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

  • Unit Introduction
  • 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 C8-LacCer/BSA stock solution (Support Protocol 1)
  • 1 mM BSA control stock solution (Support Protocol 1)
  • BODIPY-LacCer/BSA stock solution (Support Protocol 2)
  • 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., 2005)
  • 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-1-1¢-N-octanoyl-d-erythro-sphingosine (C8-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-C5-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 (Support Protocol 2)
  • 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

 Additional Materials (also see Basic Protocol 2)
  • 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 (Support Protocol 2)
  • 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., 2005)
  • 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

 Additional Materials (also see Basic Protocol 3)
  • 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 Support Protocol 2)
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Figures

  •  FigureFigure 24.1.1 Schematic representation of steps involved in endocytosis and intracellular trafficking of sphingolipids. Abbreviations: SL, sphingolipid; PM, plasma membrane.
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  •  FigureFigure 24.1.2 Human skin fibroblasts (HSFs) were incubated in buffer alone (control) or with 20 µM C8-LacCer 30 min at 10°C. Cells were washed and incubated with 2.5 µM BODIPY-LacCer 30 min at 10°C. Samples were then washed and observed by fluorescence microscopy at green and red BODIPY emission wavelengths. Samples were maintained at 10°C at all times to prevent endocytosis. The punctate structures seen in the right panel exhibited both green and red fluorescence indicating enrichment of BODIPY-LacCer in these regions of the plasma membrane. Areas outlined with white rectangles are further magnified in insets. Bar = 10 µM.
    View Image
  •  FigureFigure 24.1.3 Structures of fluorescent SL analogs. Replacement of the natural acyl group at R¢ with BODIPY yields BODIPY-SL. Addition of saccharide groups at R results in various BODIPY-glycosphingolipids (GSLs).
    View Image
  •  FigureFigure 24.1.4 BODIPY-LacCer endocytosis assay. Human skin fibroblasts cultured on glass coverslips in 35-mm dishes were washed and then incubated with 2.5 µM BODIPY-LacCer for 30 min at 10°C. Cells were rinsed and warmed for 3 min at 37°C and viewed under the microscope. Cells in the left panel were not back exchanged (–bx) and show some punctate structures, but PM labeling predominates. Cells in the middle panel were back exchanged (bx) with defatted BSA at 10°C to remove PM labeling. Punctate labeling of endosomes is now clearly visible. In the right panel is a cell sample treated as in the middle panel, except that the cells were pretreated with the src kinase inhibitor PP2 (see Tables 24.1.1 and 24.1.2) before and during lipid incubations. Reduced intracellular labeling is observed in the right panel because endocytosis is inhibited. The dotted line indicates the perimeter of a cell in this field traced from a phase micrograph of the same field. Cell samples were viewed under the fluorescence microscope using a 100× objective. Bar = 10 µM.
    View Image
  •  FigureFigure 24.1.5 Co-localizatiion of BODIPY-LacCer with fluorescent albumin vs. dextran in rat fibroblasts (RF). RFs were incubated with BODIPY-LacCer along with either (A) AF594-albumin (30 µg/ml) for 30 min at 10°C, and further incubated for 3 min at 37°C, or (B) cascade blue dextran (1 mg/ml) for 30 min at 10°C, and further incubated for 5 min at 37°C. Cells were then back exchanged to remove PM labeling of BODIPY-LacCer and observed under the fluorescence microscope. Images were acquired of green (BODIPY-LacCer), and red (albumin or dextran) fluorescence. Panels to the right show regions within cells from images taken at 1000×. Arrows in the upper panels indicate points of co-localizatiion of BODIPY-LacCer and albumin. Arrowheads in the lower panels indicate LacCer-positive structures which do not co-localize with dextran.
    View Image
  •  FigureFigure 24.1.6 Overexpression of dominant-negative (DN) rab9 inhibits the Golgi targeting of BODIPY-LacCer. Cells were transfected with wild type (WT) or DN DsRed-labeled rab9 constructs. After 48 hrs, the cells were incubated with 5 µM BODIPY-LacCer for 45 min at 37°C, rinsed, and then chased in medium for 60 min at 37°C. The same cells were viewed in red (DsRed rab) and green (BODIPY-LacCer) channels (left and right images, respectively). Cells were either transfected (t) with rab9 or untransfected (u). The transfected cell in the lower left panel exhibits diffuse fluorescence because the dominant negative rab9 protein is diffusely distributed, unlike the wild type form (upper left panel). Note lack of perinuclear Golgi targeting of BODIPY-LacCer in the cell transfected with DN rab9 (lower right panel). Bar = 10 µM.
    View Image

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