Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking

Raman Deep Singh1, David L. Marks1, Richard E. Pagano1

1 Mayo Clinic College of Medicine, Rochester, Minnesota
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 24.1
DOI:  10.1002/0471143030.cb2401s35
Online Posting Date:  June, 2007
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Abstract

Sphingolipids (SLs), including glycosphingolipids, are found on the plasma membrane where they play important roles in a wide variety of cell functions, including cell‐cell communication, cell growth and differentiation, host‐pathogen interactions, and cell‐signaling events. This unit illustrates the use of fluorescent SL analogs to identify the mechanisms underlying SL endocytosis and subsequent intracellular trafficking. Techniques used to study SL domain formation at the plasma membrane, endocytic mechanisms and intracellular transport steps are highlighted. The use of biochemical treatments and dominant‐negative protein expression to block specific steps in lipid trafficking are also discussed.

Keywords: lipid; sphingolipid; lipid trafficking

     
 
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Table of Contents

  • Basic Protocol 1: Visualization of Sphingolipids at Plasma Membrane Microdomains
  • Support Protocol 1: Preparation of C8‐LacCer/BSA and BSA Control Stock Solutions
  • Support Protocol 2: Preparation of Fluorescent Sphingolipid Analogs: Bodipy‐LacCer/BSA Complex
  • Basic Protocol 2: Studying Endocytic Mechanisms with Fluorescent Lipid Analogs
  • Alternate Protocol 1: Detection of Endocytosis in Association with Dominant‐Negative Proteins
  • Basic Protocol 3: Intracellular Localization and Transport: Co‐Localization of Bodipy‐LacCer with Albumin but not Dextran
  • Alternate Protocol 2: Using Dominant‐Negative rab Proteins to Identify Transport Steps
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Visualization of Sphingolipids at Plasma Membrane Microdomains

  Materials
  • Human skin fibroblasts (HSFs; Coriell Institute for Medical Research)
  • EMEM containing 10% FBS (EMEM‐10; see recipe)
  • HEPES‐buffered minimal essential medium + glucose (HMEM+G; see recipe)
  • 1 mM C 8‐LacCer/BSA stock solution ( protocol 2)
  • 1 mM BSA control stock solution ( protocol 2)
  • BODIPY‐LacCer/BSA stock solution ( protocol 3)
  • 25‐mm acid‐etched coverslips (Fisher), sterilized by immersing in 70% ethanol and flaming
  • 35‐mm tissue culture dishes
  • 10°C cooling block (described in Marks et al., )
  • Depression slides
  • Fluorescence microscope with high magnification (e.g., 1000×), image acquisition capability, and a stage maintained at 10°C
  • Additional reagents and equipment for culturing cells (unit 1.1)

Support Protocol 1: Preparation of C8‐LacCer/BSA and BSA Control Stock Solutions

  Materials
  • D‐lactosyl‐β 1‐1′‐N‐octanoyl‐D‐erythro‐sphingosine (C 8‐LacCer) powder (mol. wt. 749.98; Avanti Polar Lipids)
  • 2:1 (v/v) ethanol/DMSO
  • Fatty‐acid‐free bovine serum albumin (dfBSA; Sigma)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 5‐ml glass tube
  • Dialysis tubing (12,000 to 14,000 MWCO)
  • Sonicator (optional)
  • Ultracentrifuge and appropriate tubes

Support Protocol 2: Preparation of Fluorescent Sphingolipid Analogs: Bodipy‐LacCer/BSA Complex

  Materials
  • 250 nmol BODIPY‐LacCer (Invitrogen), dissolved in 19:1 (v/v) chloroform/ethanol
  • Nitrogen gas
  • HMEM−G (see recipe) or phosphate‐buffered saline (PBS; appendix 2A)
  • Absolute ethanol
  • 1 mM (66 mg/ml) fatty‐acid‐free bovine serum albumin (dfBSA; mol. wt. 66,000; Sigma)
  • BODIPY standards (e.g., BODIPY‐C 5‐sphingomyelin; Invitrogen)
  • 5‐ml glass tubes
  • Dialysis tubing (12,000 to 14,000 MWCO)
  • 1‐ml ultracentrifuge tubes
  • Fluorometer

Basic Protocol 2: Studying Endocytic Mechanisms with Fluorescent Lipid Analogs

  Materials
  • Human skin fibroblasts (HSFs; Coriell Institute for Medical Research)
  • EMEM containing 10% FBS (EMEM‐10; see recipe)
  • HMEM+G and −G (see recipe)
  • BODIPY‐LacCer/BSA stock solution ( protocol 3)
  • 5% (w/v) fatty‐acid‐free bovine serum albumin (dfBSA) in HMEM−G (see recipe)
  • Inhibitors of endocytosis (Table 24.1.2)
  • 25‐mm glass coverslips, sterile
  • 35‐mm tissue culture dishes
  • Fluorescence microscope with image‐acquisition capability
  • Image analysis software (e.g., Metamorph; Molecular Devices)
  • Additional reagents and equipment for culturing cells (unit 1.1)

Alternate Protocol 1: Detection of Endocytosis in Association with Dominant‐Negative Proteins

  • pDsRed‐Nuc plasmid (0.3 µg/dish; Clontech)
  • Construct for dominant‐negative inhibitor of endocytosis (e.g., AP180 DN; gift from H.T. MacMahon)
  • FuGENE 6 (Roche Applied Sciences)
  • 10 µg/ml AF 488 labeled transferrin (Tfn; Invitrogen)
  • Serum‐free medium (e.g., EMEM without serum)
  • HMEM−G (see recipe)
  • HMEM−G (see recipe), adjusted to pH 3.5 with acetic acid

Basic Protocol 3: Intracellular Localization and Transport: Co‐Localization of Bodipy‐LacCer with Albumin but not Dextran

  Materials
  • Human skin fibroblasts (HSFs; Coriell Institute for Medical Research)
  • EMEM containing 10% FBS (EMEM‐10; see recipe)
  • HMEM+G and −G (see recipe)
  • 1 µM BODIPY‐LacCer/BSA complex ( protocol 3)
  • 10 µg/ml Alexa‐fluor 594 albumin (AF594‐albumin) or 1 mg/ml Alexa‐fluor 594 dextran (AF594‐dextran)
  • 5% fatty‐acid‐free bovine serum albumin (dfBSA) in HMEM−G (see recipe)
  • 25‐mm glass coverslips, sterile
  • 35‐mm tissue culture dishes
  • 10°C cooling block (described in Marks et al., )
  • Fluorescence microscope with image acquisition capability
  • Graphics program (e.g., Adobe Photoshop) or image analysis program (e.g., Metamorph; Molecular Devices)
  • Additional reagents and equipment for culturing cells (unit 1.1)

Alternate Protocol 2: Using Dominant‐Negative rab Proteins to Identify Transport Steps

  • EMEM containing 1% and 10% FBS (EMEM‐1 and EMEM‐10; see recipe)
  • Wild‐type rab9 plasmid and dominant‐negative (DN) DsRed‐rab9 plasmid (Addgene)
  • FuGENE 6 (Roche Applied Sciences)
  • BODIPY‐LacCer/BSA (see protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
   Aoki, T., Nomura, R., and Fujimoto, T. 1999. Tyrosine phosphorylation of caveolin‐1 in the endothelium. Exp. Cell Res. 253:629‐636.
   Benmerah, A., Bayrou, M., Cerf‐Bensussan, N., and Dautry‐Varsat, A. 1999. Inhibition of clathrin‐coated pit assembly by an Eps15 mutant. J. Cell Sci. 112:1303‐1311.
   Chatterjee, S. 1998. Sphingolipids in atherosclerosis and vascular biology. Arterioscler. Thromb. Vasc. Biol. 18:1523‐1533.
   Chen, C.S., Martin, O.C., and Pagano, R.E. 1997. Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells. Biophys. J. 72:37‐50.
   Chen, C.S., Bach, G., and Pagano, R.E. 1998. Abnormal transport along the lysosomal pathway in mucolipidosis, type IV disease. Proc. Natl. Acad. Sci. U.S.A. 95:6373‐6378.
   Cheng, Z.J., Deep Singh, R., Marks, D.L., and Pagano, R.E. 2006. Membrane microdomains, caveolae, and caveolar endocytosis of sphingolipids. Mol. Membr. Biol. 23:101‐110.
   Choudhury, A., Dominguez, M., Puri, V., Sharma, D.K., Narita, K., Wheatley, C.W., Marks, D.L., and Pagano, R.E. 2002. Rab proteins mediate Golgi transport of caveola‐internalized glycosphingolipids and correct lipid trafficking in Niemann‐Pick C cells. J. Clin. Invest. 109:1541‐1550.
   Choudhury, A., Sharma, D.K., Marks, D.L., and Pagano, R.E. 2004. Elevated endosomal cholesterol levels in Niemann‐Pick cells inhibit rab4 and perturb membrane recycling. Mol. Biol. Cell 15:4500‐4511.
   Damke, H., Baba, T., Warnock, D.E., and Schmid, S.L. 1994. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J. Cell Biol. 127:915‐934.
   Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K.H., Magnusson, K.E., and Stralfors, P. 1999. Localization of the insulin receptor in caveolae of adipocyte plasma membrane. FASEB J. 13:1961‐1971.
   Hansen, G., Niels‐Christiansen, L.‐L., Thorsen, E., Immerdal, L., and Danielsen, E. 2000. Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking. J. Biol. Chem. 275:5136‐5142.
   Harder, T., Scheiffele, P., Verkade, P., and Simons, K. 1998. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. 141:929‐942.
   Henley, J.R., Krueger, E.W., Oswald, B.J., and McNiven, M.A. 1998. Dynamin‐mediated internalization of caveolae. J. Cell Biol. 141:85‐99.
   Herskovits, J.S., Burgess, C.C., Obar, R.A., and Vallee, R.B. 1993. Effects of mutant rat dynamin on endocytosis. J. Cell Biol. 122:565‐578.
   Kolesnick, R.N., Goni, F.M., and Alonso, A. 2000. Compartmentalization of ceramide signaling: Physical foundations and biological effects. J. Cell Physiol. 184:285‐300.
   Kuerschner, L., Ejsing, C.S., Ekroos, K., Shevchenko, A., Anderson, K.I., and Thiele, C. 2005. Polyene‐lipids: A new tool to image lipids. Nat. Methods 2:39‐45.
   Lamaze, C., Dujeancourt, A., Baba, T., Lo, C.G., Benmerah, A., and Dautry‐Varsat, A. 2001. Interleukin 2 receptors and detergent‐resistant membrane domains define a clathrin‐independent endocytic pathway. Mol. Cell 7:661‐671.
   Larkin, J.M., Brown, M.S., Goldstein, J.L., and Anderson, R.G.W. 1983. Depletion of intracellular potassium arrests coated pit formation and receptor‐mediated endocytosis in fibroblasts. Cell 33:273‐285.
   Liu, P. and Anderson, R.G. 1999. Spatial organization of EGF receptor transmodulation by PDGF. Biochem. Biophys. Res. Commun. 261:695‐700.
   Mallard, F., Antony, C., Tenza, D., Salamero, J., Goud, B., and Johannes, L. 1998. Direct pathway from early/recycling endosome to the Golgi apparatus revealed through the study of shiga toxin B‐fragment transport. J. Cell Biol. 143:973‐990.
   Marks, D.L., Singh, R.D., Choudhury, A., Wheatley, C.L., and Pagano, R.E. 2005. Use of fluorescent sphingolipid analogs to study lipid transport along the endocytic pathway. Methods 36:186‐195.
   Martin, O.C. and Pagano, R.E. 1994. Internalization and sorting of a fluorescent analog of glucosylceramide to the Golgi apparatus of human skin fibroblasts: Utilization of endocytic and nonendocytic transport mechanisms. J. Cell Biol. 125:769‐781.
   Martin, O.C., Comly, M.E., Blanchette‐Mackie, E.J., Pentchev, P.G., and Pagano, R.E. 1993. Cholesterol deprivation affects the fluorescence properties of a ceramide analog at the Golgi apparatus of living cells. Proc. Natl. Acad. Sci. U.S.A. 90:2661‐2665.
   McIntyre, J.C. and Sleight, R.G. 1991. Fluorescence assay for phospholipid membrane asymmetry. Biochemistry 30:11819‐11827.
   Miaczynska, M. and Zerial, M. 2002. Mosaic organization of the endocytic pathway. Exp. Cell Res. 272:8‐14.
   Mitchell, J.S., Kanca, O., and McIntyre, B.W. 2002. Lipid microdomain clustering induces a redistribution of antigen recognition and adhesion molecules on human T lymphocytes. J Immunol. 168:2737‐2744.
   Mohrmann, K. and van der Sluijs, P. 1999. Regulation of membrane transport through the endocytic pathway by rabGTPases. Mol. Membr. Biol. 16:81‐87.
   Oh, P., McIntosh, D.P., and Schnitzer, J.E. 1998. Dynamin at the neck of cavolae mediates their budding to form transport vesicles by GTP‐driven fission from the plasma membrane of endothelium. J. Cell Biol. 141:101‐104.
   Okamoto, Y., Ninomiya, H., Miwa, S., and Masaki, T. 2000. Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin‐coated pits in Chinese hamster ovary cells. J. Biol. Chem. 275:6439‐6446.
   Orlandi, P.A. and Fishman, P.H. 1998. Filipin‐dependent inhibition of cholera toxin: Evidence for toxin internalization and activation through caveolae‐like domains. J. Cell Biol. 141:905‐915.
   Pagano, R.E., Martin, O.C., Kang, H.C., and Haugland, R.P. 1991. A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J. Cell Biol. 113:1267‐1279.
   Parton, R.G. 2003. Caveolae–from ultrastructure to molecular mechanisms. Nat. Rev. Mol. Cell Biol. 4:162‐167.
   Puri, V., Watanabe, R., Singh, R.D., Dominguez, M., Brown, J.C., Wheatley, C.L., Marks, D.L., and Pagano, R.E. 2001. Clathrin‐dependent and ‐independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J. Cell Biol. 154:535‐547.
   Riederer, M.A., Soldati, T., Shapiro, A.D., Lin, J., and Pfeffer, S.R. 1994. Lysosome biogenesis requires rab9 function and receptor recycling from endosomes to the trans‐Golgi network. J. Cell Biol. 125:573‐582.
   Rodal, S.K., Skretting, G., Garred, Ø., Vilhardt, F., van Deurs, B., and Sandvig, K. 1999. Extraction of cholesterol with methyl‐β‐cyclodextrin perturbs formation of clathrin‐coated endocytic vesicles. Mol. Biol. Cell 10:961‐974.
   Rothberg, K.G., Heuser, J.E., Donzell, W.C., Ying, Y.‐S., Glenney, J.R., and Anderson, R.G.W. 1992. Caveolin, a protein component of caveolae membrane coats. Cell 68:673‐682.
   Sabharanjak, S., Sharma, P., Parton, R.G., and Mayor, S. 2002. GPI‐anchored proteins are delivered to recycling endosomes via a distinct cdc42‐regulated, clathrin‐independent pinocytic pathway. Develop. Cell 2:411‐423.
   Sandvig, K., and van Deurs, B. 1996. Endocytosis, intracellular transport, and cytotoxic action of Shiga toxin and ricin. Physiol. Rev. 76:949‐966.
   Schapiro, F., Lingwood, C., Furuya, W., and Grinstein, S. 1998. pH‐independent retrograde targeting of glycolipids to the Golgi complex. Am. J. Physiol. 274:C319‐C332.
   Seabra, M.C., Mules, E.H., and Hume, A.N. 2002. Rab GTPases, intracellular traffic and disease. Trends Molec. Med. 8:23‐30.
   Sharma, D.K., Choudhury, A., Singh, R.D., Wheatley, C.L., Marks, D.L., and Pagano, R.E. 2003. Glycosphingolipids internalized via caveolar‐related endocytosis rapidly merge with the clathrin pathway in early endosomes and form microdomains for recycling. J. Biol. Chem. 278:7564‐7572.
   Sharma, D.K., Brown, J.C., Choudhury, A., Peterson, T.E., Holicky, E., Marks, D.L., Simari, R., Parton, R.G., and Pagano, R.E. 2004. Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol. Mol. Biol. Cell 15:3114‐3122.
   Sharma, D.K., Brown, J.C., Cheng, Z., Holicky, E.L., Marks, D.L., and Pagano, R.E. 2005. The glycosphingolipid, lactosylceramide, regulates beta1‐integrin clustering and endocytosis. Cancer Res. 65:8233‐8241.
   Simons, K. and Vaz, W.L. 2004. Model systems, lipid rafts, and cell membranes. Annu. Rev. Biophys. Biomol. Struct. 33:269‐295.
   Singh, R.D., Puri, V., Valiyaveettil, J.T., Marks, D.L., Bittman, R., and Pagano, R.E. 2003. Selective caveolin‐1‐dependent endocytosis of glycosphingolipids. Mol. Biol. Cell 14:3254‐3265.
   Sleight, R.G. and Pagano, R.E. 1985. Transmembrane movement of a fluorescent phosphatidylethanolamine analogue across the plasma membranes of cultured mammalian cells. J. Biol. Chem. 260:1909‐1916.
   Subtil, A., Gaidarov, I., Kobylarz, K., Lampson, M.A., Keen, J.H., and McGraw, T.E. 1999. Acute cholesterol depletion inhibits clathrin‐coated pit budding. Proc. Natl. Acad. Sci. U.S.A. 96:6775‐6780.
   Thomas, J.L., Holowka, D., Baird, B., and Webb, W.W. 1994. Large‐scale co‐aggregation of fluorescent lipid probes with cell surface proteins. J. Cell Biol. 125:795‐802.
   Torgersen, M.L., Skretting, G., van Deurs, B., and Sandvig, K. 2001. Internalization of cholera toxin by different endocytic mechanisms. J. Cell Sci. 114:3737‐3742.
   Upla, P., Marjomaki, V., Kankaanpaa, P., Ivaska, J., Hyypia, T., vand der Goot, F.G., and Heino, J. 2004. Clustering induces a lateral redistribution of α2β1 integrin from membrane rafts to caveolae and subsequent PKC‐dependent internalization. Mol. Biol. Cell, in press.
   Wolf, A.A., Jobling, M.G., Wimer‐Mackin, S., Ferguson‐Maltzman, M., Madara, J.L., Holmes, R.K., and Lencer, W.I. 1998. Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae‐like membrane domains in polarized epithelia. J. Cell Biol. 141:917‐927.
   Woodman, P.G. 2000. Biogenesis of the sorting endosome: The role of rab5. Traffic 1:695‐701.
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