Analysis of Membrane Traffic in Polarized Epithelial Cells

Joshua H. Lipschutz1, Lucy Erin O'Brien1, Yoram Altschuler1, Dana Avrahami1, Yen Nguyen1, Kitty Tang1, Keith E. Mostov1

1 University of California, San Francisco, San Francisco, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.5
DOI:  10.1002/0471143030.cb1505s12
Online Posting Date:  November, 2001
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Abstract

Spatial asymmetry is fundamental to the structure and function of most eukaryotic cells. A basic aspect of this polarity is that the cell's plasma membrane is divided into discrete domains. The best studied and simplest example of this occurs in epithelial cells, which line exposed body surfaces. Epithelial cells use two pathways to send proteins to the cell surface. Newly made proteins can travel directly from the transā€Golgi network (TGN) to either the apical or basolateral surface. Alternatively, proteins can be sent to the basolateral surface and then endocytosed and transcytosed to the apical surface. Epithelial cells grown on porous filters adopt a typical polarized morphology; transfected epithelial cells can be used to biosynthetically characterize the trafficking patterns of a given protein. These cells can also be used to study delivery to a particular surface and to localize the protein by immunofluorescence.

     
 
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Table of Contents

  • Basic Protocol 1: Transfection of Polarized Epithelial Cells in Suspension and Selection of Resistant Clones
  • Support Protocol 1: Picking Stably Transfected Clones
  • Support Protocol 2: Culture of Epithelial Cells on Filters
  • Support Protocol 3: Determining the Leakiness of a Monolayer of Cells Grown on a Filter
  • Basic Protocol 2: Pulse‐Chase Experiments in Polarized Epithelial Cells
  • Basic Protocol 3: Biotinylation of Newly Synthesized Epithelial Cell Surface Proteins
  • Basic Protocol 4: Indirect Immunofluorescence of Proteins in Polarized Epithelial Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transfection of Polarized Epithelial Cells in Suspension and Selection of Resistant Clones

  Materials
  • Confluent cells grown on 10‐cm tissue culture dish
  • HEPES buffered saline (HeBS), pH 7.05 (see recipe; also see appendix 2A)
  • 20 µg plasmid DNA in 1 to 20 µl H 2O
  • 2 M CaCl 2: filter sterilize and store up to 6 months at 4°C
  • CMF‐DPBS ( appendix 2A)
  • Trypsin/EDTA solution—e.g., 0.25% (w/v) trypsin/0.2% (w/v) EDTA (unit 1.1)
  • MEM medium ( appendix 2B) with and without 5% (v/v) FBS ( appendix 2A)
  • 20 mM chloroquine in water: filter sterilize and store up to 6 months at 4°C
  • 15% (w/v) glycerol in HeBS: filter sterilize and store up to 6 months at 4°C
  • Selection medium: MEM/5% FBS containing eukaryotic antibiotic (e.g., G418; see recipe)
  • Nontransfected cells
  • 10‐cm dishes
  • Additional reagents and equipment for picking clones (see protocol 2)

Support Protocol 1: Picking Stably Transfected Clones

  • ∼16‐day‐old 10‐cm dishes containing diluted transfected and nontransfected cells in selection medium (see protocol 1)
  • Calcium‐ and magnesium‐free DPBS (CMF‐DPBS; appendix 2A)
  • Medium‐sized glass cloning ring, sterile (Bellco Glass)
  • 0.5% (w/v) SDS lysis buffer
  • 12% (w/v) slurry of CL‐2B beads
  • 12‐well tissue culture plates

Support Protocol 2: Culture of Epithelial Cells on Filters

  • 10‐cm dish of confluent epithelial cells (Table 15.5.1)
  • 12‐mm Transwell filters and appropriate dishes
  • IEC clinical centrifuge with 12 × 15 rotor
  • Additional reagents and equipment for determining the tightness of epithelial monolayers (see protocol 4)

Support Protocol 3: Determining the Leakiness of a Monolayer of Cells Grown on a Filter

  • Pasteur pipet connected to a vacuum system

Basic Protocol 2: Pulse‐Chase Experiments in Polarized Epithelial Cells

  Materials
  • 4‐ to 7‐day‐old epithelial cell cultures growing on 12‐mm Transwell filters (see protocol 3)
  • Dulbecco's phosphate‐buffered saline (DPBS; appendix 2A)
  • Starvation medium: MEM medium ( appendix 2B) lacking the radioactive tracer amino acid
  • Radioactive amino acid (e.g., 1175 Ci/mmol [35S]methionine)
  • MEM medium
  • 0.5% (w/v) SDS lysis buffer (see recipe)
  • Parafilm
  • Humid box: plastic box with 2 charcoal bags and a piece of wet Whatman filter paper
  • 12‐well Transwell tissue culture plate (Table 15.5.2)
  • Phosphorimager
  • Scalpel
  • Additional reagents and equipment for gel electrophoresis (Chapter 6)

Basic Protocol 3: Biotinylation of Newly Synthesized Epithelial Cell Surface Proteins

  Materials
  • Radioactively labeled amino acid
  • 4‐day‐old epithelial cultures growing on 12‐mm Transwell filters (see protocol 3)
  • Hanks' balanced salt solution (HBSS; appendix 2A), 4°C
  • 20 mg/ml sulfo‐NHS‐biotin (Pierce) in anhydrous DMSO: prepare just before use
  • 10 mM Tris buffered saline, pH 7.4 (TBS; appendix 2A)
  • 0.5% (v/v) SDS lysis buffer (see recipe)
  • 2.5% (v/v) Triton dilution buffer (see recipe)
  • Protein A–Sepharose beads without and with rabbit antimouse and the monoclonal antibody of choice, or rabbit polyclonal antibodies
  • Streptavidin beads (e.g., Pierce)
  • Mixed micelle wash buffer (see recipe)
  • Final wash buffer (see recipe)
  • 5% (w/v) SDS
  • 2× loading buffer (see recipe)
  • Platform rocker
  • Orbital shaker (e.g., Bellco)
  • Phosphorimager
  • Additional equipment and reagents for radioactively labeling epithelial cultures (see protocol 5, steps to ) and denaturing (SDS) gel electrophoresis (unit 6.1)

Basic Protocol 4: Indirect Immunofluorescence of Proteins in Polarized Epithelial Cells

  Materials
  • 5‐ to 7‐day‐old cells growing on 12‐mm Transwell filters (see protocol 3)
  • DPBS ( appendix 2A), ice cold
  • 16% or 40% (v/v) paraformaldehyde
  • Quenching solution, fresh (see recipe)
  • Permeabilizing solution, fresh (see recipe)
  • Primary antibody
  • Secondary antibody coupled to fluorophore
  • DPBS/0.1% (v/v) Triton: add 250 µl of 20% (v/v) Triton X‐100 to 49.75 ml DPBS ( appendix 2A), store up to 6 months at 4°C
  • Antifade mounting solution
  • Metal board
  • Orbital shaker (e.g., Bellco)
  • Humid box: plastic box with a piece of damp Whatman filter paper
  • Aluminum foil
  • Scalpel
  • Inverted fluorescent microscope with appropriate glass slide and cover slip
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Figures

Videos

Literature Cited

Literature Cited
   Aroeti, B., Kosen, P.A., Kuntz, I.D., Cohen, F.E., and Mostov, K.E. 1993. Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor. J. Cell Biol. 123:1149‐1160.
   Barth, A.I.M., Pollack, A.L., Altschuler, Y., Mostov, K.E., and Nelson, W.J. 1997. NH2‐terminal deletion of β‐catenin results in stable colocalization of mutant β‐catenin with adenomatous polyposis coli protein and altered MDCK cell adhesion. J. Cell Biol. 136:693‐706.
   Breitfeld, P., Casanova, J.E., Hsarris, J.M., Simister, N.E., and Mostov, K.E. 1989. Expression and analysis of the polymeric immunoglobulin receptor. Methods Cell Biol. 32:329‐337.
   Brewer, C.B. and Roth, M.G. 1991. A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin. J. Cell Biol. 114:413‐421.
   Drubin, D.G. and Nelson, W.J. 1996. Origins of cell polarity. Cell 84:335‐344.
   LeBivic, A., Real, F.X., and Rodriguez‐Boulan, E. 1989. Vector targeting of apical and basolateral proteins in human adenocarcinoma cell line. Proc. Natl. Acad. Sci. U.S.A. 86:9313‐9317.
   Lipshutz, J.H., Guo, W., O'Brien, L.E., Nguyen, Y.H., Novick, P., and Mostov, K.E. 2000. The exocyst is involved in epithelial cytogenesis and tubulogenesis and acts by modulating syntheses and delivery of basolateral plasma membrane and secretory proteins. Mol. Cell Biol. 11:4259‐4275.
   Louvard, D. 1980. Apical membrane aminopeptidase appears at site of cell‐cell contact in cultured kidney epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 77:4132‐4136.
   Luton, F. and Mostov, K.E. 1999. Transduction of basolateral‐to‐apical signals across epithelial cells: Ligand stimulated transcytosis of the polymeric immunoglobulin receptor requires two signals. Mol. Biol. Cell 10:1409‐1427.
   Matter, K. and Mellman, I. 1994. Mechanisms of cell polarity: Sorting and transport in epithelial cells. Curr. Opin. Cell Biol. 6:545‐554.
   Mostov, K.E., ter Beest, M.B.A., and Chapin, S.J. 1999. Catch the µ1B train to the basolateral surface. Cell 99:121‐122.
   Mostov, K.E., Verges, M., and Altschuler, Y. 2000. Membrane traffic in polarized epithelial cells. Curr. Opin. Cell Biol. 12:483‐490.
   Weimbs, T., Low, S.‐H., Chapin, S.J., and Mostov, K.E. 1997. Apical targeting in polarized epithelial cells: There's more afloat than rafts. Trends Cell Biol. 7:393‐399.
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