Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

The pir gene of Erwinia chrysanthemi EC16 regulates hyperinduction of pectate lyase virulence genes in response to plant signals

The plant pathogenic bacterium Erwinia chrysanthemi secretes pectate lyase proteins that are important virulence factors attacking the cell walls of plant hosts. Bacterial production of these enzymes is induced by the substrate polypectate-Na (NaPP) and further stimulated by the presence of plant extracts. The bacterial regulator responsible for induction by plant extracts was identified and purified by using a DNA-binding assay with the promoter region of pelE that encodes a major pectate lyase. A novel bacterial protein, called Pir, was isolated that produced a specific gel shift of the pelE promoter DNA, and the corresponding pir gene was cloned and sequenced. The Pir protein contains 272 amino acids with a molecular mass of 30 kDa and appears to function as a dimer. A homology search indicates that Pir belongs to the IclR family of transcriptional regulators. Pir bound to a 35-bp DNA sequence in the promoter region of pelE. This site overlaps that of a previously described negative regulator, KdgR. Gel shift experiments showed that the binding of either Pir or KdgR interfered with binding of the other protein.

Transduction of Basolateral-to-Apical Signals across Epithelial Cells: Ligand-stimulated Transcytosis of the Polymeric Immunoglobulin Receptor Requires Two Signals

Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA–pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++ from inositol trisphosphate–sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 → S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate

The β-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4+ target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4+ CCR-5+ HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the “nonsyncytium-inducing,” primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the “syncytium-inducing” HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the β-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1β (MIP-1β) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these β-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and β-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.

HSP70 homolog functions in cell-to-cell movement of a plant virus

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.

Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.

Cytoskeletal Reorganization Induced by Engagement of the NG2 Proteoglycan Leads to Cell Spreading and Migration

Cells expressing the NG2 proteoglycan can attach, spread, and migrate on surfaces coated with NG2 mAbs, demonstrating that engagement of NG2 can trigger the cytoskeletal rearrangements necessary for changes in cell morphology and motility. Engagement of different epitopes of the proteoglycan results in distinct forms of actin reorganization. On mAb D120, the cells contain radial actin spikes characteristic of filopodial extension, whereas on mAb N143, the cells contain cortical actin bundles characteristic of lamellipodia. Cells that express NG2 variants lacking the transmembrane and cytoplasmic domains are unable to spread or migrate on NG2 mAb-coated surfaces, indicating that these portions of the molecule are essential for NG2-mediated signal transduction. Cells expressing an NG2 variant lacking the C-terminal half of the cytoplasmic domain can still spread normally on mAbs D120 and N143, suggesting that the membrane-proximal cytoplasmic segment is responsible for this process. In contrast, this variant migrates poorly on mAb D120 and exhibits abnormal arrays of radial actin filaments decorated with fascin during spreading on this mAb. The C-terminal portion of the NG2 cytoplasmic domain, therefore, may be involved in regulating molecular events that are crucial for cell motility.

Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20–100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

Repression of a matrix metalloprotease gene by E1A correlates with its ability to bind to cell type-specific transcription factor AP-2.

Adenovirus E1A 243-amino acid protein can repress a variety of enhancer -linked viral and cellular promoters. This repression is presumed to be mediated by its interaction with and sequestration of p3OO, a transcriptional coactivator. Type IV 72-kDa collagenase is one of the matrix metalloproteases that has been implicated in differentiation, development, angiogenesis, and tumor metastasis. We show here that the cell type-specific transcription factor AP-2 is an important transcription factor for the activation of the type IV 72-kDa collagenase promoter and that adenovirus E1A 243-amino acid protein represses this promoter by targeting AP-2. Glutathione S-transferase-affinity chromatography studies show that the E1A protein interacts with the DNA binding/dimerization region of AP-2 and that the N-terminal amino acids of E1A protein are required for this interaction. Further, E1A deletion mutants which do not bind to p3OO can repress this collagenase promoter as efficiently as the wildtype E1A protein. Because the AP-2 element is present in a variety of viral and cellular enhancers which are repressed by E1A, these studies suggest that E1A protein can repress cellular and viral promoter/enhancers by forming a complex with cellular transcription factors and that this repression mechanism may be independent of its interaction with p3OO.

Anti-idiotypic antibodies mimicking glycoprotein D of herpes simplex virus identify a cellular protein required for virus spread from cell to cell and virus-induced polykaryocytosis.

Glycoprotein D (gD) of herpes simplex virus 1 (HSV-1) is required for stable attachment and penetration of the virus into susceptible cells after initial binding. We derived anti-idiotypic antibodies to the neutralizing monoclonal antibody HD1 to gD of HSV-1. These antibodies have the properties expected of antibodies against a gD receptor. Specifically, they bind to the surface of HEp-2, Vero, and HeLa cells susceptible to HSV infection and specifically react with a Mr 62,000 protein in these and other (143TK- and BHK) cell lines. They neutralize virion infectivity, drastically decrease plaque formation by impairing cell-to-cell spread of virions, and reduce polykaryocytosis induced by strain HFEM, which carries a syncytial (syn-) mutation. They do not affect HSV growth in a single-step cycle and plaque formation by an unrelated virus, indicating that they specifically affect the interaction of HSV gD) with a cell surface receptor. We conclude that the Mr 62,000 cell surface protein interacts with gD to enable spread of HSV-1 from cell to cell and virus-induced polykaryocytosis.

Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness

Growth factors can influence lineage determination of neural crest stem cells (NCSCs) in an instructive manner, in vitro. Because NCSCs are likely exposed to multiple signals in vivo, these findings raise the question of how stem cells would integrate such combined influences. Bone morphogenetic protein 2 (BMP2) promotes neuronal differentiation and glial growth factor 2 (GGF2) promotes glial differentiation; if NCSCs are exposed to saturating concentrations of both factors, BMP2 appears dominant. By contrast, if the cells are exposed to saturating concentrations of both BMP2 and transforming growth factor β1 (which promotes smooth muscle differentiation), the two factors appear codominant. Sequential addition experiments indicate that NCSCs require 48–96 hrs in GGF2 before they commit to a glial fate, whereas the cells commit to a smooth muscle fate within 24 hr in transforming growth factor β1. The delayed response to GGF2 does not reflect a lack of functional receptors; however, because the growth factor induces rapid mitogen-activated protein kinase phosphorylation in naive cells. Furthermore, GGF2 can attenuate induction of the neurogenic transcription factor mammalian achaete-scute homolog 1, by low doses of BMP2. This short-term antineurogenic influence of GGF2 is not sufficient for glial lineage commitment, however. These data imply that NCSCs exhibit cell-intrinsic biases in the timing and relative dosage sensitivity of their responses to instructive factors that influence the outcome of lineage decisions in the presence of multiple factors. The relative delay in glial lineage commitment, moreover, apparently reflects successive short-term and longer-term actions of GGF2. Such a delay may help to explain why glia normally differentiate after neurons, in vivo.

Telomere lengthening and telomerase activation during human B cell differentiation

The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activity in vitro in response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.

c-Myc enhances protein synthesis and cell size during B lymphocyte development

Members of the myc family of nuclear protooncogenes play roles in cell proliferation, differentiation, and apoptosis. Moreover, inappropriate expression of c-myc genes contributes to the development of many types of cancers, including B cell lymphomas in humans. Although Myc proteins have been shown to function as transcription factors, their immediate effects on the cell have not been well defined. Here we have utilized a murine model of lymphomagenesis (Eμ-myc mice) to show that constitutive expression of a c-myc transgene under control of the Ig heavy-chain enhancer (Eμ) results in an increase in cell size of normal pretransformed B lymphocytes at all stages of B cell development. Furthermore, we show that c-Myc-induced growth occurs independently of cell cycle phase and correlates with an increase in protein synthesis. These results suggest that Myc may normally function by coordinating expression of growth-related genes in response to mitogenic signals. Deregulated c-myc expression may predispose to cancer by enhancing cell growth to levels required for unrestrained cell division.