Myopodin is an F-actin bundling protein with multiple independent actin-binding regions
Anja LinnemannPadmanabhan VakeelEduardo BezerraZacharias OrfanosKristina Djinović‐CarugoPeter F. M. van der VenGregor KirfelDieter O. Fürst
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Keywords:
Actin-binding protein
Myofilament
Actin remodeling
Actinin
Actina
Nebulin
MDia1
Myofibril
Tropomyosin
Actinin
Actina
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Gelsolin
MDia1
Actina
Actin-binding protein
Actinin
Actin remodeling
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N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. To discover more about the role of N-RAP in myofibril assembly, we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell. From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: alpha-actinin, filamin-2, and Krp1 (also called sarcosin). In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved. Three regions of N-RAP were expressed as His-tagged recombinant proteins, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal LIM domain (N-RAP-LIM), and the region of N-RAP in between the super repeat region and the LIM domain (N-RAP-IB). We detected significant alpha-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to co-localize with alpha-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly.
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Nebulin
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Lasp-2 binds to actin filaments and concentrates in the actin bundles of filopodia and lamellipodia in neural cells and focal adhesions in fibroblastic cells. Lasp-2 has three structural regions: a LIM domain, a nebulin-repeat region, and an SH3 domain; however, the region(s) responsible for its interactions with actin filaments and focal adhesions are still unclear. In this study, we revealed that the N-terminal fragment from the LIM domain to the first nebulin-repeat module (LIM-n1) retained actin-binding activity and showed a similar subcellular localization to full-length lasp-2 in neural cells. The LIM domain fragment did not interact with actin filaments or localize to actin filament bundles. In contrast, LIM-n1 showed a clear subcellular localization to filopodial actin bundles. Although truncation of the LIM domain caused the loss of F-actin binding activity and the accumulation of filopodial actin bundles, these truncated fragments localized to focal adhesions. These results suggest that lasp-2 interactions with actin filaments are mediated through the cooperation of the LIM domain and the first nebulin-repeat module in vitro and in vivo. Actin filament binding activity may be a major contributor to the subcellular localization of lasp-2 to filopodia but is not crucial for lasp-2 recruitment to focal adhesions.
Filopodia
Lamellipodium
LIM domain
Nebulin
MDia1
Actin remodeling
Actinin
Pseudopodia
Actin-binding protein
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The distribution of contractile and cytoskeletal proteins in smooth muscle has been mapped by immunocytochemical methods, with special reference to the localization of the actin-binding protein, filamin. Immunolabeling of ultrathin sections of polyvinylalcohol-embedded smooth muscle distinguished two domains in the smooth muscle cell: (a) actomyosin domains, made up of continuous longitudinal arrays of actin and myosin filaments, and (b) longitudinal, fibrillar, intermediate filament domains, free of myosin but containing actin and alpha-actinin-rich dense bodies. Filamin was found to be localized specifically in the latter intermediate filament-actin domains, but was excluded from the core of the dense bodies. Filamin was also localized close to the cell border at the inner surface of the plasmalemma-associated plaques. In isolated cells the surface filamin label showed a rib-like distribution similar to that displayed by vinculin. It is speculated that the two domains distinguished in these studies may reflect the existence of two functionally distinct systems: an actomyosin system required for contraction and an intermediate filament-actin system, with associated gelation proteins, that is responsible, at least in part, for the slow relaxation and tone peculiar to smooth muscle.
Actinin
Immunolabeling
Actina
Vinculin
Treadmilling
FLNA
Trachealis muscle
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Tropomyosin
Actinin
Actina
Alpha (finance)
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Actina
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We ascertained by the stopped flow method the overall association rate constant, k +1 , of filamin and α‐actinin to fluorescently labelled filamentous actin of ~ 1.3 × 10 6 M −1 · s −1 and ~ 1.0 × 10 6 M −1 · s −1 as well as the overall dissociation rate constant, k −1 of ~ 0.6s −1 and ~ 0.4s −1 , respectively. The overall equilibrium constant, K , for filamin and α‐actinin to actin deduced from the relation K = agree well with published data.
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Actina
Dissociation constant
Constant (computer programming)
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From the low salt-extracted debris of bovine stomach smooth muscle, a protein having a molecular mass of 60 kDa in SDS-PAGE was newly isolated. Co-sedimentation assay with actin filaments and several actin binding proteins such as filamin, alpha-actinin, caldesmon and fodrin showed that this protein co-sediments with actin only in the presence of filamin. Falling ball viscometric assay showed that this protein increases the viscosity of actin-filamin solution in a dose-dependent manner. Immunoblotting analysis showed specific localization of this protein in smooth and striated muscles.
Actin-binding protein
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The interaction between alpha-actinin and actin was further characterized using natural and synthetic peptides of actin together with anti-actin antibodies of known specificity. We demonstrated that two alpha-actinin binding sequences on actin are located within residues 112-125 and 360-372. Each peptide was shown to directly bind alpha-actinin and was able to dissociate the alpha-actinin-actin complex using solid phase binding assays and cosedimentation experiments. Taking into account the three-dimensional structure of actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37-44), we postulate that these two segments, proximal in the actin structure, are part of the same site. In addition, we compared these two segments with those recently found for filamin (Mejean, C., Lebart, M. C., Boyer, M., Roustan, C., and Benyamin, Y. (1992) Eur. J. Biochem. 209, 555-562), Egan, S., Stewart, M., Stossel, T. P., Kwiatkowski, D. J., and Hartwig, J. H. (1990) J. Cell Biol. 111, 1089-1105), and concluded that the two actin-binding proteins interact with closely spaced or overlapping but not identical sequences of actin subdomain 1.
Actin-binding protein
Actinin
Actina
Actin remodeling
Characterization
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