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PubMed2003

Papillomaviruses infect cells via a clathrin-dependent pathway.

In this study we have examined the pathway by which papillomaviruses infect cells, using bovine papillomavirus (BPV) virions and mouse C127 cells as the model system. By confocal microscopy, the entry of BPV virions, BPV virus-like particles (VLPs), and HPV16 VLPs were very similar. In dually exposed cells, HPV-16 VLPs and BPV virions colocalized intracellularly. BPV VLPs colocalized with AP-2, a clathrin adapter molecular and a marker of the clathrin-dependent endocytic pathway; and also with transferrin receptor, a marker of early endosomes; and Lamp-2, a marker of late endosomes and lysosomes. BPV infection was detected within 12 h of virion cell-surface binding, as measured by an RT-PCR assay. Infection was prevented by several pharmacologic inhibitors, including chlorpromazine, which blocks clathrin-dependent endocytosis and the lysosomotropic agent, bafilomycin A. By contrast, two inhibitors of caveolae-dependent uptake, filipin and nystatin, did not prevent BPV infection. We conclude that papillomaviruses infect cells via clathrin-dependent receptor-mediated endocytosis. Surprisingly, the kinetics of internalization were unusually slow for this mechanism, with the t(1/2) of entry of BPV-1 being approximately 4 h versus 5-15 min for a typical ligand.

Animals↗
PubMed1998

Uptake of suramin by human microvascular endothelial cells.

We have demonstrated for the first time that suramin is taken up by human dermal microvascular endothelial (HMEC-1) cells by an active process involving the caveolae system. The uptake of suramin was time-dependent and reduced by more than 90% when incubated in the presence of albumin or at 4 degrees C. Suramin uptake was also inhibited when incubated in the presence of filipin and digitonin, both potent cholesterol-binding agents, but not in the presence of probenecid. The [3H]suramin taken up by the HMEC-1 cells was located primarily within the nucleus, followed by the cytoplasmic fraction. The presence of suramin in these cellular compartments suggests that this drug may act through intracellular mechanisms.

Antineoplastic Agents↗
PubMed2003

Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes.

BACKGROUND: In contrast to the intense attention devoted to research on intracellular sterol trafficking in animal cells, knowledge about sterol transport in plant cells remains limited, and virtually nothing is known about plant endocytic sterol trafficking. Similar to animals, biosynthetic sterol transport occurs from the endoplasmic reticulum (ER) via the Golgi apparatus to the plasma membrane. The vesicle trafficking inhibitor brefeldin A (BFA) has been suggested to disrupt biosynthetic sterol transport at the Golgi level. RESULTS: Here, we report on early endocytic sterol trafficking in Arabidopsis root epidermal cells by introducing filipin as a tool for fluorescent sterol detection. Sterols can be internalized from the plasma membrane and localize to endosomes positive for the early endosomal Rab5 GTPase homolog ARA6 fused to green fluorescent protein (GFP) (ARA6-GFP). Early endocytic sterol transport is actin dependent and highly BFA sensitive. BFA causes coaccumulation of sterols, endocytic markers like ARA6-GFP, and PIN2, a polarly localized presumptive auxin transport protein, in early endosome agglomerations that can be distinguished from ER and Golgi. Sterol accumulation in such aggregates is enhanced in actin2 mutants, and the actin-depolymerizing drug cytochalasin D inhibits sterol redistribution from endosome aggregations. CONCLUSIONS: Early endocytic sterol trafficking involves transport via ARA6-positive early endosomes that, in contrast to animal cells, is actin dependent. Our results reveal sterol-enriched early endosomes as targets for BFA interference in plants. Early endocytic sterol trafficking and recycling of polar PIN2 protein share a common pathway, suggesting a connection between plant endocytic sterol transport and polar sorting events.

Actins↗
PubMed2002

Caveolae and caveolin isoforms in rat peritoneal macrophages.

Caveolea are special (highly hydrophobic) plasma membrane invaginations with a diameter of 50-100 nm. Their characteristic features are the flask- or omega-shape and the lack of basket-like coat composed of clathrin. Caveolin-an integral membrane protein-is the principal component of caveolae membranes in vivo. Multiple forms of caveolin have been identified: caveolin-1alpha, caveolin-1beta, caveolin-2 and caveolin-3. They differ in their specific properties and tissue distribution. In this paper we summarize the morphological and biochemical data providing strong evidence about the existence and function of caveolae in rat peritoneal macrophages. When studied electron microscopically, the surface of both resident and elicited macrophages exhibited omega- or flask-shaped plasma membrane invaginations. There was a significant difference, however, in the number of these profiles: whereas in resident cells only a small amount of them was found on the cell surface, in elicited cells they were abundantly present on the plasma membrane. Using an antibody against the VIP21/caveolin-1 isoform we showed that these plasma membrane pits were indeed caveolae. The number and the appearance of caveolae were found to be in close correlation with the functional activity of these phagocytotic cells, indicating that the formation of caveolae is a highly regulated process. Using Western blot analysis two different proteins ( approximately 29 and approximately 20 kDa)-both labelled with anti-caveolin antibodies-were identified in resident and elicited macrophages that have been isolated from rat peritoneal cavity. The approximately 20 kDa protein was labelled specifically only by anti-VIP21/caveolin-1, while the approximately 29 kDa protein was labelled by both anti-VIP21/caveolin-1 and anti-caveolin-2 antibodies. The presence of the approximately 29 kDa protein was highly characteristic of resident cells, and only a small amount of approximately 20 kDa protein was detected in these cells. Elicitation has resulted in a significant increase in the amount of approximately 20 kDa protein labeled only with anit-VIP21/caveolin-1. Our morphological (confocal and electron microscopical) studies have shown that in resident cells caveolin was present in the cytoplasm, in smaller vesicles and multivesicular bodies around the Golgi area. Only a very small amount of caveolae was found on the cell surface of these cells. In elicited macrophages, caveolae (labelled with anti-VIP21/caveolin-1 antibody) appeared in large numbers on the cell surface, but caveolin detected by anti-caveolin-2 was also found in small vesicles and multivesicular bodies. These data support the idea that the expression of the approximately 29 kDa (caveolin-related) protein is insufficient for caveolae formation in resident cells, it can function as a modified, macrophage-specific caveolin-2 isoform. Our results strongly suggest that caveolin-1 plays a crucial role in the formation of caveolae: it is the amount of caveolin-1 that regulates the appearance of caveolae on the plasma membrane. Studying the endocytotic processes of resident and elicited macrophages we have found that elicited macrophages bound and internalized significantly larger amounts of fluid phase marker (HRP) and immune complex (peroxidase-antiperoxidase-PAP) than resident cells. Serial section analysis, double labelled immunocytochemistry, and filipin treatment were used to demonstrate that caveolae can pinch off from the plasma membrane and can take part in endocytotic processes as alternative carriers in elicited macrophages.

Animals↗
PubMed1994

Cholesterol distribution in renal epithelial cells LLC-PK1 as determined by cholesterol oxidase: evidence that glutaraldehyde fixation masks plasma membrane cholesterol pools.

Treatment with cholesterol oxidases has shown that cholesterol is heterogeneously distributed in brush border membranes isolated from the apical domain of the renal and intestinal epithelial cells [Bloj, B., & Zilversmit, D. B. (1982) J. Biol. Chem. 257, 7608-7614; El Yandouzi, E. H., & Le Grimellec, C. (1992) Biochemistry 31, 547-551]. Cholesterol distribution between plasma membrane and intracellular membranes of the corresponding cells remains unexplored. The effects of Brevibacterium sp. cholesterol oxidase on the cholesterol content of LLC-PK1 cells, an epithelial cell line with multiple differentiated characteristics of the renal proximal tubule, were investigated. In confluent living cells grown as a monolayer on solid support, a small but significant fraction (13%) of the cholesterol was oxidized during the first hour of the oxidase treatment. Glutaraldehyde fixation prior to treatment resulted in a nearly complete (86.1 +/- 1.8) oxidation of the cellular cholesterol according to first-order kinetics. Filipin labeling and oxidation at 15 degrees C confirmed that cholesterol was essentially confined to the plasma membrane in LLC-PK1 cells. When adding the oxidase either on the apical or on the basolateral side of cells grown on permeant support and fixed with glutaraldehyde, a comparable monophasic oxidation of cholesterol was observed, despite the presence of efficient tight junctions. Adding the oxidase to both sides simultaneously did not increase the rate of oxidation. Finally, fixation of isolated renal brush border membranes with glutaraldehyde rendered undiscernible their cholesterol pools. We conclude that glutaraldehyde fixation, a commonly used process in the analysis of cholesterol distribution in cells, can mask the existence of cholesterol pools in plasma membranes.

Brevibacterium↗
PubMed1981

Effects of sterol structure and exogenous lipids on the transbilayer distribution of sterols in the membrane of Mycoplasma capricolum.

Stopped-flow kinetic measurements of the association of filipin with sterols in intact cells and isolated membranes of Mycoplasma capricolum were used to study the effects of varying the phospholipid in the membrane. The phospholipid composition and content of the membrane were varied by growing cells in an albumin-containing medium with cholesterol, palmitic and oleic acids, and various concentrations of exogenous phospholipids. The exogenous phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidic acid) were incorporated up to levels of approximately 50% of the total membrane phospholipids but had no effect on the distribution of cholesterol between the two halves of the membrane bilayer. The sterol structure was varied by growing the cells with 10/micrograms/mL of either cholesterol, beta-cholestanol, 4,6-cholestadien-3 beta-ol, ergosterol, beta-sitosterol, or stigmasterol. With cholesterol, beta-cholestanol, and 4,6-cholestadien-3 beta-ol, approximately 65% of the sterol was found to be present in the outer half of the lipid bilayer. With ergosterol, beta-sitosterol, and stigmasterol, about 89% of the sterol is localized in the outer half of the membrane bilayer. Thus, the behavior of the alkyl-substituted sterols differs from that of cholesterol. The extent to which a sterol is distributed asymmetrically between the two halves of the bilayer is not related to the extent to which maximum growth is produced. These results suggest that growth-supporting sterols need not be translocated extensively.

Cell Membrane↗
PubMed1997

Cholesterol as modulator of receptor function.

The modulatory effect of cholesterol on the function of two structurally related peptide receptors, the oxytocin receptor and the brain cholecystokinin receptor in plasma membranes as well as in intact cells, was analyzed. Different approaches for cholesterol modification were applied: (i) depletion and reloading of cholesterol mediated by methyl-beta-cyclodextrin and cholesterol-methyl-beta-cyclodextrin, respectively, in a reversible manner; (ii) mild treatment of the plasma membranes with cholesterol oxidase under control of the membrane fluidity as measured by fluorescence anisotropy of diphenylhexatriene; and (iii) filipin pretreatment of membranes. The results allowed us to distinguish two mechanisms of cholesterol affecting the ligand-binding function of receptors: changes of the membrane fluidity as demonstrated for the cholecystokinin receptor, or a putatively specific cholesterol-receptor interaction as shown for the oxytocin receptor. This was confirmed in a structure-activity analysis with a variety of sterol analogues substituting for cholesterol in the membranes. While the agonist binding of the cholecystokinin receptor was supported by each of the tested steroids and was well correlated with the corresponding fluorescence anisotropy values, a stringent and unique requirement of the oxytocin receptor's affinity state for structural features of the sterol molecule was found. The molecular requirements differ both from those postulated for sterol-phospholipid interactions and from those known to be necessary for the functional activity of other proteins. The different behavior of both peptide receptors concerning the cholesterol dependence of their ligand binding was also present in vivo at the level of signal transduction. The results suggest that cholesterol can modulate receptor function by two distinct mechanisms, by changes of the membrane fluidity, and/or by a highly specific molecular interaction.

Animals↗
PubMed1999

Membrane cholesterol modulates galanin-GalR2 interaction.

The neuropeptide galanin mediates a number of diverse physiological and pathophysiological actions via interaction with membrane-bound receptors. The role that membrane cholesterol plays in modulating the interaction between galanin and one of the three cloned galanin receptor subtypes (GalR2) expressed in Chinese hamster ovary (CHO) cells was examined. Reduction of membrane cholesterol by treatment with methyl-beta-cyclodextrin (CD) or by culturing cells in lipoprotein-deficient serum markedly decreased galanin binding to the receptor. Addition of cholesterol back to CD-treated, cholesterol-depleted membranes restored galanin binding to control levels. Hill analysis suggests that the GalR2 binds multiple molecules of cholesterol (n >/= 3) in a positively cooperative manner. This interaction appears to be cholesterol-specific as only cholesterol and a limited number of cholesterol analogues were able to rescue galanin binding. The inability of some of these analogues to rescue the binding activity also suggests that binding of galanin to GalR2 is independent of membrane fluidity as, like cholesterol, cholesterol analogues generally rigidize membranes. In addition, treatment of the membranes with other modulators of membrane fluidity, e.g. ethanol, did not affect galanin binding to the GalR2. In contrast, treatment of membranes, with filipin, a molecule that clusters cholesterol within the membranes, or with cholesterol oxidase resulted in markedly reduced galanin binding. Incubation of membranes with 100 microM GTP-gamma-S did not alter the IC(50) for CD in the prebinding assay treatment suggesting that the effect of cholesterol was independent of G protein interaction. Preincubation of intact cells with CD also drastically impaired the ability of galanin to activate intracellular inositol phosphate accumulation in GalR2-transfected CHO cells. These data detail a new mechanism for the regulation of galanin receptor signaling which may link altered functions of GalRs with abnormal cholesterol metabolism.

Animals↗
PubMed2000

Effect of synthetic sialyl 2-->1 sphingosine and other glycosylsphingosines on the structure and function of the "glycosphingolipid signaling domain (GSD)" in mouse melanoma B16 cells.

Mouse melanoma B16 cells are characterized by a high concentration of GM3 ganglioside, which has been identified as a melanoma-associated antigen and is present as a clustered microdomain organized with major signal transducers, c-Src, small G-protein (Rho A), and focal adhesion kinase (FAK), to form a "glycosphingolipid signaling domain" or "glycosignaling domain" (GSD) separable from cholesterol- and caveolin-enriched microdomain, "caveolae." Cholesterol-binding reagents, filipin and nystatin, disrupt the structure and function of caveolae, but have no effect on GSD function [Iwabuchi, K., et al. (1998) J. Biol. Chem. 273, 33766-33773]. In this study, we searched for compounds which disrupt the structure and function of GSD in B16 cells. Such compounds should have structural features analogous to those of GM3, destroy or reduce clustering of GM3 in GSD, and inhibit GM3-dependent adhesion and signaling. The simplest compound so far found with these properties is sialyl alpha2-->1 sphingosine (Sph). We describe the synthesis of this compound and its analogues, and their effects on GM3 expression pattern and GSD function, in comparison with effects of lyso-GM3 and other lyso compounds, in B16 cells. Incubation of B16 cells with 0.5-10 microM sialyl alpha2-->1 Sph or 1-5 microM lyso-GM3 reduced GM3 clustering and GM3-dependent adhesion, and inhibited adhesion-dependent cellular FAK activity. The c-Src activation response of GSD isolated from B16 cells was inhibited strongly by sialyl alpha2-->1 Sph. Substitution of the Sph amino group with a chloroacetyl or N,N-dimethyl group strongly reduced the inhibitory effect of sialyl alpha2-->1 Sph on GM3-dependent adhesion, FAK, and c-Src response. Other lyso compounds such as lyso-phosphatidylcholine, galactosyl-Sph (psychosine), and lactosyl-Sph at 0.5-10 microM did not show the same effect as sialyl alpha2-->1 Sph. Thus, adhesion coupled with signal transduction, initiated by clusters of GM3 in GSD, is blocked by sialyl alpha2-->1 Sph or lyso-GM3. Analogues with N-substitution of Sph in sialyl alpha2-->1 Sph, other lyso-phospholipids, and galactosyl- or lactosyl-Sph did not block such adhesion, coupled with activation of c-Src and FAK.

Animals↗
PubMed2000

Pentosan polysulfate regulates scavenger receptor-mediated, but not fluid-phase, endocytosis in immortalized cerebral endothelial cells.

1. Effects of pentosan polysulfate (PPS) and the structurally related sulfated polyanions dextran sulfate, fucoidan, and heparin on the scavenger receptor-mediated and fluidphase endocytosis in GP8 immortalized rat brain endothelial cells were investigated. 2. Using 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarboxyamine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL), we found a binding site with high affinity and low binding capacity, and another one with low affinity and high binding capacity. Increasing ligand concentrations could not saturate DiI-AcLDL uptake. DiI-AcLDL uptake, but not binding, was sensitive to pretreatment with filipin, an inhibitor of caveola formation. 3. PPS (20-200 microg/ml) significantly reduced the binding of DiI-AcLDL after coincubation for 3 hr, though this effect was less expressed after 18 hr. Among other polyanions, only fucoidan decreased the DiI-AcLDL binding after 3 hr, whereas dextran sulfate significantly increased it after 18 hr. PPS treatment induced an increase in DiI-AcLDL uptake, whereas other polysulfated compounds caused a significant reduction. 4. Fluid-phase endocytosis determined by the accumulation of Lucifer yellow was concentration and time dependent in GP8 cells. Coincubation with PPS or other sulfated polyanions could not significantly alter the rate of Lucifer yellow uptake. 5. In conclusion. PPS decreased the binding and increased the uptake of DiI-AcLDL in cerebral endothelial cells, an effect not mimicked by the other polyanions investigated.

Animals↗
PubMed1999

Bacteria-host cell interaction mediated by cellular cholesterol/glycolipid-enriched microdomains.

Gram negative bacterial infection is a leading cause of fatality and is attributed, at least in part, to the bacteria's capacity to persist in the host in spite of appropriate antibiotic therapy. It has been suggested that bacteria evade antibiotics by hiding within host cells. We sought to investigate this important aspect of infections in mast cells, which are inflammatory cells found in close proximity to the host-environment interface and which have recently been reported to play a crucial role in the early innate immune response to bacteria. We examined mast cell interactions with FimH-expressing E. coli, one of the major opportunistic pathogens of humans. We determined that in serum free conditions, these bacteria were able to trigger mast cell uptake without loss of bacterial viability. CD48, a mannose containing GPI (glycosylphosphatidylinositol)-linked molecule was found to be the receptor of FimH-expressing E. coli in mouse mast cells. We found that the internalization via CD48 was blocked by filipin, a cholesterol binding drug known to disrupt cholesterol/glycolipid-enriched microdomains and the bacteria-encasing vacuoles were rich in cholesterol inside cells. Interestingly, we found that mast cells subsequently expelled majority of the intracellular bacteria in 24 hours. This expulsion process was blocked by lovastatin/cyclodextrin treatment, which is known to inhibit cellular trafficking of cholesterol/glycolipid-enriched microdomains. Thus, the bacterial entry into and expulsion from mast cells were critically dependent on cholesterol/glycolipid-enriched microdomains, which represents a novel mode of tussle between the pathogen and the mast cell occurring in opsonin deficient sites in the body or even at other sites in naive or immunocompromised hosts which have low systemic levels of E. coli specific antibody.

Adhesins, Bacterial↗
PubMed2002

Uptake of FITC-chitosan nanoparticles by A549 cells.

PURPOSE: The objective of this study was to evaluate the extent and mechanism of uptake of fluorescent chitosan nanoparticles by the A549 cells, a human cell line derived from the respiratory epithelium. METHODS: Covalent conjugation with fluorescein-5-isothiocyanate yielded stably labeled chitosan molecules, which were successfully formulated into nanoparticles by ionotropic gelation. Uptake of fluorescein-5-isothiocyanate-chitosan nanoparticles and chitosan molecules by confluent A549 cells was quantified by fluorometry. RESULTS: Cellular uptake of chitosan nanoparticles was concentration and temperature dependent, having Km and Vmax of 3.84 microM and 58.14 microg/mg protein/h, respectively. Uptake of chitosan nanoparticles was up to 1.8-fold higher than that of chitosan molecules alone and was not inhibited by excess unlabeled chitosan molecules. Hyperosmolarity, chlorpromazine and K+ depletion inhibited by 65, 34, and 54%, respectively, the uptake of chitosan nanoparticles at 37 degrees C, but filipin had no influence on the uptake. Confocal imaging confirmed the internalization of the chitosan nanoparticles by the A549 cells at 37 degrees C. CONCLUSIONS: Formulation of chitosan into nanoparticles significantly improved its uptake by the A549 cells. Internalization of chitosan nanoparticles by the cells seems to occur predominantly by adsorptive endocytosis initiated by nonspecific interactions between nanoparticles and cell membranes, and was in part mediated by clathrin-mediated process.

Chitin↗
PubMed1999

U18666A inhibits intracellular cholesterol transport and neurotransmitter release in human neuroblastoma cells.

To determine if neurochemical function might be impaired in cell models with altered cholesterol balance, we studied the effects of U18666A (3-beta-[(2-diethyl-amino)ethoxy]androst-5-en-17-one) on intracellular cholesterol metabolism in three human neuroblastoma cell lines (SK-N-SH, SK-N-MC, and SH-SY5Y). U18666A (< or =0.2 microg/ml) completely inhibited low density lipoprotein (LDL)-stimulated cholesterol esterification in SK-N-SH cells, while cholesterol esterification stimulated by 25-hydroxycholesterol or bacterial sphingomyelinase was unaffected or partially inhibited, respectively. U18666A also blocked LDL-stimulated downregulation of LDL receptor and caused lysosomal accumulation of cholesterol as measured by filipin staining. U18666A treatment for 18 h resulted in 70% inhibition of K+-evoked norepinephrine release in phorbol ester-differentiated SH-SY5Y cells, while release stimulated by the calcium ionophore A23187 was only slightly affected. These results suggest that U 18666A may preferentially block a voltage-regulated Ca2+ channel involved in norepinephrine release and that alterations in neurotransmitter secretion might be a feature of disorders such as Niemann-Pick Type C, in which intracellular cholesterol transport and distribution are impaired.

Androstenes↗
PubMed2002

Niemann-Pick disease type C in adults.

Although it is often perceived as a paediatric disorder, significant numbers of patients with Niemann-Pick disease type C present for the first time in adult life or survive into adult life. The presentation in these patients differs from that seen in the classical juvenile form of the disease. Adult patients are often referred to clinicians with psychosis or other major psychiatric problems. The dystonia with preserved intellectual functioning can be mistaken for other basal ganglia disorders such as Wilson disease. The presence of vertical gaze palsy is an important clinical clue and, in the presence of a modest increase in plasma chitotriosidase activity, can be very helpful in the differential diagnosis. The diagnosis should be confirmed in suspected cases by filipin staining of cultured fibroblasts, as well as cholesterol esterification studies and DNA mutation analysis.

Adolescent↗
PubMed2003

Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles.

PURPOSE: To evaluate the uptake of chitosan molecules (fCS) and nanoparticles (fNP), and their ability to mediate insulin transport in Caco-2 cell monolayers. METHODS: Cell-associated fCS and fNP were evaluated by fluorometry, trypan blue quenching, and confocal microscopy using FITC-labeled chitosan. Chitosan-mediated transport of FITC-labeled insulin was studied in Caco-2 cell monolayers cultured on permeable inserts. RESULTS: Caco-2 cells showed twofold higher association with fNP than fCS after 2-h incubation with 1 mg/ml samples. fNP uptake was a saturable (Km 1.04 mg/ml; Vmax 74.15 microg/mg/h), concentration- and temperature-dependent process that was inhibited by coadministered chlorpromazine. fCS uptake was temperature dependent, but was less sensitive to concentration and was inhibited by filipin. Postuptake quenching with 100 microg/ml of trypan blue suggests a significant amount of intracellular fNP, although the bulk of fCS was extracellular. Internalized fNP were located by confocal microscopy at 15 microm from the apical membrane, but there was no apparent breaching of the basal membrane. This might explain the failure of the nanoparticles to mediate significant insulin transport across the Caco-2 cell monolayer. CONCLUSIONS: Formulation of chitosan into nanoparticles transforms its extracellular interactions with the Caco-2 cells to one of cellular internalization via clathrin-mediated endocytosis.

Caco-2 Cells↗
PubMed1997

Survival of FimH-expressing enterobacteria in macrophages relies on glycolipid traffic.

Strains of Escherichia coli persist within the human gut as normal commensals, but are frequent pathogens and can cause recurrent infection. Here we show that, in contrast to E. coli subjected to opsonic interactions stimulated by the host's immune response, E. coli that bind to the macrophage surface exclusively through the bacterial lectin FimH can survive inside the cell following phagocytosis. This viability is largely due to the attenuation of intracellular free-radical release and of phagosome acidification during FimH-mediated internalization, both of which are triggered by antibody-mediated internalization. This different processing of non-opsonized bacteria is supported by morphological evidence of tight-fitting phagosomes compared with looser, antibody-mediated phagosomes. We propose that non-opsonized FimH-expressing E. coli co-opt internalization of lipid-rich microdomains following binding to the FimH receptor, the glycosylphosphatidylinositol-linked protein CD48, because (1) the sterol-binding agents filipin, nystatin and methyl beta-cyclodextrin specifically block FimH-mediated internalization; (2) CD48 and the protein caveolin both accumulate on macrophage membranes surrounding bacteria; and (3) antibodies against CD48 inhibit FimH-mediated internalization. Our findings bring the traditionally extracellular E. coli into the realm of opportunistic intracellular parasitism and suggest how opportunistic infections with FimH-expressing enterobacteria could occur in a setting deprived of opsonizing antibodies.

Adhesins, Bacterial↗
PubMed2001

Structure and composition of drusen associated with glomerulonephritis: implications for the role of complement activation in drusen biogenesis.

PURPOSE: The ocular fundi of many patients with membranoproliferative glomerulonephritis type II (MPGN-II) are characterised by the presence of deposits within Bruch's membrane that resemble drusen, hallmark lesions associated with age-related macular degeneration (AMD). Glomerulonephritis (GN)-associated drusen appear at a younger age, however, than do drusen in individuals with AMD. In light of recent evidence that immune-mediated events participate in drusen biogenesis and AMD, we examined the structure and composition of drusen in eyes obtained from human donors with two distinct glomerulopathies, both of which involve complement deposition within glomeruli. These features were compared with those of drusen from patients with clinically documented AMD. METHODS: Eyes obtained from two human human donors diagnosed with membranous and post-streptococcal GN, respectively, were analysed histochemically, immunohistochemically and ultrastructurally. RESULTS: Subretinal pigment epithelial (RPE) deposits in both types of GN are numerous and indistinguishable, both structurally and compositionally, from drusen in donors with AMD. GN-associated drusen exhibit sudanophilia, bind filipin, and react with antibodies directed against vitronectin, complement C5 and C5b-9 complexes, TIMP-3 and amyloid P component. Drusen from the membranous GN donor, but not the post-streptococcal GN donor, reacted with peanut agglutinin and antibodies directed against MHC class II antigens and IgG. The ultrastructural characteristics of these deposits were also identical with those of AMD-associated drusen. CONCLUSIONS: The composition and structure of ocular drusen associated with membranous and post-streptococcal/segmental GN are generally similar to those of drusen in individuals with AMD. In view of the recent data supporting the involvement of complement activation in drusen biogenesis and the pathobiology of AMD, further studies of the biological relationships between AMD and diseases associated with complement activation are warranted.

Complement Activation↗
PubMed2000

Differences in caveolae dynamics in vascular smooth muscle cells of different phenotypes.

Vascular smooth muscle cells shift between two major differentiated states with distinct morphological and functional properties, a contractile and a synthetic phenotype. Here, primary cultures were used to study caveolae expression and dynamics in these cells. The results demonstrate that caveolae are more numerous and more actively interact with intracellular organelles in contractile than in synthetic cells. Immunohistochemistry showed that caveolin-1 was mainly localized to caveolae in contractile cells and partly shifted to Golgi-associated vesicles in synthetic cells, whereas caveolin-2 chiefly appeared in cytoplasmic vesicles in both cases. Cholera toxin B subunit, a ligand of GM1 ganglioside, was internalized via caveolae and carried to endosomes and Golgi-associated vesicles. In contractile cells, it later moved into Golgi and endoplasmic reticulum (ER) cisternae and thus had access to the entire endocytic and exocytic pathways. In contrast, in synthetic cells, the tracer was restricted to the endocytic pathway. Filipin staining similarly disclosed that cholesterol was more widely distributed in contractile than in synthetic cells, with strong labeling of both caveolae and adjacent ER portions. Although no direct continuity between caveolae and ER was detected, it is suggested that cholesterol and other molecules may be translocated between these compartments. The observed differences in caveolae expression and dynamics are likely to be significant for the differences in proliferative capacity and cholesterol transport between contractile and synthetic smooth muscle cells.

Animals↗