Related Experiment Video
Updated: May 21, 2026
Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins
Published on: May 3, 2022
The polarized total internal reflection fluorescence microscopy (polTIRFM) twirling filament assay
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.
Insights
This study introduces the polarized total internal reflection fluorescence microscopy (polTIRFM) twirling filament assay to measure actin filament rotation. The method tracks individual fluorescent probes on actin filaments translocated by myosin V, revealing rotational dynamics.
Area of Science:
- Biophysics
- Molecular Biology
- Microscopy
Background:
- Single-molecule biophysics requires methods to determine molecular orientation and dynamics.
- Polarized total internal reflection fluorescence microscopy (polTIRFM) offers a way to probe the 3D orientation of single fluorescent molecules.
- Understanding actin filament rotation is crucial for motor protein function.
Purpose of the Study:
- To develop and describe a novel assay for measuring the rotational dynamics of single actin filaments.
- To investigate the 'twirling' motion of actin filaments about their own axis during myosin V translocation.
- To adapt polTIRFM for observing the spatial orientation of individual fluorophores along a moving filament.
Main Methods:
- Construction of a gliding filament assay using sparsely labeled actin filaments (0.3% IATR-labeled actin monomers).
- Translocation of actin filaments by a surface-immobilized field of unlabeled myosin V in the presence of ATP.
- Utilizing polTIRFM to measure the spatial angle and orientation of individual fluorescent probes attached to actin as a function of time.
- Observation of approximately 50 isolated, moving points of fluorescence per microscopic field.
Main Results:
- The polTIRFM twirling assay successfully visualizes individual fluorophores moving along with translocated actin filaments.
- The assay allows for the measurement of the spatial angle of fluorescent probes, providing data on filament rotation.
- Successful assays demonstrate the translocation of actin filaments by myosin V, indicated by the movement of fluorescent points.
Conclusions:
- The polTIRFM twirling filament assay is a viable method for investigating the rotational dynamics of single actin filaments.
- This technique provides insights into the complex movements of actin during motor protein-mediated transport.
- The assay's ability to resolve individual probe orientations opens new avenues for studying macromolecular motion.
