Free‐Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes

Renate Fuchs1, Isabella Ellinger1

1 University of Vienna, Vienna
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.11
DOI:  10.1002/0471143030.cb0311s14
Online Posting Date:  May, 2002
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Abstract

Endosomes constitute a functionally, morphologically, and biochemically heterogeneous population of intracellular organelles that play a major role in sorting of incoming ligands, receptors, membrane, and lumenal content. This unit provides protocols for labeling and isolation of endosomes of polarized rat hepatocytes. By application of free‐flow zone electrophoresis functional endosomal subcompartments involved in transports to lysosomes and transcytosis are resolved from each other. Methods to analyze the protein composition or acidification properties of these highly purified endosomes are also described.

     
 
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Table of Contents

  • Basic Protocol 1: Free‐Flow Electrophoresis of Hepatocyte Endosomes
  • Alternate Protocol 1: Preparative FFE
  • Support Protocol 1: Determination of Protein, Endosomal Marker, and Marker Enzymes
  • Support Protocol 2: Characterization of Proteins in Endosome Subpopulations
  • Support Protocol 3: In Vitro Acidification of Isolated Endosomes
  • Support Protocol 4: Preparation of Asialoorosomucoid (ASOR)
  • Support Protocol 5: Coupling of Fluorescein Isothiocyanate (FITC) to ASOR
  • Support Protocol 6: Preparation of 125I‐Labeled Human Polymeric Immunoglobulin A ([125I]pIgA)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Free‐Flow Electrophoresis of Hepatocyte Endosomes

  Materials
  • 150‐ to 200‐g male Sprague‐Dawley rats (e.g., Taconic Farms, The Jackson Laboratory)
  • Urethane anesthetic solution: 10 g urethane ethyl carbamate/10 ml 0.9% (w/v) NaCl (prepare immediately before use)
  • 0.25 M, 1 M, 1.15 M, and 2.5 M sucrose in TEA buffer, ice cold (see reciperecipes)
  • Endocytic tracers: 220 µg FITC‐ASOR (see protocol 6Support Protocols 4 and protocol 75) and 1.4 × 106 cpm [125I]pIgA (see protocol 8) per 500 µl phosphate‐buffered saline (PBS; appendix 2A)
  • Protease inhibitor solutions (unit 3.4)
  • 1× TEA buffer (see recipe)
  • FFE chamber buffer (see recipe)
  • FFE electrode buffer (see recipe)
  • 1 mg/ml trypsin solution (trypsin‐TPCK treated; Worthington), freshly prepared
  • 2 mg/ml trypsin inhibitor solution (trypsin inhibitor, Type I‐S, from soybean; Sigma‐Aldrich), freshly prepared
  • 2‐ml syringes and 25‐G needles
  • Plastic dissecting trays (∼15 cm width × 30 cm length) with legs (∼8 cm height)
  • Plastic basins (∼35 cm width × 35 cm height × 15 cm depth)
  • 500‐ml buffer reservoir with outlet at bottom and connecting tube to fit the 18‐G cannula (e.g., 500 ml squibb, separatory pear‐shaped funnel with stopcock; Cole Parmer No. P‐34506‐04) or 500 ml aspirator bottle)
  • Cannula (e.g., 18‐G plastic cannula)
  • Dissecting instruments including: microscissors, scissors, and curved forceps
  • 4‐0 surgical silk
  • 15‐ml and 40‐ml Dounce homogenizers (Kontes Glass) with loose‐ and tight‐fitting pestles (size A and B, respectively)
  • 50‐ml thick‐walled polycarbonate centrifuge tubes for Beckman J6‐B centrifuge
  • Low‐speed centrifuge with swinging bucket‐rotor for 50‐ml tubes (e.g., Beckman J6‐B)
  • 30‐ml thick‐walled polycarbonate centrifuge tubes for Sorvall SS‐34 rotor
  • High‐speed centrifuge (e.g., Sorvall RC‐5B) with fixed‐angle rotor (e.g., Sorvall SS‐34 rotor) for 30‐ml tubes
  • 14‐ml Ultraclear ultracentrifuge tubes
  • Ultracentrifuge (e.g., Beckman L8‐55) with swinging‐bucket rotor (e.g SW 40 or equivalent)
  • 1‐ml and 2‐ml syringes with blunt‐ended 1.8 × 18–mm needles (for underlaying gradients)
  • Zeiss Refractometer
  • 5‐ml syringe with blunt‐ended 1.8 × 10–mm needle (for collecting fractions from gradients)
  • 2‐ml syringe with 0.6 × 30–mm blunt‐ended needle (for resuspending pellets)
  • Conductivity meter
  • FFE apparatus (e.g., Elphor VaP 22, Bender and Hobein)
  • Additional reagents and equipment for determining the protein concentration according to Bradford ( appendix 3B)

Alternate Protocol 1: Preparative FFE

  Materials
  • FFE fractions enriched in endosomes or other organelles (see protocol 1 or protocol 2)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • PBS, pH 7.4 ( appendix 2A) containing 0.2% (v/v) Triton X‐100
  • 0.25 M sucrose in TEA buffer (see recipe)
  • Disposable plastic cuvettes for spectrofluorometer
  • Spectrofluorometer (e.g., Jasco FP 777)
  • 2.0 ml scintillation vials
  • γ‐counter

Support Protocol 1: Determination of Protein, Endosomal Marker, and Marker Enzymes

  Materials
  • 1 mg/ml trypsin
  • 2 mg/ml soybean trypsin inhibitor
  • FFE fractions enriched in endosomes or other organelles (Interfaces I and II; see protocol 1 or protocol 2)
  • 0.25 M sucrose in TEA buffer, ice cold (see recipe)
  • 1× SDS sample buffer (unit 6.1)
  • Ultracentrifuge with swinging‐bucket rotor (e.g., Beckman SW 40 or equivalent) and 14‐ml Ultraclear tubes
  • Additional reagents and equipment for protein determination ( appendix 3B), SDS‐PAGE (unit 6.1), and immunoblotting (unit 6.2)

Support Protocol 2: Characterization of Proteins in Endosome Subpopulations

  Materials
  • FFE fractions from microsomes (Interface I, see protocol 1, step ) containing FITC‐ASOR labeled endosomes (see protocol 1), treated with reduced quantity of trypsin (see protocol 1, steps and )
  • Acidification buffer (see recipe)
  • pH calibration buffers (see recipe)
  • ATP stock (see recipe)
  • 500 µM nigericin stock (see recipe)
  • 1 mg/ml rabbit anti‐fluorescein antibody: IgG(H+L)‐fraction (Molecular Probes)
  • Spectrofluorometer (e.g., Jasco FP 777)
  • Disposable plastic cuvettes for spectrofluorometer (e.g., Sarstedt)

Support Protocol 3: In Vitro Acidification of Isolated Endosomes

  Materials
  • Insoluble neuraminidase (type X‐A, attached to beaded agarose; Sigma‐Aldrich)
  • 0.1 M sodium acetate buffer, pH 5.5 ( appendix 2A)
  • 2% (w/v) sodium azide in H 2O, prepare before use
  • Human α1‐acid glycoprotein (Sigma‐Aldrich)
  • 0.1 M sodium borate buffer, pH 9.3
  • Sephadex G‐25 M column (prepacked; Pharmacia Biotech PD‐10; bed volume 9.1 ml)
  • Low‐speed centrifuge with swinging bucket rotor for 50‐ml tubes (e.g., Beckman JA‐20 rotor or equivalent)
  • 50‐ml conical centrifuge tubes

Support Protocol 4: Preparation of Asialoorosomucoid (ASOR)

  Materials
  • 20 mg ASOR (see protocol 6) in 1 ml of 0.1 M sodium borate buffer, pH 9.3
  • 40 mg FITC on celite (Isomer I on celite; ∼10% FITC; Sigma‐Aldrich): use 2 mg FITC on celite/mg protein for coupling
  • TBS, pH 7.38 (see recipe)
  • PBS, pH 7.38 ( appendix 2A)
  • Thermomixer for microcentrifuge tubes (e.g., Eppendorf thermomixer)
  • Dialysis tubing (Spectra/Por 2; 0.32 ml/cm; MWCO, 12,000 to 14,000 Da)
  • Sephadex G‐25 M column (prepacked; Pharmacia Biotech PD‐10; bed volume 9.1 ml)
  • Spectrofluorometer (e.g., Jasco FP 777)
  • Microcuvettes for spectrofluorometer
  • Additional reagents and equipment for dialysis ( appendix 3C)

Support Protocol 5: Coupling of Fluorescein Isothiocyanate (FITC) to ASOR

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 0.1% (w/v) bovine serum albumin (BSA)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Iodo‐Beads (iodination reagent; Pierce)
  • Na[125I] (17 mCi/ml; NEN Life Sciences)
  • Human polymeric immunoglobulin A (pIgA), purified from human myeloma serum (Vaerman and Lemaitre‐Coelho, ) and kindly provided by Jean‐Pierre Vaerman and Pierre J. Courtoy (Catholic University of Louvain, Brussels, Belgium).
  • TBS ( appendix 2A)
  • 10% (w/v) trichloroacetic acid (TCA)
  • Pre‐packed anion‐exchange cartridges (e.g., Bio‐Rad Econo‐Pac Q; bed volume 5 ml)
  • 5‐ml conical‐bottom screw‐cap glass reaction vials
  • 2.0‐ml scintillation vials
  • γ counter (e.g., Beckman Instruments)
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Figures

Videos

Literature Cited

Literature Cited
   Courtoy, P.J. 1991. Dissection of endosomes. In Intracellular Trafficking of Proteins (C. Steer and C.J. Hanover, eds.) pp. 103‐156. Cambridge University Press, Cambridge, U.K.
   Exton, J.H. 1975. The perfused rat liver. Methods Enzymol. 39:25‐36.
   Fuchs, R., Male, P., and Mellman, I. 1989. Acidification and ion permeabilities of highly purified rat liver endosomes. J. Biol. Chem. 264:2212‐2220.
   Giffroy, D., Langendries, A., Maurice, M., Daniel, F., Lardeux, B., Courtoy, P.J., and Vaerman, J.‐P. 1998. In vivo stimulation of polymeric Ig receptor transcytosis by circulating polymeric IgA in rat liver. Int. Immunol. 10:347‐354.
   Hoppe, C.A., Connolly, T.P., and Hubbard, A.L. 1985. Transcellular transport of polymeric IgA in the rat hepatocyte: Biochemical and morphological characterization of the transport pathway. J. Cell Biol. 101:2113‐2123.
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   Krivankova, L. and Bocek, P. 1998. Continuous free‐flow electrophoresis. Electrophoresis. 19:1064‐74.
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