Oxidative Stress Induces Caveolin 1 Degradation and Impairs Caveolae Functions in Skeletal Muscle Cells

Increased level of oxidative stress, a major actor of cellular aging, impairs the regenerative capacity of skeletal muscle and leads to the reduction in the number and size of muscle fibers causing sarcopenia. Caveolin 1 is the major component of caveolae, small membrane invaginations involved in signaling and endocytic trafficking. Their role has recently expanded to mechanosensing and to the regulation of oxidative stress-induced pathways. Here, we increased the amount of reactive oxidative species in myoblasts by addition of hydrogen peroxide (H₂O₂) at non-toxic concentrations. The expression level of caveolin 1 was significantly decreased as early as 10 min after 500 μM H₂O₂ treatment. This reduction was not observed in the presence of a proteasome inhibitor, suggesting that caveolin 1 was rapidly degraded by the proteasome. In spite of caveolin 1 decrease, caveolae were still able to assemble at the plasma membrane. Their functions however were significantly perturbed by oxidative stress. Endocytosis of a ceramide analog monitored by flow cytometry was significantly diminished after H₂O₂ treatment, indicating that oxidative stress impaired its selective internalization via caveolae. The contribution of caveolae to the plasma membrane reservoir has been monitored after osmotic cell swelling. H₂O₂ treatment increased membrane fragility revealing that treated cells were more sensitive to an acute mechanical stress. Altogether, our results indicate that H₂O₂ decreased caveolin 1 expression and impaired caveolae functions. These data give new insights on age-related deficiencies in skeletal muscle.     

Caveolin Transfection Results in Caveolae Formation but Not Apical Sorting of Glycosylphosphatidylinositol (GPI)-anchored Proteins in Epithelial Cells

Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The “raft” hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI- anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI- anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42–53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI- anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.     

Caveolae as portals of entry for microbes

Many pathogens, including many traditionally extracellular microbes, now appear capable of entry into host cells with limited loss of viability. A portal of entry shared by some bacteria, bacterial toxins, viruses and parasites are caveolae (or lipid rafts), which are involved in the import and intracellular translocation of macromolecules in host cells. A requirement for caveolae-mediated endocytosis of microbes appears to be that the respective receptor is a constituent of caveolae or must move to caveolae following ligation.     

Caveolae as potential mediators of MCH-signaling pathways

The melanin-concentrating hormone receptor (MCHR) 1 is a G protein-coupled receptor involved in the regulation of appetite and energy expenditure in mammals. Here, we show that MCHR1 partitions to lipid rafts in stably expressing Chinese hamster ovary cells. In addition to co-fractionating with lipid rafts containing caveolin-1 on sucrose gradients, caveolin-1 was present in MCHR1 immunoprecipitates, suggesting that MCHR1 complexes with caveolae. The cholesterol-depleting drug methyl-β-cyclodextrin impaired MCH-mediated ERK signaling. These data suggest that a functional interaction between MCHR1 and caveolin-1 in lipid rafts exists and provide a basis for further biochemical studies to understand the significance on MCH-mediated signal transduction events.     

Ultrastructural and Biochemical Changes in Cotyledon Reserve Tissues3

Changes in major protein, lipid and carbohydrate reserves duringthe germination of Citrus limon (L.) Burm. f. seeds have beenstudied. The rate of release of amino acids and soluble sugarshas been evaluated. Mobilization of protein reserves began 4 d after the onset ofimbibition. The main period of hydrolysis occurred between 8d and 24 d after the start of germination. Ultrastructural studies showed the presence of protein bodiesin quiescent cotyledon cells. These bodies virtually disappeared14 d after the start of germination. The nitrogenous compoundsthat were liberated and subsequently translocated were predominantlyin the form of asparagine, arginine, and proline. The cotyledonshad a lipid content representing 51.7% of their dry weight.Lipid reserves in quiescent cotyledons were laid down in theform of oil-bodies. These organelles rapidly disappeared asgermination progressed, and were replaced by vacuoles. The starch content of quiescent cotyledons is very low, butit increased considerably up to 20 d after germination started. Key words: Proteins, lipids, carbohydrates     

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white-matter regions of rat brain, and in Vibratome sections as well as paraffin sections.     

Caveolae and caveolae constituents in mechanosensing

Studies in modeled microgravity or during orbital space flights have clearly demonstrated that endothelial cell physiology is strongly affected by the reduction of gravity. Nevertheless, the molecular mechanisms by which endothelial cells may sense gravity force remain unclear. We previously hypothesized that endothelial cell caveolae could be a mechanosensing system involved in hypergravity adaptation of human endothelial cells. In this study, we analyzed the effect on the physiology of human umbilical vein endothelial cell monolayers of short exposure to modeled microgravity (24–48h) obtained by clinorotation. For this purpose, we evaluated the levels of compounds, such as nitric oxide and prostacyclin, involved in vascular tone regulation and synthesized starting from caveolae-related enzymes. Furthermore, we examined posttranslational modifications of Caveolin (Cav)-1 induced by simulated microgravity. The results we collected clearly indicated that short microgravity exposure strongly affected endothelial nitrix oxide synthase activity associated with Cav-1 (Tyr 14) phosphorylation, without modifying the angiogenic response of human umbilical vein endothelial cells. We propose here that one of the early molecular mechanisms responsible for gravity sensing of endothelium involves endothelial cell caveolae and Cav-1 phosphorylation.     

Seed Development in Podocarpus henkelii: an Ultrastructural and Biochemical Study

Biochemical and fine-structural studies were undertaken on theembryo and female gametophyte of seeds of Podocarpus henkeliiduring the 24-week period of post-fertilization growth and development. Although the d. wt of the embryo and female gametophyte showeda steady increase during development, the levels of sugars,amino acids, proteins and lipids differed between seed partsand showed changes during the four sampling intervals. Onlystarch was seen to increase steadily throughout development.Lipid levels were high in the mature embryo and free amino acidsshowed a steady increase until the seeds were shed. At thisstage seeds were fully hydrated, possessed abundant reservesand all organelles appeared fully functional. This was interpretedas part of the development strategy of neotonous (recalcitrant)seeds, namely, the maintenance of full metabolic competencefor continued growth in the absence of development arrest whichfollows drying in orthodox seeds. Podocarpus henkelii, yellow wood, embryo and endosperm growth, neotonous seed, recalcitrance     

Seed Germination in Podocarpus henkelii: An Ultrastructural and Biochemical Study

Biochemical and ultrastructural studies were undertaken on theembryo and female gametophyte of neotonous (recalcitrant) seedsof Podocarpus henkelii over a 9-d period following scarificationand incubation on a moist substrate. After 3 days incubationat 25 °C, cells of the root tip were characterized at theelectron microscope level by increased vacuolation, numerousamyloplasts and lipid mobilization. By d 6 measurable embryonicgrowth was noted and ultrastructural evidence of synthetic activitywas suggested by abundant endoplasmic reticulum (ER), ribosomesand dictyosomes. Fine-structural changes suggestive of reservemobilization were also observed in the female gametophyte. Biochemicalstudies indicated a gradual decline in lipid and protein inboth the embryo, and female gametophyte, over the 9 d of incubation.A decline in embryonic starch levels contrasted with the increaseseen in the female gametophyte at d 6. Small changes in thesugar levels of the female gametophyte contrasted with increasesin the embryonic tissues between d 3 and 6, a period coincidingwith the first records of germination. The maintenance of highmoisture contant and the evidence of metabolic activity obtainedfrom biochemical and ultrastructural observations suggest that,following scarification, the transition between maturation andgermination is characterized by a continuation of earlier syntheticactivity and reserve interconversions. Podocarpus henkelii Stapf, yellow-wood, embryo, endosperm, overgrown seed, neotonous, recalcitrant     

Caveolae and lipid trafficking in adipocytes

The abundance of caveolae in adipocytes suggests a possible cell-specific role for these structures, and because these cells take up and release fatty acids as their quantitatively most robust activity, modulation of fatty acid movement is one such role that is supported by substantial in vitro and in vivo data. In addition, caveolae are particularly rich in cholesterol and sphingolipids, and indeed, fat cells harbor more cholesterol than any other tissue. In this article, we review the role of adipocyte caveolae with regard to these important lipid classes.     

Immunological Approach to the Detection of Taurine and Immunocytochemical Results

An immunological approach to the detection of taurine resulted in antibodies specific enough to be used for immunocytochemical studies. The experimental conditions were similar to those previously described for raising antibodies against some small-sized neurotransmitter molecules: antisera were obtained from rabbits immunized with taurine conjugated to carrier proteins via glutaraldehyde and purified by adsorption on the glutaraldehyde-treated protein carriers. Antibody affinity and specificity were determined in competition experiments between conjugated taurine and other conjugated amino acids or derivatives by enzyme-linked immunosorbent assay. The resulting cross-reactivity ratios, calculated at half-displacement, showed conjugated taurine to be the best recognized compound. Given the molecular structure of taurine and the method used to prepare the conjugate, it seemed necessary to perform an oxidation step. However, adsorption of antisera on reoxidized or nonreoxidized taurine conjugates suggested that reoxidation did not make a significant difference. Immunocytochemical application of the sera revealed populations of strongly immunopositive nerve cells in the cerebellum, striatum, and septum. The results confirmed that antitaurine antibodies can be used as specific tools for a better understanding of the role of taurine in the central nervous system.     

Immunocytochemical Detection of Anti-Hippocampal Antibodies in Alzheimer's Disease and Normal Cerebrospinal Fluid

Immunocytochemical staining was performed to investigate the presence of anti-hippocampal antibodies in cerebrospinal fluid (CSF) from patients with probable Alzheimer's disease (AD) (n = 19), aged normal controls (n = 9), and young normal controls (n = 10). Marked staining of neurons in the granule cell layer of the dentate gyrus and in pyramidal neurons in CA1-3 of the rat hippocampus was observed in 5 AD CSF samples (26%), 1 aged control sample (11%), and 1 young control sample (10%). These differences were not statistically significant. One of the immunoreactive AD CSF specimens also contained high concentrations of C5b-9, the membrane attack complex. The infrequent occurrence of anti-hippocampal antibodies in AD CSF, and the detection of similar immunoreactivity in control CSF specimens, suggest that these antibodies are unlikely to play a role in the neurodegenerative process in most individuals with AD. However, elevated C5b-9 concentration in an AD CSF specimen with marked immunoreactivity to hippocampal neurons suggests the possibility that anti-neuronal antibodies may contribute to complement activation in some AD patients.     

Immunocytochemical Detection of Structural and Regulatory Proteins in Rat Adrenal Nuclear Matrix

The nuclear matrix is a specific cell structure consisting of a residual nucleoskeleton that extends from the nucleoli to the nuclear envelope. The nuclear matrix of steroido-genic cells was isolated previously from a purified nuclear fraction. We present here an in situ extraction method, modified Lutz's method, for rat glandular adrenal cell nuclear matrix. This residual organelle was characterized and studied using immunocytochemical methods. The adrenal glands were removed, the cells prepared in suspension and deposited by cytospin onto Poly-L-lysine glass slides. The nuclear matrix was extracted with Nonidet P-40, DNase I and high and low ionic strength buffers. Structural proteins, nuclear lamins, coilin and fibrillarin were detected immunocytochemically. The adrenal fasciculata cells were easily identified by this method because of their large nuclei and abundant lipid droplets in the cytoplasm. After immunocytochemical detection by antibodies against lamins A and C, a marked brown layer at the periphery of the nucleus was observed. The intensity of the staining was lower using the antibody against nuclear lamin B. Immunocytochemical detection of the protein coilin revealed punctuated stained areas, 2-6 per nucleus, that probably correspond to the coiled bodies. The protein fibrillarin was detected at the nucleolus and coiled bodies. Our technique is simple, reveals well preserved adrenal nuclear matrices, and may be a useful method for immunocytochemical analysis and in situ hybridization.     

Internalization of Echovirus 1 in Caveolae

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor alpha 2 beta 1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, alpha 2 beta 1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.     

Caveolae and caveolin-3 in muscular dystrophy

Caveolae are vesicular invaginations of the plasma membrane, and function as 'message centers' for regulating signal transduction events. Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolar membrane domains in skeletal muscle and in the heart. Several mutations within the coding sequence of the human caveolin-3 gene (located at 3p25) have been identified. Mutations that lead to a loss of approximately 95% of caveolin-3 protein expression are responsible for a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans. By contrast, upregulation of the caveolin-3 protein is associated with Duchenne muscular dystrophy (DMD). Thus, tight regulation of caveolin-3 appears essential for maintaining normal muscle health and homeostasis.     

Caveolae as an additional route for influenza virus endocytosis in MDCK cells

Clathrin-mediated endocytosis has been described as the primary internalization pathway for many viruses, including the influenza virus. However, caveolae, an alternative clathrin-independent endocytotic pathway, has also been described as mediating the entry of some molecules, including viruses. To address the question of pathway selection by the influenza virus, we have investigated whether the virus is internalized via clathrin-coated pits and/or caveolae in Madin Darby canine kidney (MDCK) cells. By applying pharmacological manipulations to selectively disrupt the cell internalization pathways, we found that, in MDCK cells, the influenza virus may be internalized via caveolae in addition to entry by clathrin-mediated endocytosis. However, a small contribution by another mode of entry, as recently proposed, cannot be excluded.     

Caveolin scaffolding region and cholesterol-rich domains in membranes

A protein that constitutes a good marker for a type of cholesterol-rich domain in biological membranes is caveolin. A segment of this protein has a sequence that corresponds to a cholesterol recognition/interaction amino acid consensus (CRAC) motif; this motif has been suggested to cause the incorporation of proteins into cholesterol-rich domains. We have studied the interaction of two peptides containing the CRAC motif of caveolin-1 by differential scanning calorimetry, fluorescence, circular dichroism and magic angle spinning NMR. These peptides promote the segregation of cholesterol into domains from mixtures of the sterol with phosphatidylcholine, as shown by depletion of cholesterol from a portion of the membrane and enrichment of cholesterol in another domain. Cholesterol passes its solubility limit in the cholesterol-rich domain, resulting in the formation of cholesterol crystallites, suggesting that not all of the cholesterol recruited to this domain is bound to the peptide. NMR studies show that the peptides insert somewhat more deeply into membranes when cholesterol is present, but their strongest interaction takes place with the interfacial region of the membrane. We conclude that the peptides we studied containing CRAC sequences are more effective in promoting the formation of cholesterol-rich domains than are shorter peptides of this region of caveolin, which although they contain several aromatic amino acids, they have no CRAC motif. The presence or absence of a CRAC motif, however, is not a sufficient criterion to determine the extent to which a protein will promote the segregation of cholesterol in membranes.     

An Ultrastructural and Immunocytochemical Study of Gastrodermal Cell Types in Microstomum lineare (Turbellaria,Macrostomida)

The gastrodermal cell types of Microstomum lineare (Turbellaria, Macrostomida) were studied by electron microscopy. Their immunoreactivity (IR) to bovine pancreatic peptide (BPP), FMRF-amide and vasotocin, somatostatin, neurotensin, ACTH, CCK, bombesin, secretin, gastrin/CCK and insulin antisera was tested by light microscopic immunocytochemical methods. In addition to granular club cells and phagocytic cells, neurons and neoblasts occur in the gastroderm of this turbellarian species. This is the first observation of neurons in the gastroderm of a flatworm. Dense-core vesicles (70–100 nm diameter), electron lucent cytoplasm and numerous Golgi complexes characterize the neurons. Unpolarized two-way synapses, neuromuscular junctions and polarized chemical synapses can be observed in the gastroderm. Neoblasts with large nuclei and scanty cytoplasm and differentiating cells containing clusters of basal bodies occur next to the basal lamina of the gastroderm. BPP-like, FMRF-amide-like and vasotocin-like immunoreactivity is demonstrated in the gastroderm. Both BPP and FMRF-amide IR is restricted to the basal cytoplasm of the granular club cells, while a different location for IR to vasotocin antiserum is observed. The status of the neuronal cell in the gastroderm of M. lineare is discussed in relation to endocrine (paracrine) cells and neurons in the gastroderm of invertebrates.