Leiomodin 3 and tropomodulin 4 have overlapping functions during skeletal myofibrillogenesis.

Precise regulation of thin filament length is essential for optimal force generation during muscle contraction. The thin filament capping protein tropomodulin (Tmod) contributes to thin filament length uniformity by regulating elongation and depolymerization at thin filament ends. The leiomodins (Lmod1-3) are structurally related to Tmod1-4 and also localize to actin filament pointed ends, but in vitro biochemical studies indicate that Lmods act instead as robust nucleators. Here, we examined the roles of Tmod4 and Lmod3 during Xenopus skeletal myofibrillogenesis. Loss of Tmod4 or Lmod3 resulted in severe disruption of sarcomere assembly and impaired embryonic movement. Remarkably, when Tmod4-deficient embryos were supplemented with additional Lmod3, and Lmod3-deficient embryos were supplemented with additional Tmod4, sarcomere assembly was rescued and embryonic locomotion improved. These results demonstrate for the first time that appropriate levels of both Tmod4 and Lmod3 are required for embryonic myofibrillogenesis and, unexpectedly, both proteins can function redundantly during in vivo skeletal muscle thin filament assembly. Furthermore, these studies demonstrate the value of Xenopus for the analysis of contractile protein function during de novo myofibril assembly.

Preparation of developing Xenopus muscle for sarcomeric protein localization by high-resolution imaging.

Mutations in several sarcomeric proteins have been linked to various human myopathies. Therefore, having an in vivo developmental model available that develops quickly and efficiently is key for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; this is the pivotal foundation for deciphering disease mechanisms as well as the development of myopathy-related therapeutics. Although striated muscle cell culture studies have been extremely informative in providing clues to both the distribution and functions of sarcomeric proteins, myocytes in vivo develop in an irreproducible 3D environment. Xenopus laevis (frog) embryos are cost effective, compliant to protein level manipulations and develop relatively quickly (⩽ a week) in a petri dish, thus providing a powerful system for de novo myofibrillogenesis studies. Although fluorophore-conjugated phalloidin labeling is the gold standard approach for investigating actin-thin filament architecture, it is well documented that phalloidin-labeling can be challenging and inconsistent within Xenopus embryos. Therefore we highlight several techniques that can be utilized to preserve both antibody and fluorophore-conjugated phalloidin labeling within Xenopus embryos for high-resolution fluorescence microscopy.