Analysis of Fibronectin Matrix Assembly

Iwona Wierzbicka‐Patynowski1, Yong Mao1, Jean E. Schwarzbauer1

1 Princeton University, Princeton, New Jersey
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.12
DOI:  10.1002/0471143030.cb1012s25
Online Posting Date:  December, 2004
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Abstract

The extracellular matrix acts as a framework for tissue architecture and dynamically regulates many cellular functions. Fibronectin is a ubiquitous extracellular matrix component that plays critical roles in matrix structure and in directing cell behaviors. Fibronectin is synthesized and secreted by many cell types including fibroblasts, endothelial cells, myoblasts, and astrocytes. Upon secretion, cells assemble fibronectin into a fibrillar network. During assembly, fibronectin is initially organized into fine cellā€associated fibrils and, through continued accumulation of fibronectin, these fibrils are converted into a dense network of detergentā€insoluble fibrils. Differential solubility in the detergent deoxycholate is the principle for biochemical fractionation of fibronectin matrix. Fibril assembly and organization can also be examined by immunofluorescence staining. In this unit, basic methods of detection, quantification, and visualization of fibrillar fibronectin matrix are described.

Keywords: fibronectin; extracellular matrix; fibroblasts; immunofluorescence; quantification; detergent extracts

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Matrix Assembly Using A Doc‐Solubility Assay
  • Alternate Protocol 1: Quantification of Matrix Assembly Using 125I‐Labeled Protein A
  • Alternate Protocol 2: Analysis of Assembly of Exogenous FN
  • Alternate Protocol 3: Analysis of Metabolically Labeled FN
  • Alternate Protocol 4: Direct Detection of Matrix Assembly by Incorporation of Fluorescently Labeled Fibronectin
  • Alternate Protocol 5: Detection of Matrix Assembly by Indirect Immunofluorescence Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Analysis of Matrix Assembly Using A Doc‐Solubility Assay

  Materials
  • Sub‐confluent (80% confluent) fibroblasts in a 10‐cm tissue culture dish
  • PBS ( appendix 2A)
  • Trypsin/EDTA solution (GIBCO, Invitrogen)
  • Culture medium containing 10% FN‐depleted serum (see unit 10.5 for FN‐depletion)
  • DOC lysis buffer (see recipe)
  • SDS‐solubilization buffer (see recipe)
  • BCA protein assay kit (Pierce Chemical)
  • 2× SDS sample buffer (see recipe)
  • 15‐ml screw‐cap tube
  • 24‐well tissue culture plate
  • Rubber policeman
  • 1‐ml syringe and 26‐G, 3/8‐in. needle
  • Additional reagents and equipment for cell culture (unit 1.1), gel electrophoresis (unit 6.1), and immunoblotting (unit 6.2)

Alternate Protocol 1: Quantification of Matrix Assembly Using 125I‐Labeled Protein A

  Materials
  • Samples of DOC‐soluble and ‐insoluble FN from cultures (see protocol 1, steps to )
  • 5% (w/v) BSA in TBS buffer (see appendix 2A for TBS)
  • Primary anti‐FN antibody (e.g., HFN7.1, ATCC)
  • Rabbit secondary antibody (e.g., unconjugated rabbit anti‐mouse IgG, Pierce Chemical)
  • 125I‐labeled protein A (10 µCi/µg,specific activity; MP Biomedicals)
  • Buffer A (see recipe)
  • Plastic wrap
  • Phosphorimager screen (cassette) and scanner
  • ImageQuant software
  • Additional reagents and equipment for gel electrophoresis (unit 6.1) and immunoblotting (unit 6.2)

Alternate Protocol 2: Analysis of Assembly of Exogenous FN

  Materials
  • Purified plasma FN (unit 10.5)
  • 125I‐labeled FN (∼1 µCi/µg; MP Biomedicals; optional)
  • Additional reagents and equipment for trypsinization and collection of cells, and isolation and analysis of DOC‐insoluble and DOC‐soluble FN (see protocol 1)

Alternate Protocol 3: Analysis of Metabolically Labeled FN

  Materials
  • Cell cultures for labeling
  • Culture medium containing FN‐depleted serum (see unit 10.5 for FN‐depletion)
  • Labeling medium (see recipe)
  • 35S‐methionine (>1000 Ci/mmol)
  • IP buffer (see recipe)
  • Protein A–Sepharose beads
  • 35‐mm tissue culture dish or 6‐well plate
  • Phosphorimager screen and scanner
  • ImageQuant software
  • Additional reagents and equipment for cell preparation (see protocol 1), IP protocol (unit 7.2)

Alternate Protocol 4: Direct Detection of Matrix Assembly by Incorporation of Fluorescently Labeled Fibronectin

  Materials
  • Purified FN (unit 10.5)
  • 50 mM sodium bicarbonate, pH 8.
  • Sulfo‐NHS‐rhodamine or fluorescein (Pierce Chemical)
  • CAPS‐NaCl solution
  • Cell cultures (see protocol 1, steps to )
  • Culture medium containing FN‐depleted serum (see unit 10.5 for FN‐depletion)
  • PBS/Mg (PBS containing 0.5 mM MgCl 2)
  • 3.7% (v/v) formaldehyde in PBS/Mg
  • 0.5% NP‐40 (v/v) in PBS/Mg
  • FluoroGuard (Bio‐Rad)
  • Nail polish
  • Spectrophotometer
  • 12‐mm circular coverslips
  • 24‐well plate
  • Fine‐tip forceps
  • Beakers
  • Paper towels
  • Kimwipes
  • Glass microscope slides

Alternate Protocol 5: Detection of Matrix Assembly by Indirect Immunofluorescence Staining

  Materials
  • Cell cultures
  • Primary anti‐FN antibody
  • 2% (w/v) ovalbumin in PBS/Mg solution
  • Fluorescein‐conjugated or rhodamine‐conjugated goat anti‐mouse IgG (or anti‐rabbit IgG)
  • Petri dishes
  • Fluorescence microscope
  • Additional reagents and equipment for detection of FN matrix (see protocol 5)
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Figures

Videos

Literature Cited

Literature Cited
   Hynes, R.O. 1990. Fibronectins. Springer‐Verlag, New York.
   McKeown‐Longo, P.J. and Mosher, D.F. 1983. Binding of plasma fibronectin to cell layers of human skin fibroblasts. J. Cell Biol. 97:466‐472.
   Pankov, R. and Yamada, K.M. 2002. Fibronectin at a glance. J. Cell Sci. 115:3861‐3863.
   Quade, B.J. and McDonald, J.A. 1988. Fibronectin's amino‐terminal matrix assembly site is located within the 29‐kDa amino‐terminal domain containing five type I repeats. J. Biol. Chem. 263:19602‐19609.
   Schwarzbauer, J.E. 1991. Alternative splicing of fibronectin: Three variants, three functions. BioEssays 13:527‐533.
   Schwarzbauer, J.E. and Sechler, J.L. 1999. Fibronectin fibrillogenesis: A paradigm for extracellular matrix assembly. Curr. Opin. Cell Biol. 11:622‐627.
   Wierzbicka‐Patynowski, I. and Schwarzbauer, J.E. 2003. The ins and outs of fibronectin matrix assembly. J. Cell Sci. 116:3269‐3276.
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