Analysis of Polarized Membrane Traffic in Hepatocytes and Hepatic Cell Lines

Julie G. In1, Gudrun Ihrke2, Pamela L. Tuma1

1 Department of Biology, The Catholic University of America, Washington, DC, 2 Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.17
DOI:  10.1002/0471143030.cb1517s54
Online Posting Date:  March, 2012
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Abstract

The protocols described in this unit were developed to monitor membrane traffic in cultured cell monolayers that display hepatic polarity. In general, the assays are designed to visualize and/or quantitate membrane trafficking by monitoring the fates of antibodies bound to specific membrane proteins. We first describe how to infect cells with recombinant adenovirus, the preferred method for introducing exogenous genes into hepatic cells. We next provide a morphological assay to monitor basolateral to apical transcytosis. In a supporting protocol, we describe how to visualize apical recycling and/or retention. In an additional supporting protocol, we provide a semi‐quantitative method to measure the relative extents of apical delivery. Finally, we describe quantitative assays to measure basolateral internalization and recycling. The methods presented in this unit provide a relatively simple, yet powerful approach to examining hepatic membrane traffic. Curr. Protoc. Cell Biol. 54:15.17.1‐15.17.19. © 2012 by John Wiley & Sons, Inc.

Keywords: hepatocytes; apical; basolateral; transcytosis; membrane; traffic

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Recombinant Adenovirus Infection
  • Basic Protocol 2: Basolateral to Apical Transcytosis Assay
  • Support Protocol 1: Semi‐Quantitative Method to Measure Extent of Apical Delivery
  • Support Protocol 2: Morphology‐Based Recycling/Apical Retention Assay
  • Basic Protocol 3: Quantitative Basolateral Internalization and Recycling Assays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Recombinant Adenovirus Infection

  Materials
  • Purified adenovirus or lentivirus (see protocol Introduction)
  • Ice
  • Serum‐free medium (e.g., Coon's or Khaighn's modified F12 medium; Sigma‐Aldrich)
  • Cells grown on 22 × 22–mm glass coverslips in 35‐mm dishes or 6‐well dishes
  • Complete medium [e.g., Coon's or Khaighn's modified F12 medium (Sigma) supplemented with fetal bovine serum (Gemini Bio‐Products)]
  • Ice bucket
  • Analog vortex mixer
  • 1.5‐ml microcentrifuge tubes
  • Vacuum/aspirator

Basic Protocol 2: Basolateral to Apical Transcytosis Assay

  Materials
  • Cells grown on 22 × 22–mm glass coverslips in 35‐mm dishes or 6‐well dishes
  • Complete medium [e.g., Coon's or Khaighn's modified F12 medium (Sigma) supplemented with fetal bovine serum (Gemini Bio‐Products)]
  • Ice
  • Primary antibodies specific to antigen(s) of choice (e.g., anti‐CD59, AbCam; anti‐dipeptidyl peptidase, Life Span Biosciences; or anti‐scavenger receptor B1, Novus Biologicals)
  • 1× PBS (see recipe)
  • 4% paraformaldehyde/PBS (see recipe)
  • Methanol (production grade)
  • 1% bovine serum albumin/PBS (see recipe)
  • Fluorophore‐conjugated secondary antibodies (e.g., Alexa 488 or 568‐conjugated antibodies; Invitrogen)
  • 0.2% bovine serum albumin/PBS (see recipe)
  • Mounting medium (e.g., Fluoromount from Electron Microscopy Sciences or ProLongGold from Invitrogen)
  • 1.5‐ml microcentrifuge tubes
  • 6‐well dishes
  • Ice bucket
  • Vacuum/aspirator
  • Stainless steel forceps, super‐fine tip
  • Humidified chamber
  • Glass slides
  • Epifluorescence microscope equipped with 60× or 100× objectives
  • Digital camera (e.g., Coolsnap HQ2 digital camera from Photometrics) and image capture software (e.g., IPLabs from Biovision)

Support Protocol 1: Semi‐Quantitative Method to Measure Extent of Apical Delivery

  • Slides prepared using protocol 2
  • Image analysis software (e.g., ImageJ, National Institutes of Health)
  • Additional reagents and equipment for principles of imaging an analysis (Asai, )

Support Protocol 2: Morphology‐Based Recycling/Apical Retention Assay

  • Cells on coverslips (see step 8 from protocol 2)
  • Isoglycine, pH 2.5 (see recipe)

Basic Protocol 3: Quantitative Basolateral Internalization and Recycling Assays

  Materials
  • 0.5 M Tris base
  • Cells grown on 22 × 22–mm glass coverslips in 35‐mm or 6‐well dishes
  • Primary antibodies specific to antigen(s) of choice
  • Complete medium (with serum)
  • Ice
  • 1× PBS (see recipe), ice‐cold
  • Isoglycine, pH 2.5 (see recipe)
  • Low pH lysis buffer, pH 2.5 (see recipe)
  • High pH lysis buffer, pH 7.75 (see recipe)
  • 0.4% Trypan blue stain (Invitrogen)
  • Wash buffer (see recipe)
  • Biotinylated affinity‐purified antibodies or total IgG purified from serum or ascites
  • Neutralized isoglycine (see recipe)
  • HRP‐conjugated secondary antibodies (e.g., anti‐rabbit or mouse IgG peroxidase conjugates from Sigma‐Aldrich)
  • HRP Substrate Detection kit (Bio‐Rad Laboratories)
  • Stainless steel forceps, super‐fine tip
  • Kimwipes
  • 37°C incubator
  • 1.5‐ml microcentrifuge tubes
  • Cell scraper
  • 35‐mm dishes
  • Light microscope equipped with a low‐powered objective
  • Reacti‐Bind Neutravidin‐Coated Polystyrene Plates (Thermo Fisher Scientific)
  • Humidified chamber (see protocol 2)
  • Microplate reader
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Figures

Videos

Literature Cited

Literature Cited
   Asai, D.J. 2008. Immunofluorescence microscopy. Curr. Protoc. Essential Lab. Tech.. 9.2.1‐2.21.
   Bartles, J.R. and Hubbard, A.L. 1988. Plasma membrane protein sorting in epithelial cells: Do secretory pathways hold the key? Trends Biochem. Sci. 13:181‐184.
   Bartles, J.R., Feracci, H.M., Stieger, B., and Hubbard, A.L. 1987. Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. J. Cell Biol. 105:1241‐1251.
   Bastaki, M., Braiterman, L.T., Johns, D.C., Chen, Y.H., and Hubbard, A.L. 2002. Absence of direct delivery for single transmembrane apical proteins or their “Secretory” forms in polarized hepatic cells. Mol. Biol. Cell 13:225‐237.
   Boyer, J.L. 1997. Isolated hepatocyte couplets and bile duct units‐novel preparations for the in vitro study of bile secretory function. Cell Biol. Toxicol. 13:289‐300.
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   Griffo, G., Hamon‐Benais, C., Angrand, P.O., Fox, M., West, L., Lecoq, O., Povey, S., Cassio, D., and Weiss, M. 1993. HNF4 and HNF1 as well as a panel of hepatic functions are extinguished and reexpressed in parallel in chromosomally reduced rat hepatoma‐human fibroblast hybrids. J. Cell Biol. 121:887‐898.
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   Schell, M.J., Maurice, M., Stieger, B., and Hubbard, A.L. 1992. 5′nucleotidase is sorted to the apical domain of hepatocytes via an indirect route. J. Cell Biol. 119:1173‐1182.
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   Tuma, P., Nyasae, L., and Hubbard, A. 2002. Nonpolarized cells selectively sort apical proteins from cell surface to a novel compartment, but lack apical retention mechanisms. Mol. Biol. Cell 13:3400‐3415.
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