Actin Retrograde Flow in Permeabilized Cells: Myosin-II Driven Centripetal Movement of Transverse Arcs

Materials and Reagents

1. 35-mm ibidi’s hydrophobic uncoated μ-dishes for cell culture (ibidi GmbH, catalog number: 80131 )
   
2. 1 x 1 cm polydimethylsiloxane (PDMS) stamp containing microfeatures of circles (area, 1,800 μm²; center-to-center distance, 100 μm)
   Note: For a detailed protocol on preparation of PDMS stamps and micro-contact printing see Théry and Piel (2009) and Tee et al., (2015).
   
3. Nunc^TM Cell Culture Treated Flasks with Filter Caps (Thermo Fisher Scientific, catalog number: 136196 )
   
4. Human foreskin fibroblast (ATCC, catalog number: SCRC-1041 )
   
5. Growth medium: Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Thermo Fisher Scientific, catalog number: 11965-092 ), supplemented with
   
   1. 10% fetal bovine serum (Thermo Fisher Scientific, catalog number: 10438-026 )
      
   2. 1 mM sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360-070 )
      
   3. 10 U/ml penicillin and streptomycin (Thermo Fisher Scientific, catalog number: 15140148 )
6. TrypLE^TM Express Enzyme (Thermo Fisher Scientific, catalog number: 12604013 )
   
7. PBS (1x), pH 7.4 (Thermo Fisher Scientific, catalog number: 10010-023 )
   
8. Fibronectin (Merck Millipore Corporation, catalog number: 341635 )
   
9. Imidazole (Sigma-Aldrich, catalog number: I15513 )
   
10. KCl (First BASE Laboratories Sdn Bhd, catalog number: BIO-1300 )
    
11. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
    
12. EDTA (First BASE Laboratories Sdn Bhd, catalog number: BIO-1050 )
    
13. Ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
    
14. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
    
15. Triton^TM X-100 (Sigma-Aldrich, catalog number: X100 )
    
16. Polye (ethylene glycol) MW35,000 (PEG) (Sigma-Aldrich, catalog number: 81310 )
    
17. Protease inhibitors cocktail (for use with mammalian cell and tissue extracts, DMSO solution) (Sigma-Aldrich, catalog number: P8340 )
    
18. N, N-Dimethylformamide (DMF) (Sigma-Aldrich, catalog number: 227056 )
    
19. Methanol (Thermo Fisher Scientific, catalog number: M/4000/17 )
    
20. Sterile Milli-Q water
    
21. Alexa Fluor^® 488 Phalloidin (Thermo Fisher Scientific, catalog number: A12379 ) (see Recipes)
    
22. Dark phalloidin [Phalloidin from Amanita phalloides (≥90%)] (Sigma-Aldrich, catalog number: P2141 ) (see Recipes)
    
23. Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A6419 ) (see Recipes)
    
24. Extraction buffer A (see Recipes)
    
25. Extraction buffer B (see Recipes)
    
26. Staining solution (see Recipes)
    
27. Contractility buffer (see Recipes)
    

Equipment

1. Confocal microscope equipped with 100x oil immersion objective
   
2. 37 °C on-stage incubation chamber
   

Software

1. Image J software [National Institutes of Health (NIH)] (http://imagej.nih.gov/ij/)

Procedure