Generation of force and movement by actomyosin cross-bridges is the molecular basis of muscle contraction, but generally accepted ideas about cross-bridge properties have recently been questioned. Of the utmost significance, evidence for nonlinear cross-bridge elasticity has been presented. We here investigate how this and other newly discovered or postulated phenomena would modify cross-bridge operation, with focus on post-power-stroke events. First, as an experimental basis, we present evidence for a hyperbolic [MgATP]-velocity relationship of heavy-meromyosin-propelled actin filaments in the in vitro motility assay using fast rabbit skeletal muscle myosin (28-29°C). As the hyperbolic [MgATP]-velocity relationship was not consistent with interhead cooperativity, we developed a cross-bridge model with independent myosin heads and strain-dependent interstate transition rates. The model, implemented with inclusion of MgATP-independent detachment from the rigor state, as suggested by previous single-molecule mechanics experiments, accounts well for the [MgATP]-velocity relationship if nonlinear cross-bridge elasticity is assumed, but not if linear cross-bridge elasticity is assumed. In addition, a better fit is obtained with load-independent than with load-dependent MgATP-induced detachment rate. We discuss our results in relation to previous data showing a nonhyperbolic [MgATP]-velocity relationship when actin filaments are propelled by myosin subfragment 1 or full-length myosin. We also consider the implications of our results for characterization of the cross-bridge elasticity in the filament lattice of muscle.
Semiconductor nanowire arrays offer significant potential for biosensing applications with optical read-out due to their high surface area and due to the unique optical properties of one-dimensional materials. A challenge for optical read-out of analyte-binding to the nanowires is the need to efficiently collect and detect light from a three-dimensional volume. Here we show that light from fluorophores attached along several μm long vertical Al2O3 coated gallium phosphide nanowires couples into the wires, is guided along them and emitted at the tip. This enables effective collection of light emitted by fluorescent analytes located at different focal planes along the nanowire. We unequivocally demonstrate the light-guiding effect using a novel method whereby the changes in emitted fluorescence intensity are observed when fluorescent cytoskeletal filaments are propelled by molecular motors along the wires. The findings are discussed in relation to nanobiosensor developments, other nanotechnological applications, and fundamental studies of motor function.
Portable biosensor systems would benefit from reduced dependency on external power supplies as well as from further miniaturization and increased detection rate. Systems built around self-propelled biological molecular motors and cytoskeletal filaments hold significant promise in these regards as they are built from nanoscale components that enable nanoseparation independent of fluidic pumping. Previously reported microtubule-kinesin based devices are slow, however, compared to several existing biosensor systems. Here we demonstrate that this speed limitation can be overcome by using the faster actomyosin motor system. Moreover, due to lower flexural rigidity of the actin filaments, smaller features can be achieved compared to microtubule-based systems, enabling further miniaturization. Using a device designed through optimization by Monte Carlo simulations, we demonstrate extensive myosin driven enrichment of actin filaments on a detector area of less than 10 μm², with a concentration half-time of approximately 40 s. We also show accumulation of model analyte (streptavidin at nanomolar concentration in nanoliter effective volume) detecting increased fluorescence intensity within seconds after initiation of motor-driven transportation from capture regions. We discuss further optimizations of the system and incorporation into a complete biosensing workflow.
BackgroundWhile overall physical activity is clearly associated with a better short-term and long-term health, prolonged strenuous physical activity may result in a rise in acute levels of blood-biomarkers used in clinical practice for diagnosis of various conditions or diseases. In this study, we explored the acute effects of a full Ironman-distance triathlon on biomarkers related to heart-, liver-, kidney- and skeletal muscle damage immediately post-race and after one week’s rest. We also examined if sex, age, finishing time and body composition influenced the post-race values of the biomarkers.MethodsA sample of 30 subjects was recruited (50% women) to the study. The subjects were evaluated for body composition and blood samples were taken at three occasions, before the race (T1), immediately after (T2) and one week after the race (T3). Linear regression models were fitted to analyse the independent contribution of sex and finishing time controlled for weight, body fat percentage and age, on the biomarkers at the termination of the race (T2). Linear mixed models were fitted to examine if the biomarkers differed between the sexes over time (T1-T3).ResultsBeing male was a significant predictor of higher post-race (T2) levels of myoglobin, CK, and creatinine levels and body weight was negatively associated with myoglobin. In general, the models were unable to explain the variation of the dependent variables. In the linear mixed models, an interaction between time (T1-T3) and sex was seen for myoglobin and creatinine, in which women had a less pronounced response to the race.ConclusionOverall women appear to tolerate the effects of prolonged strenuous physical activity better than men as illustrated by their lower values of the biomarkers both post-race as well as during recovery.
Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm−1). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.
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