Actin‐Based Motility Assay

Christophe Le Clainche1, Marie‐France Carlier1

1 Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Gif‐sur‐Yvette
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.7
DOI:  10.1002/0471143030.cb1207s24
Online Posting Date:  October, 2004
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Abstract

Actin‐based movement can be reconstituted by using microspheres functionalized with the enzymes N‐WASP or ActA, which use the Arp2/3 complex and actin to catalyze the formation of a branched actin filament network that is maintained in rapid turnover by three proteins (capping protein, profilin, and ADF). The particles continuously initiate filament assembly at their surface and are propelled, mimicking bacteria or the leading edge of motile cells. This biomimetic assay offers advantages over approaches based on living cells and cell extracts, because the physical‐chemical parameters are under control. The biomimetic motility assay offers the opportunity to test the function of proteins involved in signaling pathways or actin dynamics. It is a powerful tool to understand the physical mechanism of force production and has the potential to support high‐throughput screens for drugs, inhibitors of motility, or therapeutic agents in metastatic states in which motility is impaired.

Keywords: motility; actin; Arp2/3; N‐WASP; ADF; profilin; capping proteins

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Actin‐Based Motility Assay
  • Support Protocol 1: Data Analysis
  • Support Protocol 2: Profilin Purification
  • Support Protocol 3: ADF Purification
  • Support Protocol 4: Gelsolin Purification
  • Support Protocol 5: N‐WASP Purification
  • Support Protocol 6: ARP2/3 Purification
  • Support Protocol 7: Preparation of N‐WASP‐Functionalized Microspheres
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Actin‐Based Motility Assay

  Materials
  • Profilin (see protocol 3)
  • ADF (see protocol 4)
  • Gelsolin (see protocol 5) or other capping protein
  • VASP (for use with Listeria or ActA‐functionalized microspheres only; prepare as for N‐WASP, see protocol 6)
  • Arp2/3 complex or Alexa488‐Arp2/3 complex (see protocol 7)
  • Assay buffer (see recipe) containing 1% (w/v) bovine serum albumin (BSA)
  • 10% (w/v) bovine serum albumin (BSA) in distilled water
  • 5 µM α‐actinin (Sigma) in distilled H 2O
  • 48 µM Mg‐F‐actin (see recipe)
  • 1% (w/v) methylcellulose (see recipe)
  • ATP/DABCO/DTT mix (see recipe)
  • 40 µM rhodamine‐G‐actin (Cytoskeleton, Inc.) in assay buffer/1% BSA (store up to 1 month at −80°C)
  • 4 × 109 microspheres/ml suspension of N‐WASP‐functionalized microspheres (see protocol 8) or 4 × 109 bacteria/ml suspension of Listeria or E. coli IcsA in assay buffer/1% BSA
  • Valap (unit 13.1)
  • Glass slides (Superfrost Plus, Fisher, or Menzel‐Gläser; 25 × 75 × /mm)
  • Glass coverslips (22 × 22 mm)
  • Microscope, phase‐contrast or fluorescence (with appropriate filters; unit 4.2)

Support Protocol 1: Data Analysis

  Materials
  • Bovine spleen (see )
  • Profilin buffer A (see recipe) containing 0.5% (v/v) Triton X‐100
  • 200 mM PMSF stock in absolute ethanol
  • 4 M and 7 M urea in profilin buffer A (see recipe for buffer)
  • Profilin buffer B (see recipe)
  • Liquid nitrogen
  • Centrifuge with Sorvall SLA 1500 rotor (or equivalent)
  • Cheescloth
  • Ultracentrifuge with Sorvall A641 rotor (or equivalent)
  • 15‐cm length × 2.5‐cm inner diameter poly‐L‐proline‐Sepharose column (see recipe)
  • 10,000‐kDa MWCO dialysis membrane
  • Centriprep 10 centrifugal concentrator (Millipore; also see appendix 3C)
  • Ultracentrifuge with Beckman TLA 100.3 rotor (or equivalent)
  • Additional reagents and equipment for dialysis and concentration of protein solutions ( appendix 3C) and determination of protein concentration ( appendix 3B)

Support Protocol 2: Profilin Purification

  Materials
  • E. coli BL21 DE3 strain transformed with pET16‐ADF (see appendix 3A for transformation of E. coli with plasmid)
  • LB medium ( appendix 2A) containing 0.2 mg/ml ampicillin
  • ADF buffers A, B, C, D, and E (see reciperecipes)
  • 10 mg/ml lysozyme
  • 250 mM EDTA
  • 200 mM PMSF in absolute ethanol
  • 1 M MgCl 2
  • 2 mg/ml DNase I
  • Liquid nitrogen
  • Centrifuge with Sorvall GSA rotor (or equivalent)
  • Probe sonicator (Fisher)
  • Centrifuge with Sorvall SS‐34 rotor (or equivalent)
  • 10,000‐kDa MWCO dialysis membrane
  • Ultracentrifuge with Beckman TL‐100 rotor (or equivalent)
  • DEAE (DE‐52; Whatman) column (20 × 2.5 cm; unit 10.11)
  • Stirred cell with Diaflo PM 10 ultrafiltration membrane (Amicon)
  • SP trisacryl column (15 × 1.5 cm; BioSepra)
  • Additional reagents and equipment for transforming and growing bacteria and induction of protein expression with IPTG ( appendix 3A), dialysis ( appendix 3C), and determination of protein concentration ( appendix 3B)

Support Protocol 3: ADF Purification

  Materials
  • E. coli BL21 DE3 strain expressing gelsolin (see appendix 3A for transformation of E. coli)
  • Gelsolin buffers A, B, C, and D (see reciperecipes)
  • 1 M MgCl 2
  • 2 mg/ml DNase I
  • 500 mM NaCl in gelsolin buffer B (see recipe for buffer)
  • Anti‐gelsolin antibody (Sigma)
  • 500 mM NaCl in gelsolin buffer C (see recipe for buffer)
  • Liquid nitrogen
  • Probe sonicator (Fisher)
  • Refrigerated centrifuge
  • Centriprep 30 centrifugal concentrator
  • 30,000‐kDa MWCO dialysis membrane
  • 2.5 × 20–cm DEAE‐cellulose (DE‐52; Whatman) column (unit 10.11)
  • Gradient maker
  • 11 × 1.25–cm SP trisacryl column (BioSepra)
  • Additional reagents and equipment for transforming and growing bacteria and induction of protein expression with IPTG ( appendix 3A), dialysis ( appendix 3C), and determination of protein concentration ( appendix 3B), and immunodetection (unit 6.2)

Support Protocol 4: Gelsolin Purification

  Materials
  • Insect (Sf9) cells expressing N‐WASP (see appendix 3A for transformation of insect cells)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • PBS ( appendix 2A) containing 0.5 mM PMSF (add from 200 mM PMSF stock in absolute ethanol)
  • N‐WASP buffers A, B, C, D, E, and F (see reciperecipes)
  • NaCl
  • Imidazole
  • 50% (w/v) suspension of Ni‐NTA resin (Qiagen)
  • Liquid nitrogen
  • Cell scraper
  • Clinical (tabletop) centrifuge
  • Dounce homogenizer
  • Probe sonicator (Fisher)
  • Ultracentrifuge with Beckman TL‐100 rotor (or equivalent)
  • 2.5 × 20–cm DEAE‐cellulose (DE‐52; Whatman) column (unit 10.11)
  • 1 × 5 cm, 10‐ml chromatography column
  • 30,000‐kDa MWCO SpectraPor 2 dialysis membrane (Spectrum)
  • Additional reagents and equipment for protein expression in insect cells ( appendix 3A) and dialysis ( appendix 3C)

Support Protocol 5: N‐WASP Purification

  Materials
  • Bovine brain (see )
  • Arp2/3 buffers A, B, C, D, E, F, G, and H (see reciperecipes)
  • 2 M sucrose
  • 100 µM Alexa488‐C5‐maleimide (Molecular Probes) in Arp2/3 buffer H (see recipe for buffer)
  • DTT
  • Hammer
  • Waring blender
  • Refrigerated centrifuge
  • 30,000‐kDa MWCO dialysis membrane
  • SP trisacryl column (4 × 30 cm; BioSepra)
  • Sepharose GSH‐GST‐VCA as described by Egile et al. ( ) 2‐ml column containing
  • Disposable NAP‐10 column (Amersham Biosciences)
  • Additional reagents and equipment for dialysis ( appendix 3C) and determination of protein concentration ( appendix 3B)

Support Protocol 6: ARP2/3 Purification

  Materials
  • Polystyrene carboxylated microspheres of diameters from 0.2 to 3 µm (Polysciences)
  • N‐WASP buffers A and C (see reciperecipes)
  • 4 µM N‐WASP (see protocol 6)
  • 10 mg/ml BSA
  • Refrigerated centrifuge
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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.
   Boujemaa‐Paterski, R., Gouin, E., Hansen, G., Samarin, S., Le Clainche, C., Didry, D., Dehoux, P., Cossart, P., Kocks, C., Carlier, M.F., and Pantaloni, D. 2001 Listeria protein ActA mimics WASp family proteins: It activates filament barbed end branching by Arp2/3 complex. Biochemistry 40:11390‐11404.
   Carlier, M.F., Laurent, V., Santolini, J., Melki, R., Didry, D., Xia, G.X., Hong, Y., Chua, N.H., and Pantaloni, D. 1997. Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: Implication in actin‐based motility. J. Cell Biol. 136:1307‐1322.
   Carlier, M.F., Nioche, P., Broutin‐L'Hermite, I., Boujemaa, R., Le Clainche, C., Egile, C., Garbay, C., Ducruix, A., Sansonetti, P., and Pantaloni, D. 2000. GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott‐Aldrich syndrome protein (N‐WASp) with actin‐related protein (ARP2/3) complex. J. Biol. Chem. 275:21946‐21952.
   Egile, C., Loisel, T.P., Laurent, V., Li, R., Pantaloni, D., Sansonetti, P.J., and Carlier, M.F. 1999. Activation of the CDC42 effector N‐WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin‐based motility. J. Cell Biol. 146:1319‐1332.
   Higgs, H.N., Blanchoin, L., and Pollard, T.D. 1999. Influence of the C terminus of Wiskott‐Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization. Biochemistry 38:15212‐15222.
   Laurent, V., Loisel, T.P., Harbeck, B., Wehman, A., Grobe, L., Jockusch, B.M., Wehland, J., Gertler, F.B., and Carlier, M.F. 1999. Role of proteins of the Ena/VASP family in actin‐based motility of Listeria monocytogenes. J. Cell Biol. 144:1245‐1258.
   Loisel, T.P., Boujemaa, R., Pantaloni, D., and Carlier, M.F. 1999. Reconstitution of actin‐based motility of Listeria and Shigella using pure proteins. Nature 401:613‐616.
   Mullins, R.D., Heuser, J.A., and Pollard, T.D. 2000. The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc. Natl. Acad. Sci. U.S.A. 95:6181‐6186.
   Pantaloni, D., Le Clainche, C., and Carlier, M.F. 2001. Mechanism of actin‐based motility. Science 292:1502‐1506.
   Perelroizen, I., Didry, D., Christensen, H., Chua, N.H., and Carlier, M.F. 1996. Role of nucleotide exchange and hydrolysis in the function of profilin in actin assembly. J. Biol. Chem. 271:12302‐12309.
   Rohatgi, R., Ma, L., Miki, H., Lopez, M., Kirchhausen, T., Takenawa, T., and Kirschner, M.W. 1999. The interaction between N‐WASP and the Arp2/3 complex links Cdc42‐dependent signals to actin assembly. Cell 97:221‐231.
   Rohatgi, R., Nollau, P., Ho, H.Y., Kirschner, M.W., and Mayer, B.J. 2001. Nck and phosphatidylinositol 4,5‐bisphosphate synergistically activate actin polymerization through the N‐WASP‐Arp2/3 pathway. J. Biol. Chem. 276:26448‐26452.
   Samarin, S., Romero, S., Kocks, C., Didry, D., Pantaloni, D., and Carlier, M.F. 2003. How VASP enhances actin‐based motility. J. Cell Biol. 163:131‐142.
   Theriot, J.A., Rosenblatt, J., Portnoy, D.A., Goldschmidt‐Clermont, P.J., and Mitchison, T.J. 1994. Involvement of profilin in the actin‐based motility of L. monocytogenes in cells and in cell‐free extracts. Cell 76:505‐517.
   Uruno, T., Liu, J., Zhang, P., Fan, Y.X., Egile, C., Li, R., Mueller, S.C., and Zhan, X. 2001. Activation of Arp2/3 complex‐mediated actin polymerization by cortactin. Nat. Cell Biol. 3:259‐266.
   Weaver, A.M., Karginov, A.V., Kinley, A.W., Weed, S.A., Li, Y., Parsons, J.T., and Cooper, J.A. 2001. Cortactin promotes and stabilizes Arp2/3‐induced actin filament network formation. Curr. Biol. 11:370‐374.
   Welch, M.D., Iwamatsu, A., and Mitchison, T.J. 1997. Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature 385:265‐269.
   Welch, M.D., Rosenblatt, J., Skoble, J., Portnoy, D.A., and Mitchison, T.J. 1998. Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science 281:105‐108.
   Wiesner, S., Helfer, E., Didry, D., Ducouret, G., Lafuma, F., Carlier, M.F., and Pantaloni, D. 2003. A biomimetic motility assay provides insight into the mechanism of actin‐based motility. J. Cell Biol. 160:387‐398.
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