Polarization-resolved microscopy reveals a muscle myosin motor-independent mechanism of molecular actin ordering during sarcomere maturation

Abstract

Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.

Myofibril and mitochondria morphogenesis are coordinated by a mechanical feedback mechanism in muscle

“…7c,d). This finding is also supported by electron microscopy data that found mitochondria present between assembled myofibrils at 32 h APF 32 . This shows that mitochondria and myofibrils are present in close proximity directly after myofibrils have been assembled, suggesting a potential role of mitochondrial intercalation for fibrillar flight muscle morphogenesis.…”

“…Transport could be achieved by microtubule motors, since they have been described to transport mitochondria in various other cell types, particularly in neurons 36,37 . Interestingly, microtubules have been found in close proximity to the freshly assembled myofibrils in flight muscles 32,38 and hence ideally placed to mediate mitochondria intercalation and alignment with the myofibrils.…”

Flow-independent accumulation of motor-competent non-muscle myosin II in the contractile ring is essential for cytokinesis

“…S1C). This suggests that myosin motor activity is not required for actin organization in adult muscles, which agrees with recent findings in D. melanogaster (Loison et al, 2018) . We conclude that mutating the highly conserved residues S240 and R241 results in non-functional UNC-54 in vivo , in agreement with the effects of the mutations on myosin motor activity reported in vitro for D. discoideum non-muscle myosin II.…”

The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover

“…Under these fixation and staining conditions, the error in Ψ and ρ is only ∼2° (Kress et al, 2013). Previous studies on sarcomeres and contractile actin rings using polarization-resolved microscopy validated that a 5–10° change in Ψ corresponds to a major changes in F-actin organization (Mavrakis et al, 2014; Loison et al, 2018).…”