Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphodynamics

Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogeneous and micropatterned substrates. We find that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. The knockout of NM IIC has no detectable effects. A scale-bridging mathematical model explains our observations by relating actin fiber stability to the molecular rates of the myosin crossbridge cycle. We also find that NM IIA initiates and guides co-assembly of NM IIB into heterotypic minifilaments. We finally use mathematical modeling to explain the different exchange dynamics of NM IIA and B in minifilaments, as measured in FRAP experiments.

Effect of Mechanical Length Oscillations on Airways Smooth Muscle Reactivity and Crossbridge Cycling

Superimposition of length fluctuations on contracted ASM have shown to reduce active force and stiffness. This effect is usually attributed to disruption of the actomyosin crossbridge cycle; however no direct experimental data is available to support this hypothesis. This in vitro study investigated the effect of the mechanical strains on 1) the ASM reactivity and 2) on the actin-myosin crossbridges. Experiments were carried out on maximally contracted bovine ASM subjected to length strains at various frequency in the range from 10 to 100Hz, superimposed on normal tidal stretches (frequency 0.33Hz, amplitude 4%). An organ bath system was used to apply strains and measure the force; immunofluorescence technique was performed to assess the crossbridges. The results show that superimposed length strains increase breathing relaxation effect with an optimal effect obtained at 50Hz. The cholinergic stimulation promotes actin-myosin connection, and length stretches promote the detachment of those crossbridges.

Force suppression and the crossbridge cycle in swine carotid artery

Cyclic nucleotides can relax arterial smooth muscle without reductions in crossbridge phosphorylation, a process termed force suppression. There are two potential mechanisms for force suppression: 1) phosphorylated crossbridges binding to thin filaments could be inhibited or 2) the attachment of thin filaments to anchoring structures could be disrupted. These mechanisms were evaluated by comparing histamine-stimulated swine arterial smooth muscle with and without forskolin-induced force suppression and with and without latrunculin-A-induced actin filament disruption. At matched force, force suppression was associated with higher crossbridge phosphorylation and shortening velocity at low loads when compared with tissues without force suppression. Shortening velocity at high loads, noise temperature, hysteresivity, and stiffness did not differ with and without force suppression. These data suggest that crossbridge phosphorylation regulates the crossbridge cycle during force suppression. Actin disruption with latrunculin-A was associated with higher crossbridge phosphorylation when compared with tissues without actin disruption. Shortening velocity, noise temperature, hysteresivity, and stiffness did not differ with and without actin disruption. These data suggest that actin disruption interferes with regulation of crossbridge cycling by crossbridge phosphorylation. Stiffness was linearly dependent on stress, suggesting that the force per attached crossbridge was not altered with force suppression or actin disruption. These data suggest a difference in the mechanical characteristics observed during force suppression and actin disruption, implying that force suppression does not mechanistically involve actin disruption. These data are most consistent with a model where force suppression involves the inhibition of phosphorylated crossbridge binding to thin filaments.