Dynamin‐mediated lipid acquisition is essential for Chlamydia trachomatis development

Chlamydia trachomatis is an obligate intracellular pathogen responsible for a high burden of human disease. Here, a loss‐of‐function screen using a set of lentivirally transduced shRNAs identified 14 human host cell factors that modulate C. trachomatis infectivity. Notably, knockdown of dynamin, a host GTPase, decreased C. trachomatis infectivity. Dynamin functions in multiple cytoplasmic locations, including vesicle formation at the plasma membrane and the trans‐Golgi network. However, its role in C. trachomatis infection remains unclear. Here we report that dynamin is essential for homotypic fusion of C. trachomatis inclusions but not for C. trachomatis internalization into the host cell. Further, dynamin activity is necessary for lipid transport into C. trachomatis inclusions and for normal re‐differentiation from reticulate to elementary bodies. Fragmentation of the Golgi apparatus is proposed to be an important strategy used by C. trachomatis for efficient lipid acquisition and replication within the host. Here we show that a subset of C. trachomatis‐infected cells displayed Golgi fragmentation, which was concurrent with increased mitotic accumulation. Golgi fragmentation was dispensable for dynamin‐mediated lipid acquisition into C. trachomatis inclusions, irrespective of the cell cycle phase. Thus, our study reveals a critical role of dynamin in host‐derived lipid acquisition for C. trachomatis development.     

Intracellular location regulates calcium-calmodulin-dependent activation of organelle-restricted eNOS

Mislocalization of endothelial nitric oxide (NO) synthase (eNOS) in response to oxidized low-density lipoprotein, cholesterol depletion, elevated blood pressure, and bound eNOS interacting protein/NOS traffic inducer is associated with reduced NO release via unknown mechanisms. The proper targeting of eNOS to the plasma membrane or intracellular organelles is an important regulatory step controlling enzyme activity. Previous studies have shown that plasma membrane eNOS is constitutively phosphorylated on serine 1179 and highly active. In contrast, the activity of eNOS targeted to intracellular organelles is more complex. The cis-Golgi eNOS is fully activated by Akt-dependent phosphorylation. However, eNOS targeted to the trans-Golgi is decidedly less active in response to all modes of activation, including mutation to the phosphomimetic aspartic acid. In this study, we establish that when expressed within other intracellular organelles, such as the mitochondria and nucleus, the activity of eNOS is also greatly reduced. To address the mechanisms underlying the impaired catalytic activity of eNOS within these locations, we generated subcellular-targeted constructs that express a calcium-independent NOS isoform, iNOS. With the use of organelle specific (plasma membrane, cis- vs. trans-Golgi, plasma membrane, and Golgi, nucleus, and mitochondria) targeting motifs fused to the wild-type iNOS, we measured NO release from intact cells. With the exception of the Golgi lumen, our results showed no impairment in the ability of targeted iNOS to synthesize NO. Confirmation of correct targeting was obtained through confocal microscopy using identical constructs fused to the green fluorescent protein. We conclude that the reduced activation of eNOS within discrete cytoplasmic regions of the Golgi, the mitochondria and the nucleus is primarily due to insufficient access to calcium-calmodulin. nitric oxide; Akt; Golgi     

Targeting of endothelial nitric-oxide synthase to the cytoplasmic face of the Golgi complex or plasma membrane regulates Akt- versus calcium-dependent mechanisms for nitric oxide release

The heterogeneous localization of endothelial nitricoxide synthase (eNOS) on the Golgi complex versus the plasma membrane has made it difficult to dissect the regulation of each pool of enzyme. Here, we generated fusion proteins that specifically target the plasma membrane or cytoplasmic aspects of the Golgi complex and have assessed eNOS activation. Plasma membrane-targeted eNOS constructs were constitutively active, phosphorylated, and responsive to transmembrane calcium fluxes, yet were insensitive to further activation by Akt-mediated phosphorylation. In contrast, cis-Golgi complex-targeted eNOS behaved similarly to wild-type eNOS and was less sensitive to calcium-dependent activation and highly responsive to Akt-dependent phosphorylation compared with plasma membrane versions. In plasma membrane- and Golgi complex-targeted constructs, Ser1179 is critical for NO production. This study provides clear evidence for functional roles of plasma membrane- and Golgi complex-localized eNOS and supports the concept that proteins thought to be regulated and to function exclusively in the plasma membrane of cells can indeed signal and be regulated in internal Golgi membranes.     

Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells

Background information. The GA (Golgi apparatus) has an essential role in membrane trafficking, determining the assembly and delivery of UPs (uroplakins) to the APM (apical plasma membrane) of superficial UCs (uroepithelial cells) of urinary bladder. UPs are synchronously and uniformly delivered from the GA to the APM by DFVs (discoidal‐ or fusiform‐shaped vesicles); however, the mechanism of UP delivery is not known. We have used the culture model of UCs with the capacity to undergo terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved. Results. By three‐dimensional localization using confocal microscopy of immunofluorescence‐labelled GA‐related markers [GM130 (cis‐Golgi matrix protein of 130 kDa), GS15 (Golgi Snare 15 kDa), GS28 and giantin], uroepithelial differentiation‐related markers (UPs), MTs (microtubules; α‐tubulin) and intermediate filaments [CK7 (cytokeratin 7) and CK20], we found that in non‐differentiated, UP‐negative UCs the GA is mostly organized as a single ribbon‐like structure close to the nucleus, whereas in differentiated, UP‐positive UCs the GA is fragmented and spread almost through the entire cell. The FRAP (fluorescence recovery after photobleaching) experiments on the UCs transfected with GalT (trans‐Golgi/TGN enzyme β1,4‐galactosyltransferase) fused to fluorescent protein showed that Golgi‐resident enzyme cycles freely within ribbon‐like GA but not within fragmented GA. By CLEM (correlative light—electron microscopy), we examined the GA fragments in cells expressing UPs. We found that GA fragments are fully functional and similar to the GA fragments that are formed after nocodazole treatment. Furthermore, we demonstrated that the reorganization of GA into a fragmented form is associated with the impairment of the MT organization in the basal, central and subapical cytoplasm and the accumulation of intermediate filaments in the apical cytoplasm that could affect the kinetics of MT star leading to the peripheral fragmentation of the GA in the differentiated UCs. Conclusions. The fragmentation of the GA and the subsequent spreading of GA to the cell periphery represent one of the key events that promote the uniform delivery of UPs over the entire APM of differentiating UCs and thus are of major importance in the final proper formation and maintenance of the blood—urine barrier.     

M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen Chlamydia pneumoniae

Intracellular pathogens have developed various strategies to escape immunity to enable their survival in host cells, and many bacterial pathogens preferentially reside inside macrophages, using diverse mechanisms to penetrate their defenses and to exploit their high degree of metabolic diversity and plasticity. Here, we characterized the interactions of the intracellular pathogen Chlamydia pneumoniae with polarized human macrophages. Primary human monocytes were pre-differentiated with granulocyte macrophage colony-stimulating factor or macrophage colony-stimulating factor for 7 days to yield M1-like and M2-like macrophages, which were further treated with interferon-γ and lipopolysaccharide or with interleukin-4 for 48 h to obtain fully polarized M1 and M2 macrophages. M1 and M2 cells exhibited distinct morphology with round or spindle-shaped appearance for M1 and M2, respectively, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophage polarization did not influence uptake of C. pneumoniae, since comparable copy numbers of chlamydial DNA were detected in M1 and M2 at 6 h post infection, but an increase in chlamydial DNA over time indicating proliferation was only observed in M2. Accordingly, 72±5% of M2 vs. 48±7% of M1 stained positive for chlamydial lipopolysaccharide, with large perinuclear inclusions in M2 and less clearly bordered inclusions for M1. Viable C. pneumoniae was present in lysates from M2, but not from M1 macrophages. The ability of M1 to restrict chlamydial replication was not observed in M1-like macrophages, since chlamydial load showed an equal increase over time for M1-like and M2-like macrophages. Our findings support the importance of macrophage polarization for the control of intracellular infection, and show that M2 are the preferred survival niche for C. pneumoniae. M1 did not allow for chlamydial proliferation, but failed to completely eliminate chlamydial infection, giving further evidence for the ability of C. pneumoniae to evade cellular defense and to persist in human macrophages.     

Chlamydophila pneumoniae downregulates MHC‐class II expression by two cell type‐specific mechanisms

Chlamydophila pneumoniae was shown to prevent IFNγ‐inducible upregulation of MHC‐class II molecules by secreting chlamydial protease‐like activity factor (CPAF) into the cytosol of those host cells which support the complete bacterial replication cycle. CPAF acts by degrading upstream stimulatory factor 1 (USF‐1). However, in cells like bone marrow‐derived macrophages (BMM), which restrict chlamydial replication, we show that CPAF expression is barely detectable and the expression of USF‐1 is induced upon infection with C. pneumoniae. Nevertheless, the infection still reduced base line and prevented IFNγ‐inducible MHC‐class II expression. Similar results were obtained with heat‐inactivated C. pneumoniae. In contrast, reduction of MHC‐class II molecules was not observed in MyD88‐deficient BMM. Reduction of IFNγ‐inducible MHC‐class II expression by C. pneumoniae in BMM was mediated in part by the MAP‐kinase p38. Infection of murine embryonic fibroblasts (MEF) with C. pneumoniae, which allow chlamydial replication, induced the expression of CPAF and decreased USF‐1 and MHC‐class II expression. Treatment of these cells with heat‐inactivated C. pneumoniae reduced USF‐1 and MHC‐class II expression to a much lower extent. In summary, C. pneumoniae downregulates MHC‐class II expression by two cell type‐specific mechanisms which are either CPAF‐independent and MyD88‐dependent like in BMM or CPAF‐dependent like in MEFs.     

Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase

Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.     

Coordinated regulation of endothelial nitric oxide synthase activity by phosphorylation and subcellular localization

Endothelial nitric oxide synthase (eNOS) is regulated by multiple mechanisms including Ca(2+)/calmodulin binding, protein-protein interactions, phosphorylation, and subcellular locations. Emerging evidence suggests that these seemingly independent mechanisms may be closely correlated. In the present study, the interplay between membrane targeting and phosphorylation of eNOS was investigated by using various mutants designed to target specific subcellular locations or to mimic different phospho states. Phospho-mimicking mutations of wild-type eNOS at S635 and S1179 synergistically activated the enzyme. The targeted eNOS mutants to plasma membrane and Golgi complex exhibited higher NO production activities than that of a myristoylation-deficient cytosolic mutant. Phospho-mimicking mutations at S635 and S1179 rescued the activity of the cytosolic mutant and increased those of the plasma membrane- and Golgi-targeted mutants. In contrast, phospho-deficient mutations at these sites led to inactivation of eNOS. Unlike the other targeted mutants, the cytosolic eNOS mutant was unresponsive to cAMP, indicating that membrane association and phosphorylation are required for eNOS activation. These findings suggest that the coordinated interplay between phosphorylation and subcellular localization of eNOS plays an important role in regulating NO production in endothelial cells.     

Infection of epithelial cells with Chlamydia trachomatis inhibits TNF‐induced apoptosis at the level of receptor internalization while leaving non‐apoptotic TNF‐signalling intact

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of medical importance. C. trachomatis develops inside a membranous vacuole in the cytosol of epithelial cells but manipulates the host cell in numerous ways. One prominent effect of chlamydial infection is the inhibition of apoptosis in the host cell, but molecular aspects of this inhibition are unclear. Tumour necrosis factor (TNF) is a cytokine with important roles in immunity, which is produced by immune cells in chlamydial infection and which can have pro‐apoptotic and non‐apoptotic signalling activity. We here analysed the signalling through TNF in cells infected with C. trachomatis. The pro‐apoptotic signal of TNF involves the activation of caspase‐8 and is controlled by inhibitor of apoptosis proteins. We found that in C. trachomatis‐infected cells, TNF‐induced apoptosis was blocked upstream of caspase‐8 activation even when inhibitor of apoptosis proteins were inhibited or the inhibitor of caspase‐8 activation, cFLIP, was targeted by RNAi. However, when caspase‐8 was directly activated by experimental over‐expression of its upstream adapter Fas‐associated protein with death domain, C. trachomatis was unable to inhibit apoptosis. Non‐apoptotic TNF‐signalling, particularly the activation of NF‐κB, initiates at the plasma membrane, while the activation of caspase‐8 and pro‐apoptotic signalling occur subsequently to internalization of TNF receptor and the formation of a cytosolic signalling complex. In C. trachomatis‐infected cells, NF‐κB activation through TNF was unaffected, while the internalization of the TNF–TNF‐receptor complex was blocked, explaining the lack of caspase‐8 activation. These results identify a dichotomy of TNF signalling in C. trachomatis‐infected cells: Apoptosis is blocked at the internalization of the TNF receptor, but non‐apoptotic signalling through this receptor remains intact, permitting a response to this cytokine at sites of infection.     

Effector proteins of chlamydiae

This review summarizes the recently published data on the molecular mechanisms of Chlamydiae-host cell interaction, first of all, on chlamydial effector proteins. Such proteins, along with type III transport system proteins, which transfer many effector proteins into the host cytoplasm, are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. The C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of infection. The pathogen proteins that are involved in the next stage, which is the intracellular inclusion traffic to the centrosome, are C. trachomatis CT229 and C. pneumoniae Cpn0585, which interact with cell Rab GTPases. In C. trachomatis, IncA plays a key role in the fusion of chlamydial inclusions, CT847 modulates the life cycle of the host cell, and LDA3 is essential for the acquisition of nutrients. The protease CPAF and the inclusion membrane proteins IncG and CADD are involved in suppressing apoptosis of infected cells. The proteases CPAF and CT441 and the deubiquitinating protein ChlaDub1 help the pathogen to evade the immune response.     

NOSIP, a novel modulator of endothelial nitric oxide synthase activity.

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+-calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl-terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin-1 scaffolding domain. NO production was significantly reduced in eNOS-expressing CHO cells (CHO-eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergent-insoluble to the detergent-soluble fractions of CHO-eNOS, and this translocation was completely prevented by transient coexpression of NOSIP in CHO-eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO-eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin-rich membrane fractions of CHO-eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.     

The role of endoplasmic reticulum‐related BiP/GRP78 in interferon gamma‐induced persistent Chlamydia pneumoniae infection

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN‐γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN‐γ‐induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN‐γ‐induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN‐γ‐induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor‐2α (eIF2α) and down‐regulation of the vesicle‐associated membrane protein‐associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN‐γ‐induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER‐related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN‐γ‐induced persistent infection.     

The novel chlamydial adhesin CPn0473 mediates the lipid raft‐dependent uptake of Chlamydia pneumoniae

Chlamydiae are Gram‐negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down‐stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473‐coupled fluorescent latex beads adhere to human epithelial HEp‐2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp‐2 cells with rCPn0473 does not attenuate adhesion but promotes dose‐dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473‐dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae.     

Chlamydia trachomatis infection prevents front–rear polarity of migrating HeLa cells

Chlamydiae are obligate intracellular bacterial pathogens that cause trachoma, sexually transmitted diseases and respiratory infections in humans. Fragmentation of the host cell Golgi apparatus (GA) is essential for chlamydial development, whereas the consequences for host cell functions, including cell migration are not well understood. We could show that Chlamydia trachomatis‐infected cells display decelerated migration and fail to repopulate monolayer scratch wounds. Furthermore, infected cells lost the ability to reorient the fragmented GA or the microtubule organization centre (MTOC) after a migratory stimulus. Silencing of golgin‐84 phenocopied this defect in the absence of the infection. Interestingly, GA stabilization via knockdown of Rab6A and Rab11A improved its reorientation in infected cells and it was fully rescued after inhibition of Golgi fragmentation with WEHD‐fmk. These results show that C. trachomatis infection perturbs host cell migration on multiple levels, including the alignment of GA and MTOC.     

Glucosylceramide Biosynthesis is Involved in Golgi Morphology and Protein Secretion in Plant Cells

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that d ,l ‐threo‐1‐phenyl‐2‐decanoyl amino‐3‐morpholino‐propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high‐pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.     

<Emphasis Type="Italic">Chlamydophila pneumoniae</Emphasis> derived from inclusions late in the infectious cycle induce aponecrosis in human aortic endothelial cells

Background  

Atherosclerosis is still the leading cause of death in the western world. Besides known risk factors studies demonstrating Chlamydophila pneumoniae (C. pneumoniae) to be implicated in the progression of the disease, little is known about C. pneumoniae infection dynamics. We investigated whether C. pneumoniae induce cell death of human aortic endothelial cells, a cell type involved in the initiation of atherosclerosis, and whether chlamydial spots derive from inclusions.     

Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture

We have recently demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases endothelial nitric oxide synthase (eNOS) phosphorylation, NOS activity, and nitric oxide (NO) synthesis in cultured human umbilical vein endothelial cells (HUVEC), without inducing apoptotic cell death. Although an important factor that regulates eNOS activity is its localization within the cells, little is known about the role of TRAIL in the regulation of eNOS trafficking among cellular compartments and the cytoskeleton involvement in this machinery. Then, we did both quantitative and semi-quantitative evaluations with biochemical assays and immune fluorescence microscopy in the presence of specific inhibitors of NOS activity as well as of cytoskeletal microtubule structures. In our cellular model, TRAIL treatment not only increased NO levels but also caused a time-dependent NO migration of fluorescent spots from the plasma membrane to the inner part of the cells. In unstimulated cells, most of the eNOS was localized at the cell membranes. However, within 10 min following addition of TRAIL, nearly all the cells showed an increased cytoplasm localization of eNOS which appeared co-localized with the Golgi apparatus at a higher extent than in unstimulated cells. These effects were associated to an increased formation of trans-cytoplasm stress fibers with no significant changes of the microtubule network. Conversely, microtubule disruption and Golgi scattering induced with Nocodazole treatment inhibited TRAIL-increased NOS activity, indicating that, on cultured HUVEC, TRAIL ability to affect NO production by regulating eNOS sub-cellular distribution is mediated by cytoskeleton and Golgi complex modifications.     

Pathogen–host reorganization during Chlamydia invasion revealed by cryo‐electron tomography

Invasion of host cells is a key early event during bacterial infection, but the underlying pathogen–host interactions are yet to be fully visualized in three‐dimensional detail. We have captured snapshots of the early stages of bacterial‐mediated endocytosis in situ by exploiting the small size of chlamydial elementary bodies (EBs) for whole‐cell cryo‐electron tomography. Chlamydiae are obligate intracellular bacteria that infect eukaryotic cells and cause sexually transmitted infections and trachoma, the leading cause of preventable blindness. We demonstrate that Chlamydia trachomatis LGV2 EBs are intrinsically polarized. One pole is characterized by a tubular inner membrane invagination, while the other exhibits asymmetric periplasmic expansion to accommodate an array of type III secretion systems (T3SSs). Strikingly, EBs orient with their T3SS‐containing pole facing target cells, enabling the T3SSs to directly contact the cellular plasma membrane. This contact induces enveloping macropinosomes, actin‐rich filopodia and phagocytic cups to zipper tightly around the internalizing bacteria. Once encapsulated into tight early vacuoles, EB polarity and the T3SSs are lost. Our findings reveal previously undescribed structural transitions in both pathogen and host during the initial steps of chlamydial invasion.     

The First 35 Amino Acids and Fatty Acylation Sites Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of Cells: A Green Fluorescent Protein Study

Catalytically active endothelial nitric oxide synthase (eNOS) is located on the Golgi complex and in the caveolae of endothelial cells (EC). Mislocalization of eNOS caused by mutation of the N-myristoylation or cysteine palmitoylation sites impairs production of stimulated nitric oxide (NO), suggesting that intracellular targeting is critical for optimal NO production. To investigate the molecular determinants of eNOS targeting in EC, we constructed eNOS–green fluorescent protein (GFP) chimeras to study its localization in living and fixed cells. The full-length eNOS–GFP fusion colocalized with a Golgi marker, mannosidase II, and retained catalytic activity compared to wild-type (WT) eNOS, suggesting that the GFP tag does not interfere with eNOS localization or function. Experiments with different size amino-terminal fusion partners coupled to GFP demonstrated that the first 35 amino acids of eNOS are sufficient to target GFP into the Golgi region of NIH 3T3 cells. Additionally, the unique (Gly-Leu)₅ repeat located between the palmitoylation sites (Cys-15 and -26) of eNOS is necessary for its palmitoylation and thus localization, but not for N-myristoylation, membrane association, and NOS activity. The palmitoylation-deficient mutants displayed a more diffuse fluorescence pattern than did WT eNOS–GFP, but still were associated with intracellular membranes. Biochemical studies also showed that the palmitoylation-deficient mutants are associated with membranes as tightly as WT eNOS. Mutation of the N-myristoylation site Gly-2 (abolishing both N-myristoylation and palmitoylation) caused the GFP fusion protein to distribute throughout the cell as GFP alone, consistent with its primarily cytosolic nature in biochemical studies. Therefore, eNOS targets into the Golgi region of NIH 3T3 cells via the first 35 amino acids, including N-myristoylation and palmitoylation sites, and its overall membrane association requires N-myristoylation but not cysteine palmitoylation. These results suggest a novel role for fatty acylation in the specific compartmentalization of eNOS and most likely, for other dually acylated proteins, to the Golgi complex.