Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB.

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II paralogs A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modeling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between paralogs A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-localizes with pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two paralogs form a balanced system for regulated force generation.

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 homogenously coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals 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. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.

Mechanosensitive self-assembly of myosin II minifilaments.

Self-assembly and force generation are two central processes in biological systems that usually are considered in separation. However, the signals that activate nonmuscle myosin II molecular motors simultaneously lead to self-assembly into myosin II minifilaments as well as progression of the motor heads through the cross-bridge cycle. Here we investigate theoretically the possible effects of coupling these two processes. Our assembly model, which builds on a consensus architecture of the minifilament, predicts a critical aggregation concentration at which the assembly kinetics slows down dramatically. The combined model predicts that increasing actin filament concentration and force both lead to a decrease in the critical aggregation concentration. We suggest that due to these effects, myosin II minifilaments in a filamentous context might be in a critical state that reacts faster to varying conditions than in solution. We finally compare our model to experiments by simulating fluorescence recovery after photobleaching.