Analyzing FAK and Pyk2 in Early Integrin Signaling Events

Joie A. Bernard‐Trifilo1, Ssang‐Taek Lim1, Shihe Hou1, David D. Schlaepfer1, Dusko Ilic2

1 The Scripps Research Institute, La Jolla, 2 Stem Life Line, Inc., San Carlos
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.7
DOI:  10.1002/0471143030.cb1407s30
Online Posting Date:  April, 2006
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Abstract

Integrins are a family of heterodimeric α/β transmembrane cell adhesion receptors that play important roles in the regulation of cell migration, proliferation, and survival. Integrins do not possess intrinsic catalytic activity, and signaling events are mediated by their lateral association with other cell surface receptors or clustering of their cytoplasmic domains with signaling proteins. Rapid activation of protein‐tyrosine kinases is one of the first signaling events associated with integrin binding to the extracellular matrix protein fibronectin. The intracellular focal adhesion kinase (FAK) is recruited to sites of integrin clustering, and this unit describes the methods with which to analyze FAK phosphorylation, activity, and localization within fibroblasts. Additional methods on how to grow primary FAK+/+ and FAK−/− fibroblasts and measure integrin‐stimulated cell motility are described as well as methods for evaluating the activity of the FAK‐related kinase, Pyk2, which is expressed in FAK−/− cells.

Keywords: FAK; Pyk2; Src; tyrosine phosphorylation; focal adhesions; cell motility

     
 
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Table of Contents

  • Basic Protocol 1: Replating Assays for Signaling Studies
  • Support Protocol 1: Growth of FAK+/+ and FAK−/− Fibroblasts
  • Support Protocol 2: Serum Starvation of Cells
  • Support Protocol 3: Preparation of Cell Lysates
  • Support Protocol 4: Immunoprecipitation of FAK, Pyk2, and c‐Src
  • Basic Protocol 2: In Vitro Kinase Assay
  • Alternate Protocol 1: FAK‐PYK2 Poly Glu:Tyr Phosphorylation
  • Alternate Protocol 2: Measurements of Src‐Associated Kinase Activity
  • Basic Protocol 3: Immunoblotting with FAK/PYK2 Phospho‐Specific Antibodies
  • Basic Protocol 4: Haptotaxis Motility Assay: Matrix‐Stimulated Migration
  • Alternate Protocol 3: Analyzing Cell Motility Using Plasmid‐Transfected Cells
  • Basic Protocol 5: Scratch‐Wound Healing Assay With Time‐Lapse Imaging
  • Basic Protocol 6: Immunolocalization of Focal Adhesion Proteins
  • Alternate Protocol 4: Immunolocalization of Actin Stress Fibers
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Replating Assays for Signaling Studies

  Materials
  • Fibronectin (from bovine plasma; Sigma‐Aldrich)
  • Poly‐L‐lysine (mol. wt. 70,000 to 150,000 or 150,000 to 300,000; Sigma‐Aldrich)
  • Replating and migration medium (see recipe)
  • FAK+/+ and FAK−/− MEFs, serum‐starved ( protocol 3)
  • Phosphate buffered saline (PBS; appendix 2A)
  • Trypsin/EDTA: 0.25% (w/v) trypsin/1 mM EDTA (Invitrogen)
  • Trypsin inhibitor solution (see recipe)
  • 10‐cm plastic tissue culture plates (Falcon)
  • 4°C incubator
  • 15‐ml centrifuge tubes (Corning)
  • Tabletop centrifuge
  • 10‐ml transfer pipets, sterile
  • 50‐ml conical centrifuge tubes (Corning)
  • Light microscope

Support Protocol 1: Growth of FAK+/+ and FAK−/− Fibroblasts

  Materials
  • Gelatin
  • Phosphate buffered saline (PBS; appendix 2A)
  • FAK+/+ mouse embryo fibroblasts (ATCC CRL‐2645) FAK−/− mouse embryo fibroblasts (ATCC CRL‐2644): grown on plates as per ATCC product sheet
  • Trypsin/EDTA: 0.25% (w/v) trypsin/1 mM EDTA (Invitrogen), 37°C
  • Cell growth medium (see recipe), warmed to 37°C
  • 42°C water bath
  • 0.22‐µm GP Express Plus filter membrane (Millipore)
  • 10‐cm plastic tissue culture plates (Falcon)
  • 10‐ml transfer pipets, sterile
  • Light microscope
  • 15‐ml conical centrifuge tubes (Corning)

Support Protocol 2: Serum Starvation of Cells

  • Starvation medium: prepared by making cell growth medium (see recipe) with FBS reduced to 0.5%.

Support Protocol 3: Preparation of Cell Lysates

  Materials
  • Experimentally treated, control adherent, and control suspended cells ( protocol 1)
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • RIPA cell lysis buffer (see recipe), 4°C
  • 50% (w/v) Sephadex G‐100 (Sigma G100‐120) slurry in PBS ( appendix 2A)
  • Cell scraper (Corning)
  • Refrigerated centrifuge
  • 1.5‐ml microcentrifuge tubes
  • 1‐ml disposable syringes
  • 21‐G Luer‐Lok needles
  • Tube rotation device (e.g., Labquake, Barnstadt‐Thermolyne)
  • Microcentrifuge
  • −80°C freezer (optional)

Support Protocol 4: Immunoprecipitation of FAK, Pyk2, and c‐Src

  Materials
  • Primary antibody (see Table 14.7.1 and Table 14.7.2)
  • Lysate of treated or control cells ( protocol 4)
  • 50% (w/v) protein A‐agarose beads (fast flow immobilized protein A; Repligen, http://www.repligen.com) in PBS ( appendix 2A)
  • 50% (w/v) protein G‐Plus‐agarose beads (Calbiochem) in PBS ( appendix 2A)
  • Triton lysis buffer (see recipe), 4°C
  • HNTG buffer (see recipe), 4°C
  • 1.5‐ml microcentrifuge tubes
  • Tube rotation device (e.g., Labquake, Barnstadt‐Thermolyne)
  • Centrifuge
  • Transfer pipets
    Table 4.7.1   Materials   Commercially Available Antibodies to FAK, Pyk2, and c‐Src a   Commercially Available Antibodies to FAK, Pyk2, and c‐Src   Commercially Available Phospho‐Specific Antibodies to FAK, Pyk2, and c‐Src b   Commercially Available Phospho‐Specific Antibodies to FAK, Pyk2, and c‐Src

    Antibody Company Source Application Catalog number
    FAK, clone 4.47 Upstate mouse (mAb) WB, IP, IC, IH 05‐537
    FAK clone 2A7 Upstate mouse (mAb) IP, IC 05‐182
    FAK Upstate rabbit WB, IP, IH 06‐543
    FAK, BC3 Upstate rabbit IP, IC 06‐446
    FAK BioSource rabbit IP, WB AHO0502
    FAK BD PharMingen rabbit WB, IP 556368
    FAK BD Transduction mouse (mAb) WB, IP, IC, IH 610087
    FAK Cell Signaling rabbit WB, IH 3285
    FAK BioSource rabbit WB, IP, IC AH0502
    FAK BioSource rabbit WB, IP, IC AMO0672
    FAK Chemicon rabbit IP AB1605
    FAK Chemicon mouse (mAb) WB, IP, IC MAB2156
    FAK (H‐1) Santa Cruz mouse (mAb) WB, IP, IC, IH sc‐1688
    FAK (A‐17) Santa Cruz rabbit WB, IP, IC sc‐557
    FAK (C‐20) Santa Cruz rabbit WB, IP, IC sc‐558
    FAK (C‐903) Santa Cruz rabbit WB, IP, IC, IH sc‐932
    Pyk2 Upstate rabbit WB, IP, IC 06‐559
    Pyk2, clone 74 Upstate mouse (mAb) WB, IP 05‐488
    Pyk2 Upstate rabbit WB, IP, IC 07‐437
    Pyk2 Cell Signaling rabbit WB, IP 3292
    Pyk2 BD Transduction mouse (mAb) WB, IP, IC, IH 610548
    Pyk2 (H‐102) Santa Cruz mouse (mAb) WB, IP, IC sc‐9019
    Pyk2 (N‐19) Santa Cruz rabbit WB, IP, IC sc‐1514
    Pyk2 (C‐19) Santa Cruz rabbit WB, IP, IC sc‐1515
    c‐Src, clone GD11 Upstate mouse WB, IP 05‐184
    c‐Src, clone N6L Upstate rabbit (mAb) WB 05‐889
    c‐Src, clone NL19 Upstate rabbit (mAb) WB, IP 05‐772
    c‐Src BioSource mouse (mAb) WB AHO1152
    c‐Src BioSource rabbit WB 44‐655G
    c‐Src BioSource rabbit WB 44‐656G
    c‐Src Chemicon sheep WB, IP CB769
    c‐Src, clone 36D10 Cell Signaling rabbit (mAb) WB, IP, IC, IH 2109
    c‐Src, clone L4A1 Cell Signaling mouse (mAb) WB, IP 2110
    c‐Src Cell Signaling rabbit WB, IP, IC, IH 2108
    c‐Src (H‐12) Santa Cruz mouse (mAb) WB, IP, IC sc‐5266
    c‐Src (B‐12) Santa Cruz mouse (mAb) WB, IP, IC sc‐8056
    c‐Src (N‐16) Santa Cruz rabbit WB, IP, IC sc‐19
    c‐Src (Src 2) Santa Cruz rabbit WB, IP, IC sc‐18
    Antibody Company Source Application Catalog number
    FAK pY397 BioSource rabbit WB, IC, IH 44‐624G
    FAK pY397 clone 141‐9 BioSource rabbit (mAb) WB, IC 44‐625G
    FAK pY407 BioSource rabbit WB, IC, IH 44‐650G
    FAK pY576 BioSource rabbit WB 44‐652G
    FAK pY577 BioSource rabbit WB, IC 44‐614G
    FAK pS722 BioSource rabbit WB 44‐588
    FAK pS732 BioSource rabbit WB 44‐590G
    FAK pS843 BioSource rabbit WB 44‐594
    FAK pY861 BioSource rabbit WB, IC 44‐626G
    FAK pS910 BioSource rabbit WB 44‐596
    FAK pY397 clone 14 BD/Transduction mouse (mAb) WB, IC 611722
    FAK pY397 clone 18 BD/Transduction mouse (mAb) WB, IC 611806
    FAK pY576/pY577 Cell Signaling rabbit WB 3281
    FAK pY397 Santa Cruz rabbit WB, IC sc‐11765‐R
    FAK pY397 Santa Cruz rabbit WB, IC sc‐21868‐R
    FAK pY407 Santa Cruz goat WB, IC sc‐16664
    FAK pY576 Santa Cruz rabbit WB, IC sc‐16563‐R
    FAK pY477 Santa Cruz goat WB, IC sc‐16665
    FAK pY576/pY577 Santa Cruz rabbit WB, IC sc‐21831‐R
    FAK pY576/pY577 Santa Cruz goat WB, IC sc‐21831
    FAK pS722 Santa Cruz goat WB, IC sc‐16662
    FAK pY861 Santa Cruz goat WB, IC sc‐16663
    FAK pS910 Santa Cruz goat WB, IC sc‐16666
    FAK pY925 Santa Cruz goat WB, IC sc‐11766
    FAK pY397 Chemicon mouse (mAb) WB, IC MAB1144
    FAK pY397 Upstate rabbit WB, IC 07‐012
    FAK pY576 Upstate rabbit WB, IC 07‐157
    Pyk2 pY402 BioSource rabbit WB, IH 44‐618G
    Pyk2 pY579 BioSource rabbit WB, IC 44‐632G
    Pyk2 pY579/pY580 BioSource rabbit WB 44‐636G
    Pyk2 pY580 BioSource rabbit WB 44‐634G
    Pyk2 pY881 BioSource rabbit WB, IH 44‐620
    Pyk2 pY402 Cell Signaling rabbit WB, IP 3291
    Pyk2 pY402 Santa Cruz rabbit WB, IC sc‐11767‐R
    Pyk2 pY579 Santa Cruz goat WB, IC sc‐16822
    Pyk2 pY579/pY580 Santa Cruz goat WB, IC sc‐16824
    Pyk2 pY580 Santa Cruz goat WB, IC sc‐16823
    Pyk2 pY881 Santa Cruz goat WB, IC sc‐16825
    Pyk2 pY402 clone RR102 Upstate mouse (mAb) IP 05‐679
    Active Src clone 28 BioSource mouse (mAb) WB, IC, IH AHO0051
    Src pY416 BioSource rabbit WB, IC 44‐660G
    Src pY527 BioSource rabbit WB, IH 44‐662G
    Src pY527 clone 31 BD/Transduction mouse (mAb) WB 612668
    Src pY416 Cell Signaling rabbit WB, IC, IH 2101
    Src pY416 clone 7G9 Cell Signaling mouse (mAb) WB, IP 2102
    Non‐phospho Src pY527 Cell Signaling rabbit WB 2107
    Src pY527 Cell Signaling rabbit WB, IH 2105
    Src pY527 Santa Cruz goat WB, IC sc‐16846
    Src pY416 clone 2N8 Upstate rabbit (mAb) WB 05‐857
    Src pY416 clone 9A6 Upstate mouse (mAb) WB 05‐677

     aAbbreviation: mAb, monoclonal antibody; WB, western (immuno)blot; IP, immunoprecipitation; IC, immunocytochemistry; IH, immunohistochemistry.
    Table 4.7.2   Materials   Commercially Available Antibodies to FAK, Pyk2, and c‐Src a   Commercially Available Antibodies to FAK, Pyk2, and c‐Src   Commercially Available Phospho‐Specific Antibodies to FAK, Pyk2, and c‐Src b   Commercially Available Phospho‐Specific Antibodies to FAK, Pyk2, and c‐Src

    Antibody Company Source Application Catalog number
    FAK, clone 4.47 Upstate mouse (mAb) WB, IP, IC, IH 05‐537
    FAK clone 2A7 Upstate mouse (mAb) IP, IC 05‐182
    FAK Upstate rabbit WB, IP, IH 06‐543
    FAK, BC3 Upstate rabbit IP, IC 06‐446
    FAK BioSource rabbit IP, WB AHO0502
    FAK BD PharMingen rabbit WB, IP 556368
    FAK BD Transduction mouse (mAb) WB, IP, IC, IH 610087
    FAK Cell Signaling rabbit WB, IH 3285
    FAK BioSource rabbit WB, IP, IC AH0502
    FAK BioSource rabbit WB, IP, IC AMO0672
    FAK Chemicon rabbit IP AB1605
    FAK Chemicon mouse (mAb) WB, IP, IC MAB2156
    FAK (H‐1) Santa Cruz mouse (mAb) WB, IP, IC, IH sc‐1688
    FAK (A‐17) Santa Cruz rabbit WB, IP, IC sc‐557
    FAK (C‐20) Santa Cruz rabbit WB, IP, IC sc‐558
    FAK (C‐903) Santa Cruz rabbit WB, IP, IC, IH sc‐932
    Pyk2 Upstate rabbit WB, IP, IC 06‐559
    Pyk2, clone 74 Upstate mouse (mAb) WB, IP 05‐488
    Pyk2 Upstate rabbit WB, IP, IC 07‐437
    Pyk2 Cell Signaling rabbit WB, IP 3292
    Pyk2 BD Transduction mouse (mAb) WB, IP, IC, IH 610548
    Pyk2 (H‐102) Santa Cruz mouse (mAb) WB, IP, IC sc‐9019
    Pyk2 (N‐19) Santa Cruz rabbit WB, IP, IC sc‐1514
    Pyk2 (C‐19) Santa Cruz rabbit WB, IP, IC sc‐1515
    c‐Src, clone GD11 Upstate mouse WB, IP 05‐184
    c‐Src, clone N6L Upstate rabbit (mAb) WB 05‐889
    c‐Src, clone NL19 Upstate rabbit (mAb) WB, IP 05‐772
    c‐Src BioSource mouse (mAb) WB AHO1152
    c‐Src BioSource rabbit WB 44‐655G
    c‐Src BioSource rabbit WB 44‐656G
    c‐Src Chemicon sheep WB, IP CB769
    c‐Src, clone 36D10 Cell Signaling rabbit (mAb) WB, IP, IC, IH 2109
    c‐Src, clone L4A1 Cell Signaling mouse (mAb) WB, IP 2110
    c‐Src Cell Signaling rabbit WB, IP, IC, IH 2108
    c‐Src (H‐12) Santa Cruz mouse (mAb) WB, IP, IC sc‐5266
    c‐Src (B‐12) Santa Cruz mouse (mAb) WB, IP, IC sc‐8056
    c‐Src (N‐16) Santa Cruz rabbit WB, IP, IC sc‐19
    c‐Src (Src 2) Santa Cruz rabbit WB, IP, IC sc‐18
    Antibody Company Source Application Catalog number
    FAK pY397 BioSource rabbit WB, IC, IH 44‐624G
    FAK pY397 clone 141‐9 BioSource rabbit (mAb) WB, IC 44‐625G
    FAK pY407 BioSource rabbit WB, IC, IH 44‐650G
    FAK pY576 BioSource rabbit WB 44‐652G
    FAK pY577 BioSource rabbit WB, IC 44‐614G
    FAK pS722 BioSource rabbit WB 44‐588
    FAK pS732 BioSource rabbit WB 44‐590G
    FAK pS843 BioSource rabbit WB 44‐594
    FAK pY861 BioSource rabbit WB, IC 44‐626G
    FAK pS910 BioSource rabbit WB 44‐596
    FAK pY397 clone 14 BD/Transduction mouse (mAb) WB, IC 611722
    FAK pY397 clone 18 BD/Transduction mouse (mAb) WB, IC 611806
    FAK pY576/pY577 Cell Signaling rabbit WB 3281
    FAK pY397 Santa Cruz rabbit WB, IC sc‐11765‐R
    FAK pY397 Santa Cruz rabbit WB, IC sc‐21868‐R
    FAK pY407 Santa Cruz goat WB, IC sc‐16664
    FAK pY576 Santa Cruz rabbit WB, IC sc‐16563‐R
    FAK pY477 Santa Cruz goat WB, IC sc‐16665
    FAK pY576/pY577 Santa Cruz rabbit WB, IC sc‐21831‐R
    FAK pY576/pY577 Santa Cruz goat WB, IC sc‐21831
    FAK pS722 Santa Cruz goat WB, IC sc‐16662
    FAK pY861 Santa Cruz goat WB, IC sc‐16663
    FAK pS910 Santa Cruz goat WB, IC sc‐16666
    FAK pY925 Santa Cruz goat WB, IC sc‐11766
    FAK pY397 Chemicon mouse (mAb) WB, IC MAB1144
    FAK pY397 Upstate rabbit WB, IC 07‐012
    FAK pY576 Upstate rabbit WB, IC 07‐157
    Pyk2 pY402 BioSource rabbit WB, IH 44‐618G
    Pyk2 pY579 BioSource rabbit WB, IC 44‐632G
    Pyk2 pY579/pY580 BioSource rabbit WB 44‐636G
    Pyk2 pY580 BioSource rabbit WB 44‐634G
    Pyk2 pY881 BioSource rabbit WB, IH 44‐620
    Pyk2 pY402 Cell Signaling rabbit WB, IP 3291
    Pyk2 pY402 Santa Cruz rabbit WB, IC sc‐11767‐R
    Pyk2 pY579 Santa Cruz goat WB, IC sc‐16822
    Pyk2 pY579/pY580 Santa Cruz goat WB, IC sc‐16824
    Pyk2 pY580 Santa Cruz goat WB, IC sc‐16823
    Pyk2 pY881 Santa Cruz goat WB, IC sc‐16825
    Pyk2 pY402 clone RR102 Upstate mouse (mAb) IP 05‐679
    Active Src clone 28 BioSource mouse (mAb) WB, IC, IH AHO0051
    Src pY416 BioSource rabbit WB, IC 44‐660G
    Src pY527 BioSource rabbit WB, IH 44‐662G
    Src pY527 clone 31 BD/Transduction mouse (mAb) WB 612668
    Src pY416 Cell Signaling rabbit WB, IC, IH 2101
    Src pY416 clone 7G9 Cell Signaling mouse (mAb) WB, IP 2102
    Non‐phospho Src pY527 Cell Signaling rabbit WB 2107
    Src pY527 Cell Signaling rabbit WB, IH 2105
    Src pY527 Santa Cruz goat WB, IC sc‐16846
    Src pY416 clone 2N8 Upstate rabbit (mAb) WB 05‐857
    Src pY416 clone 9A6 Upstate mouse (mAb) WB 05‐677

     bAbbreviations: mAb, monoclonal antibody; WB, western (immuno)blot; IP Immunoprecipitation; IC, immunocytochemistry; IH, immunohistochemistry.

Basic Protocol 2: In Vitro Kinase Assay

  Materials
  • Immunoprecipitated samples ( protocol 5, step )
  • FAK‐Pyk2 kinase buffer (see recipe), 4°C
  • Src kinase buffer (see recipe), 4°C
  • 10 µCi/µl [γ32P]ATP (>3,000 Ci/mmol; PerkinElmer)
  • Magnesium/ATP cocktail (Upstate Biotechnology or see recipe)
  • 2× Laemmli SDS buffer (see recipe)
  • Coomassie blue stain (see recipe)
  • Molecular weight marker (Precision Plus, Bio‐Rad)
  • Destaining solution (see recipe)
  • Microcentrifuge
  • 1.5‐ml microcentrifuge tubes
  • 32°C water bath
  • Plexiglas shielding
  • Gloves
  • Geiger counter
  • Plexiglas box
  • Micro tube cap locks (RPI 145063; http://www.rpicorp.com)
  • Whatman 3MM filter paper
  • Gel dryer
  • Immobilon PVDF membrane (Millipore IPFL 000‐10)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), autoradiography (unit 6.3), and transfer of proteins to membranes for immunoblotting (unit 6.2)
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in appropriately designated areas, following guidelines provided by the local radiation safety officer (also see appendix 1A).

Alternate Protocol 1: FAK‐PYK2 Poly Glu:Tyr Phosphorylation

  • Immunoprecipitated samples (see protocol 5, step )
  • 10 mg/ml poly(Glu:Tyr; 4:1) mol. wt. 20,000 to 50,000: sodium salt (Sigma‐Aldrich) prepared in PBS and stored up to 2 years at −20°C in 1‐ml aliquots
  • Magnesium/ATP cocktail (Upstate Biotechnology or see recipe)
  • 10 µCi/µl [γ32P]ATP (>3,000 Ci/mmol; PerkinElmer)
  • FAK‐Pyk2 kinase buffer
  • 0.75% phosphoric acid
  • 100% acetone
  • Scintillation fluid (Sigma)
  • 2 × 2–cm Whatman 3MM filter paper squares
  • Conical 50‐ml centrifuge tube
  • Scintillation vials
  • Scintillation counter
  • Additional reagents and equipment for preparing immunoprecipitated samples ( protocol 5)
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in appropriately designated areas, following guidelines provided by the local radiation safety officer (also see appendix 1A).

Alternate Protocol 2: Measurements of Src‐Associated Kinase Activity

  • 10 mg/ml GST‐FAK 853‐1052 (Schlaepfer lab)
  • Enolase (Sigma‐Aldrich E‐0379), optional
  • 50 mM HCl, optional
  • 1 M PIPES, pH 7, optional
  • 30°C water bath, optional
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in appropriately designated areas, following guidelines provided by the local radiation safety officer (also see appendix 1A).

Basic Protocol 3: Immunoblotting with FAK/PYK2 Phospho‐Specific Antibodies

  Materials
  • 2× Laemmli SDS buffer (see recipe)
  • Immunoprecipitated samples ( protocol 5, step )
  • 100% and 20% methanol
  • Transfer buffer (see unit 6.2)
  • EZBlue gel staining reagent (Sigma)
  • BSA blocking buffer (see recipe)
  • Primary antibody (see Tables 14.7.1 and 14.7.2)
  • Tris‐buffered saline with Tween (TBST, see recipe)
  • Secondary antibody: Horseradish peroxidase (HRP)‐conjugated appropriate species (Pierce)
  • Enhanced chemiluminescence (ECL) western detection solution (Amersham RPN 2132)
  • Western blot stripping buffer (see recipe)
  • Immobilon PVDF membrane (Millipore)
  • Rotating shaker
  • Additional reagents and equipment for gel electrophoresis (unit 6.1) and electrophoretic transfer (unit 6.2)

Basic Protocol 4: Haptotaxis Motility Assay: Matrix‐Stimulated Migration

  Materials
  • FAK−/− and FAK+/+ cells, subconfluent (see protocol 2)
  • Human plasma fibronectin (Sigma‐Aldrich F2006): 2 to 10 µg/ml in replating and migration medium (see recipe), 37°C
  • BSA‐coating control: 10 µg BSA/ml in replating and migration medium (see recipe)
  • Trypsin/EDTA: 0.25% (w/v) trypsin/1 mM EDTA (Invitrogen)
  • Trypsin inhibitor solution (see recipe)
  • PBS++: phosphate‐buffered saline (PBS; appendix 2A) with 0.1 g/liter CaCl 2 and 0.5 mM MgCl 2
  • Cell fixative: PBS with 1.85% (v/v) formaldehyde and 0.05% (v/v) glutaraldehyde
  • 10% (w/v) crystal violet stain (Sigma) in ethanol
  • 0.1 M sodium borate, pH 9.0
  • Parafilm
  • Millicell PCF chamber inserts, 8‐µm pore (Millipore PITP01250)
  • Flat‐tipped forceps
  • 15‐ml centrifuge tube
  • Tabletop centrifuge
  • Hemacytometer
  • 24 well‐tissue culture dishes (Costar)
  • Cotton swabs
  • Inverted light microscope
  • Spectrophotometer with A 600 capability, optional
  • Additional reagents and equipment for serum‐starving cells ( protocol 3) and counting cells (unit 1.1)

Alternate Protocol 3: Analyzing Cell Motility Using Plasmid‐Transfected Cells

  • pcDNA3.1 FAK (contact Schlaepfer lab)
  • FAK−/− cells (see protocol 2)
  • pcDNA3.1 LacZ (Invitrogen)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • PBS++: phosphate‐buffered saline ( appendix 2A) with 0.1 g/liter CaCl 2 and 0.5 mM MgCl 2
  • Opti‐MEM I reduced‐serum medium (Invitrogen)
  • PLUS reagent (Invitrogen)
  • Lipofectamine (Invitrogen)
  • Cell growth medium (see recipe) containing 20% (v/v) FBS (instead of 10%)
  • Starvation medium: Cell growth medium (see recipe) with FBS reduced to 0.5%
  • Lac Z staining solution (see recipe)
  • Additional reagents and equipment for bacterial transformation (Seidman et al., ), plasmid miniprep (Engebrecht et al., ), and DNA quantification ( appendix 3D)

Basic Protocol 5: Scratch‐Wound Healing Assay With Time‐Lapse Imaging

  Materials
  • Extracellular matrix molecule (ECM) of interest (e.g., 2 µg fibronectin/ml PBS)
  • 70% confluent 24‐hr serum‐starved cells (see protocol 3)
  • DMEM with and without 0.5 µg/ml mitomycin‐C
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Serum, optional
  • Mitomycin‐C (Sigma)
  • Medium 199 (Invitrogen) with 0.5 µg/ml mitomycin‐C
  • Mineral oil (Sigma)
  • 25‐mm glass coverslips (1 oz., Fisher) and 6‐well tissue culture plates or
  • 35‐mm Bioptechs delta‐T dishes (Fisher)
  • Forceps
  • 1‐ to 10‐µl micropipet tips
  • Transfer pipets, sterile
  • Inverted microscope with 20× objective, 37°C heated stage, and acquisition/analysis software (e.g., Improvision Openlab; https://www.improvision.com)
  • Etched‐grid coverslips (Bellco), optional

Basic Protocol 6: Immunolocalization of Focal Adhesion Proteins

  Materials
  • Matrix‐coating substrates: prepared according to manufacturer's directions and diluted (commonly to 10 µg/ml) in PBS ( appendix 2A)
    • Fibronectin, human plasma (Roche)
    • Laminin, human placenta (Sigma)
    • Vitronectin, human plama (Sigma)
    • Poly‐L‐lysine (70,000–150,000 or 150,000–300,000; Sigma)
    • Collagen, Type I, human placenta (Calbiochem)
    • Collagen, Type II, bovine (Calbiochem)
    • Collagen, Type IV, human placenta (Calbiochem)
    • Collagen, Type V, human (Calbiochem)
  • FAK−/− and FAK+/+ cells (see protocol 2)
  • Replating and migration medium (see recipe), warm or growth medium (see recipe), 37°C
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 3.8% (w/v) paraformaldehyde fixative (see recipe)
  • Acetone, cold (stored at −20°C) or
  • 0.5% (v/v) Triton X‐100/0.05% (v/v) Tween 20/PBS or
  • 0.2% Triton X‐100/PBS or
  • 0.2% (v/v) Triton X‐100/3.8% (w/v) paraformaldehyde/PBS or
  • 0.1% (v/v) Triton X‐100/0.1% (w/v) sodium citrate or
  • Methanol, cold
  • Blocking antibody (e.g., ChromPure donkey IgG, unconjugated; Jackson Immunoresearch) or
  • 2% (w/v) BSA in PBS
  • 1 to 10 µg/ml PBS ( appendix 2A) primary antibodies for focal adhesion markers
    • anti‐paxillin (ZO35; Zymed/Invitrogen)
    • anti‐vinculin (VIN‐11‐5; Sigma)
    • anti‐FAK (clone #77; BD‐Transduction)
    • anti‐FAK (Ab‐1; LabVision)
    • anti‐phospho Y397FAK (BioSource)
    • anti‐active Src family members (clone #28; BioSource)
  • Secondary antibodies: e.g., fluorescein (FITC)‐conjugated donkey antibodies (excitation/emission maxima 492/520 nm; Jackson Immunoresearch) or FITC‐conjugate
    • anti‐mouse IgG
    • anti‐mouse IgM
    • anti‐rabbit IgG
    • anti‐goat IgG
    • anti‐rat IgG
  • Rhodamine X (RRX)‐conjugated donkey antibodies (excitation/emission maxima 570/590 nm; Jackson Immunoresearch) or Rhodamine X (RRX)‐conjugated
    • anti‐mouse IgG
    • anti‐mouse IgM
    • anti‐rabbit IgG
    • anti‐goat IgG
    • anti‐rat IgG
  • Hoechst 33342 (excitation/emission maxima 350/460; Molecular Probes)
  • Vectashield mounting medium (Vector)
  • Nail polish, clear
  • 12‐mm round coverslips, German glass (Bellco Glass)
  • 4‐well tissue culture plates (Nunc)
  • 12‐well tissue culture plates (Costar)
  • 18‐ to 22‐G needle with a slightly bent tip
  • Flat‐ended forceps with beveled, unserrated tips, stainless steel (Millipore)
  • 6‐well tissue culture plates
  • Rotating platform shaker
  • Vacuum source
  • Porcelain spot plates with 12 cavities (CoorsTek)
  • Light microscope with fluorescence excitation and detecting capability

Alternate Protocol 4: Immunolocalization of Actin Stress Fibers

  • 0.4 to 1 U/ml (10 to 30 nM) fluorescein‐conjugated phalloidin (Molecular Probes) or rhodamine‐conjugated phalloidin (Molecular Probes) in PBS
  • Rotating platform shaker
  • Vacuum source
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Literature Cited

Internet Resources
   http://www.cellmigration.org/index.shtml
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