Functional dissection of nebulette demonstrates actin binding of nebulin-like repeats and Z-line targeting of SH3 and linker domains

Abstract

Nebulette, a 107 kDa protein associated with the I‐Z‐I complex of cardiac myofibrils, may play an important role in the assembly of the Z‐line. Determination of the complete primary structure of 1011 residue human fetal nebulette reveals a four‐domain layout similar to skeletal muscle nebulin: a short N‐terminal domain, followed by 22 nebulin‐like repeats that are linked to a C‐terminal Src homology 3 (SH3) domain via a short linker domain. To elucidate the mechanisms of assembly for nebulette in the Z‐line, the complete coding sequence or fusions of nebulette domains with green fluorescent protein (GFP) were expressed in cardiomyocytes and fibroblasts. The complete protein localized to Z‐lines in cardiac cells and to dense bodies in nonmuscle cells. The GFP‐repeat domain forms bundles that are associated with actin filaments in both cell types and disrupts the microfilament network. In contrast, the GFP‐repeat plus linker shows limited interaction with dense bodies in nonmuscle cells and the Z‐lines of cardiomyocytes. Interestingly, the tagged linker or SH3 is diffusely distributed in nonmuscle cells, but localizes to the Z‐lines in cardiomyocytes. Supporting the cellular localization work, recombinant nebulette fragments bind to actin, tropomyosin, and α‐actinin in in vitro binding assays. These results suggest the repeat domain contains actin binding functions and that the linker domain may target this interaction to Z‐lines and dense bodies. Our data also indicate that the linker and SH3 domains can distinguish between dense bodies and Z‐lines, suggesting that the ligands for their interactions are specific to these muscular substructures. Cell Motil. Cytoskeleton 44:1–22, 1999. © 1999 Wiley‐Liss, Inc.

Interactions between Nebulin-like Motifs and Thin Filament Regulatory Proteins

“…This was in contrast to the MSN fragment, which was confined to the inclusion bodies upon large scale expression in E. coli (data not shown), similar to previously published data on mammalian nebulin fragments (6,8,24). A mouse polyclonal antiserum was generated using the purified CN5 protein as the immunogen.…”

“…Evidence has been mounted by studies showing the interaction of nebulin or nebulette fragments with F-actin (6,8), protein constituents of the Z-line (6,9), and with tropomodulin at the pointed ends of the thin filaments (11). Correlations between nebulin/nebulette expression and Z-line thickness as well as nebulin protein size and actin filament length in skeletal muscle sarcomeres (5,26) have all contributed to the large body of evidence in support of a structural role for nebulin/nebulette.…”

“…At the Z-line, the nebulin/nebulette interactions are probably mediated by contributions from the linker domain as well as interactions of the SH3 domains with ␣-actinin (6,9). However, results from Ojima and coworkers (10) suggest that neither the serine-rich linker domain nor the SH3 domain are obligatory elements for the incorporation of nebulin and presumably nebulette into the Z-line of striated muscle sarcomeres.…”

“…cDNA sequences of nebulin and nebulette have shown that both proteins are primarily composed of ϳ35 amino acid repeats termed nebulin-like repeats or motifs, each marked by a conserved SXXXY sequence (2,3). Both nebulin and nebulette are capped by a unique region at the amino terminus in addition to serine-rich linker and SH3 domains at the carboxyl terminus (2)(3)(4)(5)(6). Immunoelectron microscopy has demonstrated that a single nebulin molecule extends from the Z-line (COOH terminus of the protein) to the pointed ends of the thin filament at its NH 2 terminus (7).…”

“…Experiments using in vitro systems have formed a consensus that nebulin-like motifs from both nebulin and nebulette associate with F-actin (4,6,8). Additional experiments have extended this observation to demonstrate that nebulin-like motifs may interact directly with tropomyosin (Tm) 1 and troponin (Tn) (4) along the striated muscle thin filament.…”