UNC-87 Is an Actin-bundling Protein

Abstract

The Caenorhabditis elegans unc-87 gene product is essential for the maintenance of the nematode body wall muscle where it is found colocalized with actin in the I band. The molecular domain structure of the protein reveals similarity to the C-terminal repeat region of the smooth muscle actin-binding protein calponin. In this study we investigated the in vitro function of UNC-87 using both the full-length recombinant molecule and several truncated mutants. According to analytical ultracentrifugation UNC-87 occurs as a monomer in solution. UNC-87 cosedimented with both smooth and skeletal muscle F-actin, but not with monomeric G-actin, and exhibited potent actin filament bundling activity. Actin binding was independent of the presence of tropomyosin and the actin cross-linking proteins filamin and ␣-actinin. Consistent with its actin bundling activity in vitro, UNC-87 tagged with green fluorescent protein associated with and promoted the formation of actin stress fiber bundles in living cells. These data identify UNC-87 as an actin-bundling protein and highlight the calponin-like repeats as a novel actin-binding module.The interaction of actin and myosin to produce force is an essential prerequisite for a variety of cellular processes including muscle contraction (1), cell motility, and anchorage (2). The organization of contractile and motile systems based on actin relies on a large family of actin-associated proteins that regulate and define the assembly of actin into filaments and then into filament arrays (3, 4). To date, more than 60 different proteins directly interacting with actin have been identified, but the majority of F-actin-binding proteins populate partially overlapping regions on the filament (5, 6). Despite the large number of actin-binding proteins, functional diversity is reflected by a limited number of basic structural modules (7). Most actin cross-linking proteins exhibit two independent actin-binding domains, each individual actin-binding domain commonly composed of a tandem arrangement of the calponin homology domain module (8) and other modular elements defining the distance between and the relative orientation of the two actin-binding domains, often involving parallel or antiparallel dimerization (7).We have shown recently that a unique sequence motif found in the C-terminal third of the calponin (CaP) 1 molecule and other members of the CaP family of actin-associated proteins (9), namely a 23-amino acid residue repeat, which we will refer to from now on as the CLIK-23 repeat, forms an independent actin-binding site (10). This finding was corroborated by Mino et al. (11) who demonstrated the direct interaction of a peptide corresponding to the first CaP repeat with actin in vitro. A survey of the available data bases identified other proteins with CLIK-23 repeats, in particular the Caenorhabditis elegans body wall muscle protein UNC-87 that exhibits seven tandem CLIK-23 repeats (12). A protein with a similar molecular structure has also been described by Irvine et al. (13) in the filar...

UNC-87, a calponin-related protein in C. elegans, antagonizes ADF/cofilin-mediated actin filament dynamics

“…Recombinant UNC-87 and turkey smooth muscle tropomyosin have been shown to simultaneously bind to Factin in vitro (Kranewitter et al, 2001), and we confirmed that UNC-87 and CeTM bind to C. elegans actin (Ce-actin) in a similar manner (Fig. 4A).…”

“…Although UNC-87 bundles actin filaments in vitro (Kranewitter et al, 2001), this bundling activity is not required for the actin-stabilizing effect. Immobilized actin filaments, which are decorated but not bundled by UNC-87, are still protected from severing by ADF/cofilin (Fig.…”

“…The monomeric UNC-87 protein is capable of bundling actin filaments (Kranewitter et al, 2001), suggesting that multiple actin-binding sites are present in a single molecule. When we tested the binding of 1.0-20 M UNC-87 to 10 M F-actin by the co-sedimentation assay, binding did not reach saturation within this concentration range (Fig.…”

“…UNC-87 is a calponinrelated protein with seven calponin-like (CLIK) repeats, but it lacks a calponin-homology (CH) domain, which is present in the calponin and SM22/transgelin families of actin binding proteins (Gimona et al, 2002). UNC-87 localizes to the thin filaments (Goetinck and Waterston, 1994a) and directly binds to actin filaments in vitro (Kranewitter et al, 2001). However, the precise mode of actin binding and the nature of the actin stabilizing function of UNC-87 are not clearly understood.…”

The identification of a new actin-binding region in p57

“…p57 297-461 without GST tag was used to cross-link pre-assembled actin filaments and a low speed co-sedimentation assay was utilized to test the effect of cross-linking [23]. At low speed (15,000 × g), neither F-actin nor p57 297-461 alone was pelleted upon centrifugation ( Figure 5).…”