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• W2000930446 abstract "Lipid analogs with dialkylindocarbocyanine (DiI) head groups and short or unsaturated hydrocarbon chains (e.g. DiIC12 and FAST DiI) enter the endocytic recycling compartment efficiently, whereas lipid analogs with long, saturated tails (e.g. DiIC16 and DiIC18) are sorted out of this pathway and targeted to the late endosomes/lysosomes (Mukherjee, S., Soe, T. T., and Maxfield, F. R. (1999) J. Cell Biol. 144, 1271-1284). This differential trafficking of lipid analogs with the same polar head group was interpreted to result from differential partitioning to different types of domains with varying membrane order and/or curvature. Here we investigate the system further by monitoring the trafficking behavior of these lipid analogs under conditions that alter domain properties. There was a marked effect of cholesterol depletion on the cell-surface distribution and degree of internalization of the lipid probes. Furthermore, instead of going to the late endosomes/lysosomes as in control cells, long chain DiI analogs, such as DiIC16, were sorted to the recycling pathway in cholesterol-depleted cells. We confirmed that this difference was due to a change in overall membrane properties, and not cholesterol levels per se, by utilizing a Chinese hamster ovary cell line that overexpressed transfected stearoyl-CoA desaturase 1, a rate-limiting enzyme in the production of monounsaturated fatty acids. These cells have a decrease in membrane order because they contain a much larger fraction of unsaturated fatty acids. These cells showed alteration of DiI trafficking very similar to cholesterol-depleted cells. By using cold Triton X-100 extractability of different lipids as a criterion to determine the membrane properties of intracellular organelles, we found that the endocytic recycling compartment has abundant detergent-resistant membranes, in contrast to the late endosomes and lysosomes. Lipid analogs with dialkylindocarbocyanine (DiI) head groups and short or unsaturated hydrocarbon chains (e.g. DiIC12 and FAST DiI) enter the endocytic recycling compartment efficiently, whereas lipid analogs with long, saturated tails (e.g. DiIC16 and DiIC18) are sorted out of this pathway and targeted to the late endosomes/lysosomes (Mukherjee, S., Soe, T. T., and Maxfield, F. R. (1999) J. Cell Biol. 144, 1271-1284). This differential trafficking of lipid analogs with the same polar head group was interpreted to result from differential partitioning to different types of domains with varying membrane order and/or curvature. Here we investigate the system further by monitoring the trafficking behavior of these lipid analogs under conditions that alter domain properties. There was a marked effect of cholesterol depletion on the cell-surface distribution and degree of internalization of the lipid probes. Furthermore, instead of going to the late endosomes/lysosomes as in control cells, long chain DiI analogs, such as DiIC16, were sorted to the recycling pathway in cholesterol-depleted cells. We confirmed that this difference was due to a change in overall membrane properties, and not cholesterol levels per se, by utilizing a Chinese hamster ovary cell line that overexpressed transfected stearoyl-CoA desaturase 1, a rate-limiting enzyme in the production of monounsaturated fatty acids. These cells have a decrease in membrane order because they contain a much larger fraction of unsaturated fatty acids. These cells showed alteration of DiI trafficking very similar to cholesterol-depleted cells. By using cold Triton X-100 extractability of different lipids as a criterion to determine the membrane properties of intracellular organelles, we found that the endocytic recycling compartment has abundant detergent-resistant membranes, in contrast to the late endosomes and lysosomes. Lipids and proteins associated with the cell surface vary in their lateral and transbilayer distribution, as well as the rate at which they are internalized from the plasma membrane. Once inside the cell, they can potentially be delivered to a large variety of organelles by selective partitioning in a series of sorting steps associated with vesicle or tubule formation (1Mukherjee S. Soe T.T. Maxfield F.R. J. Cell Biol. 1999; 144: 1271-1284Google Scholar). Although many specific peptide motifs and protein-protein interactions that determine the distribution and trafficking of transmembrane proteins have been characterized (1Mukherjee S. Soe T.T. Maxfield F.R. J. Cell Biol. 1999; 144: 1271-1284Google Scholar, 2Dell'Angelica E.C. Trends Cell Biol. 2001; 11: 315-318Google Scholar), the principles underlying lipid sorting and trafficking remain relatively unclear. Although the intracellular destinations and sorting decisions for a variety of lipids and lipid analogs have been investigated in recent years (1Mukherjee S. Soe T.T. Maxfield F.R. J. Cell Biol. 1999; 144: 1271-1284Google Scholar, 3Marks D.L. Pagano R.E. Trends Cell Biol. 2002; 12: 605-613Google Scholar), a coherent general set of sorting rules for lipids has yet to emerge. Part of the difficulty in understanding lipid sorting and distribution in the cell arises from the fact that this is the result of a complex interplay between the specific chemistries of individual lipid molecules (e.g. their size, hydrophobicity, head group to acyl chain cross-sectional ratio, charge on the head group, acyl chain unsaturation, etc.) as well as the biophysical properties of the membrane bilayer as a whole (e.g. its composition, thickness, tension, fluidity, and curvature) (reviewed in Refs. 4Maier O. Ait Slimane T. Hoekstra D. Cell. Dev. Biol. 2001; 12: 149-161Google Scholar and 5Mukherjee S. Maxfield F.R. Traffic. 2000; 1: 203-211Google Scholar). Adding to the complexity is the fact that a typical biological membrane is composed of hundreds of different lipid classes, with varying permutations of head groups and acyl chains, in addition to varying amounts of rigid, relatively planar structures like cholesterol. In addition, membranes contain a variety of proteins, both transmembrane and peripheral, that vary in shapes, sizes, charge distribution, and propensity for aggregation among themselves or with other proteins and/or lipids. As a final measure of the complexity, many of these components are distributed non-randomly in the bilayer, varying both in the lateral and the transbilayer dimensions. Biophysical studies in model membrane systems of precise composition have clarified to a great extent the ways in which classes of lipid molecules interact among themselves and with other classes of lipids or cholesterol (6Gennis R.B. Biomembranes: Molecular Structure and Function. Springer-Verlag, New York1989Google Scholar). However, these studies were carried out in relatively simple, well defined, two- or three-component systems. Thus, although there is no doubt that similar principles are at play in various biological membranes, these membranes are too complex to allow a simple extrapolation of the insights obtained from model systems. In contrast to the plasma membrane, very little is known about the biophysical properties of most intracellular membranes. Although lateral membrane domains or “rafts” have been shown in several studies to exist on the plasma membranes of mammalian cells, whether such domains exist in endocytic organelles as well and, if they do, how their properties compare with the domains on the cell surface remain open questions. Glycolipid and cholesterol-enriched rafts have been proposed to play a role in biosynthetic protein and lipid sorting (7Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Google Scholar). Also, experiments have utilized the ability of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-S-indacene)-labeled lipid analogs to form excimers in a concentration-dependent manner to suggest a redistribution of lipids within seconds after the initiation of endocytosis (8Chen C.S. Martin O.C. Pagano R.E. Biophys, J. 1997; 72: 37-50Google Scholar). Lipid rafts have been shown to exist as early in the biosynthetic pathway as the endoplasmic reticulum (7Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Google Scholar, 9Watanabe R. Funato K. Venkataraman K. Futerman A.H. Riezman H. J. Biol. Chem. 2002; 277: 49538-49544Google Scholar). In the current study, we have utilized several of the fluorescent lipid analogs of the dialkylindocarbocyanine (DiI) 1The abbreviations used are: DiI, dialkylindocarbocyanine; DAF, decay-accelerating factor; DHE, dehydroergosterol; ERC, endocytic recycling compartment; GPI, glycosylphosphatidylinositol; LDL, low density lipoprotein; LE/LY, late endosomes/lysosomes; MβCD, methyl-β-cyclodextrin; SCD1, stearoyl-CoA desaturase1; Tf, transferrin; CHO, Chinese hamster ovary; BSA, bovine serum albumin; TfR, Tf receptor. 1The abbreviations used are: DiI, dialkylindocarbocyanine; DAF, decay-accelerating factor; DHE, dehydroergosterol; ERC, endocytic recycling compartment; GPI, glycosylphosphatidylinositol; LDL, low density lipoprotein; LE/LY, late endosomes/lysosomes; MβCD, methyl-β-cyclodextrin; SCD1, stearoyl-CoA desaturase1; Tf, transferrin; CHO, Chinese hamster ovary; BSA, bovine serum albumin; TfR, Tf receptor. series, whose trafficking behavior in normal fibroblasts was investigated previously (1Mukherjee S. Soe T.T. Maxfield F.R. J. Cell Biol. 1999; 144: 1271-1284Google Scholar). In the previous study, we found that, in general, lipids that have a propensity to partition into the more disordered lipid domains, or ones with a propensity to enter membranes of concave curvature, trafficked preferentially to the endocytic recycling compartment (ERC), whereas those with opposite propensities trafficked predominantly to the late endosomes/lysosomes (LE/LY). In order to explain the results of the above studies, we had proposed a working hypothesis based on differential partitioning of endocytosed membrane-associated molecules into coexisting membrane domains, as defined by varying fluidities and/or curvatures. It is well known that both membrane fluidity (or “membrane order”) as well as curvature are strongly modulated by the amount of cholesterol present in the bilayer (10Yeagle P.L. Biochimie (Paris). 1991; 73: 1303-1310Google Scholar, 11Feigenson G.W. Buboltz J.T. Biophys. J. 2001; 80: 2775-2788Google Scholar, 12Xu X. London E. Biochemistry. 2000; 39: 843-849Google Scholar, 13Julicher F. Lipowsky R. Physical Rev. E. 1996; 53: 2670-2683Google Scholar). Thus, in this paper, we test our working hypothesis by following the trafficking of the DiI analogs in cells whose membrane properties have been altered by depleting the amount of cholesterol in the bilayer. In order to ensure that the effect of cholesterol depletion was an overall alteration of membrane structure and dynamics, rather than a specific cholesterol interaction effect, we utilized an alternate method to alter membrane fluidity, without changing its cholesterol content. This was achieved by utilizing a cell line (CHO-SCD1 cells) overexpressing an enzyme, stearoyl-CoA desaturase 1 (SCD1), that alters the amount of monounsaturated fatty acid chains in the membrane lipids (35Ntambi J.M. Miyazaki M. Curr. Opin. Lipidol. 2003; 14: 255-261Google Scholar). In addition to following the trafficking itineraries of the lipid analogs in this study, we also investigate the membrane properties of intracellular endocytic organelles, namely the ERC and the LE/LY, by monitoring their insolubility in cold Triton X-100. Resistance to cold Triton extraction is a criterion often used in cell biology research as phenomenological evidence for molecules residing in membrane domains, termed rafts, that are believed to exist in the so-called liquid ordered or Lo phase (14Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Google Scholar, 15London E. Brown D.A. Biochim. Biophys. Acta. 2000; 1508: 182-195Google Scholar). Our interest in this study is to look for any general sorting rules for lipids that may emerge from these comparative studies and also to investigate the types of lateral lipid distributions (domains) that may occur in various endocytic organelles. In these studies, we are able to follow the fate of cholesterol directly, by using the fluorescent cholesterol analog dehydroergosterol (DHE) (16Mukherjee S. Zha X. Tabas I. Maxfield F.R. Biophys. J. 1998; 75: 1915-1925Google Scholar, 17Hao M. Lin S.X. Karylowski O.J. Wustner D. McGraw T.E. Maxfield F.R. J. Biol. Chem. 2002; 277: 609-617Google Scholar). All fluorescent probes and anti-Alexa 488 were obtained from Molecular Probes Inc. (Eugene, OR). Human Tf was obtained from Sigma. It was iron-loaded and passed through a Sephacryl S-300 gel filtration system as described previously (18Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Google Scholar). Alexa 488 was then conjugated to the iron-loaded Tf following manufacturers' instructions. Labeled transferrin was dialyzed thoroughly to remove the unbound dye. Monoclonal antibody against DAF was provided by Dr. S. Tomlinson (Medical University of South Carolina) (19Song H. He C. Knaak C. Guthridge J.M. Holers V.M. Tomlinson S. J. Clin. Investig. 2003; 111: 1875-1885Google Scholar). DHE-loaded MβCD was prepared as described previously (17Hao M. Lin S.X. Karylowski O.J. Wustner D. McGraw T.E. Maxfield F.R. J. Biol. Chem. 2002; 277: 609-617Google Scholar). DiI-LDL was a gift from Dr. Ira Tabas (Columbia University, New York). Lipid analogs and free fatty acids were transferred as monomers from fatty acid-free BSA carriers (1Mukherjee S. Soe T.T. Maxfield F.R. J. Cell Biol. 1999; 144: 1271-1284Google Scholar). All tissue culture supplies were from Invitrogen. All other chemicals were from Sigma. TRVb-1 is a modified CHO cell line that lacks endogenous Tf receptor and expresses the human Tf receptor (20McGraw T.E. Greenfield L. Maxfield F.R. J. Cell Biol. 1987; 105: 207-214Google Scholar). DAFTb-1 cells are a derivative of TRVb-1 cells. In addition to the human Tf receptors, they also express the GPI-linked DAF (21Mayor S. Maxfield F.R. Mol. Biol. Cell. 1995; 6: 929-944Google Scholar). They were grown in bicarbonate buffered Ham's F-12 medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 200 μg/ml geneticin as a selection for the transfected Tf receptors. The CHO-SCD cell line was established by transfecting CHO cells with pcDNA3.1/Hygro-mSCD1 using LipofectAMINE 2000 (Invitrogen) according to manufacturer's instruction. Hygromycin-resistant clones were pooled for experiments. CHO and CHO-SCD cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin. 200 units/ml hygromycin was used as a selection for the transfected SCD1. All cells were kept in a 5% CO2 environment in humidified incubators at 37 °C. Cells for microscopy were grown for 2 days on 35-mm plastic tissue culture dishes whose bottoms were replaced with poly-d-lysine-coated coverslips (22Salzman N.H. Maxfield F.R. J. Cell Biol. 1989; 109: 2097-2104Google Scholar). All experimental manipulations as well as microscopy were carried out in these dishes. Metabolic Depletion—TRVb-1 cells were grown for 2 days in metabolic depletion medium (Ham's F-12 medium similar to the growth medium but with 5% lipoprotein-deficient serum in place of fetal bovine serum, supplemented with 200 μm mevalonate, and 10 μm mevastatin (23Mayor S. Sabharanjak S. Maxfield F.R. EMBO J. 1998; 17: 4626-4638Google Scholar) to block cholesterol synthesis and deplete cholesterol stores (24Rothberg K.G. Ying Y.S. Kamen B.A. Anderson R.G.W. J. Cell Biol. 1990; 111: 2931-2938Google Scholar). Depletion by MβCD—Cells were incubated with 10 mm MβCD in Medium 1 (150 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 2 g/liter glucose, and 20 mm Hepes, pH 7.4) for 1 h prior to labeling. Control TRVb-1 cells were labeled with 10 nm DiI labeling solution for 2 min at 37 °C, rinsed several times with Medium 1, and then incubated with 5 μg/ml Alexa 488-Tf for 30 min at 37 °C. For cholesterol depletion experiments, cells were prelabeled with 5 μg/ml Alexa 488-Tf for 1 h at 37 °C, extracted with 10 mm MβCD for 1 h at 37 °C, labeled with 50 nm DiI for 2 min, and chased for 30 min at 37 °C. Alexa 488-Tf was present in all the steps except for the 2 min labeling with DiI. Cells that were cholesterol-depleted metabolically were singly labeled with DiI. At the end of the chase period, the cells were rinsed with Medium 1 and fixed with 2% paraformaldehyde. The effects of free saturated long chain fatty acids (heptadecanoic (C17:0) and nonadecanoic acid (C19:0) on DiIC16 trafficking were monitored in both TRVb-1 and CHO-SCD cells, under conditions of growth in normal media as well as after acute cholesterol depletion using MβCD. For these experiments, free fatty acids were first loaded onto 1% w/v fatty acid-free BSA, at a final stock concentration of 0.3 mm fatty acid, by using established protocols (25Johnson R.A. Hamilton J.A. Worgall T.S. Deckelbaum R.J. Biochemistry. 2003; 42: 1637-1645Google Scholar). The cells were then cholesterol-depleted (or not for control) with MβCD for 30 min, rinsed, labeled for 1 min with DiIC16, rinsed, and incubated with or without the fatty acid for 45 min at 37 °C, followed by imaging of the live cells. Fluorescence microscopy and digital image acquisition were carried out using a Leica DMIRB microscope (Leica Mikroscopie und Systeme GmbH, Germany) equipped with a Princeton Instruments (Princeton, NJ) cooled CCD camera driven by Image 1/MetaMorph Imaging System software (Universal Imaging Corp.). All images were acquired using a high magnification oil immersion objective (63×, 1.4 NA). Alexa 488-conjugated proteins were imaged using a standard fluorescein filter cube [470-nm (20-nm bandpass) excitation filter, 510-nm longpass dichromatic filter, and 537-nm (23-nm bandpass) emission filter], DiI probes using a standard rhodamine filter cube [535-nm (50-nm bandpass) excitation filter, 565-nm longpass dichromatic filter, and 610-nm (75-nm bandpass) emission filter], and DHE using a filter cube obtained from Chroma Technology Corp. (Brattleboro, VT) [335-nm (20-nm bandpass) excitation filter, 365-nm longpass dichromatic filter, and 405-nm (40-nm bandpass) emission filter] (17Hao M. Lin S.X. Karylowski O.J. Wustner D. McGraw T.E. Maxfield F.R. J. Biol. Chem. 2002; 277: 609-617Google Scholar). Fluorescence crossover was measured using single-labeled samples of each probe. Images were corrected for background (26Hao M. Maxfield F.R. J. Biol. Chem. 2000; 275: 15279-15286Google Scholar) and crossover (1Mukherjee S. Soe T.T. Maxfield F.R. J. Cell Biol. 1999; 144: 1271-1284Google Scholar). Confocal microscopy was performed using an Axiovert 100M inverted microscope equipped with an LSM 510 laser scanning unit and a 63× 1.4 NA plan Apochromat objective (Carl Zeiss, Inc.). Cells labeled with DiI were excited with a 1.0-milliwatt helium/neon laser emitting at 543 nm, and a 560-nm long pass filter was used for collecting emissions. Alexa 488-conjugated proteins were excited with a 25-milliwatt argon laser emitting at 488 nm and a 505-530 bandpass filter was used for emissions. The two channels were scanned alternately in a line-by-line fashion, having only one laser line and one detector channel on at each time. Summation projection of all background corrected confocal slices was produced using the MetaMorph software. Correlation Measurements—Six images were selected in which each of the cells was double-labeled with Alexa 488-Tf and FAST DiI, DiIC12, DiIC16, or DiIC18 in normal media, and DiIC16 or DiIC18 in cholesterol depletion media. They were first background corrected by subtracting the average fluorescence in regions within the image that contained no cells from the overall fluorescence (26Hao M. Maxfield F.R. J. Biol. Chem. 2000; 275: 15279-15286Google Scholar). A threshold was then applied to each image such that only cell-associated pixels were used in calculating the correlation coefficient. A correlation plot and a correlation coefficient for all selected pixels above the threshold for each image were generated by MetaMorph. Cholesterol Depletion Interferes with the Normal Trafficking of the DiI Analogs—Endocytic fates of the DiI derivatives were determined after their initial incorporation in the plasma membrane. Fluorescent Tf, bound to its receptor (TfR), was used as a marker for the ERC (27Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. 1997; 77: 759-803Google Scholar, 28Dunn K.W. McGraw T.E. Maxfield F.R. J. Cell Biol. 1989; 109: 3303-3314Google Scholar). Exit from the ERC is the slowest step in the endocytic recycling itinerary of the TfR, resulting in the ERC being the most brightly labeled structure at steady state (18Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Google Scholar). It has been shown previously (29Subtil A. Gaidarov I. Kobylarz K. Lampson M.A. Keen J.H. McGraw T.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6775-6780Google Scholar) that cholesterol depletion severely inhibits the internalization of TfR from the cell surface. Thus, under these conditions, most of the Tf remains at the cell surface, and the bright cell surface fluorescence impedes the detection of the central ERC fluorescence. In order to obtain a clear definition of the ERC in cholesterol-depleted cells, we labeled the cells first with Alexa 488-Tf, allowing enough time to load the ERC, followed by cholesterol extraction and labeling with the DiI analog. Fig. 1 shows control (normal growth medium) and cholesterol-depleted cells that were labeled for 2 min with DiIC16 (Fig. 1, panels A-D) and chased for 30 min. Our previous work showed that DiIC16, which has some preference for ordered membrane domains due to its long and saturated tails, was sorted away from the endocytic recycling pathway and delivered to the LE/LY after 30 min of internalization (1Mukherjee S. Soe T.T. Maxfield F.R. J. Cell Biol. 1999; 144: 1271-1284Google Scholar) (Fig. 1, panels A and B). Several changes in lipid traffic were seen upon cholesterol depletion. First, a very large fraction of DiIC16 in cholesterol-depleted cells was found in the plasma membrane (the ring stain in Fig. 1, panel D). Second, most of the internalized DiIC16 localized to the ERC, indicated by extensive colocalization with internalized Tf (Fig. 1, panels C and D). This was clearly different from the route taken by DiIC16 in control cells that resulted in its accumulation in the LE/LY (Fig. 1, panels A and B). Even after 30 min of chase, very few punctate structures representing the LE/LY were seen labeled with DiIC16 in cholesterol-depleted cells. To be sure that the change in intracellular trafficking upon cholesterol depletion was related to the order preferences of the hydrocarbon tails, we confirmed these results with DiIC18, which has a higher affinity for the ordered domains than DiIC16 (30Spink C.H. Yeager M.D. Feigenson G.W. Biochim. Biophys. Acta. 1990; 1023: 25-33Google Scholar). Cells labeled with DiIC16 and DiIC18 behaved identically, both under normal growth conditions and upon cholesterol depletion (data not shown). In order to avoid possible artifacts, cholesterol depletion was carried out in two different ways. The approach used for the cells shown in Fig. 1 was to treat cells grown in normal medium with MβCD, an efficient cholesterol chelator known to form soluble inclusion complexes with cholesterol (31Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Google Scholar). In CHO cells, incubation with 10 mm MβCD for 30 min or longer reduced total cellular cholesterol by 40-50% (29Subtil A. Gaidarov I. Kobylarz K. Lampson M.A. Keen J.H. McGraw T.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6775-6780Google Scholar, 32Hao M. Mukherjee S. Maxfield F.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13072-13077Google Scholar). The other method to reduce cholesterol was metabolic depletion, in which the cells were grown for 2 days in medium containing lipid-depleted serum, an inhibitor of cholesterol biosynthesis (mevastatin), and supplemented with low levels of mevalonate in order to maintain basic metabolism of other essential isoprenoids (23Mayor S. Sabharanjak S. Maxfield F.R. EMBO J. 1998; 17: 4626-4638Google Scholar, 24Rothberg K.G. Ying Y.S. Kamen B.A. Anderson R.G.W. J. Cell Biol. 1990; 111: 2931-2938Google Scholar). Both methods produced similar degrees of cholesterol depletion and resulted in similar trafficking alterations (not shown). Cholesterol depletion of the cells shown in all figures here was performed using MβCD. In CHO cells, the ERC is a collection of narrow tubular elements that appears as an unresolved central spot by epifluorescence microscopy (Fig. 1). We used confocal scanning microscopy to examine in more detail the extent of colocalization between DiIC16 and Tf in cholesterol-depleted cells. Shown in Fig. 2 are single plane images (panels A-C) and summation projections of all focal planes (panels D-F). In cholesterol-depleted cells, DiIC16 was seen to colocalize extensively with Tf in ERC tubules in the juxtanuclear region of the cell. To make sure that the morphology of LE/LY was not disrupted by cholesterol depletion, we double-labeled the cholesterol-depleted cells to steady state with DiI-LDL and Tf. The LE/LY, appearing as small punctate dots labeled by LDL (Fig. 2, panel G), distributed throughout the cell and separated from Tf (Fig. 2, panel H) in a fashion identical to that in control cells. Unlike the control cells, however, these LDL-containing endosomes did not contain significant amounts of DiIC16. Fig. 3 shows control and cholesterol-depleted cells that were labeled for 2 min with either FAST DiI (Fig. 3, panels A-D) or DiIC12 (Fig. 3, panels E-H) and chased for 30 min. As reported previously, both FAST DiI and DiIC12 are delivered to the ERC in control cells, and they show significant codistribution with Tf (Fig. 3, panels A and B, E and F) (1Mukherjee S. Soe T.T. Maxfield F.R. J. Cell Biol. 1999; 144: 1271-1284Google Scholar). However, when cholesterol was depleted, labeling of the ERC by FAST DiI and DiIC12 was reduced, and most of the label was retained at the plasma membrane (Fig. 3, panels C and D, G and H). Many patches of fluorescence were seen, especially in the FAST DiI (Fig. 3, panel D), and these patches on the plasma membrane were relatively stable over time (at least up to 20 min). We have reported previously (32Hao M. Mukherjee S. Maxfield F.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13072-13077Google Scholar) patching of DiIC12 and C6-NBD-SM into the more disordered regions of the plasma membrane upon cholesterol depletion. To verify that the patches of FAST DiI were indeed on the plasma membrane, we utilized a confocal microscope to optically section through a cholesterol-depleted cell labeled with FAST DiI (Fig. 4, panels A-D). At each section, the fluorescent patches were seen at or near the cell border, consistent with patching on the cell surface. Very little FAST DiI was seen inside the cells.Fig. 4FAST DiI distribution in cholesterol-depleted and -repleted cells. Cells were incubated with 10 mm MβCD for 1 h, labeled with FAST DiI for 2 min, and chased for 30 min. Optical sections were then taken through the cells by scanning confocal microscopy. Panel A shows a summation projection of all the focal planes. Planes B-D show three representative planes through the cell (panel B, bottom focal plane; panel D, top). Panels E and F shows the reversibility of cholesterol depletion (Chol-depl) on the trafficking of FAST DiI. Cells were incubated with 10 mm MβCD for 1 h, labeled with FAST DiI for 2 min, and chased for 30 min in Medium 1 (panel E) or Medium 1 supplemented with cholesterol-loaded MβCD (panel F). They were then imaged using a wide-field microscope. Bar, 10 μm.View Large Image Figure ViewerDownload (PPT) The effect of cholesterol depletion on the DiI trafficking is reversible (Fig. 4, panels E and F). Cells were first incubated with MβCD for 1 h. They were then labeled with FAST DiI for 2 min, chased for 30 min in either Medium 1 (Fig. 4, panel E) or Medium 1 supplemented with cholesterol-loaded MβCD (Fig. 4, panel F). Repletion of cholesterol with an exogenous source resulted in a uniform plasma membrane distribution and intracellular accumulation for FAST DiI, demonstrating that the change in FAST DiI distribution upon cholesterol depletion treatment was truly an effect of reduction in cholesterol levels. Quantification of the Overlap of the Distribution of Different DiI Analogs Relative to Tf—Although Fig. 1 shows the redirection of DiIC16 trafficking from the LE/LY to the ERC visually, we quantified this redirection to give statistical validity to our results. Fig. 5, panel A, shows the correlation plots of the distribution of different DiI derivatives relative to Tf, for control and cholesterol-depleted cells. Fig. 5, panel B, shows a plot of the correlation coefficients obtained from an average of several such correlation measurements, for each experimental condition. Correlation data in cholesterol-depleted cells (relative to Tf) for the disordered domain preferring DiI analogs (DiIC12 and FAST DiI) were not calculated because these analogs were primarily retained on the plasma membrane upon cholesterol depletion. The correlation coefficients for the DiIC16 and DiIC18 compared with Tf increase significantly upon cholesterol depletion. Retention of Disorder-preferring Lipids on the Plasma Membrane of Cholesterol-depleted Cells Is Due to Impaired Internalization—In the pulse-chase protocols" @default.
• W2000930446 created "2016-06-24" @default.
• W2000930446 creator A5031849699 @default.
• W2000930446 creator A5053648650 @default.
• W2000930446 creator A5063711122 @default.
• W2000930446 creator A5078599028 @default.
• W2000930446 date "2004-04-01" @default.
• W2000930446 modified "2023-10-16" @default.
• W2000930446 title "Effects of Cholesterol Depletion and Increased Lipid Unsaturation on the Properties of Endocytic Membranes" @default.
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