Cadherin‐Dependent Cell‐Cell Adhesion

Masatoshi Takeichi1, Shinichi Nakagawa1

1 Kyoto University, Kyoto
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 9.3
DOI:  10.1002/0471143030.cb0903s00
Online Posting Date:  May, 2001
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Abstract

Differential treatment of cells with trypsin can be used to distinguish Ca2+‐dependent adhesion (CDS) from Ca2+‐independent adhesion (CIDS). Cadherins appear to be a unique family of molecules whose structure and function as adhesion molecules are protected from trypsin in the presence of Ca2+. This unit provides protocols for preparation and analysis of cells for cadherin‐dependent adhesion in short‐term and long‐term aggregation assays. The functions of different cadherins can be assessed in mixed aggregate assays. Fluorescence antibody‐based assays are used to identify specific cadherins and their associated catenins, and transformation of cells with specific constructs can be used to assay adhesion in cells with loss of cadherin activity.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Short‐Term Aggregation Culture
  • Alternate Protocol 1: Long‐Term Aggregation Culture
  • Basic Protocol 2: Mixed‐Cell Aggregation Culture
  • Support Protocol 1: Dissociation of Fibroblasts by TC Treatment
  • Support Protocol 2: Dissociation of Embryonic Cells by TC Treatment
  • Support Protocol 3: Dissociation of Cells by LTE or TE Treatment
  • Basic Protocol 3: Detection of Cadherins and Catenins
  • Basic Protocol 4: Inhibition of Cadherin Function
  • Basic Protocol 5: Restoration of Cadherin Activity in Cadherin‐ or Catenin‐Deficient Cell Lines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Short‐Term Aggregation Culture

  Materials
  • 1% (w/v) BSA (see recipe)
  • HCMF (see recipe)
  • Cells of interest (see s protocol 4 to protocol 6), suspended in ice‐cold recipeHCMF at 0.5–1 × 105 cells/ml for cell lines, or at 2–5 × 106 cells/ml for early embryonic neurons
  • 100 mM CaCl 2 ( appendix 2A)
  • 8% (w/v) paraformaldehyde in recipeHCMF
  • 24‐well tissue culture plate (Falcon)
  • Gyrating shaker (New Brunswick Scientific, Model G2)
  • BSA‐coated Pasteur pipets (see recipe)
  • Additional reagents and equipment for counting cell aggregates with a hemacytometer (unit 1.1) or Coulter counter

Alternate Protocol 1: Long‐Term Aggregation Culture

  • 1% (w/v) agar (e.g., Noble agar, Difco) in standard culture medium
  • Cells of interest (see protocol 6), suspended at 0.5–1 × 105 cells/ml for cell lines, or at 2–5 × 106 cells/ml for early embryonic neurons, in standard cell culture medium (e.g., supplemented Dulbecco's modified Eagle medium, DMEM; appendix 2A)

Basic Protocol 2: Mixed‐Cell Aggregation Culture

  Materials
  • Cell cultures
  • Standard cell culture medium (e.g., supplemented Dulbecco's modified Eagle medium, DMEM; appendix 2A) with 10% FBS ( appendix 2A)
  • Fluorescent dye(s): e.g., 3 mg/ml 3,3′‐dioctadecyloxacarbocyanine perchlorate (DiO) in dimethylsulfoxide
  • HEPES buffer (see recipe)
  • 8% (w/v) paraformaldehyde in HCMF (see recipe for HCMF)
  • Glycerol‐based mounting medium containing antifading reagents (e.g., 1 mg/ml p‐phenylenediamine in 90% glycerol)
  • BSA‐coated microcentrifuge tubes (see recipe)
  • Additional reagents and equipment for dispersing cultures (see protocol 4 to protocol 6), for counting cells (unit 1.1), and for short‐term or long‐term aggregation culture (see protocol 1 or protocol 2)

Support Protocol 1: Dissociation of Fibroblasts by TC Treatment

  Materials
  • Semiconfluent monolayer of the cells of interest
  • HMF (see recipe)
  • TC solution (see recipe)
  • 0.5% (w/v) soybean trypsin inhibitor solution (see recipe)
  • HCMF (see recipe), ice cold
  • BSA‐coated 10‐ml test tubes and Pasteur pipets (see recipe)
  • Additional reagents and equipment for disaggregation by TE treatment (see protocol 6) and for counting (unit 1.1)

Support Protocol 2: Dissociation of Embryonic Cells by TC Treatment

  • Embryo(s)
  • HEPES buffer (see recipe), ice cold
  • DNase I
  • 1 M MgCl 2 ( appendix 2A)
  • BSA‐coated 5‐cm petri dish (see recipe)

Support Protocol 3: Dissociation of Cells by LTE or TE Treatment

  • TE solution (see recipe)
  • LTE solution (see recipe)
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Figures

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Literature Cited

Literature Cited
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