In Vitro Motility Assay to Study Translocation of Actin by Myosin

James R. Sellers1

1 National Heart, Lung, and Blood Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 13.2
DOI:  10.1002/0471143030.cb1302s00
Online Posting Date:  May, 2001
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Abstract

A basic property of myosin is its ability to interact with and translocate actin. This unit describes an in vitro motility assay that can be used to study the translocation, or sliding, of actin filaments by myosin bound to a coverslip. The assay makes use of the ability to image single F‐actin filaments labeled with rhodamine phalloidin, a high‐affinity fluorescent ligand using fluorescence microscopy. The system is fast, easy to set up and maintain, uses only small amounts of protein, and yields quantitative results.

     
 
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Table of Contents

  • Basic Protocol 1: Analyzing Actin Translocation by Myosin
  • Support Protocol 1: Preparation of Flow Cells
  • Support Protocol 2: Purification of Actin
  • Support Protocol 3: Preparation of Rhodamine Phalloidin–Labeled Actin
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Analyzing Actin Translocation by Myosin

  Materials
  • 0.2 mg/ml myosin monomers (see Table 13.2.1)
  • 1 mg/ml recipeBSA in G‐actin buffer (see recipe)
  • Wash buffer (see recipe)
  • recipeWash buffer containing 1 mM recipeATP and 5 µM actin (see protocol 3)
  • recipeWash buffer containing 20 nM rhodamine phalloidin–labeled actin (see protocol 4)
  • Assay buffer (see recipe)
  • Flow cell (see protocol 2)
  • Fluorescent microscope, with high‐numerical‐aperture objective (60× to 100×, 1.3 to 1.4 NA) and 100‐W mercury lamp
  • SIT camera or an intensified CCD
  • VHS or sVHS video recorder
  • Image processor
  • Cell Trak image analysis system (Motion Analysis)

Support Protocol 1: Preparation of Flow Cells

  Materials
  • 1% (w/v) nitrocellulose (Ernest F. Fullham) in amyl acetate
  • Apiezon M grease (Thomas; optional)
  • Double‐sided tape
  • 18‐mm2 no. 1 coverslips
  • Glass microscope slides
  • 24 × 60–mm no. 0 coverslips (optional)

Support Protocol 2: Purification of Actin

  Materials
  • Back and leg muscles of freshly sacrificed 350‐g rabbit
  • 0.1 M KCl/0.15 M potassium phosphate, pH 6.5
  • 0.05 M NaHCO 3
  • 1 mM EDTA, pH 7.0
  • Acetone
  • G‐actin buffer (see recipe)
  • 2 M KCl (stock solution)
  • 1 M MgCl 2 (stock solution)
  • Meat grinder, prechilled
  • Cheesecloth
  • Filter paper
  • Sorvall centrifuge and SS‐34 rotor (or equivalent)
  • Beckman ultracentrifuge and 55 Ti rotor (or equivalent)
  • Potter‐Elvehjem tissue grinder
  • Additional reagents and equipment for dialysis ( appendix 3A)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.NOTE: All procedures are performed at 4°C unless otherwise specified and all buffers should be prechilled to this temperature.

Support Protocol 3: Preparation of Rhodamine Phalloidin–Labeled Actin

  Materials
  • Rhodamine phalloidin (Molecular Probes)
  • Methanol
  • Labeling buffer (see recipe)
  • Actin (see protocol 3, step )
  • Wash buffer (see recipe)
  • Speed‐Vac evaporator (Savant) or equivalent
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Figures

Videos

Literature Cited

Literature Cited
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