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Endocytosis Assays in Intact and Permeabilized Cells

Andrew Osborne1,  Alexander Flett2,  Elizabeth Smythe2

1Harvard Medical School, Boston, Massachusetts
2University of Sheffield, Sheffield, England


Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
UNIT 11.18
DOI: 
10.1002/0471143030.cb1118s27
Online Posting Date: 
July, 2005
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Abstract

Clathrin-coated pits and vesicles represent the major ports of entry into most eukaryotic cells. As well as performing housekeeping functions (e.g., allowing cells to take up essential nutrients), the endocytic pathway participates in a number of tissue-specific events such as synaptic-vesicle recycling, control of morphogen gradients during development, downregulation of receptor tyrosine kinases, and immune surveillance. To understand the role played by clathrin-mediated uptake, it is therefore essential to have robust endocytosis assays in intact cells. The clathrin-coated vesicle cycle requires a complicated interplay of proteins and lipids that is regulated in space and time. Reconstitution assays in permeabilized cells provide a powerful approach to understanding how this complex process is regulated.

Keywords: endocytosis; reconstitution; intact cells; permeabilized cells; ELISA; clathrin; vesicles

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Measurement of Receptor-Mediated Endocytosis in Intact Cells
  • Basic Protocol 2: Measurement of Receptor-Mediated Endocytosis in Permeabilized Cells
  • Stage-Specific Assays using Permeabilized Cells
  • Alternate Protocol 1: Coat Protein–Dependent Cargo Sequestration
  • Alternate Protocol 2: Further Manipulation of Endocytosis in Permeabilized Cells
  • Support Protocol 1: Preparation of Biotinylated Transferrin
  • Support Protocol 2: Preparation of Elisa Plates
  • Support Protocol 3: Methods for Permeabilization of Cells
  • Support Protocol 4: Preparation of Cytosol from Tissue
  • Support Protocol 5: Preparation of Cytosol from Tissue Culture Cells
  • Support Protocol 6: Preparation of an ATP-Regenerating System
  • Support Protocol 7: Preparation of an ATP-Depleting System
  • Support Protocol 8: Preparation of Coat Proteins
  • Support Protocol 9: Lactate Dehydrogenase Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
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Materials

Basic Protocol 1: Measurement of Receptor-Mediated Endocytosis in Intact Cells

 Materials
  • 35-mm tissue culture dishes of A431 cells (or other adherent cells; ~80% confluent) or cell suspension (~1 × 106 cells/200 µl)
  • Serum-free medium: DMEM containing 1 mg/ml BSA
  • PBS/BSA: CMF-DPBS (appendix 2A) containing 0.2% (w/v) BSA, ice cold and 31°C
  • 1 mg/ml biotinylated transferrin (B-SS-Tfn; see Support Protocol 1)
  • 50 µg/ml avidin (Sigma) in PBS/BSA, for avidin assay only
  • 1 mg/ml biocytin (Sigma), for avidin assay only
  • 10 and 100 mM sodium 2-mercaptoethanesulfonate (MesNa; Sigma) in MesNa buffer (see recipe), prepare fresh (for MesNa assay only)
  • 500 mM iodoacetamide (Sigma), prepare fresh (for MesNa assay only)
  • Blocking buffer (see recipe)
  • ELISA plates coated with anti-transferrin antibodies (see Support Protocol 2)
  • 0.1 U/ml streptavidin–horseradish peroxidase (HRP; Pierce) in blocking buffer
  • Dulbecco's PBS (appendix 2A)
  • o-Phenylenediamine tablets (Sigma)
  • H2O2 (Sigma)
  • HRP assay buffer: 51 mM phosphate/27 mM citrate, pH 5.0
  • 2 M H2SO4 (see recipe)
  • 31°C incubator or water bath with metal tray to support tissue culture dishes
  • Microscope suitable for examining cells in culture, for adherent cells only
  • ELISA plate reader with 492-nm filter

Basic Protocol 2: Measurement of Receptor-Mediated Endocytosis in Permeabilized Cells

 Materials
  • 15-cm tissue culture dishes of A431 cells (~80% confluent)
  • Serum-free medium: DMEM containing 1 mg/ml BSA
  • KSHM buffer (see recipe), ice cold
  • 4 µg/ml biotinylated transferrin (B-SS-Tfn; see Support Protocol 1) in KSHM containing 0.2% (w/v) BSA (KSHM/BSA)
  • ATP-regenerating system (see Support Protocol 6)
  • ATP-depleting system (see Support Protocol 7)
  • Cytosol (see Support Protocols 4 and 5)
  • 50 µg/ml avidin (Sigma) in KSHM/BSA, for avidin assay only
  • 1 mg/ml biocytin (Sigma), for avidin assay only
  • 10 mM and 50 mM sodium 2-mercaptoethanesulfonate (MesNa; Sigma) in MesNa buffer (see recipe), prepare fresh (for MesNa assay only)
  • 500 mM iodoacetamide (IAA; Sigma), prepare fresh (for MesNa assay only)
  • Blocking buffer (see recipe)
  • ELISA plates coated with anti-transferrin antibodies (see Support Protocol 2)
  • 0.1 U/ml streptavidin–horseradish peroxidase (HRP; Pierce) in blocking buffer
  • Dulbecco's PBS (appendix 2A)
  • o-Phenylenediamine tablets (Sigma)
  • H2O2 (Sigma)
  • HRP assay buffer: 51 mM phosphate/27 mM citrate, pH 5.0
  • 2 M H2SO4 (see recipe)
  • Microcentrifuge, 4°C
  • ELISA plate reader with 492-nm filter
  • Additional reagents and equipment for permeabilizing cells (see Support Protocol 3)

Alternate Protocol 2: Further Manipulation of Endocytosis in Permeabilized Cells

 Additional Materials (also see Basic Protocol 2 and see Alternate Protocol 1)
  • Purified GST fusion protein containing an SH3 domain of interest

Support Protocol 1: Preparation of Biotinylated Transferrin

 Materials
  • Transferrin (Sigma)
  • Dulbecco's PBS (appendix 2A)
  • NHS-SS-biotin: sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate (EZ-Link Sulfo-NHS-SS-Biotin; Pierce)
  • Dimethyl sulfoxide (DMSO; Sigma)
  • 500 mM Tris×Cl, pH 7.5 (appendix 2A)
  • PBS/BSA: PBS containing 1 mg/ml BSA
  • Liquid nitrogen
  • Dialysis tubing (10,000 MWCO) or buffer exchangers (e.g., Vivaspins; Vivascience)
  • Quartz cuvettes
  • Spectrophotometer with UV source (280 nm)

Support Protocol 2: Preparation of Elisa Plates

 Materials
  • Anti-transferrin antibodies (available as sheep serum from Alba Bioscience)
  • 100 mM sodium bicarbonate, pH 8.3
  • Dulbecco's PBS (appendix 2A)
  • Blocking buffer (see recipe)
  • Flat-bottomed ELISA microtiter plates

Support Protocol 4: Preparation of Cytosol from Tissue

 Materials
  • KSHM buffer (see recipe), ice cold
  • Tissue (brain or liver), freshly harvested or stored at –80°C
  • Liquid nitrogen
  • Motor-driven 50-ml Dounce homogenizer with Teflon restle
  • Medium-speed centrifuge and rotor (e.g., Beckman J2-21 with JA 25.5 rotor), 4°C, and centrifuge tubes
  • Ultracentrifuge (e.g., Beckman LE-80K with 45Ti rotor), 4°C, and ultracentrifuge tubes

Support Protocol 5: Preparation of Cytosol from Tissue Culture Cells

 Additional Materials (also see Support Protocol 4)
  • Cells grown in suspension
  • KSHM buffer (see recipe) containing protease inhibitors (e.g., Complete EDTA-Free Protease Inhibitor Cocktail; Roche), ice cold
  • 10-ml Dounce homogenizer with Teflon pestle, optional

Support Protocol 6: Preparation of an ATP-Regenerating System

 Materials
  • ATP (Sigma)
  • Phosphocreatine (Sigma)
  • Creatine phosphokinase (Sigma)
  • 50% (v/v) glycerol in KSHM buffer (see recipe)

Support Protocol 7: Preparation of an ATP-Depleting System

 Materials
  • Ammonium sulfate suspension of hexokinase (Sigma)
  • 500 mM glucose
  • KSHM buffer (see recipe)
  • Microcentrifuge, 4°C

Support Protocol 8: Preparation of Coat Proteins

 Materials
  • Bovine or porcine brains
  • Buffer A (see recipe)
  • 12.5% (w/v) Ficoll/12.5% (w/v) sucrose in buffer A
  • Buffer B: 1 vol buffer A and 3 vol of 1 M Tris·Cl, pH 8 (appendix 2A)
  • Ammonium sulfate
  • 1 M DTT (appendix 2A)
  • 100 mM PMSF in 100% ethanol or isopropanol, store at –20°C
  • Staining solution: 40% (v/v) methanol/7% (v/v) acetic acid/25 g/liter Coomassie brilliant blue R250
  • Destaining solution: 15% (v/v) methanol/7% (v/v) acetic acid in water
  • 10 mM Tris·Cl, pH 7.5 (appendix 2A), for AP mix purification only
  • KSHM buffer (see recipe), for clathrin purification only
  • Liquid nitrogen
  • Kitchen blender
  • Medium-speed centrifuge and rotor (e.g., Beckman J2-21 with JLA 10.5 and JA 25.5 rotors), 4°C, and 500- and 50-ml centrifuge tubes
  • Ultracentrifuge (e.g., Beckman LE-80K with 45Ti rotor), 4°C, and 65-ml ultracentrifuge tubes
  • 50-ml Dounce homogenizer with Teflon pestle
  • 2-liter graduated cylinder
  • Sephacryl S-400 column (26 mm × 1 m)
  • Fraction collector and tubes
  • 50-ml Vivaspin concentrators (Vivascience)
  • Dialysis membrane (10,000 MWCO)
  • Cryovials
  • Additional reagents and equipment for SDS–polyacrylamide gel electrophoresis (unit 6.1)

NOTE: All procedures are carried out at 4°C and all solutions should be ice cold.

Support Protocol 9: Lactate Dehydrogenase Assay

 Materials
  • Cell suspension samples, unpermeabilized and permeabilized with method of choice (see Support Protocol 3)
  • KSHM buffer (see recipe)
  • 0.5% (v/v) Triton X-100 in PBS (appendix 2A)
  • LDH reaction buffer (see recipe)
  • Microcentrifuge, 4°C
  • Spectrophotometer with UV detector (340 nm) and quartz cuvettes
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Figures

  • Figure 11.18.1
    Principles of the avidin and sodium 2-mercaptoethanesulfonate (MesNa) assays. Permeabilized cells are incubated with transferrin (Tfn) that has been biotinylated via a cleavable disulfide linkage (B-SS-Tfn) under conditions where endocytosis can occur (1). Some of the B-SS-Tfn is sequestered into deeply invaginated coated pits or internalized into coated vesicles (2). The permeabilized cell mixes are then chilled and treated with avidin (3). Cell surface B-SS-Tfn is accessible to avidin, whereas sequestered B-SS-Tfn (either in deeply invaginated coated pits or internalized in coated vesicles) is inaccessible. Excess binding sites on avidin are quenched by the addition of biocytin (4). The cells are then lysed and applied to ELISA plates coated with anti-transferrin antibodies (anti-Tfn Ab). Sequestered and internalized B-SS-Tfn may be detected by binding of streptavidin–horseradish peroxidase (HRP; 5). Alternatively, following the endocytosis reaction, cell surface B-SS-Tfn is reduced using the small membrane-impermeant reducing agent MesNa. MesNa can access B-SS-Tfn sequestered in deeply invaginated coated pits, whereas B-SS-Tfn internalized in coated vesicles is resistant to MesNa reduction (6). Iodoacetamide (IAA) is used to quench excess MesNa (7). The cells are solubilized, and internalized B-SS-Tfn is detected using streptavidin-HRP (8).

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Literature Cited

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