Probing Myosin Ensemble Mechanics in Actin Filament Bundles using Optical Tweezers.

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping. These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments. These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Myosin II Adjusts Motility Properties and Regulates Force Production Based on Motor Environment.

Introduction: Myosin II has been investigated with optical trapping, but single motor-filament assay arrangements are not reflective of the complex cellular environment. To understand how myosin interactions propagate up in scale to accomplish system force generation, we devised a novel actomyosin ensemble optical trapping assay that reflects the hierarchy and compliancy of a physiological environment and is modular for interrogating force effectors. Methods: Hierarchical actomyosin bundles were formed in vitro. Fluorescent template and cargo actin filaments (AF) were assembled in a flow cell and bundled by myosin. Beads were added in the presence of ATP to bind the cargo AF and activate myosin force generation to be measured by optical tweezers. Results: Three force profiles resulted across a range of myosin concentrations: high force with a ramp-plateau, moderate force with sawtooth movement, and baseline. The three force profiles, as well as high force output, were recovered even at low solution concentration, suggesting that myosins self-optimize within AFs. Individual myosin steps were detected in the ensemble traces, indicating motors are taking one step at a time while others remain engaged in order to sustain productive force generation. Conclusions: Motor communication and system compliancy are significant contributors to force output. Environmental conditions, motors taking individual steps to sustain force, the ability to backslip, and non-linear concentration dependence of force indicate that the actomyosin system contains a force-feedback mechanism that senses the local cytoskeletal environment and communicates to the individual motors whether to be in a high or low duty ratio mode. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00731-1.

Determination of vibrational band positions in the E-hook of ß-tubulin.

Raman spectral characterization of the ß-TUBB2A E-hook hexapeptide, EGEDEA, is determined through experimental analysis combined with full geometry optimizations and corresponding harmonic vibrational frequency computations employing DFT methods. The hexapeptide is first broken down into di- and tetrapeptide fragments which are analyzed both quantum chemically and experimentally, and then combined to achieve an energetic minimum of the large EGEDEA hexapeptide. The Raman spectral characterization of EGEDEA band positions are then verified via the literature and comparison to the small fragment's similarly located band positions. The approach employed provides further evidence for the use of fragments as a helpful tool in characterization of the vibrational band positions of large peptides. STATEMENT OF SIGNIFICANCE: To investigate ß-TUBB2A E-hook hexapeptide, a unique approach is employed whereby the hexapeptide is broken into fragments, EG, ED, EA, EGED, and EDEA and analyzed via experimental Raman spectroscopy of the crystalline solids. The experimentally observed vibrational band positions are compared to those computed using and scaled from DFT methods and Pople's 6-311+G(2df,2pd) basis set. The reported vibrational band positions are also confirmed by previously reported bands of similar peptides in the literature. This methodology facilitates differentiation between the behaviors of various side chains and their influence on the structure of the hexapeptide, providing insight into not only the nature of the peptide but also defining regions for potential protein and cytoplasmic interactions, without requiring excessive computing resources or overly-sensitive experimental methods.