin was labeled separately with bacterial aadA gene sequences. Then, a single zygote with or without cp nucleoids was isolated under direct observation by using optical tweezers and investigated by nested PCR analysis of the aadA sequences. This demonstrated that cpDNA molecules are digested completely during the preferential disappearance of mt− cp nucleoids within 10 min, whereas mt+ cpDNA and mitochondrial DNA are protected from the digestion. These results indicate that the non-Mendelian transmission pattern of organelle genes is determined immediately after zygote formation.

Constitutive and impaired signaling of leptin receptors containing the Gln → Pro extracellular domain fatty mutation

Leptin (OB), an adipocyte-secreted circulating hormone, and its receptor (OB-R) are key components of an endocrine loop that regulates mammalian body weight. In this report we have analyzed signal transduction activities of OB-R containing the fatty mutation [OB-R(fa)], a single amino acid substitution at position 269 (Gln → Pro) in the OB-R extracellular domain that results in the obese phenotype of the fatty rat. We find that this mutant receptor exhibits both ligand-independent transcriptional activation via interleukin 6 and hematopoietin receptor response elements and ligand-independent activation of signal transducer and activator of transcription (STAT) proteins 1 and 3. However, OB-R(fa) is unable to constitutively activate STAT5B and is highly impaired for ligand induced activation of STAT5B compared with OB-R(wt). Introduction of the fatty mutation into a OB-R/G-CSF-R chimera generates a receptor with constitutive character that is similar but distinct from that of OB-R(fa). Constitutive mutant OB-R(fa) receptor signaling is repressed by coexpression of OB-R(wt). The implications of an extracellular domain amino acid substitution generating a cytokine receptor with a partially constitutive phenotype are discussed both in terms of the mechanism of OB-R triggering and the biology of the fatty rat.

A peptide derived from an extracellular domain selectively inhibits receptor internalization: Target sequences on insulin and insulin-like growth factor 1 receptors

Certain peptides derived from the α1 domain of the major histocompatibility class I antigen complex (MHC-I) inhibit receptor internalization, increasing the steady-state number of active receptors on the cell surface and thereby enhancing the sensitivity to hormones and other agonists. These peptides self-assemble, and they also bind to MHC-I at the same site from which they are derived, suggesting that they could bind to receptor sites with significant sequence similarity. Receptors affected by MHC-I peptides do, indeed, have such sequence similarity, as illustrated here by insulin receptor (IR) and insulin-like growth factor-1 receptor. A synthetic peptide with sequence identical to a certain extracellular receptor domain binds to that receptor in a ligand-dependent manner and inhibits receptor internalization. Moreover, each such peptide is selective for its cognate receptor. An antibody to the IR peptide not only binds to IR and competes with the peptide but also inhibits insulin-dependent internalization of IR. These observations, and binding studies with deletion mutants of IR, indicate that the sequence QILKELEESSF encoded by exon 10 plays a key role in IR internalization. Our results illustrate a principle for identifying receptor-specific sites of importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists.

Effects of extracellular Ca2+ concentration on hair-bundle stiffness and gating-spring integrity in hair cells

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca2+ is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca2+ chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca2+ also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca2+ concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca2+ concentration; the maximal stiffness of ≈1200 μN⋅m−1 occurred when bundles were bathed in solutions containing 250 μM Ca2+, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca2+ concentration below ≈100 nM, hair-bundle stiffness fell to ≈200 μN⋅m−1 and no longer exhibited Ca2+-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca2+ concentration.

Proteinase inhibitors I and II from potatoes specifically block UV-induced activator protein-1 activation through a pathway that is independent of extracellular signal-regulated kinases, c-Jun N-terminal kinases, and P38 kinase

Solar UV irradiation is the causal factor for the increasing incidence of human skin carcinomas. The activation of the transcription factor activator protein-1 (AP-1) has been shown to be responsible for the tumor promoter action of UV light in mammalian cells. We demonstrate that proteinase inhibitor I (Inh I) and II (Inh II) from potato tubers, when applied to mouse epidermal JB6 cells, block UV-induced AP-1 activation. The inhibition appears to be specific for UV-induced signal transduction for AP-1 activation, because these inhibitors did not block UV-induced p53 activation nor did they exhibit any significant influence on epidermal growth factor-induced AP-1 transactivation. Furthermore, the inhibition of UV-induced AP-1 activity occurs through a pathway that is independent of extracellular signal-regulated kinases and c-Jun N-terminal kinases as well as P38 kinases. Considering the important role of AP-1 in tumor promotion, it is possible that blocking UV-induced AP-1 activity by Inh I or Inh II may be functionally linked to irradiation-induced cell transformation.

An extracellular signaling component in propagation of astrocytic calcium waves

Focally evoked calcium waves in astrocyte cultures have been thought to propagate by gap-junction-mediated intercellular passage of chemical signal(s). In contrast to this mechanism we observed isolated astrocytes, which had no physical contact with other astrocytes in the culture, participating in a calcium wave. This observation requires an extracellular route of astrocyte signaling. To directly test for extracellular signaling we made cell-free lanes 10–300 μm wide in confluent cultures by deleting astrocytes with a glass pipette. After 4–8 hr of recovery, regions of confluent astrocytes separated by lanes devoid of cells were easily located. Electrical stimulation was used to initiate calcium waves. Waves crossed narrow (<120 μm) cell-free lanes in 15 of 36 cases, but failed to cross lanes wider than 120 μm in eight of eight cases. The probability of crossing narrow lanes was not correlated with the distance from the stimulation site, suggesting that cells along the path of the calcium wave release the extracellular messenger(s). Calculated velocity across the acellular lanes was not significantly different from velocity through regions of confluent astrocytes. Focal superfusion altered both the extent and the direction of calcium waves in confluent regions. These data indicate that extracellular signals may play a role in astrocyte–astrocyte communication in situ.

Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively reflecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.

Extracellular antigen processing and presentation by immature dendritic cells

In antigen presentation to CD4+ T cells, proteins are degraded to peptide fragments and loaded onto class II MHC molecules in a process involving the peptide exchange factors H-2M (murine) or HLA-DM (human). In many antigen-presenting cells these processes occur in intracellular endosomal compartments, where peptides are generated and loaded onto class II MHC proteins for subsequent transport to the surface and presentation to T cells. Here, we provide evidence for an additional antigen-processing pathway in immature dendritic cells (DC). Immature DC express at the cell surface empty or peptide-receptive class II MHC molecules, as well as H-2M or HLA-DM. Secreted DC proteases act extracellularly to process intact proteins into antigenic peptides. Peptides produced by such activity are efficiently loaded onto cell surface class II MHC molecules. Together these elements comprise an unusual extracellular presentation pathway in which antigen processing and peptide loading can occur entirely outside of the cell.

Interaction of Fibroblast Growth Factor-2 (FGF-2) with Free Gangliosides: Biochemical Characterization and Biological Consequences in Endothelial Cell Cultures

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF–2/GM1 interaction occurs with a Kd equal to 6 μM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112–129) and, to a lesser extent, FGF-2(130–155), whereas peptides FGF-2(10–33), FGF-2(39–59), FGF-2(86–96), and the basic peptide HIV-1 Tat(41–60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.

Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55–65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.

Integrin α6Aβ1 Induces CD81-dependent Cell Motility without Engaging the Extracellular Matrix Migration Substrate

It is well established that integrins and extracellular matrix (ECM) play key roles in cell migration, but the underlying mechanisms are poorly defined. We describe a novel mechanism whereby the integrin α6β1, a laminin receptor, can affect cell motility and induce migration onto ECM substrates with which it is not engaged. By using DNA-mediated gene transfer, we expressed the human integrin subunit α6A in murine embryonic stem (ES) cells. ES cells expressing α6A (ES6A) at the surface dimerized with endogenous β1, extended numerous filopodia and lamellipodia, and were intensely migratory in haptotactic assays on laminin (LN)-1. Transfected α6A was responsible for these effects, because cells transfected with control vector or α6B, a cytoplasmic domain α6 isoform, displayed compact morphology and no migration, like wild-type ES cells. The ES6A migratory phenotype persisted on fibronectin (Fn) and Ln-5. Adhesion inhibition assays indicated that α6β1 did not contribute detectably to adhesion to these substrates in ES cells. However, anti-α6 antibodies completely blocked migration of ES6A cells on Fn or Ln-5. Control experiments with monensin and anti-ECM antibodies indicated that this inhibition could not be explained by deposition of an α6β1 ligand (e.g., Ln-1) by ES cells. Cross-linking with secondary antibody overcame the inhibitory effect of anti-α6 antibodies, restoring migration or filopodia extension on Fn and Ln-5. Thus, to induce migration in ES cells, α6Aβ1 did not have to engage with an ECM ligand but likely participated in molecular interactions sensitive to anti-α6β1 antibody and mimicked by cross-linking. Antibodies to the tetraspanin CD81 inhibited α6Aβ1-induced migration but had no effect on ES cell adhesion. It is known that CD81 is physically associated with α6β1, therefore our results suggest a mechanism by which interactions between α6Aβ1 and CD81 may up-regulate cell motility, affecting migration mediated by other integrins.

Defining Extracellular Integrin α-Chain Sites That Affect Cell Adhesion and Adhesion Strengthening without Altering Soluble Ligand Binding

It was previously shown that mutations of integrin α4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in α4β1-dependent cell adhesion. Some reports have suggested that α-chain “EF-hand” sites may interact directly with ligands. However, we show here that mutations of three different α4 “EF-hand” sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on α4β1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin α4 “EF-hand-like” sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.