Abstract
Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes the twirling filament assay, so named because actin sometimes twirls about its own axis as it is translocated by myosin. A gliding filament assay is constructed in which a sparsely labeled actin filament (0.3% of the actin monomers contain 6′-iodoacetamidotetramethylrhodamine [IATR]) is translocated by a field of unlabeled myosin V fixed to the surface. The polTIRFM twirling assay differs from a standard gliding filament assay in that full filaments are not visible, but rather individual fluorophores are spaced along each filament. The goal is to investigate possible rotational motions of the actin filament about its axis (i.e., twirling) by measuring the spatial angle of the fluorescent probe as a function of time. Successful assays contain microscopic fields of approximately 50 isolated points of fluorescence that move across the field in the presence of ATP. Actin is usually translocated by more than one myosin molecule, depending on the filament length and the myosin surface density. Sparsely labeled filaments are required because the orientation of only one probe can be resolved at a time.
MATERIALS
It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.
RECIPES: Please see the end of this article for recipes indicated by <R>. Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
ATP (2 mm)
Dilute ATP daily in M5 buffer from 100 mm stock (stored at −20°C, thawed shortly before use).
Bovine serum albumin (BSA; 5 mg/mL) (Sigma-Aldrich B2518), prepared monthly in M5 buffer
Prepare a 1:10 dilution of BSA in M5+ daily.
F-actins
-
Labeled (0.3%) with 6′-IATR (1 μm) (see Preparation of Filamentous Actin for Polarized Total Internal Reflection Fluorescence Microscopy [polTIRFM] Motility Assays [Beau-sang et al. 2012a])
Prepare a 1:500 dilution of 0.3% 6′-IATR-labeled F-actin in M5+ daily. -
Unlabeled (70 μm stock) (prepared similarly)
Prepare a 1:20 dilution of unlabeled F-actin in M5+ daily. Shear the actin by rapidly passing it through a 26-gauge needle 10 times.
M5 buffer <R>
M5+ <R>
Motility buffer for actin <R>
Myosin V, unlabeled (~0.2 mg/mL)
Poly-l-lysine HBr (1.0 mg/mL; MP Biomedicals 71120)
Equipment
Filter paper (2-ply; Kettenbach 30761 or similar)
Flow chamber, prepared as in Construction of Flow Chambers for Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM) Motility Assays (Beausang et al. 2012b), but without poly (methyl methacrylate) (PMMA)
Pipette tip (cut; see Step 9)
polTIRFM and analysis software
For details, see the section entitled The Principles of polTIRFM in Orientation and Rotational Motions of Single Molecules by Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM) (Beausang et al. 2012c).
METHOD
When possible, all solutions should be prepared using 0.2-μm-filtered, deionized water in a clean hood while wearing gloves to prevent sample contamination.
Using a flow chamber prepared without PMMA, flow 20 μL of poly-l-lysine. Incubate for 1 min.
Flow 20 μL of M5+.
Flow 20 μL of unlabeled myosin V. Incubate for 2 min.
-
Flow 20 μL of the BSA dilution twice. Incubate for 1 min.
This blocks exposed glass or poly-l-lysine. -
Flow 20 μL of diluted, sheared, unlabeled F-actin. Incubate for 1 min.
This blocks the inactive myosin heads. Repeat Step 5.
-
Flow 20 μL of 2 mm ATP.
This releases the unlabeled actin from the active myosin heads. -
Immediately flow 20 μL of M5+ twice.
This washes out the ATP and the unbound actin. -
Use a cut pipette tip to flow 20 μL of the 0.3% 6′-IATR-labeled F-actin dilution. Incubate for 1 min.
The cut pipette tip minimizes the shearing of the actin. -
Flow 20 μL of motility buffer for actin to initiate gliding. Record the data using the polTIRFM and analysis software as described in The Acquisition and Analysis of Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM) Data (Beausang et al. 2012d).
An assay that examines myosin movement along a stationary actin filament is described in The Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM) Processive Motility Assay for Myosin V (Beausang et al. 2012e).
RECIPES
M5 Buffer
20 mm HEPES (pH 7.6)
2 mm MgCl2
25 mm KCl
1 mm EGTA
M5+
10 mm DTT (prepare 1 m DTT stock in M5 buffer daily)
100 μg/mL wild-type calmodulin (WT-CaM)
Prepare daily in M5 buffer.
Motility Buffer for Actin
5–50 μm ATP
10 mm phosphocreatine (stocks stored at −20 °C and thawed daily; Sigma-Aldrich P7936)
0.3 mg/mL creatine phosphokinase (prepared daily from powder; Sigma-Aldrich C3755)
50 mm DTT (prepare 1 m DTT stock in M5+ daily)
Prepare daily in M5+.
ACKNOWLEDGMENTS
This work was funded by National Institutes of Health grants AR26846 and AR51174 to the Pennsylvania Muscle Institute and by the National Science Foundation (NSF) NSEC Nano/Bio Interface Center (NBIC) DMR 04-25780. J.F.B. is supported by an NSF IGERT fellowship through the NBIC DGE 0221664. We thank Drs. Philip C. Nelson and Jody A. Dantzig-Brody for review and useful comments on the manuscript.
REFERENCES
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