Reduced GM1 ganglioside in CFTR-deficient human airway cells results in decreased ß1-integrin signaling and delayed wound repair.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function reduces chloride secretion and increases sodium uptake, but it is not clear why CFTR mutation also results in progressive lung inflammation and infection. We previously demonstrated that CFTR-silenced airway cells migrate more slowly during wound repair than CFTR-expressing controls. In addition, CFTR-deficient cells and mouse models have been reported to have altered sphingolipid levels. Here, we investigated the hypothesis that reduced migration in CFTR-deficient airway epithelial cells results from altered sphingolipid composition. We used cell lines derived from a human airway epithelial cell line (Calu-3) stably transfected with CFTR short hairpin RNA (CFTR-silenced) or nontargeting short hairpin RNA (controls). Cell migration was measured by electric cell substrate impedance sensing (ECIS). Lipid analyses, addition of exogenous glycosphingolipids, and immunoblotting were performed. We found that levels of the glycosphingolipid, GM1 ganglioside, were ~60% lower in CFTR-silenced cells than in controls. CFTR-silenced cells exhibited reduced levels of activated ß1-integrin, phosphorylated tyrosine 576 of focal adhesion kinase (pFAK), and phosphorylation of Crk-associated substrate (pCAS). Addition of GM1 (but not GM3) ganglioside to CFTR-silenced cells restored activated ß1-integrin, pFAK, and pCAS to near control levels and partially restored (~40%) cell migration. Our results suggest that decreased GM1 in CFTR-silenced cells depresses ß1-integrin signaling, which contributes to the delayed wound repair observed in these cells. These findings have implications for the pathology of cystic fibrosis, where altered sphingolipid levels in airway epithelial cells could result in a diminished capacity for wound repair after injury.

Viral attachment induces rapid recruitment of an innate immune sensor (TRIM5α) to the plasma membrane.

TRIM5α (tripartite motif 5α) acts as a pattern recognition receptor specific for the retrovirus capsid lattice and blocks infection by HIV-1 immediately after entry. However, the precise mechanisms underlying this rapid recognition of viral components remain elusive. Here, we analyzed the influence of viral exposure on TRIM5α. Total internal reflection fluorescence microscopy and lipid flotation assays revealed rapid recruitment of a TRIM5α subpopulation to the plasma membrane (PM) upon exposure to vesicular stomatitis virus-G-pseudotyped HIV-1 viral-like particles (VLPs), but not to envelope (Env)-less HIV-1 VLPs. TRIM5α signals were frequently colocalized with those of HIV-1 capsid at the PM. Exposure to HIV-1 Env-pseudotyped HIV-1 vectors also triggered translocation of endogenous TRIM5α to lipid microdomains within human T cells. Similarly, clustering of lipid microdomains by a glycosphingolipid stereoisomer resulted in rapid TRIM5α recruitment to the PM. Of note, recruitment of endogenous rhesus TRIM5α to the PM prior to HIV-1 infection significantly increased the potency of viral restriction. Our data therefore suggest the importance of TRIM5α recruitment to the PM for TRIM5α-mediated innate immune sensing and restriction of retroviral infection.

Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis.

BACKGROUND: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins that bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. AIM: To characterize the effects of Prom-2 expression on PM microdomain organization. METHODS: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. RESULTS: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. CONCLUSIONS: Prom2 protrusions primarily localize to lipid rafts and recruit cholesterol into protrusions and away from caveolae, leading to increased phosphorylation of caveolin-1, which inhibits Cdc42-dependent endocytosis. This study provides a new insight for the role for prominins in the regulation of PM lipid organization.

Glycosphingolipids mediate pneumocystis cell wall ß-glucan activation of the IL-23/IL-17 axis in human dendritic cells.

Pneumocystis species are opportunistic fungal organisms that cause severe pneumonia in immune-compromised hosts, with resultant high morbidity and mortality. Recent work indicates that IL-17 responses are important components of host defense against fungal pathogens. In the present study, we demonstrate that cell-surface ß-glucan components of Pneumocystis (PCBG) stimulate human dendritic cells (DCs) to secrete IL-23 and IL-6. These cytokines are well established to stimulate a T helper-17 (Th17) phenotype. Accordingly, we further observe that PCBG-stimulated human DCs interact with lymphocytes to drive the secretion of IL-17 and IL-22, both Th17-produced cytokines. The activation of DCs was shown to involve the dectin-1 receptor with a downstream activation of the Syk kinase and subsequent translocation of both the canonical and noncanonical components of the NF-κB transcription factor family. Finally, we demonstrate that glycosphingolipid-rich microdomains of the plasma membrane participate in the activation of DCs by PCBG through the accumulation of lactosylceramide at the cell surface during stimulation with PCBG. These data strongly support the idea that the ß-glucan surface components of Pneumocystis drive the activation of the IL-23/IL-17 axis during this infection, through a glycosphingolipid-initiated mechanism.

Primary alveolar epithelial cell surface membrane microdomain function is required for Pneumocystis ß-glucan-induced inflammatory responses.

Intense lung inflammation characterizes respiratory failure associated with Pneumocystis pneumonia. Our laboratory has previously demonstrated that alveolar epithelial cells (AECs) elaborate inflammatory cytokines and chemokines in response to the Pneumocystis carinii cell wall constituent ß-(1→3)-glucan (PCBG), and that these responses require lactosylceramide, a prominent glycosphingolipid constituent of certain cell membrane microdomains. The relevance of membrane microdomains, also termed plasma membrane lipid rafts, in cell signaling and macromolecule handling has been increasingly recognized in many biologic systems, but their role in P. carinii-induced inflammation is unknown. To investigate the mechanisms of microdomain-dependent P. carinii-induced inflammation, we challenged primary rat AECs with PCBG with or without pre-incubation with inhibitors of microdomain function. Glycosphingolipid and cholesterol rich microdomain inhibition resulted in significant attenuation of P. carinii-induced expression of TNF-α and the rodent C-X-C chemokine MIP-2, as well as their known inflammatory secondary signaling pathways. We have previously shown that protein kinase C (PKC) is activated by PCBG challenge and herein show that PKC localizes to AEC microdomains. We also demonstrate by conventional microscopy, fluorescence microscopy, confocal microscopy and spectrophotofluorimetry that AECs internalize fluorescently-labeled PCBG by microdomain-mediated mechanisms, and that anti-microdomain pretreatments prevent internalization. Taken together, these data suggest an important role for AEC microdomain function in PCBG-induced inflammatory responses. This offers a potential novel target for therapeutics for a condition that continues to exert unacceptable morbidity and mortality among immunocompromised populations.

Role of protein kinase d in Golgi exit and lysosomal targeting of the transmembrane protein, Mcoln1.

The targeting of lysosomal transmembrane (TM) proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of mucolipin-1 (Mcoln1), the TM protein defective in the autosomal recessive disease, mucolipidosis type IV, was studied by overexpressing full-length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53-amino acid C-terminal region of Mcoln1 is required for efficient exit from the Golgi. Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of Mcoln1 into characteristic ∼35-kDa fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that the co-expression of full-length Mcoln1 with kinase-inactive protein kinase D (PKD) 1 or 2 inhibited Mcoln1 Golgi exit and transport to lysosomes and decreased Mcoln1 cleavage. These studies suggest that PKDs play a role in the delivery of some lysosomal resident TM proteins from the Golgi to the lysosomes.

Endocytic response of type I alveolar epithelial cells to hypertonic stress.

We present plasma membrane (PM) internalization responses of type I alveolar epithelial cells to a 50 mosmol/l increase in tonicity. Our research is motivated by interest in ATI repair, for which endocytic retrieval of PM appears to be critical. We validated pharmacological and molecular tools to dissect the endocytic machinery of these cells and used these tools to test the hypothesis that osmotic stress triggers a pathway-specific internalization of PM domains. Validation experiments confirmed the fluorescent analogs of lactosyl-ceramide, transferrin, and dextran as pathway-specific cargo of caveolar, clathrin, and fluid-phase uptake, respectively. Pulse-chase experiments indicate that hypertonic exposure causes a downregulation of clathrin and fluid-phase endocytosis while stimulating caveolar endocytosis. The tonicity-mediated increase in caveolar endocytosis was associated with the translocation of caveolin-1 from the PM and was absent in cells that had been transfected with dominant-negative dynamin constructs. In separate experiments we show that hypertonic exposure increases the probability of PM wound repair following micropuncture from 82 ± 4 to 94 ± 2% (P < 0.01) and that this effect depends on Src pathway activation-mediated caveolar endocytosis. The therapeutic and biological implications of our findings are discussed.

Spatiotemporal dynamics of actin remodeling and endomembrane trafficking in alveolar epithelial type I cell wound healing.

Alveolar epithelial type I cell (ATI) wounding is prevalent in ventilator-injured lungs and likely contributes to pathogenesis of "barotrauma" and "biotrauma." In experimental models most wounded alveolar cells repair plasma membrane (PM) defects and survive insults. Considering the force balance between edge energy at the PM wound margins and adhesive interactions of the lipid bilayer with the underlying cytoskeleton (CSK), we tested the hypothesis that subcortical actin depolymerization is a key facilitator of PM repair. Using real-time fluorescence imaging of primary rat ATI transfected with a live cell actin-green fluorescent protein construct (Lifeact-GFP) and loaded with N-rhodamine phosphatidylethanolamine (PE), we examined the spatial and temporal coordination between cytoskeletal remodeling and PM repair following micropuncture. Membrane integrity was inferred from the fluorescence intensity profiles of the cytosolic label calcein AM. Wounding led to rapid depolymerization of the actin CSK near the wound site, concurrent with accumulation of endomembrane-derived N-rhodamine PE. Both responses were sustained until PM integrity was reestablished, which typically occurs between ∼10 and 40 s after micropuncture. Only thereafter did the actin CSK near the wound begin to repolymerize, while the rate of endomembrane lipid accumulation decreased. Between 60 and 90 s after successful PM repair, after translocation of the actin nucleation factor cortactin, a dense actin fiber network formed. In cells that did not survive micropuncture injury, actin remodeling did not occur. These novel results highlight the importance of actin remodeling in ATI cell repair and suggest molecular targets for modulating the repair process.

Type II transforming growth factor-beta receptor recycling is dependent upon the clathrin adaptor protein Dab2.

Transforming growth factor (TGF)-ß family proteins form heteromeric complexes with transmembrane serine/threonine kinases referred to as type I and type II receptors. Ligand binding initiates a signaling cascade that generates a variety of cell type-specific phenotypes. Whereas numerous studies have investigated the regulatory activities controlling TGF-ß signaling, there is relatively little information addressing the endocytic and trafficking itinerary of TGF-ß receptor subunits. In the current study we have investigated the role of the clathrin-associated sorting protein Disabled-2 (Dab2) in TGF-ß receptor endocytosis. Although small interfering RNA-mediated Dab2 knockdown had no affect on the internalization of various clathrin-dependent (i.e., TGF-ß, low-density lipoprotein, or transferrin) or -independent (i.e., LacCer) cargo, TGF-ß receptor recycling was abrogated. Loss of Dab2 resulted in enlarged early endosomal antigen 1-positive endosomes, reflecting the inability of cargo to traffic from the early endosome to the endosomal recycling compartment and, as documented previously, diminished Smad2 phosphorylation. The results support a model whereby Dab2 acts as a multifunctional adaptor in mesenchymal cells required for TGF-ß receptor recycling as well as Smad2 phosphorylation.

Humanin is expressed in human vascular walls and has a cytoprotective effect against oxidized LDL-induced oxidative stress.

AIMS: Humanin (HN) is a 24-amino acid peptide that has been shown to have an anti-apoptotic function against neuronal cell death caused by Alzheimer's disease. Increased oxidative stress, one of the major factors contributing to this cell death, also plays an important role in the inflammatory process of atherosclerosis. The current study was designed to test the hypothesis that HN is expressed in the human vascular wall and may protect against oxidative stress. METHODS AND RESULTS: HN expression in the vascular wall was detected by immunostaining in the endothelial cell layer of human internal mammary arteries (n = 5), atherosclerotic coronary arteries (n = 17), and sections of the greater saphenous vein (n = 3). HN mRNA was expressed in the human aortic endothelial cells (HAECs). Cytoprotective effects of HN against oxidative stress were tested in vitro in HAECs. Pre-treatment with 0.1 µM HN reduced oxidized LDL (Ox-LDL)-induced (i) formation of reactive oxygen species by 50%, (ii) apoptosis by ∼50% as determined by TUNEL staining, and (iii) formation of ceramide, a lipid second messenger involved in the apoptosis signalling cascade, by ∼20%. CONCLUSION: The current study demonstrates for the first time the expression of HN in the endothelial cell layer of human blood vessels. Exogenous addition of HN to endothelial cell cultures was shown to be effective against Ox-LDL-induced apoptosis. These findings suggest that HN may play a role and may have a protective effect in early atherosclerosis in humans.

Co-regulation of caveolar and Cdc42-dependent fluid phase endocytosis by phosphocaveolin-1.

Several clathrin-independent endocytosis mechanisms have been identified that can be distinguished by specific requirements for certain proteins, such as caveolin-1 (Cav1) and the Rho GTPases, RhoA and Cdc42, as well as by specific cargo. Some endocytic pathways may be co-regulated such that disruption of one pathway leads to the up-regulation of another; however, the underlying mechanisms for this are unclear. Cav1 has been reported to function as a guanine nucleotide dissociation inhibitor (GDI), which inhibits Cdc42 activation. We tested the hypothesis that Cav1 can regulate Cdc42-dependent, fluid phase endocytosis. We demonstrate that Cav1 overexpression decreases fluid phase endocytosis, whereas silencing of Cav1 enhances this pathway. Enhancement of Cav1 phosphorylation using a phosphatase inhibitor reduces Cdc42-regulated pinocytosis while stimulating caveolar endocytosis. Fluid phase endocytosis was inhibited by expression of a putative phosphomimetic mutant, Cav1-Y14E, but not by the phospho-deficient mutant, Cav1-Y14F. Overexpression of Cav2, or a Cav1 mutant in which the GDI region was altered to the corresponding sequence in Cav2, did not suppress fluid phase endocytosis. These results suggest that the Cav1 expression level and phosphorylation state regulates fluid phase endocytosis via the interaction between the Cav1 GDI region and Cdc42. These data define a novel molecular mechanism for co-regulation of two distinct clathrin-independent endocytic pathways.

Gangliosides and beta1-integrin are required for caveolae and membrane domains.

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin-mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo-glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol ('lipid rafts'), reduced caveolae and caveolin-1 at the plasma membrane by approximately 80%, and blunted activation of beta1-integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo-gangliosides or other sphingolipids) at 10 degrees C, suggesting that sialo-lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Igamma away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin-1 could be recapitulated by beta1-integrin knockdown. These results suggest that both gangliosides and beta1-integrin are required for maintenance of caveolae and plasma membrane domains.

Stimulation of GLUT4 (glucose transporter isoform 4) storage vesicle formation by sphingolipid depletion.

Insulin stimulates glucose transport in fat and skeletal muscle cells primarily by inducing the translocation of GLUT4 (glucose transporter isoform 4) to the PM (plasma membrane) from specialized GSVs (GLUT4 storage vesicles). Glycosphingolipids are components of membrane microdomains and are involved in insulin-regulated glucose transport. Cellular glycosphingolipids decrease during adipocyte differentiation and have been suggested to be involved in adipocyte function. In the present study, we investigated the role of glycosphingolipids in regulating GLUT4 translocation. We decreased glycosphingolipids in 3T3-L1 adipocytes using glycosphingolipid synthesis inhibitors and investigated the effects on GLUT4 translocation using immunocytochemistry, preparation of PM sheets, isolation of GSVs and FRAP (fluorescence recovery after photobleaching) of GLUT4-GFP (green fluorescent protein) in intracellular structures. Glycosphingolipids were located in endosomal vesicles in pre-adipocytes and redistributed to the PM with decreased expression at day 2 after initiation of differentiation. In fully differentiated adipocytes, depletion of glycosphingolipids dramatically accelerated insulin-stimulated GLUT4 translocation. Although insulin-induced phosphorylation of IRS (insulin receptor substrate) and Akt remained intact in glycosphingolipid-depleted cells, both in vitro budding of GLUT4 vesicles and FRAP of GLUT4-GFP on GSVs were stimulated. Glycosphingolipid depletion also enhanced the insulin-induced translocation of VAMP2 (vesicle-associated membrane protein 2), but not the transferrin receptor or cellubrevin, indicating that the effect of glycosphingolipids was specific to VAMP2-positive GSVs. Our results strongly suggest that decreasing glycosphingolipid levels promotes the formation of GSVs and, thus, GLUT4 translocation. These studies provide a mechanistic basis for recent studies showing that inhibition of glycosphingolipid synthesis improves glycaemic control and enhances insulin sensitivity in animal models of Type 2 diabetes.

Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid, C8-beta-D-lactosylceramide.

Plasma membrane (PM) microdomains, including caveolae and other cholesterol-enriched subcompartments, are involved in the regulation of many cellular processes, including endocytosis, attachment and signaling. We recently reported that brief incubation of human skin fibroblasts with the synthetic glycosphingolipid, D-erythro-octanoyl-lactosylceramide (C8-D-e-LacCer), stimulates endocytosis via caveolae and induces the appearance of micron-size microdomains on the PM. To further understand the effects of C8-D-e-LacCer treatment on PM microdomains, we used a detergent-free method to isolate microdomain-enriched membranes from fibroblasts treated +/-C8-D-e-LacCer, and performed 2-DE and mass spectrophotometry to identify proteins that were altered in their distribution in microdomains. Several proteins were identified in the microdomain-enriched fractions, including lipid transfer proteins and proteins related to the functions of small GTPases. One protein, Rho-associated protein kinase 2 (ROCK2), was verified by Western blotting to occur in microdomain fractions and to increase in these fractions after D-e-LacCer treatment. Immunofluorescence revealed that ROCK2 exhibited an increased localization at or near the PM in C8-D-e-LacCer-treated cells. In contrast, ROCK2 distribution in microdomains was decreased by treatment of cells with C8-L-threo-lactosylceramide, a glycosphingolipid with non-natural stereochemistry. This study identifies new microdomain-associated proteins and provides evidence that microdomains play a role in the regulation of the Rho/ROCK signaling pathway.

Development of a Rab9 transgenic mouse and its ability to increase the lifespan of a murine model of Niemann-Pick type C disease.

Niemann-Pick, type C (NP-C) disease is an autosomal recessive neurovisceral storage disorder in which cholesterol and sphingolipids accumulate. There is no specific treatment for this disease, which is characterized by progressive neurological deterioration, sometimes accompanied by hepatosplenomegaly. We and others have shown that overexpression of certain Rab GTPases corrects defective membrane trafficking and reduces lipid storage in cultured NP-C fibroblasts. Here, we tested the possibility that Rab protein overexpression might also have beneficial effects in vivo using a murine model of NP-C. We first generated several lines of transgenic mice that ubiquitously overexpress Rab9 up to approximately 30-fold more than endogenous levels and found that the transgene expression had no obvious effects on fertility, behavior, or lifespan in normal mice. These transgenic strains were then crossed with NP-C mutant mice to produce NP-C homozygous recessive mice with and without the Rab9 transgene. Life expectancy of the NPC1 homozygous recessive animals was extended up to 22% depending on gender and the transgenic strain that was used. Histological studies and lipid analysis of brain sections indicated that the NP-C mice carrying the Rab9 transgene had dramatically reduced storage of GM(2) and GM(3) gangliosides relative to NP-C animals lacking the transgene. These results demonstrate that Rab9 overexpression has the potential to reduce stored lipids and prolong lifespan in vivo.

Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells.

Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple "microdomains" of differing lipid and protein composition and function. These domains are sometimes enriched in cholesterol and sphingolipids and are believed to be important structures for the regulation of many biological and pathological processes. This review focuses on the use of fluorescent (Bodipy) labeled analogs of sphingolipids and cholesterol to study such domains. We discuss the similarities between the behavior of Bodipy-cholesterol and natural cholesterol in artificial bilayers and in cultured cells, and the use of Bodipy-sphingolipid analogs to visualize membrane domains in living cells based on the concentration-dependent monomer-excimer fluorescence properties of the Bodipy-fluorophore. The use of Bodipy-D-erythro-lactosylceramide is highlighted for detection of domains on the plasma membrane and endosome membranes, and the importance of the sphingolipid stereochemistry in modulating domain formation is discussed. Finally, we suggest that Bodipy-sphingolipids may be useful in future studies to examine the relationship between membrane domains at the cell surface and domains enriched in other lipids and proteins on the inner leaflet of the plasma membrane.

Pathways of clathrin-independent endocytosis.

There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. In addition to the classical clathrin-dependent mechanism of endocytosis, several pathways that do not use a clathrin coat are emerging. These pathways transport a diverse array of cargoes and are sometimes hijacked by bacteria and viruses to gain access to the host cell. Here, we review our current understanding of various clathrin-independent mechanisms of endocytosis and propose a classification scheme to help organize the data in this complex and evolving field.

Inhibition of caveolar uptake, SV40 infection, and beta1-integrin signaling by a nonnatural glycosphingolipid stereoisomer.

Caveolar endocytosis is an important mechanism for the uptake of certain pathogens and toxins and also plays a role in the internalization of some plasma membrane (PM) lipids and proteins. However, the regulation of caveolar endocytosis is not well understood. We previously demonstrated that caveolar endocytosis and beta1-integrin signaling are stimulated by exogenous glycosphingolipids (GSLs). In this study, we show that a synthetic GSL with nonnatural stereochemistry, beta-D-lactosyl-N-octanoyl-L-threo-sphingosine, (1) selectively inhibits caveolar endocytosis and SV40 virus infection, (2) blocks the clustering of lipids and proteins into GSLs and cholesterol-enriched microdomains (rafts) at the PM, and (3) inhibits beta1-integrin activation and downstream signaling. Finally, we show that small interfering RNA knockdown of beta1 integrin in human skin fibroblasts blocks caveolar endocytosis and the stimulation of signaling by a GSL with natural stereochemistry. These experiments identify a new compound that can interfere with biological processes by inhibiting microdomain formation and also identify beta1 integrin as a potential mediator of signaling by GSLs.

Regulation of endocytic recycling of KCNQ1/KCNE1 potassium channels.

Stress-dependent regulation of cardiac action potential duration is mediated by the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. It is accompanied by an increased magnitude of the slow outward potassium ion current, I(Ks). KCNQ1 and KCNE1 subunits coassemble to form the I(Ks) channel. Mutations in either subunit cause long QT syndrome, an inherited cardiac arrhythmia associated with an increased risk of sudden cardiac death. Here we demonstrate that exocytosis of KCNQ1 proteins to the plasma membrane requires the small GTPase RAB11, whereas endocytosis is dependent on RAB5. We further demonstrate that RAB-dependent KCNQ1/KCNE1 exocytosis is enhanced by the serum- and glucocorticoid-inducible kinase 1, and requires phosphorylation and activation of phosphoinositide 3-phosphate 5-kinase and the generation of PI(3,5)P(2). Identification of KCNQ1/KCNE1 recycling and its modulation by serum- and glucocorticoid-inducible kinase 1-phosphoinositide 3-phosphate 5-kinase -PI(3,5)P(2) provides a mechanistic insight into stress-induced acceleration of cardiac repolarization.

Misassembled mutant DeltaF508 CFTR in the distal secretory pathway alters cellular lipid trafficking.

Most patients with cystic fibrosis (CF) have a single codon deletion (DeltaF508) in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impairs assembly of the multidomain glycoprotein. The mutant protein escapes endoplasmic reticulum (ER) quality control at low temperature, but is rapidly cleared from the distal secretory pathway and degraded in lysosomes. CF cells accumulate free cholesterol similar to Niemann-Pick disease type C cells. We show that this lipid alteration is caused by the presence of misassembled mutant CFTR proteins, including DeltaF508, in the distal secretory pathway rather than the absence of functional CFTR. By contrast, cholesterol distribution is not changed by either D572N CFTR, which does not mature even at low temperature, or G551D, which is processed normally but is inactive. On expression of the DeltaF508 mutant, cholesterol and glycosphingolipids accumulate in punctate endosomal structures and cholesterol esters are reduced, indicating a block in the translocation of cholesterol to the ER for esterification. This is overcome by Rab9 overexpression, resulting in clearance of accumulating intracellular cholesterol. Similar but less pronounced alterations in intracellular cholesterol distribution are observed on expression of a temperature-rescued mutant variant of the related ATP-binding cassette (ABC) protein multidrug resistance-associated protein 1 (MRP1). Thus, on escape from ER quality control, misassembled mutants of CFTR and MRP1 impair lipid homeostasis in endocytic compartments.