USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn2+, at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent crosslinking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

Rapid burst of H2O2 by plant growth regulators increases intracellular Ca2+ amounts and modulates CD4(+) T cell activation

The identification of small molecules that affect T cell activation is an important area of research. Three molecules that regulate plant growth and differentiation, but not their structurally similar analogs, were identified to enhance primary mouse CD4(+) T cell activation in conjunction with soluble anti-CD3 stimulation: Indoleacetic acid (natural plant auxin), 1-Napthaleneacetic acid (synthetic plant auxin) and 2,4-Dichlorophenoxyacetic acid (synthetic plant auxin and herbicide). These effects are distinct in comparison to Curcumin, the well known phenolic immunomodulator, which lowers T cell activation. An investigation into the mechanisms of action of the three plant growth regulators revealed a rapid induction of reactive oxygen species (ROS), mainly comprising H2O2 . In addition, these three molecules synergize with soluble anti-CD3 signaling to enhance intracellular Ca2+ concentrations Ca2+](i), leading to greater T cell activation, e.g. induction of CD25 and IL-2. Enhanced production of TNF alpha and IFN gamma by CD4+ T cells is also observed upon plant growth regulator treatment with soluble anti-CD3. Interestingly, maximal IL-2 production and CD4(+) T cell cycle progression are observed upon activation with soluble anti-CD3 and phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Additionally, stimulation with PMA and Ionomcyin (a Ca2+ ionophore), which activates T cells by circumventing the TCR, and plant growth regulators also demonstrated the role of the strength of signal (SOS): T cell cycle progression is enhanced with gentle activation conditions but decreased with strong activation conditions. This study demonstrates the direct effects of three plant growth regulators on CD4(+) T cell activation and cycling. (C) 2010 Elsevier B.V. All rights reserved.

Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

Coated pits,endosome CURLing and intracellular receptor--ligand trafficking

In mediating endocytosis of extracellular macromolecules; the major mechanism in which cells ingest nutrients, degrade hormones and maintain the protein and lipid compositions of their organelle membrane, the cell surface receptors encounter 'coated pits', migrate continuously from one organelle to another, deliver the 'cargo' and often recycle back to the cell surface. This article is an attempt to give an account of the recent advances in our understanding of the molecular events involved in the 'round trip itinerary' of cell surface receptors.

Intracellular Pathogen Sensor NOD2 Programs Macrophages to Trigger Notch1 Activation

Intracellular pathogen sensor, NOD2, has been implicated in regulation of wide range of anti-inflammatory responses critical during development of a diverse array of inflammatory diseases; however, underlying molecular details are still imprecisely understood. In this study, we demonstrate that NOD2 programs macrophages to trigger Notch1 signaling. Signaling perturbations or genetic approaches suggest signaling integration through cross-talk between Notch1-PI3K during the NOD2-triggered expression of a multitude of immunological parameters including COX-2/PGE(2) and IL-10. NOD2 stimulation enhanced active recruitment of CSL/RBP-Jk on the COX-2 promoter in vivo. Intriguingly, nitric oxide assumes critical importance in NOD2-mediated activation of Notch1 signaling as iNOS(-/-) macrophages exhibited compromised ability to execute NOD2-triggered Notch1 signaling responses. Correlative evidence demonstrates that this mechanism operates in vivo in brain and splenocytes derived from wild type, but not from iNOS(-/-) mice. Importantly, NOD2-driven activation of the Notch1-PI3K signaling axis contributes to its capacity to impart survival of macrophages against TNF-alpha or IFN-gamma-mediated apoptosis and resolution of inflammation. Current investigation identifies Notch1-PI3K as signaling cohorts involved in the NOD2-triggered expression of a battery of genes associated with anti-inflammatory functions. These findings serve as a paradigm to understand the pathogenesis of NOD2-associated inflammatory diseases and clearly pave a way toward development of novel therapeutics.

Characterization of a 10- to 14-kilodalton protease-sensitive mycobacterium tuberculosis H37Ra antigen that stimulates human -yi T cells

gamma delta T-cell receptor-bearing T cells (gamma delta T cells) are readily activated by intracellular bacterial pathogens such as Mycobacterium tuberculosis. The bacterial antigens responsible for gamma delta T-cell activation remain poorly characterized. We have found that heat treatment of live M. tuberculosis bacilli released into the supernatant an antigen which stimulated human gamma delta T cells, gamma delta T-cell activation was measured by determining the increase in percentage of gamma delta T cells by flow cytometry in peripheral blood mononuclear cells stimulated with antigen and by proliferation of gamma delta T-cell lines with monocytes as antigen-presenting cells. Supernatant from heat-treated M. tuberculosis was fractionated by fast-performance liquid chromatography (FPLC) on a Superose 12 column. Maximal gamma delta T-cell activation was measured for a fraction of 10 to 14 kDa. Separation of the supernatant by preparative isoelectric focusing demonstrated peak activity at a pi of <4.0. On two-dimensional gel electrophoresis, the 10- to 14-kDa FPLC fraction contained at least seven distinct molecules, of which two had a pi of <4.5. Protease treatment reduced the bioactivity of the 10- to 14-kDa FPLC fraction for both resting and activated gamma delta T cells. Murine antibodies raised to the 10- to 14-kDa fraction reacted by enzyme-linked immunosorbent assay with antigens of 10 to 14 kDa in lysate of M. tuberculosis. In addition, gamma delta T cells proliferated in response to an antigen of 10 to 14 kDa present in M. tuberculosis lysate. gamma delta T-cell-stimulating antigen was not found in culture filtrate of M. tuberculosis but was associated,vith the bacterial pellet and lysate of M. tuberculosis. These results provide a preliminary characterization of a 10- to 14-kDa, cell-associated, heat-stable, low-pI protein antigen of M. tuberculosis which is a major stimulus for human gamma delta T cells.

Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin CDNA

A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)(i)], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)(i)] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells al all stages of development exhibit a sharp transient increase in [(Ca2+)(i)] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)(i)] and show three times as large a response to cAMP stimulation as prespore cells.

Stimulation by DIF causes an increase of intracellular Ca2+ in Dictyostelium discoideum

Using fluorescence- activated cell sorting (FAGS), we have studied the effect of the differentiation-inducing factor (DIF) on cellular Ca2+ in Dictyostelium discoideum. We have shown previously that freshly starved or postaggregation amoebae are heterogenous with respect to the amounts of cellular Ca2+ that they contain; the L or ''low Ca2+'' class exhibits a prespore tendency and the H or ''high Ca2+'' class exhibits a prestalk tendency. Upon adding DIF, within 2 min there is an approximately twofold increase in the relative fraction of amoebae falling in the H class. A major part of the increase is caused by Ca2+ influx from the extracellular medium. Therefore a rise in the level of cellular Ca2+ is an early step in the signal transduction pathway following stimulation by DIF. Also, in parallel with the cellular heterogeneity in respect of Ca2+ content, there is a heterogeneity in the response to DIF, which appears to be restricted to L cells. (C) 1997 Academic Press.

Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+](i)) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline [0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoal, cGMP values (similar to 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/HCO(3)(-)antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 mu M) A protein kinase G inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR, A phospholipase A(2) inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+](i) was measured using fura-2-AM, there was an early rise 271 nM at 0.5 hi in pentoxifylline(-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR(20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-](i) and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A;A(2) and protein kinase C activity.

Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.

Acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella

During the course of infection, Salmonella has to face several potentially lethal environmental conditions, one such being acidic pH. The ability to sense and respond to the acidic pH is crucial for the survival and replication of Salmonella. The physiological role of one gene (STM1485) involved in this response, which is upregulated inside the host cells (by 90- to 113-fold) is functionally characterized in Salmonella pathogenesis. In vitro, the DSTM1485 neither exhibited any growth defect at pH 4.5 nor any difference in the acid tolerance response. The DSTM1485 was compromised in its capacity to proliferate inside the host cells and complementation with STM1485 gene restored its virulence. We further demonstrate that the surface translocation of Salmonella pathogenicity island-2 (SPI-2) encoded translocon proteins, SseB and SseD were reduced in the DSTM1485. The increase in co-localization of this mutant with lysosomes was also observed. In addition, the DSTM1485 displayed significantly reduced competitive indices (CI) in spleen, liver and mesenteric lymph nodes in murine typhoid model when infected by intra-gastric route. Based on these results, we conclude that the acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella.

Abrin Immunotoxin: Targeted Cytotoxicity and Intracellular Trafficking Pathway

Background: Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy. Methods: Protein synthesis assay using (3)H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins. Results: We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells. Conclusions: This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.