Materials and Reagents

1. Circular coverslips, German glass, 12 mm diameter, #1.5 thickness (Electron Microscopy Sciences, catalog number: 72291-02)

2. 24-well flat bottom cell culture plate (Corning Costar, Corning, catalog number: 3526, or similar)

3. 1.5–1.7 mL microcentrifuge tubes (Thomas Scientific, catalog number: 1138W14)

4. 0.5 mL open-top thickwall polycarbonate tube (Beckman Coulter, catalog number: 343776)

5. 50 mL conical tubes (Corning, catalog number: 352070)

6. 0.22 µm syringe filters (Millex, Millipore-Sigma, catalog number: SLGPR33RS)

7. 50 mL Luer tip syringes (BD Luer-Lok Tip, Fisher Scientific, catalog number: 14-820-11)

8. 10 µL pipette tips, ends cut off to make a larger orifice

9. Frosted microscope slides (Fisherbrand, Fisher Scientific, catalog number: 12-550-343)

10. Ethanol/dry ice bath or liquid nitrogen for snap-freezing protein

11. Phosphate buffered saline (PBS) (Gibco, ThermoFisher Scientific, catalog number: 20012027)

12. Poly-L-lysine, 0.01% solution (Sigma-Aldrich, MilliporeSigma, catalog number: P4707-50ML)

13. Rabbit muscle G-actin, >99% pure (Cytoskeleton, Inc., catalog number: AKL99)

    Note: Using actin prepared in-house from rabbit skeletal acetone powder is also fine, but care needs to be taken to verify that all bundling protein contaminants have been removed.

14. ASAP1 N-BAR recombinant protein, expressed and purified as described previously (Gasilina et al., 2019, 2022)

15. α-actinin (Cytoskeleton, Inc., catalog number: AT01)

    Note: It is highly recommended to use a bona fide actin bundling protein, such as α-actinin or vinculin tail, as a positive control.

16. Bovine serum albumin (BSA) (Sigma-Aldrich, Millipore Sigma, catalog number: B8667). Aliquot into 100 µL volumes and snap freeze. Store at -80 °C.

17. Methanol ≥99.9% (J.T. Baker, VWR International, catalog number: 9093-02)

18. Fluorescently labeled phalloidin [rhodamine phalloidin (Invitrogen, ThermoFisher Scientific, catalog number: R415), or Alexa Fluor 488 phalloidin (Invitrogen, ThermoFisher Scientific, catalog number: A12379)]. Reconstitute in 1.5 mL of 100% methanol and store at -20 °C.

    Note: Although any bright and photostable fluorescent phalloidin conjugate can be used to stain and visualize bundles, the use of fluorophores in the visible spectra will facilitate finding and focusing on the sample during data acquisition.

19. Adenosine 5’-triphosphate disodium salt (ATP) (Cytoskeleton, Inc., catalog number: BSA04). Reconstitute with 1 mL of cold 100 mM Tris, pH 7.5 (Recipe 7), for a 100 mM stock, aliquot into 5–100 µL portions and 10–50 µL portions, snap freeze, and store at -80 °C. Note that the ATP from cytoskeleton is lyophilized from a solution buffered to pH 7, and, consequently, the solutions do not require additional adjusting of the pH.

20. Paraformaldehyde, 16%, methanol-free (Electron Microscopy Sciences, catalog number: 15710)

21. Dako fluorescence mounting medium (Agilent, catalog number: S302380-2)

22. Calcium chloride (CaCl₂) (Sigma-Aldrich, Millipore Sigma, catalog number: 223506)

23. Potassium chloride (KCl) (Sigma-Aldrich, Millipore Sigma, catalog number: P3911)

24. Magnesium chloride (MgCl₂) (Sigma-Aldrich, Millipore Sigma, catalog number: M9272)

25. Tris-HCl, 1 M, pH 7.5 (KD Medical, catalog number: RGF-3350)

26. Dithiothreitol (DTT) Cleland’s reagent (MP Biomedicals, VWR International, catalog number: 0219482101)

27. 1× G-actin buffer (see Recipes)

28. 10× actin polymerization buffer (see Recipes)

29. 1× F-actin buffer (see Recipes)

30. 1 M CaCl₂ (see Recipes)

31. 1 M KCl (see Recipes)

32. 1 M MgCl₂ (see Recipes)

33. Tris-HCl, 1 M, pH 7.5 (see Recipes)

34. 1 M DTT (see Recipes)

Equipment

1. P10, P20, and P200 micropipettes

2. Benchtop microcentrifuge for 1.5/2 mL tubes

3. Small volume ultra-centrifuge (e.g., Thermo Scientific Sorvall MTX with S120-AT3 rotor)

4. Confocal microscope equipped with oil-immersion objective, preferably with a tile scanning option (we use a 63× objective, but given the size of the bundles, any objective between 40× and 100× is suitable.)

Software

1. Fiji/ImageJ (National Institutes of Health, imagej.nih.gov)

Procedure