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• W2108901610 abstract "We have designed a tagged probe [sphingolipid binding domain (SBD)] to facilitate the tracking of intracellular movements of sphingolipids in living neuronal cells. SBD is a small peptide consisting of the SBD of the amyloid precursor protein. It can be conjugated to a fluorophore of choice and exogenously applied to cells, thus allowing for in vivo imaging. Here, we present evidence to describe the characteristics of the SBD association with the plasma membrane. Our experiments demonstrate that SBD binds to isolated raft fractions from human neuroblastomas and insect neuronal cells. In protein-lipid overlay experiments, SBD interacts with a subset of glycosphingolipids and sphingomyelin, consistent with its raft association in neurons. We also provide evidence that SBD is taken up by neuronal cells in a cholesterol- and sphingolipid-dependent manner via detergent-resistant microdomains. Furthermore, using fluorescence correlation spectroscopy to assay the mobility of SBD in live cells, we show that SBD's behavior at the plasma membrane is similar to that of the previously described raft marker cholera toxin B, displaying both a fast and a slow component. Our data suggest that fluorescently tagged SBD can be used to investigate the dynamic nature of glycosphingolipid-rich detergent-resistant microdomains that are cholesterol-dependent. We have designed a tagged probe [sphingolipid binding domain (SBD)] to facilitate the tracking of intracellular movements of sphingolipids in living neuronal cells. SBD is a small peptide consisting of the SBD of the amyloid precursor protein. It can be conjugated to a fluorophore of choice and exogenously applied to cells, thus allowing for in vivo imaging. Here, we present evidence to describe the characteristics of the SBD association with the plasma membrane. Our experiments demonstrate that SBD binds to isolated raft fractions from human neuroblastomas and insect neuronal cells. In protein-lipid overlay experiments, SBD interacts with a subset of glycosphingolipids and sphingomyelin, consistent with its raft association in neurons. We also provide evidence that SBD is taken up by neuronal cells in a cholesterol- and sphingolipid-dependent manner via detergent-resistant microdomains. Furthermore, using fluorescence correlation spectroscopy to assay the mobility of SBD in live cells, we show that SBD's behavior at the plasma membrane is similar to that of the previously described raft marker cholera toxin B, displaying both a fast and a slow component. Our data suggest that fluorescently tagged SBD can be used to investigate the dynamic nature of glycosphingolipid-rich detergent-resistant microdomains that are cholesterol-dependent. amyloid β peptide cell line DL-DMBG2-c6 cholera toxin B dialkyl-indocarbocyanine detergent-resistant membrane fumonisin B1 fluorescence correlation spectroscopy green fluorescent protein 1,1,1,3,3,3-hexafluoro-2-propanol methyl-β-cyclodextrin Oregon green sphingolipid binding domain tetramethyl rhodamine The involvement of cholesterol and sphingolipid-rich membrane microdomains known as lipid rafts in a variety of cellular processes is well established (reviewed in Refs. 1.Simons K. Ikonen E. Functional rafts in cell membranes.Nature. 1997; 387: 569-572Crossref PubMed Scopus (8117) Google Scholar, 2.Brown D.A. London E. Functions of lipid rafts in biological membranes.Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2551) Google Scholar), and raft-borne lipids and proteins have been implicated in several pathological conditions, including neurodegeneration and inflammation (reviewed in Ref. 3.Simons K. Ehehalt R. Cholesterol, lipid rafts, and disease.J. Clin. Invest. 2002; 110: 597-603Crossref PubMed Scopus (920) Google Scholar). Previously, cholera toxin B (CtxB) was used to study the intracellular trafficking of raft-borne lipids (4.Glebov O.O. Bright N.A. Nichols B.J. Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.Nat. Cell Biol. 2006; 8: 46-54Crossref PubMed Scopus (429) Google Scholar, 5.Lencer W.I. Saslowsky D. Raft trafficking of AB5 subunit bacterial toxins.Biochim. Biophys. Acta. 2005; 1746: 314-321Crossref PubMed Scopus (91) Google Scholar, 6.Sabharanjak S. Sharma P. Parton R.G. Mayor S. GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway.Dev. Cell. 2002; 2: 411-423Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar, 7.Shogomori H. Futerman A.H. Cholesterol depletion by methyl-beta-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons.J. Neurochem. 2001; 78: 991-999Crossref PubMed Scopus (52) Google Scholar, 8.Torgersen M.L. Skretting G. Deurs B.van Sandvig K. Internalization of cholera toxin by different endocytic mechanisms.J. Cell Sci. 2001; 114: 3737-3747Crossref PubMed Google Scholar). Studies on the uptake mechanisms, intracellular itineraries, and biophysical properties of raft-associated proteins at the plasma membrane have revealed heterogeneity in their trafficking and dynamic behavior (9.Drobnik W. Borsukova H. Bottcher A. Pfeiffer A. Liebisch G. Schutz G.J. Schindler H. Schmitz G. Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains.Traffic. 2002; 3: 268-278Crossref PubMed Scopus (141) Google Scholar, 10.Gomez-Mouton C. Abad J.L. Mira E. Lacalle R.A. Gallardo E. Jimenez-Baranda S. Illa I. Bernad A. Manes S. Martinez A.C. Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization.Proc. Natl. Acad. Sci. USA. 2001; 98: 9642-9647Crossref PubMed Scopus (432) Google Scholar, 11.Ishitsuka R. Kobayashi T. Lysenin: a new tool for investigating membrane lipid organization.Anat. Sci. Int. 2004; 79: 184-190Crossref PubMed Scopus (32) Google Scholar, 12.Schade A.E. Levine A.D. Lipid raft heterogeneity in human peripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCR signal transduction.J. Immunol. 2002; 168: 2233-2239Crossref PubMed Scopus (114) Google Scholar). Currently, very little is known about how different ligands associate with raft domains, to what extent the lipid content in those domains differs, and what effect raft lipids have on intracellular targeting. To begin to answer these questions, it will be necessary to develop a diverse battery of markers to characterize the determinants of binding and trafficking behaviors. Here, we present the biochemical and biophysical characterization of a novel, fluorescently tagged sphingolipid binding raft probe, the sphingolipid binding domain (SBD), derived from the amyloid β peptide (Aβ). This motif, identified by Fantini (13.Fantini J. How sphingolipids bind and shape proteins: molecular basis of lipid-protein interactions in lipid shells, rafts and related biomembrane domains.Cell. Mol. Life Sci. 2003; 60: 1027-1032Crossref PubMed Scopus (77) Google Scholar) in several glycolipid-associated proteins, was postulated to form a V3 loop structure that interacts with the sugar rings in glycosphingolipid head groups. In a separate study (S. Steinert and E. Lee, unpublished data), we showed that fluorescent SBD is targeted to endolysosmal compartments in a cholesterol-dependent manner and that it interacts with raft-like lipid mixtures in liposome binding assays and by surface plasmon resonance. We used standard raft isolation methods in conjunction with lipid-protein overlays, live cell imaging, and fluorescence correlation spectroscopy (FCS) to describe the characteristics of SBD association with the plasma membrane. Lipid-protein overlay experiments (fat blots) suggest that SBD interacts with particular gangliosides and sphingomyelin, which are generally thought to reside in raft domains. We also demonstrate that SBD interacts predominantly with detergent-insoluble membrane fractions isolated from neuronal cells, similar to other known raft markers. Moreover, its endocytic uptake by neurons is dependent on the presence of intact microdomains, which can be disrupted by cholesterol or sphingolipid depletion. By FCS, we demonstrate that SBD displays mobility characteristics at the plasma membrane that are consistent with partial raft association and shows a distribution of diffusion times strikingly similar to that of CtxB. Pharmacological removal of cholesterol reduced the SBD association with detergent-resistant membranes (DRMs), dependent on cell type. However, in live cell labeling, uptake at the plasma membrane was cholesterol- and sphingolipid-dependent in both neuronal types tested. In summary, we suggest that SBD can serve as a useful tool for the study of cholesterol-dependent sphingolipid membrane microdomains and their trafficking. The Drosophila neuronal cell line DL-DMBG2-c6 (c6; Drosophila Genome Resource Center) (14.Ui K. Nishihara S. Sakuma M. Togashi S. Ueda R. Miyata Y. Miyake T. Newly established cell lines from Drosophila larval CNS express neural specific characteristics.In Vitro Cell. Dev. Biol. Anim. 1994; 30A: 209-216Crossref PubMed Scopus (79) Google Scholar) was grown at 25°C in Shields and Sang M3 medium (Gibco) with 10% FBS (Gibco), 0.125 IU/ml bovine insulin (Biological Industries), and 1% antibiotic/antimycotic solution (Gibco). NIH 3T3 mouse fibroblasts and SH-SY5Y neuroblastoma (American Type Culture Collection-ATCC) were grown at 37°C in DMEM (Gibco) supplemented with 10% FBS and antibiotic. One microgram of plasmid DNA was used along with Lipofectamine 2000 (Invitrogen) for transfections according to the manufacturer's instructions. Flotillin-green fluorescent protein (GFP) plasmid (a kind gift from L. Briggs and S. Sweeney) has the flotillin sequence under the control of the Act5c promoter that is constitutively active in Drosophila cells. For use in neuroblastomas, flotillin-GFP was cloned into pcDNA 3.1 (Invitrogen) with cytomegalovirus promoter. For labeling of free cholesterol with filipin, cells were fixed in 4% paraformaldehyde for 3 min followed by washes in HBSS/HEPES. Cells were incubated with 50 μg/ml filipin (Sigma) for 45 min and then washed before imaging. SBD peptide linked to an N-terminal cysteine and an inert spacer (cysteine-amino-ethoxy-ethoxy-acetyl[AEEAc]2-DAEFRHDSGYEVHHQELVFFAEDVG), thiol-coupled with Oregon green (OG) or amine-coupled with tetramethyl rhodamine (TMR) directly to the spacer, was synthesized by Bachem. Myc-tagged SBD was synthesized by GenScript Corp. A mutated sequence (DAEFAHDSGAEVHHQELVFFAEDVG) and a scrambled sequence (FYHDESEFGHAVEQFGRDVEAVHDL) were also coupled to myc as controls. To avoid aggregate formation of the peptide, SBD was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Merck), divided into aliquots, and dried. For larger volumes of peptide, evaporation was done under a supply of inert nitrogen. Lyophilized peptide was stored at −20°C and redissolved in DMSO immediately before use. Peptide was diluted to a final working concentration of 10 μM in HBSS (Gibco), supplemented with 10 mM HEPES, incubated at 25°C for 30 min at 10 μM (for Drosophila cells) or at 37°C at 5 μM (for mammalian cells), and then washed three times in HBSS. For lipid overlays/fat blot experiments, the peptide film obtained after HFIP evaporation was dissolved in DMSO and then in Tris buffer, pH 7.4 (the final concentration of DMSO in buffer did not exceed 1%). For cholesterol depletion, cells were incubated in 10 mM methyl-β-cyclodextrin (MβCD; Sigma) for 30 min (in serum-free medium for c6 cells or in medium supplemented with 1% FBS for neuroblastomas) and washed. The Amplex Red Cholesterol Assay kit (Invitrogen) was used to measure cholesterol concentrations in cell extracts before OptiPrep gradient formation and later on the DRM fractions generated. For sphingolipid depletion, cells were incubated with 10 μM fumonisin B1 (FB1; AG Scientific F1022) for 2 h at 37°C, washed three times with HBSS/HEPES before being labeled with transferrin 594 (Molecular Probes) or CtxB 594 (Molecular Probes) at 37°C for 30 min, and then washed three times with HBSS/HEPES before being imaged in phenol red-free DMEM/F12 with FB1 (10 μM). DRMs were isolated as described by Zhai, Chaturvedi, and Cumberledge (15.Zhai L. Chaturvedi D. Cumberledge S. Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine.J. Biol. Chem. 2004; 279: 33220-33227Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Briefly, cells from a confluent plate were washed with phosphate-buffered saline and then resuspended in 0.8 ml of TNET lysis buffer (100 mM Tris, pH 7.5, 20 mM EGTA, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail; Sigma). The postnuclear supernatant was diluted 1:2 with 60% OptiPrep (Accurate Chemicals and Scientific Corp.). Cell lysate-OptiPrep solution was overlaid with 7.2 ml of 30% and 2.4 ml of 5% OptiPrep solution in a Beckman SW41 tube and centrifuged at 41,000 rpm for 5 h at 4°C. Twelve fractions of 1 ml were collected from the top of the gradient and subjected to routine SDS-PAGE or dot blot analysis. For the lipid-protein overlay assay, Sphingostrips (Invitrogen) were used according to the manufacturers' instructions and protocol as described (16.Dowler S. Kular G. Alessi D.R. Protein lipid overlay assay.Sci. STKE. 2002; 2002: PL6Crossref PubMed Scopus (206) Google Scholar). Additional sphingolipids (GM1, galactocerebrosides, sphingomyelin, GD1a, GD1b, GT1b, phosphoethanolamine ceramide; Sigma) were spotted onto Hybond C nitrocellulose strips (Amersham) and allowed to dry. These strips were then exposed to 5–20 μM peptides. For dot blots, equal volumes of each fraction were blotted onto nitrocellulose membrane and exposed to antibodies against various raft and nonraft proteins, or 5–10 μM peptide solution in PBST or 1 ng/ml peroxidase-conjugated CtxB (Invitrogen) or 1 μg/ml Lysenin (Peptide Institute). The following primary antibodies were used: 8C3 (anti-syntaxin; Developmental Studies Hybridoma Bank); 9E10 (HRP-conjugated, 1:200; Santa Cruz); anti-caveolin (1:1,000; BD Pharmingen); anti-flotillin (1:1,000; Transduction Laboratories); anti-Lysenin (1:1,000; Peptide Institute); and anti-rac (1:250; BD Pharmingen). Primary antibody treatment was followed by peroxidase-conjugated secondary antibody exposure, and then blots were developed using standard chemiluminscent detection (Amersham). Intensity quantification from dot blots was carried out using Quantity-One (Bio-Rad Laboratories) software. For uptake experiments, images were acquired with a CoolsnapHQ charged coupled device camera on a Deltavision (Applied Precision) wide-field microscope with a 60×/1.42 numerical aperture oil lens (Olympus) and a standard filter set (green: excitation, 490/20, emission, 528/38; red: excitation, 555/28, emission 617/73) (Chroma). Quantification of images was performed using the MetaMorph image-processing program as described previously (17.Sharma D.K. Brown J.C. Cheng Z. Holicky E.L. Marks D.L. Pagano R. The glycosphingolipid, lactosylceramide, regulates B1-integrin clustering and endocytosis.Cancer Res. 2005; 65: 8233-8241Crossref PubMed Scopus (80) Google Scholar). Whole cell fluorescence was determined by drawing borders around individual cells, and noncellular background was subtracted. All photomicrographs in a given experiment were exposed and processed identically for a given fluorophore. The FCS instrumental setup used in this study is an Olympus FV300 confocal microscope, with which correlator and Avalanche photo detectors are coupled in house. To excite BODIPY-FL-SM and SBD-OG, a 488 nm argon laser was used, and the emission signal was detected through a 510 AF23 emission filter. Dialkyl-indocarbocyanine (DiI), CtxB-Alexa-594, and TMR-SBD were excited with a 543 nm He-Ni laser and were detected through a 595 AF60 emission filter. For all measurements, 100 μW laser power before the microscope objective was used. The measurement was carried out as follows: a cell was first imaged in transmitted light, using the XY scan of the Fluoview software of the Olympus confocal system, followed by choosing a region of interest by adjusting the proper Z plane and then performing FCS in the fluorescence point scanning mode. The Avalanche photo detector creates an intensity plot of the fluorescence signal from the sample, and the hardware correlator calculates the autocorrelation function, G(τ), expressed as {[δF(t) δF(t+τ)]/[F(t)]2}, where F(t) is the fluorescence fluctuation caused by a particle at time t and F(t+τ) is for the same particle at time point (t+τ) (18.Bacia K. Schwille P. A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy.Methods. 2003; 29: 74-85Crossref PubMed Scopus (206) Google Scholar, 19.Haustein E. Schwille P. Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy.Methods. 2003; 29: 153-166Crossref PubMed Scopus (207) Google Scholar). Igor Pro software (version 4) was used to fit the data to the FCS curve-fitting model. The equations for the G(τ) fitting models used are as follows: 2D1P1t: G(τ)=1N×1(1+τ/τD)1+Ftrip(1-Ftrip)×e-(τ/τtrip)+1 2D2P1t: G(τ)=1N(1-F2)(1+τ/τD)F2(1+τ/τD2)×1+Ftrip(1-Ftrip)×e-(τ/τtrip)+1 3D1P1t: G(τ)=1N(1+τ/τD)-11+(ττD)K-2×1+Ftrip(1-Ftrip)×e-(τ/τtrip)+1 3D2P1t: G(τ)=1N(1-F2)(1+τ/τD)-11+(τ/τD)K-2F2×(1+τ/τD2)-11+(τ/τD2)K-2×1+Ftrip(1-Ftrip)×e-(τ/τtrip)+1 Association with DRM fractions has been used as a method to detect raft association (20.Edidin M. The state of lipid rafts: from model membranes to cells.Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 257-283Crossref PubMed Scopus (1137) Google Scholar). Although DRM binding by itself is insufficient to prove raft association, it is generally considered a necessary criterion. Therefore, we used SBD conjugated to a myc tag to analyze binding to DRMs isolated from different cell types that are spotted onto membranes. Because our goal is to characterize SBD for the study of lipid trafficking in Drosophila and other cellular neurodegeneration models, we carried out experiments on Drosophila neuronal cells and mammalian neurons. It has been established that DRMs can be isolated from Drosophila embryonic membranes and cell lines and have similar properties to those isolated from mammalian cells (15.Zhai L. Chaturvedi D. Cumberledge S. Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine.J. Biol. Chem. 2004; 279: 33220-33227Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 21.Hoehne M. de Couet H.G. Stuermer C.A. Fischbach K.F. Loss- and gain-of-function analysis of the lipid raft proteins Reggie/Flotillin in Drosophila: they are posttranslationally regulated, and misexpression interferes with wing and eye development.Mol. Cell. Neurosci. 2005; 30: 326-338Crossref PubMed Scopus (42) Google Scholar, 22.Rietveld A. Neutz S. Simons K. Eaton S. Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains.J. Biol. Chem. 1999; 274: 12049-12054Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). We validate the use of SBD as a raft/sphingolipid tracer by comparing its behavior in the fly neuronal cell line c6 versus SH-SY5Y neuroblastomas, which are susceptible to Aβ toxicity (23.Li Y.P. Bushnell A.F. Lee C.M. Perlmutter L.S. Wong S.K. Beta-amyloid induces apoptosis in human-derived neurotypic SH-SY5Y cells.Brain Res. 1996; 738: 196-204Crossref PubMed Scopus (155) Google Scholar). Drosophila c6 neurons and SH-SY5Y neuroblastomas were solubilized with cold 1% Triton X-100 and fractionated by high-speed centrifugation into detergent-resistant/insoluble (DRM) and nonresistant/soluble (non-DRM) membrane fractions over an OptiPrep density gradient (see Methods). These fractions were spotted onto membranes and then incubated with SBD-myc. To verify DRM isolation from c6 cells, fractions were assayed for association with a transfected known raft protein, flotillin-GFP, and an endogenous nonraft protein, rac, by Western and dot blots. In c6 cells, flotillin is broadly distributed over DRM fractions; it is present in fraction 3 and somewhat more enriched in fractions 4 and 5 (Fig. 1A). In contrast, the nonraft protein rac (15.Zhai L. Chaturvedi D. Cumberledge S. Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine.J. Biol. Chem. 2004; 279: 33220-33227Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) is excluded from fractions 3 and 4 and is found primarily in the more soluble bottom fractions 9 and 10 (Fig. 1A). We also used the binding of another raft marker, GM1, to bind CtxB to isolated fractions as an additional criterion for rafts. CtxB binds most prominently to fraction 3 and to a lesser extent to fractions 1 and 2 (Fig. 1B). Therefore, based on the above four criteria (detergent resistance on a density gradient, presence of flotillin, binding of CtxB, and the exclusion of rac), we define fraction 3 as diagnostic for DRM/raft association in our assay (Fig. 1). By the same dot blot assay, SBD was seen to bind strongly to raft fraction 3, to a lesser extent to fractions 2 and 4, and also to the more soluble fractions 10 and 11 (Fig. 1B). In the neuroblastomas, the raft protein flotillin partitioned preferentially into fractions 3 and 4 and to the more soluble fractions 11 and 12 (Fig. 1C). This dual distribution was even more pronounced on the dot blots (Fig. 1D). The other raft marker, CtxB, also displayed interactions with fractions 1–4 from the top of the gradient and with the more soluble fractions 10 and 11 (Fig. 1D). That CtxB profiles are different for the two cell types may be attributable to CtxB binding to different target lipids in fly versus human neurons. Flies do not make GM1, the target of CtxB in mammalian cells (24.Holthuis J.C. Pomorski T. Raggers R.J. Sprong H. Meer G.Van The organizing potential of sphingolipids in intracellular membrane transport.Physiol. Rev. 2001; 81: 1689-1723Crossref PubMed Scopus (255) Google Scholar), but they do have terminal galactose-bearing glycolipids that could interact weakly with CtxB. From flotillin distribution patterns and CtxB binding, we conclude that there has been a separation of DRMs in fractions 3 and 4. Using the dot blot assay, SBD was seen to bind strongly to DRM fractions 3 and 4 (Fig. 1D) in addition to its binding to fractions 5–8. We note that sphingomyelin binding Lysenin (11.Ishitsuka R. Kobayashi T. Lysenin: a new tool for investigating membrane lipid organization.Anat. Sci. Int. 2004; 79: 184-190Crossref PubMed Scopus (32) Google Scholar, 25.Yamaji A. Sekizawa Y. Emoto K. Sakuraba H. Inoue K. Kobayashi H. Umeda M. Lysenin, a novel sphingomyelin-specific binding protein.J. Biol. Chem. 1998; 273: 5300-5306Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar), which was used as an additional standard because sphingomyelin is expected to be at least partially raft-localized (26.Ohanian J. Ohanian V. Sphingolipids in mammalian cell signalling.Cell. Mol. Life Sci. 2001; 58: 2053-2068Crossref PubMed Scopus (225) Google Scholar, 27.Puri V. Watanabe R. Singh R.D. Dominguez M. Brown J.C. Wheatley C.L. Marks D.L. Pagano R.E. Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.J. Cell Biol. 2001; 154: 535-547Crossref PubMed Scopus (278) Google Scholar), bound to non-DRM fractions of neuroblastomas (Fig. 1D). This does not, however, contradict published data on Lysenin, because its association with DRMs per se has not been reported. Next, we examined whether SBD interacts with domains on intact cells that can later be isolated into DRM fractions (Fig. 2). Cells were incubated with SBD-myc, and subsequently, DRM fractions were isolated and examined for the presence of known raft markers and SBD. Enrichment of cholesterol (Fig. 2A), the distribution of raft markers such as flotillin (Fig. 2B) and caveolin (Fig. 2E), binding to CtxB (Fig. 2C), and the distribution of the nonraft protein rac indicate successful isolation of DRMs. Based on these criteria, we define fractions 2–4 in c6 cells and fractions 1–4 in neuroblastomas to include DRMs. In both Drosophila c6 cells and neuroblastomas, SBD is taken up in DRM fractions in addition to being taken up in more soluble fractions (Fig. 2D). Its association with non-DRM fractions is not unusual among bona fide raft markers (e.g., syntaxin in Drosophila membranes, which also follows a broad distribution along the density gradient) (15.Zhai L. Chaturvedi D. Cumberledge S. Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine.J. Biol. Chem. 2004; 279: 33220-33227Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). In contrast to the neuronal cell types, SBD did not show a strong preference for uptake via DRM fractions of mammalian NIH 3T3 fibroblasts, segregating roughly equally between the caveolin-positive “raft” fractions (4.Glebov O.O. Bright N.A. Nichols B.J. Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.Nat. Cell Biol. 2006; 8: 46-54Crossref PubMed Scopus (429) Google Scholar, 5.Lencer W.I. Saslowsky D. Raft trafficking of AB5 subunit bacterial toxins.Biochim. Biophys. Acta. 2005; 1746: 314-321Crossref PubMed Scopus (91) Google Scholar, 6.Sabharanjak S. Sharma P. Parton R.G. Mayor S. GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway.Dev. Cell. 2002; 2: 411-423Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar) and the rac-positive “nonraft” fractions (7.Shogomori H. Futerman A.H. Cholesterol depletion by methyl-beta-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons.J. Neurochem. 2001; 78: 991-999Crossref PubMed Scopus (52) Google Scholar, 8.Torgersen M.L. Skretting G. Deurs B.van Sandvig K. Internalization of cholera toxin by different endocytic mechanisms.J. Cell Sci. 2001; 114: 3737-3747Crossref PubMed Google Scholar, 9.Drobnik W. Borsukova H. Bottcher A. Pfeiffer A. Liebisch G. Schutz G.J. Schindler H. Schmitz G. Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains.Traffic. 2002; 3: 268-278Crossref PubMed Scopus (141) Google Scholar, 10.Gomez-Mouton C. Abad J.L. Mira E. Lacalle R.A. Gallardo E. Jimenez-Baranda S. Illa I. Bernad A. Manes S. Martinez A.C. Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization.Proc. Natl. Acad. Sci. USA. 2001; 98: 9642-9647Crossref PubMed Scopus (432) Google Scholar, 11.Ishitsuka R. Kobayashi T. Lysenin: a new tool for investigating membrane lipid organization.Anat. Sci. Int. 2004; 79: 184-190Crossref PubMed Scopus (32) Google Scholar, 12.Schade A.E. Levine A.D. Lipid raft heterogeneity in human peripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCR signal transduction.J. Immunol. 2002; 168: 2233-2239Crossref PubMed Scopus (114) Google Scholar) (Fig. 2E). Treatments that inhibit cholesterol synthesis or that remove cholesterol from membranes are known to disrupt lipid rafts. Therefore, we used MβCD to deplete c6 and neuroblastoma cells of cholesterol and related sterols (see Methods) and looked at the effect on SBD internalization. First, we tested the effectiveness of the MβCD treatment by measuring total cholesterol levels using the Amplex Red Cholesterol Assay method (Invitrogen). On c6 cells, cholesterol was reduced by 46.7 ± 7.8% (average of two experiments) after 30 min, and in the neuroblastomas, we observed a reduction of 51.8 ± 8.4% (average of two experiments). We also examined the sterol levels across the fractions obtained by density centrifugation (Fig. 2A, line profiles). In both cell types used, MβCD treatment causes the distribution of the remaining cholesterol to become more uniform across the density gradient (Fig. 2A). Also, MβCD treatment results in a larger depletion of cholesterol from the top half than from the bottom half of the gradient. In conjunction with this, the distribution of both raft markers and nonraft markers is altered. Flotillin (Fig. 2B) appears to be excluded in c6 cells (fraction 2) or reduced in neuroblastomas (fractions 3 and 4) from certain DRM fractions relative to other fractions. CtxB's affinity for isolated DRM fractions (Fig. 2C) also appears to be affected by cholesterol depletion; in the neuroblastomas, CtxB shows diminished interaction with DRM fractions 1 and 2 upon MβCD treatment. Likewise in c6 cells, MβCD treatment causes CtxB's interaction with DRMs (fractions 1 and 2) to be weakened. In addition to the decreased association with DRM fractions 1 and 2 in c6 cells, the raft markers flotillin and CtxB both appear to concentrate in fraction 4 upon cholesterol depletion. Conversely, the nonraft marker rac (Fig. 2B), which is normally excluded from DRM fractions, appears to now distribute in these fractions (2.Brown D.A. London E. Functions of lipid rafts in biological membranes.Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2551) Google Scholar, 3.Simons K. Ehehalt R. Cholesterol, lipid rafts, and disease.J. Clin. Invest. 2002; 110: 597-603Crossref PubMed Scopus (920) Google Scholar), suggesting that cholesterol depletion has indeed affected the partitioning of both raft and nonraft proteins in the membrane of c6 cells. SBD uptake into DRMs is also affected by MβCD treatment. Cholesterol depletion by MβCD caused a reduced uptake of SBD in some of the DRM fractions of c6 cells (frac" @default.
• W2108901610 created "2016-06-24" @default.
• W2108901610 creator A5005617556 @default.
• W2108901610 creator A5009044407 @default.
• W2108901610 creator A5010414582 @default.
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• W2108901610 date "2008-05-01" @default.
• W2108901610 modified "2023-10-16" @default.
• W2108901610 title "A fluorescent sphingolipid binding domain peptide probe interacts with sphingolipids and cholesterol-dependent raft domains" @default.
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