Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

Glucagon-like peptide-1-(7-36) amide, oxyntomodulin, and glucagon interact with a common receptor in a somatostatin-secreting cell line.

Glucagon-like peptide-1(7-36)amide (tGLP-1), oxyntomodulin (OXM), and glucagon are posttranslational end products of the glucagon gene expressed in intestinal L-cells. In vivo, these peptides are potent inhibitors of gastric acid secretion via several pathways, including stimulation of somatostatin release. We have examined the receptors through which these peptides stimulate somatostatin secretion using the somatostatin-secreting cell line RIN T3. tGLP-1, OXM, and glucagon stimulated somatostatin release and cAMP accumulation in RIN T3 cells to similar maximum levels, with ED50 values close to 0.2, 2, and 50 nM and 0.02, 0.3, and 8 nM, respectively. Binding of [125I]tGLP-1, [125I]OXM, and [125I]glucagon to RIN T3 plasma membranes was inhibited by the three peptides, with relative potencies as follows: tGLP-1 > OXM > glucagon. Whatever the tracer used, the IC50 for tGLP-1 was close to 0.15 nM and was shifted rightward for OXM and glucagon by about 1 and 2-3 orders of magnitude, respectively. Scatchard analyses for the three peptides were compatible with a single class of receptor sites displaying a similar maximal binding close to 2 pmol/mg protein. In the hamster lung fibroblast cell line CCL39 transfected with the receptor for tGLP-1, binding of [125I]tGLP-1 was inhibited by tGLP-1, OXM, and glucagon, with relative potencies close to those obtained with RIN T3 membranes. Chemical cross-linking of [125I]tGLP-1, [125I]OXM, and [125I]glucagon revealed a single band at 63,000 mol wt, the intensity of which was dose-dependently reduced by all three peptides. These data suggest that in the somatostatin-secreting cell line RIN T3, OXM and glucagon stimulate somatostatin release through a tGLP-1-preferring receptor. This suggests that some biological effects, previously described for these peptides, might be due to their interaction with this receptor.

Astrocytic αVβ3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1.

Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to α(V)β(3) integrin in trans eliciting responses in astrocytes. Nonetheless, whether α(V)β(3) integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of α(V)β(3) integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous α(V)β(3) integrin restricted neurite outgrowth. Likewise, α(V)β(3)-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(-) CAD cells. In differentiating primary neurons exposed to α(V)β(3)-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, α(V)β(3)-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by α(V)β(3) integrin. Binding of α(V)β(3)-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that α(V)β(3) integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage.

Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

In common with many other plasma membrane glycoproteins of eukaryotic origin, the promastigote surface protease (PSP) of the protozoan parasite Leishmania contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. The GPI anchor of Leishmania major PSP was purified following proteolysis of the PSP and analyzed by two-dimensional 1H-1H NMR, compositional and methylation linkage analyses, chemical and enzymatic modifications, and amino acid sequencing. From these results, the structure of the GPI-containing peptide was found to be Asp-Gly-Gly-Asn-ethanolamine-PO4-6Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-(1-alkyl-2-acyl-glycerol). The glycan structure is identical to the conserved glycan core regions of the GPI anchor of Trypanosoma brucei variant surface glycoprotein and rat brain Thy-1 antigen, supporting the notion that this portion of GPIs are highly conserved. The phosphatidylinositol moiety of the PSP anchor is unusual, containing a fully saturated, unbranched 1-O-alkyl chain (mainly C24:0) and a mixture of fully saturated unbranched 2-O-acyl chains (C12:0, C14:0, C16:0, and C18:0). This lipid composition differs significantly from those of the GPIs of T. brucei variant surface glycoprotein and mammalian erythrocyte acetylcholinesterase but is similar to that of a family of glycosylated phosphoinositides found uniquely in Leishmania.

Paired activation of two components within muscarinic M3 receptor dimers is required for recruitment of beta-arrestin-1 to the plasma membrane.

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway.

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.

Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.

Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill-defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non-toxic expression. We demonstrate the power of this multicolor 'Wave' marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live-imaging and immunoelectron microscopy. Among other findings, our systematic co-localization analysis revealed that a class of plant Rab1-homologs has a much more extended localization than was previously assumed, and also localizes to trans-Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.

Increased expression of monocarboxylate transporters 1, 2, and 4 in colorectal carcinomas.

Tumour cells are known to be highly glycolytic, thus producing high amounts of lactic acid. Monocarboxylate transporters (MCTs), by promoting the efflux of the accumulating acids, constitute one of the most important mechanisms in the maintenance of tumour intracellular pH. Since data concerning MCT expression in colorectal carcinomas (CRC) are scarce and controversial, the present study aimed to assess the expressions of MCT1, 2, and 4 in a well characterized series of CRC and assess their role in CRC carcinogenesis. CRC samples (126 cases) were analyzed for MCT1, MCT2, and MCT4 immunoexpression and findings correlated with clinico-pathological parameters. Expression of all MCT isoforms in tumour cells was significantly increased when compared to adjacent normal epithelium. Remarkably, there was a significant gain of membrane expression for MCT1 and MCT4 and loss of plasma membrane expression for MCT2 in tumour cells. Plasma membrane expression of MCT1 was directly related to the presence of vascular invasion. This is the larger study on MCT expression in CRC and evaluates for the first time its clinico-pathological significance. The increased expression of these transporters suggests an important role in CRC, which might justify their use, especially MCT1 and MCT4, as targets in CRC drug therapy.

Fast subplasma membrane Ca2+ transients control exo-endocytosis of synaptic-like microvesicles in astrocytes

Astrocytes are the most abundant glial cell type in the brain. Although not apposite for long-range rapid electrical communication, astrocytes share with neurons the capacity of chemical signaling via Ca(2+)-dependent transmitter exocytosis. Despite this recent finding, little is known about the specific properties of regulated secretion and vesicle recycling in astrocytes. Important differences may exist with the neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca(2+) from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We here take advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine epifluorescence and total internal reflection fluorescence imaging to investigate with unprecedented temporal and spatial resolution, the stimulus-secretion coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles (SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in astrocytes proceeds with a time course on the millisecond time scale (tau(exocytosis) = 0.24 +/- 0.017 s; tau(endocytosis) = 0.26 +/- 0.03 s) and (2) exocytosis is controlled by local Ca(2+) microdomains. We identified submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli reaching beneath the plasma membrane and containing SLMVs at which fast (time-to-peak, approximately 50 ms) Ca(2+) events occurred in precise spatial-temporal correlation with exocytic fusion events. Overall, the above characteristics of transmitter exocytosis from astrocytes support a role of this process in fast synaptic modulation.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

Probing the interactions of NK cell receptors with ligand expressed in trans and cis.

Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2D(d) ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor-ligand pairs and simplify the analysis of NK cell receptor variants.

Selectively impaired development of intestinal T cell receptor gamma delta+ cells and liver CD4+ NK1+ T cell receptor alpha beta+ cells in T cell factor-1-deficient mice.

T cell factor-1 (Tcf-1) is a transcription factor that binds to a sequence motif present in several T cell-specific enhancer elements. In Tcf-1-deficient (Tcf-1-/-) mice, thymocyte development is partially blocked at the transition from the CD4-8+ immature single-positive stage to the CD4+8+ double-positive stage, resulting in a marked decrease of mature peripheral T cells in lymph node and spleen. We report here that the development of most intestinal TCR gamma delta+ cells and liver CD4+ NK1.1+TCR alpha beta+ (NK1+T) cells, which are believed to be of extrathymic origin, is selectively impaired in Tcf-1-/- mice. In contrast, thymic and thymus-derived (splenic) TCR gamma delta+ cells are present in normal numbers in Tcf-1-/- mice, as are other T cell subsets in intestine and liver. Collectively, our data suggest that Tcf-1 is differentially required for the development of some extrathymic T cell subsets, including intestinal TCR gamma delta+ cells and liver CD4+ NK1+T cells.

Early and Prolonged Antiretroviral Therapy Is Associated with an HIV-1-Specific T-Cell Profile Comparable to That of Long-Term Non-Progressors.

Background: Intervention with antiretroviral treatment (ART) and control of viral replication at the time of HIV-1 seroconversion may curtail cumulative immunological damage. We have therefore hypothesized that ART maintenance over a very prolonged period in HIV-1 seroconverters could induce an immuno-virological status similar to that of HIV-1 long-term non-progressors (LTNPs).Methodology/Principal Findings: We have investigated a cohort of 20 HIV-1 seroconverters on long-term ART (LTTS) and compared it to one of 15 LTNPs. Residual viral replication and reservoirs in peripheral blood, as measured by cell-associated HIV-1 RNA and DNA, respectively, were demonstrated to be similarly low in both cohorts. These two virologically matched cohorts were then comprehensively analysed by polychromatic flow cytometry for HIV-1-specific CD4(+) and CD8(+) T-cell functional profile in terms of cytokine production and cytotoxic capacity using IFN-gamma, IL-2, TNF-alpha production and perforin expression, respectively. Comparable levels of highly polyfunctional HIV-1-specific CD4(+) and CD8(+) T-cells were found in LTTS and LTNPs, with low perforin expression on HIV-1-specific CD8+ T-cells, consistent with a polyfunctional/non-cytotoxic profile in a context of low viral burden.Conclusions: Our results indicate that prolonged ART initiated at the time of HIV-1 seroconversion is associated with immuno-virological features which resemble those of LTNPs, strengthening the recent emphasis on the positive impact of early treatment initiation and paving the way for further interventions to promote virological control after treatment interruption.