Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42.

The mechanisms by which cells rapidly polarize in the direction of external signals are not understood. Helper T cells, when contacted by an antigen-presenting cell, polarize their cytoskeletons toward the antigen-presenting cell within minutes. Here we show that, in T cells, the mammalian Ras-related GTPase CDC42 (the homologue of yeast CDC42, a protein involved in budding polarity) can regulate the polarization of both actin and microtubules toward antigen-presenting cells but is not involved in other T-cell signaling processes such as those which culminate in interleukin 2 production. Although T-cell polarization appears dispensable for signaling leading to interleukin 2 production, polarization may direct lymphokine secretion towards the correct antigen-presenting cell in a crowded cellular environment. Inhibitor experiments suggest that phosphatidylinositol 3-kinase is required for cytoskeletal polarization but that calcineurin activity, known to be important for other aspects of signaling, is not. Apparent conservation of CDC42 function between yeast and T cells suggests that this GTPase is a general regulator of cytoskeletal polarity in many cell types.

Overexpressed Ly-6A.2 mediates cell-cell adhesion by binding a ligand expressed on lymphoid cells.

The Ly-6 locus encodes several cell surface proteins whose functions are unknown. Although it is hypothesized that these proteins may be receptors, there is no direct evidence that they bind a ligand. Herein we present evidence that Ly-6A.2, a Ly-6 protein expressed on T lymphocytes, binds a ligand expressed on normal thymocytes and splenic B and T cells. We find that transgenic thymocytes that overexpress Ly-6A.2 spontaneously aggregate in culture. This homotypic adhesion requires the overexpression of Ly-6A.2 because it is not observed in cultures of nontransgenic thymocytes. The aggregation of Ly-6A.2 transgenic thymocytes is inhibited by phosphatidylinositol-specific phospholipase C (which removes Ly-6A.2 and other glycosylphosphatidylinositol-anchored proteins from the membrane). Some anti-Ly-6A.2 monoclonal antibodies, including nonactivating ones and Fab' fragments, inhibit this aggregation. In contrast, other anti-Ly-6A.2 monoclonal antibodies increase the aggregation of transgenic but not nontransgenic thymocytes. To further examine whether Ly-6A.2 mediates adhesion (versus inducing another adhesion pathway) reaggregation assays were performed with paraformaldehyde-fixed Tg+ thymocytes. Paraformaldehyde-fixed Tg+ thymocytes reaggregate in culture and this aggregation is also blocked by phosphatidyl-inositol-specific phospholipase C and anti-Ly-6A.2 monoclonal antibodies. These results indicate that the homotypic adhesion of cultured Ly-6A.2 transgenic thymocytes is directly mediated by Ly-6A.2 and, more importantly, strongly suggests that Ly-6A.2 binds a ligand that is expressed on thymocytes. Tg+ thymocytes also bind to nontransgenic thymocytes, B cells, and T cells, indicating that normal cells naturally express the Ly-6A.2 ligand.

Folate receptors targeted to clathrin-coated pits cannot regulate vitamin uptake.

Potocytosis is an endocytic process that is specialized for the internalization of small molecules. Recent studies on the uptake of 5-methyltetrahydrofolate by the folate receptor have suggested that the glycosyl-phosphatidylinositol anchor on this protein causes it to cluster and be internalized by caveolae instead of coated pits. To test this hypothesis directly, we have constructed a chimeric folate receptor that has the glycosyl-phosphatidylinositol anchor replaced with the transmembrane domain and cytoplasmic tail of the low density lipoprotein receptor. The cells with wild-type receptors delivered 5-methyltetrahydrofolate to the cytoplasm more rapidly than did cells expressing the chimeric receptor. This suggests that efficient delivery to the cytoplasm depends on caveolae. In sharp contrast to cells with wild-type folate receptors, cells internalizing folate by clathrin-coated pits were unable to decrease vitamin uptake when they were either folate replete or confluent.

Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.

Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 microM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance.

Human naïve regulatory T-cells feature high steady-state turnover and are maintained by IL-7

Naïve FoxP3-expressing regulatory T-cells (Tregs) are essential to control immune responses via continuous replenishment of the activated-Treg pool with thymus-committed suppressor cells. The mechanisms underlying naïve-Treg maintenance throughout life in face of the age-associated thymic involution remain unclear. We found that in adults thymectomized early in infancy the naïve-Treg pool is remarkably well preserved, in contrast to conventional naïve CD4 T-cells. Naïve-Tregs featured high levels of cycling and pro-survival markers, even in healthy individuals, and contrasted with other circulating naïve/memory CD4 T-cell subsets in terms of their strong γc-cytokine-dependent signaling, particularly in response to IL-7. Accordingly, ex-vivo stimulation of naïve-Tregs with IL-7 induced robust cytokine-dependent signaling, Bcl-2 expression, and phosphatidylinositol 3-kinase (PI3K)-dependent proliferation, whilst preserving naïve phenotype and suppressive capacity. Altogether, our data strongly implicate IL-7 in the thymus-independent long-term survival of functional naïve-Tregs, and highlight the potential of targeting the IL-7 pathway to modulate Tregs in different clinical settings.

IRS-1 regulation in health and disease

The global incidence of diabetes is increasing at epidemic rates. Estimates suggest there are currently 150 million people with diabetes and this number is expected to double in the next 20 years. Type 2 diabetes accounts for 95% of all cases and is characterized in part by impaired sensitivity to insulin or 'insulin resistance'. Defects in the insulin signalling pathways underpin this resistance. In the current article we discuss the regulation of Insulin Receptor Substrate-1 (IRS-1), a protein that plays a pivotal role in insulin signalling and whose function is impaired in subjects with insulin resistance. Coordination of IRS-1 function is multi-faceted, involving phosphorylation of IRS-1 at multiple serine/threonine residues. This controls many aspects of IRS-1, including its interaction with the insulin receptor and subsequent tyrosine phosphorylation, as well as its subcellular distribution and targeting for degradation by the proteasome. Such tight control ensures appropriate transduction and attenuation of the insulin signal, thereby regulating insulin action in healthy individuals. Emerging evidence indicates that `diabetogenic factors' associated with insulin resistance, such as TNFalpha and elevated circulating fatty acids, impact on insulin signalling at the level of IRS-1 serine/threonine phosphorylation. The expression and/or activity of several kinases, such as IkappaB kinase beta (IKKbeta) and salt-induced kinase 2 (SIK2), and the phosphorylation of IRS-1 at key sites, such as Ser307 and Ser789, are increased in states of insulin resistance. Identifying the pathways by which such factors activate these and other kinases, and de. ning the precise roles of specific serine/threonine phosphorylation events in IRS-1 regulation, represent important goals which may eventually provide a rationale for therapeutic intervention.

Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts

The addition of insulin during in vitro culture has beneficial effects on rabbit preimplantation embryos leading to increased cell proliferation and reduced apoptosis. We have previously described the expression of the insulin receptor (IR) and the insulin-responsive glucose transporters (GLUT) 4 and 8 in rabbit preimplantation embryos. However, the effects of insulin on IR signaling and glucose metabolism have not been investigated in rabbit embryos. In the present study, the effects of 170 nM insulin on IR, GLUT4 and GLUT8 mRNA levels, Akt and Erk phosphorylation, GLUT4 translocation and methyl glucose transport were studied in cultured day 3 to day 6 rabbit embryos. Insulin stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) Erk1/2 and levels of IR and GLUT4 mRNA, but not phosphorylation of the phosphatidylinositol 3-kinase-dependent protein kinase, Akt, GLUT8 mRNA levels, glucose uptake or GLUT4 translocation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control.

Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma imembrane following EGF stimulation

Sorting nexins are a large family of proteins that contain the phosphoinositide-binding Phox homology (PX) domain. A number of sorting nexins are known to bind to PtdIns(3)P, which mediates their localization to membranes of the endocytic pathway. We show here that sorting nexin 5 (SNX5) can be recruited to two distinct membrane compartments. In non-stimulated cells, the PX domain was independently targeted to endosomal structures and colocalized with full-length SNX5. The membrane binding of the PX domain was inhibited by the PI 3-kinase inhibitor, wortmannin. Although SNX5 colocalized with a fluid-phase marker and was found predominantly within a PtdIns(3)P-rich endosomal domain, very little colocalization was observed between SNX5 and the PtdIns(3)P-binding protein, EEA1. Using liposome-based binding assays, we have shown that the PX domain of SNX5 interacts not only with PtdIns(3)P but also with PtdIns(3,4)P-2. In response to EGF stimulation, either the SNX5-PX domain or full-length SNX5 was rapidly recruited to the plasma membrane. The localization of SNX1, which does not bind PtdIns(3,4)P-2, was unaffected by EGF signalling. Therefore, SNX5 is localized to a subdomain of the early endosome distinct from EEA1 and, following EGF stimulation and elevation of PtdIns(3,4)P-2, is also transiently recruited to the plasma membrane. These results indicate that SNX5 may have functions not only associated with endosomal sorting but also with the phosphoinositide-signalling pathway.

Insulin and Oleate Promote Translocation of Inosine-5' Monophosphate Dehydrogenase to Lipid Bodies

In the present study we identify inosine-5' monophosphate dehydrogenase (IMPDH), a key enzyme in de novo guanine nucleotide biosynthesis, as a novel lipid body-associated protein. To identify new targets of insulin we performed a comprehensive 2-DE analysis of P-32-labelled proteins isolated from 3T3-L1 adipocytes (Hill et al. J Biol Chem 2000; 275: 24313-24320). IMPDH was identified by liquid chromatography/tandem mass spectrometry as a protein which was phosphorylated in a phosphatidylinositol (PI) 3-kinase-dependent manner upon insulin treatment. Although insulin had no significant effect on IMPDH activity, we observed translocation of IMPDH to lipid bodies following insulin treatment. Induction of lipid body formation with oleic acid promoted dramatic redistribution of IMPDH to lipid bodies, which appeared to be in contact with the endoplasmic reticulum, the site of lipid body synthesis and recycling. Inhibition of PI 3-kinase blocked insulin- and oleate-induced translocation of IMPDH and reduced oleate-induced lipid accumulation. However, we found no evidence of oleate-induced IMPDH phosphorylation, suggesting phosphorylation and translocation may not be coupled events. These data support a role for IMPDH in the dynamic regulation of lipid bodies and fatty acid metabolism and regulation of its activity by subcellular redistribution in response to extracellular factors that modify lipid metabolism.

Arp2/3 activity is necessary for efficient formation of E-cadherin adhesive contacts

Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.

Endosome-to-cytosol transport of viral nucleocapsids

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra- endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid ( LBPA) and its putative effector Alix/ AIP1, and is regulated by phosphatidylinositol- 3-phosphate ( PtdIns( 3) P) signalling via the PtdIns( 3) P- binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back- fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns( 3) P and their effectors.

Cell cycle alterations in biopsied olfactory neuroepithelium in schizophrenia and bipolar I disorder using cell culture and gene expression analyses

We previously demonstrated that olfactory cultures front individuals with schizophrenia had increased cell proliferation compared to Cultures from healthy controls. The aims of this study were to (a) replicate this observation in a new group Of individuals with schizophrenia, (b) examine the specificity of these findings by including individuals with bipolar I disorder and (c) explore gene expression differences that may underlie cell cycle differences in these diseases. Compared to controls (n = 10), there was significantly more mitosis in schizophrenia patient cultures (it = 8) and significantly more cell death in the bipolar I disorder patient cultures (n=8). Microarray data showed alterations to the cell cycle and phosphatidylinositol signalling pathways in schizophrenia and bipolar I disorder, respectively. Whilst caution is required in the interpretation of the array results, the study provides evidence indicating that cell proliferation and cell death in olfactory neuroepithelial cultures is differentially altered in schizophrenia and bipolar disorder. (c) 2005 Elsevier B.V. All rights reserved.

Comparison of selected methods for measuring the virulence properties of Listeria spp.

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.

Dynamic microtubules regulate the local concentration of E-cadherin at cell-cell contacts

In contrast to the well-established relationship between cadherins and the actin cytoskeleton, the potential link between cadherins and microtubules (MTs) has been less extensively investigated. We now identify a pool of MTs that extend radially into cell-cell contacts and are inhibited by manoeuvres that block the dynamic activity of MT plus-ends (e.g. in the presence of low concentrations of nocodazole and following expression of a CLIP-170 mutant). Blocking dynamic MTs perturbed the ability of cells to concentrate and accumulate E-cadherin at cell-cell contacts, as assessed both by quantitative immunofluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis, but did not affect either transport of E-cadherin to the plasma membrane or the amount of E-cadherin expressed at the cell surface. This indicated that dynamic MTs allow cells to concentrate E-cadherin at cell-cell contacts by regulating the regional distribution of E-cadherin once it reaches the cell surface. Importantly, dynamic MTs were necessary for myosin II to accumulate and be activated at cadherin adhesive contacts, a mechanism that supports the focal accumulation of E-cadherin. We propose that this population of MTs represents a novel form of cadherin-MT cooperation, where cadherin adhesions recruit dynamic MTs that, in turn, support the local concentration of cadherin molecules by regulating myosin II activity at cell-cell contacts.