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Single-molecule localization microscopy with spontaneously blinking fluorescent tags holds a promise of simplified imaging setup for live-cell application. However, robust blinking has been reported for just a few fluorescent proteins. Here we report on a comparison of spontaneous blinking for three bright green fluorescent proteins, mAvicFP1, AausFP1, and mNeonGreen. mAvicFP1 outperforms other fluorescent proteins in this list in a wide range of camera exposure times and illumination intensities. We establish imaging conditions for live-cell nanoscopy and single-particle tracking with mAvicFP1., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Probiotics exhibit beneficial functions for host homeostasis maintenance. We herein investigated the mechanism by which Lactobacillus brevis-derived poly P exhibited a beneficial function. Immunostaining indicated that poly P was captured in the plasma membrane via integrin β1 in Caco2/bbe cells. The uptake of poly P was reduced by the inhibition of integrin β1 as well as caveolin-1, a major component of lipid rafts. The function of poly P, including the induction of HSP27 and enhancement of the intestinal barrier function, was suppressed by the inhibition of caveolin-1, illustrating that the function of poly P was mediated by the endocytic pathway. High-throughput sequencing revealed that poly P induced tumor necrosis factor alpha-induced protein 3, which contributes to cytoprotection, including upregulation of the intestinal barrier function. The present study demonstrates a novel host-probiotic interaction through the uptake of bacterial substance into host cells, which is distinct from pattern recognition receptor pathways., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The extracellular Ca(2+) sensing receptor (CaSR) belongs to Class C G-protein-coupled receptors (GPCRs) which include receptors for amino acids, γ-aminobutyric acid and glutamate neurotransmitters. CaSR has been described as having an extended sequence containing a Ca(2+) binding pocket within an extracellular amino (N)-terminal domain, called a Venus Fly Trap (VFT) module. CaSR is thought to consist of three domains: 1) a Ca(2+-)sensory domain, 2) a region containing 7 transmembrane (TM) helices, and 3) a carboxy (C)-terminal tail. We find that SPOCTOPUS (a combination of hidden Markov models and artificial neural networks) predicts that Homo sapiens CaSR contains two additional TM helices ((190)D - G(210); (262)S-E(282)), with the second TM helix containing a pore-lining region ((265)K - I(280)). This predicts that the putative Ca(2+) sensory domain is within an extracellular loop, N-terminal to the highly conserved heptahelical bundle. This loop contains both the cysteine-rich domain ((537)V - C(598)) and a 14 residue "linker" sequence ((599)I - F(612)) thought to support signal transmission to the heptahelical bundle. Thus domain 1 may contain a 189 residue N-terminal extracellular region followed successively by TM-1, a short intracellular loop, TM-2 and a 329 residue extracellular loop; rather than the proposed 620 residue VFT module based on crystallography of the N-terminal region of mGluR1. Since the topologies of the two proteins differ, the published CaSR VFT model is questionable. CaSR also contains multiple caveolin-binding motifs and cholesterol-binding (CRAC/CARC) domains, facilitating localization to plasma membrane lipid rafts. Ion sensing may involve combination of pore-lining regions from CaSR dimers and CaSR-bound caveolins to form ion channels capable of monitoring ionized Ca(2+) levels., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Flavonoids are polyphenolic compounds able to favour cholesterol-lipid-raft formation and control cell signaling pathways by targeting receptors at the cell surface. Procyanidins (Pcy) are oligomeric and polymeric flavonoids formed by catechins and epicatechins monomers trigger apoptosis by activating TRAIL-death receptors in human colon adenocarcinoma SW480 cells. Here, we investigated whether the apoptotic process triggered by apple procyanidins involving the up-regulation of TRAIL-death receptors DR4/DR5 at the cell surface was dependent on cell membrane lipid-raft formation. We report that Pcy-induced apoptosis was enhanced in presence of nystatin, a cholesterol-sequestering compound inhibiting lipid-raft formation, without changing DR4/DR5 receptor expression. Treatment of SW480 cells with TRAIL caused a 3.5-fold increased level of caveolin together with a 2- to 2.5-fold increased amount of DR4/DR5 proteins in lipid rafts. Pcy-treatment did not induce any alteration in the expression of DR4/DR5 proteins as well as of caveolin present in lipid-raft fractions. Pcy induced an activation of TRAIL-death receptor-mediated apoptosis by a mechanism independent of lipid-raft formation. These results highlight the potential of Pcy as a direct activator of TRAIL-death receptors in cell membrane even in the absence of lipid rafts.

Caveolins, structural protein coats of caveolae primarily involved in membrane-related functions, have also been found associated to lipid droplets (LD), specialized organelles for fat storage. In the present study, we wanted to delineate the main features that govern the presence of caveolin-1 on adipocyte lipid droplets. Using either morphological or biochemical approaches, we found caveolins to associate to LD in 3T3-L1 adipocytes during their late maturation phase. The time course of this association could be modulated by constitutive activation of src-kinase, suggesting that the specific enrichment of caveolins in enlarged LD results from an active pathway rather than trapping of caveolins to lipid storage organelle acting as a passive sink. The fat cell size dependence of the association of organized caveolins on adipocytes LD suggests a role for these proteins in the long-term handling of lipid stores.

Muscarinic receptor extracted from porcine atria in digitonin-cholate copurified with Galpha(o), Galpha(i1-3), and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, alpha-subunits, and caveolins in various combinations. Homooligomers of alpha(i2) were detected on Western blots, and heterooligomers of alpha(i2) and alpha(o) were identified by coimmunoprecipitation; thus, a complex may contain at least two alpha-subunits. Other combinations of alpha-subunit were not detected. The ratio of total alpha-subunit to receptor was near 1, as measured by [(35)S]GTPgammaS and the antagonist [(3)H]quinuclidinylbenzilate, and the binding of [(35)S]GTPgammaS was manifestly biphasic. The ratio of alpha(o) to alpha(i1,2) also was near 1, as determined from the intensity of Western blots. Cardiac muscarinic receptors therefore can be purified as a mixture of complexes that contain caveolins and oligomers of alpha-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of alpha-subunit and the receptor.

Lipopolysaccharide (LPS) induces inflammatory activation through TLR4 (toll-like receptor-4)/MD-2 (myeloid differentiation-2)/CD14 (cluster of differentiation-14) complex. Although optimal LPS signaling is required to activate our innate immune systems against gram-negative bacterium, excessive amount of LPS signaling develops a detrimental inflammatory response in gram-negative bacterial infections. Downregulation of surface TLR4 expression is one of the critical mechanisms that can restrict LPS signaling. Here, we found that membrane-anchored CD14 is required for LPS-induced downregulation of TLR4 and MD-2 in CHO cells. Moreover, pretreatment of the cells with sterol-binding agent filipin reduced LPS-induced TLR4 downregulation, suggesting the involvement of caveolae-mediated endocytosis pathway. Involvement of caveolae in LPS-induced TLR4 endocytosis was further confirmed by immunoprecipitation. Thus, our data indicate that caveolae-dependent endocytosis pathway is involved in LPS-induced TLR4 downregulation and that this is dependent on membrane-anchored CD14 expression.

The contribution of plasma membrane-associated glucocorticoid receptor (GR) to transcriptional signaling is unclear. We observed GR in low-density detergent-resistant membrane (DRM) rafts derived from human hepatoma Hep3B cells in complexes with caveolin-1, HSP90, and STAT3. In transient transfection assays, GR-stimulated transcriptional signaling was reversibly inhibited by membrane-raft disrupters filipin III and progesterone. These data provide clear evidence for a functional contribution of DRM-associated GR to transcriptional signaling.

Insulin stimulates caveolin-1 and eNOS phosphorylation. The sulfonylurea glimepiride mimics several insulin actions by mechanisms that are poorly understood. Glimepiride induces caveolin-1 phosphorylation and activates PI3K and Akt in rat adipocytes. In this paper, we investigated the effect of glimepiride on eNOS activation in human endothelial cells. We found that glimepiride induces caveolin-1 and eNOS phosphorylation. To better understand the role of caveolin-1 in glimepiride action, we downregulated caveolin-1 expression by specific siRNA transfection. Caveolin-1 silencing did not change eNOS and Akt phosphorylation induced by glimepiride. On the contrary, LY294002, a specific PI3K inhibitor, blocked eNOS serine 1177 phosphorylation. These findings suggest that glimepiride induces eNOS phosphorylation in endothelial cells through an Akt-dependent mechanism, not regulated by caveolin-1.

The goal of this study was to investigate the cellular localization and the interaction between caveolin-1 and ABCA1 in cholesterol-loaded aortic endothelial cells after HDL incubation. Immunofluorescence confocal microscopy showed that ABCA1 was found primarily on the cell surface, whereas caveolin-1 was revealed on the cell surface and in the cytoplasm. The HDL appeared to colocalize with ABCA1 and caveolin-1 on the cell surface. No free HDL was revealed in the cytoplasm. The HDL was colocalized neither with early endosome marker (CD71) nor with late endosome marker (LAMP2). The chemical cross-linking and immunoprecipitation analysis revealed that ABCA1 binds directly to both HDL and caveolin-1, whereas HDL does not bind directly to caveolin-1. The studies provide evidence for a direct interaction between ABCA1 and HDL, ABCA1 and caveolin-1, but not HDL and caveolin-1, indicating that ABCA1 may act as a structural platform between HDL and caveolin-1 on the cell surface during cellular cholesterol efflux.

3-Phosphoinositide-dependent protein kinase 1 (PDK1), a member of the serine/threonine kinase family, has been demonstrated to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that PDK1 is associated with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of the caveolae membranes in COS-1. First, we noted the presence of two potential caveolin-1 binding motifs ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306)) in the PDK1 catalytic domain. Using a pull-down approach, we observed that PDK1 interacts physically with caveolin-1 both in vivo and in vitro. Second, we detected the co-localization of PDK1 and caveolin-1 via confocal microscopy. The localization of PDK1 to the plasma membrane was disrupted by caveolin binding. Third, in transient transfection assays, interaction with caveolin-1 induced a substantial reduction in the in vivo serine/threonine phosphorylation of PDK1, whereas the caveolin-1 binding site mutant ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306) change to (141)AFVKLAFTA(149) and (299)ADAPEFLA(306)) did not. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82-101) functionally suppressed the self-phosphorylation and kinase activities of purified recombinant PDK1 protein. Thus, our observations indicated that PDK1 binds to caveolin-1 through its caveolin-binding motifs, and also that the protein-protein interaction between PDK1 and caveolin-1 regulates PDK1 self-phosphorylation, kinase activity, and subcellular localization.

Functional coupling between the sarcolemmal membrane and the sarcoplasmic reticulum is based on distinct structures called junctional membrane complexes (JMCs). Recently, junctophilins are found to be responsible for normal formation of JMCs. In the present study, we found that junctophilin type 2 (JP-2), a unique isoform in the heart, was localized in caveolin-rich membranes, and that the expression of JP-2 was up-regulated during normal development and down-regulated in a hypertrophic or a dilated cardiomyopathic mouse model. The expression levels of JP-2 may be associated with the development of T-tubules and impaired Ca(2+)-induced Ca(2+) release in the heart.

The assembly of alpha-HL was shown to rapidly progress upon its interaction with Caveolin-1. Treatment of A431 cells with alpha-HL has resulted in clustering of Caveolin-1 at cell-cell contacts. Consistent with this observation, alpha-HL mutants devoid of assembly property have not induced the clustering of Caveolin-1. While cholesterol depletion of A431 cells completely arrests the assembly of alpha-HL, chelation of membrane cholesterol results in its retarded assembly. Interestingly, HT29 cells, with low Caveolin-1 levels, are resistant to alpha-HL attack. Clustering of Caveolin-1, as seen in case of A431 cells, was readily observed in case of HT29 cells transfected with Caveolin-1 construct, thus overexpressing the full length Caveolin-1, upon alpha-HL treatment. A model was constructed to visualize the interactions between alpha-HL and Caveolin-1 which suggests that facile penetration of alpha-HL's beta-barrel might occur through protein-protein interactions with the surrounding 7 alpha-helices of Caveolin-1.

Caveolin, the principal structural protein of caveolae membrane domains, has a cytosol-exposed N-terminal part that was cleaved off by trypsin treatment of caveolae vesicles isolated from primary human adipocytes. Sequencing of the released tryptic peptides by nanospray quadrupole time-of-flight mass spectrometry revealed that both caveolin-1alpha and caveolin-1beta were processed by excision of the starting methionines. The N-terminus of the mature caveolin-1alpha was acetylated, while caveolin-1beta was found in acetylated as well as in non-acetylated forms. Fractional phosphorylation of serine-36 in the mature caveolin-1alpha and of the homologous serine-5 in caveolin-1beta was identified. This is the first experimental evidence for in vivo phosphorylation of caveolin-1 at the consensus site for phosphorylation by protein kinase C. The phosphorylation was found in both the acetylated and non-acetylated variants of caveolin-1beta. This variability in modifications is consistent with critical involvement of the N-terminal domain of caveolin in the regulation of caveolae.

The exocrine pancreas releases secretory products essential for nutrient assimilation. In addition to digestive enzymes, the release of lipoprotein-like particles containing the membrane trafficking protein caveolin-1 from isolated pancreatic explants has been reported. The present study examined: (1) if gastrointestinal hormones induce the apical secretion of phospholipid in vivo and (2) a potential association of caveolin-1 and the lipid-soluble vitamin K analog menaquinone-4 (MK-4) with these structures. Analysis of isolated acinar cells, purified zymogen granules, and pancreatic juice collected in vivo indicated the presence a caveolin-1 immunoreactive protein that was acutely released in response hormone stimulation. Chloroform-extracted fractions of pancreatic juice also contained high concentrations of MK-4 that was secreted in parallel to protein and phospholipid. The presence of caveolin-1 and MK-4 in the phospholipid fraction of pancreatic juice places these molecules in the secretory pathway of exocrine cells and suggests a physiological role in digestive enzyme synthesis and/or processing.

Caveolae are hot spots in IGF-I signalling as suggested by the facts that IGF-I receptors localize in caveolae, directly interact with and tyrosine phosphorylate caveolin 1, the major caveolar protein. Also a number of IGF-IR substrates reside in caveolae, supporting a role of these organelles in the regulation of IGF-I action. Recently, we have demonstrated that IGF-I could specifically regulate Shc phosphorylation in caveolae. Here we show that also IRS1 localizes in this region where it is tyrosine phosphorylated in the presence of IGF-I. Moreover, IRS1 co-immunoprecipitates with caveolin 1 and the specific phosphocaveolin 1-IRS1 interaction is increased by IGF-I.

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are caused by mutations in 14 and 15 different disease genes, respectively, in a part of the patients and the disease genes for cardiomyopathy overlap in part with that for limb-girdle muscular dystrophy (LGMD). In this study, we examined an LGMD gene encoding caveolin-3 (CAV3) for mutation in the patients with HCM or DCM. A Thr63Ser mutation was identified in a sibling case of HCM. Because the mutation was found at the residue that is involved in the LGMD-causing mutations, we investigate the functional change due to the Thr63Ser mutation as compared with the LGMD mutations by examining the distribution of GFP-tagged CAV3 proteins. It was observed that the Thr63Ser mutation reduced the cell surface expression of caveolin-3, albeit the change was mild as compared with the LGMD mutations. These observations suggest that HCM is a clinical spectrum of CAV3 mutations.

Components of caveolae and lipid rafts are characterized by their buoyancy after detergent extraction. Using flotations in density gradients, we now show that non-raft membrane molecules are also associated with detergent-insoluble, buoyant assemblies. When Triton X-100 cellular extracts were spun to equilibrium in Nycodenz, only components of classical rafts floated. In contrast, with the zwitterionic detergent CHAPS, non-raft residents such as calnexin and APP also buoyed. When CHAPS extracts were spun in non-equilibrium (velocity) conditions, some raft components rapidly exited the input fractions while other raft markers and non-raft molecules remained relatively immobile. This pointed to size heterogeneities of CHAPS-insoluble complexes. Combined velocity/equilibrium gradients broadly divided CHAPS-insoluble membrane complexes into three size categories, which all contained cholesterol and the glycosphingolipid GM1. Large complexes were enriched in caveolin and ESA. Medium size complexes were enriched in PrP, whereas small complexes contained non-raft proteins, PrP, and some ESA. While Alzheimer's APP was primarily confined to small assemblies, a portion of its glycosylated form did buoy with large complexes. Large CHAPS-insoluble complexes resemble, but are not equal to, classical rafts. These findings extend considerably the range of detergent-insoluble membranal domains.

The association of the low-density lipoprotein (LDL) receptor with detergent resistant hepatic membranes was investigated using discontinuous sucrose gradients. In liver homogenates from both hamsters and rats, the fractions with the highest concentrations of LDL receptor coincided with the location of caveolin-1, a marker of the cholesterol-rich caveolae. Feeding the animals diets enriched in cholesterol slightly shifted both LDL receptor and caveolin-1 to positions of lower density. The cholesterol content of the caveolae fractions was increased 2-fold in animals fed cholesterol-supplemented diets. In homogenates of CHO cells, fractionated in the same manner, the LDL receptor was absent from the caveolae fractions but was present in denser fractions near the bottom of the gradient. Addition of caveolin-1 antibody to solubilized caveolae from liver coimmunoprecipitated the LDL receptor. These observations suggest that in liver, the LDL receptor is mainly located in caveolae. This location contrasts with the clathrin-coated pit location observed in fibroblasts and CHO cells.

Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions toward recoverin (Rec), a retina-specific Ca(2+) binding protein, and its aberrant expression in tumor cells lead to the retinal degeneration. To elucidate functional roles of the aberrantly expression in cancer cells, we performed immunoprecipitation using anti-human Rec mAb. We observed co-precipitation of G-protein-coupled receptor kinases (GRKs) and caveolin-1 with Rec from cell lysates of 293 or SSTW cells. Immunocytochemistry revealed that immunoreactivities toward Rec within the cancer cells were almost identical to those toward GRKs and caveolin-1. The present data strongly suggest that aberrantly expressed Rec should be involved in the GRK-dependent cellular regulation in cancer cells.

A profile-based search of the SWISS-PROT database reveals that most protein tyrosine phosphatases (PTPs) contain at least one caveolin-1-binding motif. To ascertain if the presence of caveolin-binding motif(s) in PTPs corresponds to their actual localization in caveolin-1-enriched membrane fractions, we performed subcellular fractionating experiments. We found that all tested PTPs (PTP1B, PTP1C, SHPTP2, PTEN, and LAR) are actually localized in caveolin-enriched membrane fractions, despite their distribution in other subcellular sites, too. More than 1/2 of LAR and about 1/4 of SHPTP2 and PTP-1C are localized in caveolin-enriched membrane fractions whereas, in these fractions, PTP-1B and PTEN are poorly concentrated. Co-immunoprecipitation experiments with antibodies specific for each tested PTP demonstrated that all five phosphatases form molecular complexes with caveolin-1 in vivo. Collectively, our findings propose that particular PTPs could perform some of their cellular actions or are regulated by recruitment into caveolin-enriched membrane fractions.

Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.

The molecular mechanisms underlying the regulation of neurotransmission has been an open question for many years. Here, we have examined an interaction between caveolin1 and SNAREs (soluble N-ethylmalemide-sensitive factor attachment protein receptor) which may contribute to the cellular mechanisms underlying changes in synaptic strength. Previously, we reported that application of 4-aminopyridine to hippocampal slices resulted in a persistent potentiation of synaptic transmission and the induction of a short-lasting and specific 40-kDa complex composed of synaptosomal associated protein of 25 kDa (SNAP25) and caveolin1. We have characterized the binding properties of these proteins and observed that in vitro caveolin1 directly associates with both SNAP25 and syntaxin. Caveolin/SNARE interactions are enhanced in the presence of ATP by a mechanism that involves phosphorylation. While caveolin has been associated with cholesterol transport, signal transduction, and transcytosis, this study provides evidence that caveolin is also a SNARE accessory protein., ((C)2002 Elsevier Science (USA).)

PDGF receptors and Src family kinases are concentrated in caveolae, where signal transduction cascades involving these molecules are thought to be organized. The Src family tyrosine kinases are cotransducers of signals emanating from the activated PDGF receptor. However, the Src family kinase substrates that are involved in PDGF-induced signaling remain to be fully elucidated. We have identified a 29-kDa protein in caveolae that was phosphorylated in response to PDGF stimulation. This protein, pp29, was tightly bound to the caveolar coat protein caveolin-1. pp29 was among the most prominent phosphoproteins observed in cells overexpressing Fyn, suggesting that it may be a Fyn substrate. Consistent with this, pp29 was among a specific subset of proteins whose PDGF-stimulated phosphorylation was blocked by expression of kinase inactive Fyn. These data indicate that pp29 lies downstream of Fyn activation in a PDGF-stimulated signaling pathway, and that pp29 is an abundant site for nucleation of signal transduction cascades., (©2002 Elsevier Science (USA).)

Caveolae are flask-shaped invaginations of the plasma membrane formed by the association of caveolin proteins with lipid rafts. In endothelial cells, caveolae function as signal transduction centers controlling NO synthesis and mechanotransduction. We now provide evidence that the endothelial volume-regulated anion channel (VRAC) is also under the control of the caveolar system. When calf pulmonary artery endothelial (CPAE) cells were transfected with caveolin-1 Delta1-81 (deletion of amino acids 1 to 81), activation of VRAC by hypotonic cell swelling was strongly impaired. Concomitantly, caveolin-1 Delta1-81 disturbed the formation of caveolin-1 containing lipid rafts as evidenced by sucrose density gradient centrifugation. In nontransfected cells, endogenous caveolin-1 typically associated with low-density, detergent-resistant lipid rafts. However, transient expression of caveolin-1 Delta1-81 caused a redistribution of endogenous caveolin-1 to high-density, detergent-soluble membrane fractions. We therefore conclude that the interaction between caveolin-1 and detergent-resistant lipid rafts is an important prerequisite for endothelial VRAC activity., (Copyright 2001 Academic Press.)

We determined the existence and role of beta(2)-adrenergic receptor in cultured BAECs through the effect of a beta-blocker having NO releasing action; 3,4-dihydro-8(2-hydroxy-3-isopropylamino)-propoxy-3-nitroxy-2H-1-benzopyran; nipradilol on eNOS and eNOS regulatory protein caveolin-1. beta(2) receptor exists in BAECs. eNOS mRNA and protein were up-regulated by its treatment whereas those of caveolin were not altered considerably. This eNOS up-regulatory action was abolished by beta(2) receptor antagonist, ICI-118551. Increase of NO metabolites, protein and mRNA of eNOS was also partially inhibited by co-treatment of NOS inhibitor, L-NA with nipradilol. This is the first investigation of the action of non-selective beta blocker on eNOS through beta(2) receptor. The drug increases NO on incubation with BAECs about 50% as a NO donor and about 50% as results of eNOS up-regulation., (Copyright 2001 Academic Press.)