CD1c molecules broadly survey the endocytic system

The ability of antigen-presenting cells to sample distinct intracellular compartments is crucial for microbe detection. Major histocompatibility complex class I and class II molecules sample the cytosol or the late endocytic compartment, allowing detection of microbial peptide antigens that arise in distinct intracellular compartments. In contrast, CD1a and CD1b molecules mediate the presentation of lipid and glycolipid antigens and differentially sample early recycling endosomes or late endocytic compartments, respectively, that contain distinct sets of lipid antigens. Here, we show that, unlike the other CD1 isoforms or major histocompatibility complex molecules that each sample restricted only intracellular compartments, CD1c is remarkable in that it distributes broadly throughout the endocytic system and is expressed in both recycling endosomes and late endocytic compartments. Further, in contrast to CD1b, which requires an acidic environment to function, antigen presentation by CD1c was able to overcome dependence on vesicular acidification. Because CD1c is expressed on essential antigen-presenting cells, such as epidermal Langerhans cells (in the absence of CD1b), or on B cells (without CD1a or -b), we suggest that CD1c molecules allow a comprehensive survey for lipid antigens throughout the endocytic system even in the absence of other CD1 isoforms.

Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate

Higher plants share with animals a responsiveness to the Ca2+ mobilizing agents inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular 45Ca2+ flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca2+-releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca2+ with a K1/2 = 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca2+ pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca2+ pathway distinguishes it from the InsP3- and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol/mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca2+ release is insensitive to heparin and 8-NH2-cADPR, specific inhibitors of the InsP3- and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca2+ release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca2+ release pathway is independent of cytosolic free Ca2+ and therefore incapable of operating Ca2+-induced Ca2+ release. In contrast to the sea urchin system, the NAADP-gated Ca2+ release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca2+ mobilization pathways and Ca2+ release sites might contribute to the generation of stimulus-specific Ca2+ signals in plant cells.

Anterograde flow of cargo across the Golgi stack potentially mediated via bidirectional “percolating” COPI vesicles

How do secretory proteins and other cargo targeted to post-Golgi locations traverse the Golgi stack? We report immunoelectron microscopy experiments establishing that a Golgi-restricted SNARE, GOS 28, is present in the same population of COPI vesicles as anterograde cargo marked by vesicular stomatitis virus glycoprotein, but is excluded from the COPI vesicles containing retrograde-targeted cargo (marked by KDEL receptor). We also report that GOS 28 and its partnering t-SNARE heavy chain, syntaxin 5, reside together in every cisterna of the stack. Taken together, these data raise the possibility that the anterograde cargo-laden COPI vesicles, retained locally by means of tethers, are inherently capable of fusing with neighboring cisternae on either side. If so, quanta of exported proteins would transit the stack in GOS 28–COPI vesicles via a bidirectional random walk, entering at the cis face and leaving at the trans face and percolating up and down the stack in between. Percolating vesicles carrying both post-Golgi cargo and Golgi residents up and down the stack would reconcile disparate observations on Golgi transport in cells and in cell-free systems.

Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs

Memory is a hallmark of immunity. Memory carried by antibodies is largely responsible for protection against reinfection with most known acutely lethal infectious agents and is the basis for most clinically successful vaccines. However, the nature of long-term B cell and antibody memory is still unclear. B cell memory was studied here after infection of mice with the rabies-like cytopathic vesicular stomatitis virus, the noncytopathic lymphocytic choriomeningitis virus (Armstrong and WE), and after immunization with various inert viral antigens inducing naive B cells to differentiate either to plasma cells or memory B cells in germinal centers of secondary lymphoid organs. The results show that in contrast to very low background levels against internal viral antigens, no significant neutralizing antibody memory was observed in the absence of antigen and suggest that memory B cells (i) are long-lived in the absence of antigen, nondividing, and relatively resistant to irradiation, and (ii) must be stimulated by antigen to differentiate to short-lived antibody-secreting plasma cells, a process that is also efficient in the bone marrow and always depends on radiosensitive, specific T help. Therefore, for vaccines to induce long-term protective antibody titers, they need to repeatedly provide, or continuously maintain, antigen in minimal quantities over a prolonged time period in secondary lymphoid organs or the bone marrow for sufficient numbers of long-lived memory B cells to mature to short-lived plasma cells.

Transcytosis of α1-acidic glycoprotein in the continuous microvascular endothelium

By using perfusions and bolus administration, coupled with postembedding immunocytochemical procedures, we have identified the structures involved in the transport of derivatized orosomucoid (α1-acidic glycoprotein) across the continuous microvascular endothelium of the murine myocardium. Our findings indicate that: (i) monomeric orosomucoid binds to the luminal surface of the endothelium; (ii) it is restricted to caveolae during its transport across the endothelium; (iii) it is detected in the perivascular spaces at early time points (by 1 min) and in larger quantities at later time points (>5 min) from the beginning of its perfusion or its intravascular administration; (iv) no orosomucoid molecules are found in the intercellular junctions or at the abluminal exits of interendothelial spaces; and (v) the vesicular transport of orosomucoid is strongly inhibited by N-ethylmaleimide (>80%). Because, by size and shape, the orosomucoid qualifies as a preferential probe for the postulated small pore system, our results are discussed in relation to the pore theory of capillary permeability.

Virus neutralization by germ-line vs. hypermutated antibodies

Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease. VSVneutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated. A comparative analysis of recombinant single-chain antibody fragments (scFv-Cκ) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV. Four somatic amino acid substitutions in VH increased by 300-fold the binding strength of monovalent scFv-Cκ. The multivalent binding avidity of germ-line scFv-Cκ was increased by more than 10-fold compared with the monovalent binding strength. In contrast, hypermutated scFv-Cκ did not show such avidity effects. Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold. This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show “affinity maturation” effects of up to 1000-fold.

Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals

We compared peripheral and mucosal primary CD8 T cell responses to inflammatory and noninflammatory forms of antigen in a T cell-adoptive transfer system. Immunization with the soluble antigen, ovalbumin (ova), administered i.p. or orally without adjuvant, activated nonmucosal CD8 T cells but did not induce cytotoxic activity. However, after activation, the transferred cells entered the intestinal mucosa and became potent antigen-specific killers. Thus, exogenous intact soluble protein entered the major histocompatibility complex class I antigen presentation pathway and induced mucosal cytotoxic T lymphocytes. Moreover, distinct costimulatory requirements for activation of peripheral versus mucosal T cells were noted in that the CD28 ligand, B7-1, was critical for activated mucosal T cell generation but not for activation of peripheral CD8 T cells. The costimulator, B7-2, was required for optimum activation of both populations. Infection with a new recombinant vesicular stomatitis virus encoding ovalbumin induced lytic activity in mucosal as well as peripheral sites, demonstrating an adjuvant effect of inflammatory mediators produced during virus infection. Generation of antiviral cytotoxic T lymphocytes was also costimulation-dependent. The results indicated that induction of peripheral tolerance via antigen administration may not extend to mucosal sites because of distinct costimulatory and inflammatory signals in the mucosa.

Lamellar lipoproteins uniquely contribute to hyperlipidemia in mice doubly deficient in apolipoprotein E and hepatic lipase

Remnants of triglyceride-rich lipoproteins containing apolipoprotein (apo) B-48 accumulate in apo E-deficient mice, causing pronounced hypercholesterolemia. Mice doubly deficient in apo E and hepatic lipase have more pronounced hypercholesterolemia, even though remnants do not accumulate appreciably in mice deficient in hepatic lipase alone. Here we show that the doubly deficient mice manifest a unique lamellar hyperlipoproteinemia, characterized by vesicular particles 600 Å–1,300 Å in diameter. As seen by negative-staining electron microscopy, these lipoproteins also contain an electron-lucent region adjacent to the vesicle wall, similar to the core of typical lipoproteins. Correlative chemical analysis indicates that the vesicle wall is composed of a 1:1 molar mixture of cholesterol and phospholipids, whereas the electron-lucent region appears to be composed of cholesteryl esters (about 12% of the particle mass). Like the spherical lipoproteins of doubly deficient mice, the vesicular particles contain apo B-48, but they are particularly rich in apo A-IV. We propose that cholesteryl esters are removed from spherical lipoproteins of these mice by scavenger receptor B1, leaving behind polar lipid-rich particles that fuse to form vesicular lipoproteins. Hepatic lipase may prevent such vesicular lipoproteins from accumulating in apo E-deficient mice by hydrolyzing phosphatidyl choline as scavenger receptor B1 removes the cholesteryl esters and by gradual endocytosis of lipoproteins bound to hepatic lipase on the surface of hepatocytes.

A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic

The small GTPase Rab4 is implicated in endocytosis in all cell types, but also plays a specific role in some regulated processes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for an effector(s) that specifically recognizes its GTP-bound form. We cloned a ubiquitous 69-kDa protein, Rabip4, that behaves as a Rab4 effector in the yeast two-hybrid system and in the mammalian cell. Rabip4 contains two coiled-coil domains and a FYVE-finger domain. When expressed in CHO cells, Rabip4 is present in early endosomes, because it is colocated with endogenous Early Endosome Antigen 1, although it is absent from Rab11-positive recycling endosomes and Rab-7 positive late endosomes. The coexpression of Rabip4 with active Rab4, but not with inactive Rab4, leads to an enlargement of early endosomes. It strongly increases the degree of colocalization of markers of sorting (Rab5) and recycling (Rab11) endosomes with Rab4. Furthermore, the expression of Rabip4 leads to the intracellular retention of a recycling molecule, the glucose transporter Glut 1. We propose that Rabip4, an effector of Rab4, controls early endosomal traffic possibly by activating a backward transport step from recycling to sorting endosomes.

Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans

Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist in Caenorhabditis elegans and Drosophila, but no CHAT mutations have been observed in humans to date. Here we report that mutations in CHAT cause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and to CHAT as one of the candidate genes. Direct sequencing of CHAT reveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, V506L, and R560H) have significantly impaired catalytic efficiencies.

Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagosomes in tumor necrosis factor p55 receptor-deficient macrophages

Tumor necrosis factor receptor (TNFR) p55-knockout (KO) mice are susceptible profoundly to Salmonella infection. One day after peritoneal inoculation, TNFR-KO mice harbor 1,000-fold more bacteria in liver and spleen than wild-type mice despite the formation of well organized granulomas. Macrophages from TNFR-KO mice produce abundant quantities of reactive oxygen and nitrogen species in response to Salmonella but nevertheless exhibit poor bactericidal activity. Treatment with IFN-γ enhances killing by wild-type macrophages but does not restore the killing defect of TNFR-KO cells. Bactericidal activity of macrophages can be abrogated by a deletion in the gene encoding TNFα but not by saturating concentrations of TNF-soluble receptor, suggesting that intracellular TNFα can regulate killing of Salmonella by macrophages. Peritoneal macrophages from TNFR-KO mice fail to localize NADPH oxidase-containing vesicles to Salmonella-containing vacuoles. A TNFR-KO mutation substantially restores virulence to an attenuated mutant bacterial strain lacking the type III secretory system encoded by Salmonella pathogenicity island 2 (SPI2), suggesting that TNFα and SPI2 have opposing actions on a common pathway of vesicular trafficking. TNFα–TNFRp55 signaling plays a critical role in the immediate innate immune response to an intracellular pathogen by optimizing the delivery of toxic reactive oxygen species to the phagosome.

Coupling of histamine H3 receptors to neuronal Na+/H+ exchange: A novel protective mechanism in myocardial ischemia

In myocardial ischemia, adrenergic nerves release excessive amounts of norepinephrine (NE), causing dysfunction and arrhythmias. With anoxia and the concomitant ATP depletion, vesicular storage of NE is impaired, resulting in accumulation of free NE in the axoplasm of sympathetic nerves. Intraneuronal acidosis activates the Na+/H+ exchanger (NHE), leading to increased Na+ entry in the nerve terminals. These conditions favor availability of the NE transporter to the axoplasmic side of the membrane, causing massive carrier-mediated efflux of free NE. Neuronal NHE activation is pivotal in this process; NHE inhibitors attenuate carrier-mediated NE release. We previously reported that activation of histamine H3 receptors (H3R) on cardiac sympathetic nerves also reduces carrier-mediated NE release and alleviates arrhythmias. Thus, H3R activation may be negatively coupled to NHE. We tested this hypothesis in individual human SKNMC neuroblastoma cells stably transfected with H3R cDNA, loaded with the intracellular pH (pHi) indicator BCECF. These cells possess amiloride-sensitive NHE. NHE activity was measured as the rate of Na+-dependent pHi recovery in response to an acute acid pulse (NH4Cl). We found that the selective H3R-agonist imetit markedly diminished NHE activity, and so did the amiloride derivative EIPA. The selective H3R antagonist thioperamide abolished the imetit-induced NHE attenuation. Thus, our results provide a link between H3R and NHE, which may limit the excessive release of NE during protracted myocardial ischemia. Our previous and present findings uncover a novel mechanism of cardioprotection: NHE inhibition in cardiac adrenergic neurons as a means to prevent ischemic arrhythmias associated with carrier-mediated NE release.

Multiple Roles of Arf1 GTPase in the Yeast Exocytic and Endocytic Pathways

ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic. However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear. We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae. By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes. arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner. Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport. These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.

Synaptic and extrasynaptic γ-aminobutyric acid type A receptor clusters in rat hippocampal cultures during development

We have simultaneously measured the expression of postsynaptic γ-aminobutyric acid type A (GABAA) receptor clusters and of presynaptic boutons in neonatal rat hippocampal cultures between days 1 and 30. GABAA receptors were labeled with antibodies recognizing the extracellular domains of β2/3 and γ2 subunits. Boutons were visualized by activity-dependent uptake of the styryl dye FM4-64, or by antibodies against the presynaptic vesicular protein SV2 or the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). GABAA receptor clusters could be seen in living neurons already 6 h after culturing, much before presynaptic markers could be identified in nerve terminals. The densities of receptor clusters that contained the β2/3 subunits were constant between days 10 and 30 in culture, whereas γ2 subunit-containing clusters fluctuated and reached a maximum on day 20. SV2 and GAD staining could be measured from day 2 onwards. Clustering of GAD in presynaptic terminals and FM4-64 uptake were observed only at day 5 and afterward. SV2 staining and FM4-64 uptake increased in parallel between days 5 and 20 and remained constant thereafter. GAD-stained boutons were fewer than those labeled with other, less specific, presynaptic stains. They reached a maximum on day 20 and fell again toward day 30. Double labeling of GABAA receptors and of presynaptic boutons in neurons during differentiation showed that, even after 30 days in culture, large fractions of GABAA receptor clusters containing β2/3 and/or γ2 subunits remained extrasynaptic.

Phosphatidylcholine Synthesis Influences the Diacylglycerol Homeostasis Required for Sec14p-dependent Golgi Function and Cell Growth

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20–40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.