Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

Abstract

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. INTRODUCTIONIn striated muscle, actin-containing thin filaments from adjacent sarcomeres overlap within the Z-disc in which their barbed ends are organized and anchored. Electron micrographs of longitudinal sections of mammalian skeletal muscle reveal that Z-discs contain an intricate network of "zigzag" bands (Rowe, 1973). Three-dimensional reconstruction and modeling of the Z-disc based on electron micrographs demonstrate that the zigzag bands are composed of sets of overlapping thin filament connectors called "Z-links," which are predicted to be composed of ␣-actinin (Luther et al., 2002). These connectors allow the Z-disc to transmit force from one sarcomere to the next along the myofibril.Drosophila melanogaster mutants that do not express ␣-actinin initially display relatively intact Z-discs in their striated muscle (Fyrberg et al., 1998). Later, severe muscle defects occur, and the larvae die. Thus, it seems that ␣-actinin is not absolutely required for Z-disc formation and function, but it is needed to maintain Z-disc stability in this organism. The giant sarcomeric protein titin has also been implicated in the assembly and maintenance of the Z-disc structure, whereas the specific contributions of other Z-disc components are currently unknown (Zou et al., 2006;Seeley et al., 2007).Z-discs contain the barbed-end capping protein CapZ. CapZ binds with high affinity (K d Ϸ 1 nM) to the barbed ends of actin filaments, in which it effectively inhibits actin polymerization and depolymerization (Caldwell et al., 1989). Capping protein is an obligate ␣/␤ heterodimer, and efficient actin capping requires the C terminus of both subunits (Casella and Torres, 1994;Wear et al., 2003). Vertebrates express three conserved isoforms of ea...

Nebulin Alters Cross-bridge Cycling Kinetics and Increases Thin Filament Activation

“…The length of the thin filament is an important functional parameter because it determines the degree of overlap between thin and thick filaments, and thereby sets the level of maximum force that a muscle can develop at a given SL. Several recent studies (8,22) have provided experimental evidence to show that nebulin specifies thin filament length via its unique N-and C-terminal sequences. The N-terminal end of nebulin interacts with tropomodulin and the C-terminal end with CapZ, both of which are thought to play a role in specifying the length of F-actin (22,23).…”

“…Several recent studies (8,22) have provided experimental evidence to show that nebulin specifies thin filament length via its unique N-and C-terminal sequences. The N-terminal end of nebulin interacts with tropomodulin and the C-terminal end with CapZ, both of which are thought to play a role in specifying the length of F-actin (22,23). Although details of the mechanism(s) of thin filament length regulation remain to be established (24), that nebulin is a major player in this process is suggested by studies on NEB KO mice (11,12) in which it was found that thin filament length is variable and on average shorter than in WT mice.…”

Nebulin: A Study of Protein Repeat Evolution

“…The recently duplicated C-terminal region in the human Nebulin gene, located inside the Z-disc, 27 is involved in binding desmin and is also a region of Nebulin that is known to be involved in different isoforms expressed in different muscle tissue types. 28 Although the location of the alternative splicing sites of nebulin is not known for most of the organisms, our results indicate that some organisms have undergone acquisition of new alternatively spliced exons in the recent past.…”

The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse

“…Within the Z-line, nebulin C-terminal modules interact with the intermediate filament desmin Tonino et al, 2010) as well as the actin barbed-end capping protein CapZ (Pappas et al, 2008). Furthermore, the extreme C-terminal end of nebulin contains a serine-rich region with several predicted phosphorylation sites preceding an Src homology 3 (SH3) domain.…”