Functional Characterization of Proteins Regulating Actin Assembly

Maud Hertzog1, Marie‐France Carlier2

1 Istituto FIRC di Oncologia Molecolare Fondazione Italiana per la Ricerca sul Cancro, Milano, Italy, 2 Laboratoire d'Enzymologie et Biochimie Structurale Centre National de la Recherche Scientifique, Gif‐sur‐Yvette, France
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 13.6
DOI:  10.1002/0471143030.cb1306s26
Online Posting Date:  April, 2005
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Abstract

A very large, ever‐increasing repertoire of actin‐binding proteins regulates the assembly dynamics and the spatial organization of actin filaments, thus orchestrating the motile behavior of the cell. The authors describe a series of biochemical functional assays that allow one to characterize the function of a putative actin‐binding protein in actin filament dynamics. These tests allow the characterization of three types of actin‐binding proteins: G‐actin‐sequestering proteins, profilin‐like proteins, and barbed‐end capping proteins. Biochemical tests include the use of sedimentation of actin filaments, polymerization assays at the barbed or pointed end of actin filaments derived from fluorescently labeled actin, thermodynamic measurements of actin assembly at steady state and during turnover of actin filaments, measurements of nucleotide exchange on G‐actin, and the use of the intrinsic or extrinsic fluorescence of actin to measure direct binding of different protein ligands to G‐actin.

Keywords: actin; polymerization; actin‐binding proteins; ADF/cofilin; profilin; actin‐sequestering β‐thymosins; capping proteins

     
 
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Table of Contents

  • Basic Protocol 1: Co‐sedimentation Assay for Measuring Binding of a Protein to F‐actin (or G‐actin)
  • Basic Protocol 2: Direct Binding to G‐actin: Fluorescence Measurements
  • Basic Protocol 3: Binding to Actin Derived from a Change in the Rate of Nucleotide Dissociation From G‐actin
  • Basic Protocol 4: Steady‐State Measurements of Actin Assembly: Critical Concentration Plots
  • Basic Protocol 5: Initial Rate of Filament Growth at Barbed or Pointed Ends
  • Basic Protocol 6: Measurement of Dilution‐Induced Depolymerization of Filaments
  • Basic Protocol 7: Measurements of the Treadmilling of Actin Filaments
  • Support Protocol 1: Actin Purification from Rabbit Muscle
  • Support Protocol 2: Preparation of Pyrenyl‐Labeled Actin
  • Support Protocol 3: Preparation of 7‐Chloro‐4‐Nitrobenzeno‐2‐Oxa‐1,3‐Diazole‐Labeled Actin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Co‐sedimentation Assay for Measuring Binding of a Protein to F‐actin (or G‐actin)

  Materials
  • 50 µM G‐actin in G‐buffer (see protocol 8), store on ice
  • 2 M KCl
  • 20 mM MgCl 2 ( appendix 2A)
  • 100 µM protein of interest in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT)
  • 10 mM Tris·Cl (pH 7.5)/1 mM DTT
  • F‐buffer (see recipe)
  • G‐buffer (see recipe)
  • 0.5‐ml polycarbonate centrifuge tubes
  • Beckman TL100 tabletop ultracentrifuge (or equivalent)
  • Densitometer
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (unit 6.1)

Basic Protocol 2: Direct Binding to G‐actin: Fluorescence Measurements

  Materials
  • 1.5 µM 100% fluorescently labeled G‐actin in G‐buffer (see Support Protocols protocol 92 and protocol 103)
  • Actin‐binding protein of interest in G‐buffer (see recipe)
  • Spectrofluorometer (time base mode) at wavelengths appropriate for label.

Basic Protocol 3: Binding to Actin Derived from a Change in the Rate of Nucleotide Dissociation From G‐actin

  Materials
  • 50% suspension of Dowex‐1‐X8 (Sigma) or AG‐1‐X8 (Bio‐Rad) strong anion exchange resin in G 0‐buffer
  • 50 µM G‐actin in G‐buffer (see protocol 8)
  • G 0‐buffer: G‐buffer (see recipe) without ATP
  • 1 mM etheno‐ATP (ɛ‐ATP), pH adjusted to 7.2 with NaOH, stock solution (Sigma)
  • 100 µM protein of interest in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT)
  • Microcentrifuge, 4°C
  • Spectrophotometer and quartz cuvettes
  • Spectrofluorometer (time base mode): λ ex = 350 nm, λ em = 410 nm, slits = 5‐ to 10‐nm band width

Basic Protocol 4: Steady‐State Measurements of Actin Assembly: Critical Concentration Plots

  Materials
  • 50 µM pyrenyl‐labeled G‐actin (see protocol 9), 10% labeled
  • G‐buffer (see recipe)
  • Gelsolin solution: 100 µM gelsolin (Sigma) in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT), for assaying actin with capped barbed ends only
  • Protein of interest
  • KM solution: 500 µl of 4 M KCl/20 µl of 1 M MgCl 2 ( appendix 2A)
  • Spectrofluorometer (time base mode): λ ex = 360 nm, λ em = 407 nm, slits = 5‐ to 10‐nm band width

Basic Protocol 5: Initial Rate of Filament Growth at Barbed or Pointed Ends

  Materials
  • Actin seeds (choose one):
    • 100 nM gelsolin‐actin (for pointed‐end growth): add 2.5 molar equiv G‐actin to gelsolin in G‐buffer
    • 5 nM spectrin‐actin isolated from human blood (Casella et al., ; for barbed‐end growth) in G‐buffer
    • 5 µM F‐actin (with or without 20 nM gelsolin)
  • KME solution (see recipe)
  • 5 µM pyrenyl‐labeled (10%) G‐actin (see protocol 9)
  • G‐buffer (see recipe)
  • 100 µM protein of interest in 10 mM Tris⋅Cl (pH 7.5)/1 mM dithiothreitol (DTT)
  • Microcuvettes suitable for spectrofluorometer
  • Spectrofluorometer (time base mode): λ ex = 366 nm, λ em = 407 nm, slits = 5‐ to 10‐nm band width
  • Additional reagents and equipment to generate a calibration curve (see protocol 4)

Basic Protocol 6: Measurement of Dilution‐Induced Depolymerization of Filaments

  Materials
  • G‐buffer (see recipe)
  • 4 M KCl
  • 100 mM MgCl 2 ( appendix 2A)
  • 50 µM G‐actin in G‐buffer (see protocol 8)
  • 50 µM G‐actin, 50% to 100% pyrenyl labeled in G‐buffer (see protocol 9)
  • Gelsolin solution: 100 µM gelsolin in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT), for assaying depolymerization from pointed ends only
  • 100 µM protein of interest in 10 mM Tris·Cl (pH 7.5)/1 mM DTT
  • F‐buffer (see recipe)
  • Microcuvettes, suitable for spectrofluorometer
  • Spectrofluorometer (time base mode): λ ex = 366 nm, λ em = 407 nm, slits = 5‐ to 10‐nm band width

Basic Protocol 7: Measurements of the Treadmilling of Actin Filaments

  Materials
  • 50% (w/v) suspension of Dowex‐1‐X8 (Sigma) or AG‐1‐X8 (Bio‐Rad) strong anion exchange resin in G 0‐buffer
  • 50 µM G‐actin in G‐ buffer (see protocol 8), store on ice
  • 5 mM etheno‐ATP (ɛ‐ATP), pH adjusted to 7.2 with NaOH, stock solution (Sigma)
  • KME solution (see recipe)
  • G‐buffer (see recipe)
  • 100 µM protein of interest in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT)
  • 200 mM ATP, pH adjusted to 7.2 with NaOH, stock solution (Sigma)
  • Microcentrifuge, 4°C
  • Spectrophotometer microcuvette, suitable for spectrofluorometer
  • Spectrofluorometer (time base mode; PM voltage adequate): λ ex = 350 nm, λ em = 410 nm, slits = 5‐ to 10‐nm band width

Support Protocol 1: Actin Purification from Rabbit Muscle

  Materials
  • Acetone powder of rabbit muscle (Cytoskeleton)
  • Extraction buffer (see recipe), 4°C
  • Solid KCl and 4 M KCl
  • Buffer D1 (see recipe)
  • 1 M MgCl 2 ( appendix 2A)
  • G‐buffer (see recipe)
  • 500‐ml beaker
  • Sorvall centrifuge and A641 rotor (or equivalent), 4°C
  • Glass wool
  • 20°C water bath
  • Large (3‐cm wide) dialysis bags (MWCO 14,000)
  • Dounce homogenizer (glass‐Teflon; 20‐ml working volume)
  • Sonicator with microtip (e.g., Vibra‐cell; Sonics & Materials)
  • 10‐ml ultracentrifuge tubes
  • Beckman ultracentrifuge and 70.1 Ti rotor (or equivalent), 4°C
  • 2.5 × 100–cm gel filtration column (Superdex‐200 prep grade; Amersham Biosciences)
  • Spectrophotometer (290 nm) and quartz cuvettes

Support Protocol 2: Preparation of Pyrenyl‐Labeled Actin

  • 4 mg/ml (∼95 µM) G‐actin in G‐buffer (see protocol 8)
  • Buffer A (see recipe)
  • N‐pyrenyliodoacetamide (NPI; Sigma)
  • Dimethylformamide
  • 0.2 M dithiothreitol (DTT; appendix 2A)
  • Rotating wheel, 4°C

Support Protocol 3: Preparation of 7‐Chloro‐4‐Nitrobenzeno‐2‐Oxa‐1,3‐Diazole‐Labeled Actin

  • 0.1 M KCl ( appendix 2A)
  • 50 µM G‐actin in G‐buffer (see protocol 8)
  • 100 mM N‐ethylmaleimide (NEM), prepare fresh
  • 20 mM dithiothreitol (DTT; appendix 2A)
  • G‐buffer (see recipe) with and without DTT and NaN 3
  • F‐buffer (see recipe) without DTT and NaN 3
  • 10 mM 7‐chloro‐4‐nitrobenzeno‐2‐oxa‐1,3‐diazole (NBD‐Cl) in dimethylformamide
  • 15°C water bath
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Figures

Videos

Literature Cited

Literature Cited
   Boquet, I., Boujemaa, R., Carlier, M.F., and Preat, T. 2000. Ciboulot regulates actin assembly during Drosophila brain metamorphosis. Cell 102:797‐808.
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   Carlier, M.F., Ressad, F., and Pantaloni, D. 1999. Control of actin dynamics in cell motility. Role of ADF/cofilin. J. Biol. Chem. 274:33827‐33830.
   Casella, J.F., Maack, D.J., and Lin, S. 1986. Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments. J. Biol. Chem. 261:10915‐10921.
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