Biology subjects

Rassier, D. E.

Publications and source records attributed to Rassier, D. E..

4 recordsLinked to original sources

bioRxiv2024

Oxidation-induced structural changes in actin and myosin evaluated by computational simulation, machine learning modeling and high-speed AFM

High levels of reactive oxygen species produced during muscle oxidative stress are implicated in the development of several muscle diseases. To better understand the mechanism behind a reduced myosin force generation under oxidizing conditions, we analyzed the structural and functional changes in the actin and actin-myosin complex using high-speed atomic force microscopy (HS-AFM), simulated HS-AFM, and molecular dynamics (MD) simulation. Computational oxidative nitration of tyrosine residues demonstrated instability in the molecular structure of the F-actin subunit. Cross-section analysis of the simulated HS-AFM images revealed a shift in the height values ([~]0.2-1.5 nm in magnitude) between the non-oxidized and oxidized actin, which correspond to the height differences observed in HS-AFM experiments with in vitro oxidized F-actin. The oxidation-induced structural alterations in actin impact myosin molecule displacement on the single-molecule level. The displacements of myosin heads along the F-actin filaments in the presence of ATP involve the binding of the myosin molecule to a specific site on the F-actin filament, followed by the rotation of the myosin lever arm, which triggers the release of inorganic phosphate (Pi). Subsequently, the myosin head detaches from the F-actin and re-binds to a new site on the filament. The formation of the SIN-1-treated F-actin-myosin complex in the presence of ATP resulted in a change in myosin head displacement size, with a significant decrease in the frequency of long displacements ([≥] 4 nm). These results suggest that oxidation decreases the pool of the weak-bound myosin molecules and shortens the long displacements related to the Pi release step, reducing the force generation by myosin motors.

biophysics↗

bioRxiv2023

New paradigms in actomyosin energy transduction: critical evaluation of non-traditional models for orthophosphate release

Release of the ATP hydrolysis product inorganic phosphate (Pi) from the active site of myosin is central in chemo-mechanical energy transduction and closely associated with the main force-generating structural change, the power-stroke. Despite intense investigations, the relative timing between Pi-release and the power-stroke remains poorly understood. This hampers in depth understanding of the production of force and motion by myosin in health and disease and also our understanding of myosin-active drugs. From the 1990s and up to today, models with the Pi-release either distinctly before or after the power-stroke, in unbranched kinetic schemes, have dominated the literature. However, in recent years, alternative models have emerged to explain apparently contradictory findings. Here, we first compare and critically analyze, three influential alternative models, either characterized by a branched kinetic scheme or by partial uncoupling of Pi-release and the power-stroke. Finally, we suggest critical tests of the models aiming for a unified picture.

biophysics↗

bioRxiv2022

Cooperativity of myosin II motors in the non-regulated and regulated thin filaments investigated with high-speed AFM

Skeletal myosins II are non-processive molecular motors, that work in ensembles to produce muscle contraction while binding to the actin filament. Although the molecular properties of myosin II are well known, there is still debate about the collective work of the motors: is there cooperativity between myosin motors while binding to the actin filaments? In this study, we used high-speed AFM to evaluate this issue. We observed that the initial binding of small arrays of myosin heads to the non-regulated actin filaments did not affect the cooperative probability of subsequent bindings to neighboring sites and did not lead to an increase in the fractional occupancy of the actin binding sites. These results suggest that myosin motors are independent force generators when connected in small arrays, and that the binding of one myosin does not alter the kinetics of other myosins. In contrast, the probability of binding of myosin heads to regulated thin filaments under activating conditions (at high Ca2+ concentration and with 2 M ATP) was increased with the initial binding of one myosin, leading to a larger occupancy of neighboring available binding sites. The result suggests that myosin cooperativity is defined by the activation status of the thin filaments. eLife digestMuscle contraction is the result of large ensembles of the molecular motor myosin II working in coordination while attached to actin. Myosin II produces the power stroke, responsible for force generation. In this paper, we used High-Speed Atomic Force Microscopy (HS-AFM) to determine the potential cooperativity between myosin motors bound to non-regulated and regulated thin filaments. Based on the direct visualization of myosin-actin interaction, probability of myosin binding, and the myosin fractional occupancy of binding sites along non-regulated and regulated actin filaments, our results show no cooperative effects over [~]100 nm of the actin filament length. In contrast, there is myosin cooperativity within the activated thin filament, that induces a high affinity of myosin heads to the filaments. Our results support the independent behaviour of myosin heads while attached to actin filaments, but a cooperative behavior when attached to regulated thin filaments.

biophysics↗

bioRxiv2019

SKELETAL MyBP-C ISOFORMS TUNE THE MOLECULAR CONTRACTILITY OF DIVERGENT SKELETAL MUSCLE SYSTEMS

Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein; localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle-type specific manner. Are the expression, localization, and Ca2+-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague-Dawley rats? By mass spectrometry, four MyBP-C isoforms (one fast-type MyBP-C and three N-terminally spliced slow-type MyBP-C) were expressed in EDL but only the three slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that only in the C-zone, MyBP-C Ca2+-sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-Cs N-terminus, as N-terminal proteolysis attenuated MyBP-Cs functional capacities. To determine each MyBP-C isoforms contribution to thin filament Ca2+-sensitization and slowing in the C-zone, we used a combination of in vitro motility assays using expressed recombinant N-terminal fragments and in silico mechanistic modeling. Our results suggest that each skeletal MyBP-C isoforms N terminus is functionally distinct and has modulatory capacities that depend on the muscle-type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.\n\nSIGNIFICANCEMyosin-binding protein C (MyBP-C) is a critical component of the skeletal muscle sarcomere, muscles smallest contractile unit. MyBP-Cs importance is evident by genetic mutations leading to human myopathies, such as distal arthrogryposis (i.e. club foot). However, the molecular basis of MyBP-Cs functional impact on skeletal muscle contractility is far from certain. Complicating matters further is the expression of fast- and slow-type MyBP-C isoforms that depend on whether the muscle is fast- or slow-twitch. Using multi-scale proteomic, biophysical and mathematical modeling approaches, we define the expression, localization, and modulatory capacities of these distinct skeletal MyBP-C isoforms in rat skeletal muscles. Each MyBP-C isoform appears to modulate muscle contractility differentially; providing the capacity to fine-tune muscles mechanical performance as physiological demands arise.

physiology↗