Carol C Gregorio

Efficient muscle contraction requires regulation of actin filament lengths. In one highly cited model, the giant protein nebulin has been proposed to function as a molecular ruler specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of nebulin (mini-nebulin). When endogenous nebulin was replaced with mini-nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-nebulin were remarkably maintained at lengths either matching or longer than mini-nebulin. This indicates that mini-nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of nebulin also resulted in more dynamic populations of thin filament components, whereas expression of mini-nebulin decreased the dynamics at both filament ends (i.e., recovered loss of endogenous nebulin). Thus, nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.

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.

Desmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the desmin-E245D mutant in myocytes displaces endogenous desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated desmin mutations.

Sarcomeres of cardiac muscle are comprised of numerous proteins organized in an elegantly precise order. The exact mechanism of how these proteins are assembled into myofibrils during heart development is not yet understood, although existing in vitro and in vivo model systems have provided great insight into this complex process. It has been proposed by several groups that the giant elastic protein titin acts as a "molecular template" to orchestrate sarcomeric organization during myofibrillogenesis. Titin's highly modular structure, composed of both repeating and unique domains that interact with a wide spectrum of contractile and regulatory ligands, supports this hypothesis. Recent functional studies have provided clues to the physiological significance of the interaction of titin with several titin-binding proteins in the context of live cardiac cells. Improved models of cardiac myofibril assembly, along with the application of powerful functional studies in live cells, as well as the characterization of additional titin ligands, is likely to reveal surprising new functions for the titin third filament system.

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant "molecular ruler" nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160-164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160-164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.