Emergence of Xin demarcates a key innovation in heart evolution

The mouse Xin repeat-containing proteins (mXinalpha and mXinbeta) localize to the intercalated disc in the heart. mXinalpha is able to bundle actin filaments and to interact with beta-catenin, suggesting a role in linking the actin cytoskeleton to N-cadherin/beta-catenin adhesion. mXinalpha-null mouse hearts display progressively ultrastructural alterations at the intercalated discs, and develop cardiac hypertrophy and cardiomyopathy with conduction defects. The up-regulation of mXinbeta in mXinalpha-deficient mice suggests a partial compensation for the loss of mXinalpha. To elucidate the evolutionary relationship between these proteins and to identify the origin of Xin, a phylogenetic analysis was done with 40 vertebrate Xins. Our results show that the ancestral Xin originated prior to the emergence of lamprey and subsequently underwent gene duplication early in the vertebrate lineage. A subsequent teleost-specific genome duplication resulted in most teleosts encoding at least three genes. All Xins contain a highly conserved beta-catenin-binding domain within the Xin repeat region. Similar to mouse Xins, chicken, frog and zebrafish Xins also co-localized with beta-catenin to structures that appear to be the intercalated disc. A putative DNA-binding domain in the N-terminus of all Xins is strongly conserved, whereas the previously characterized Mena/VASP-binding domain is a derived trait found only in Xinalphas from placental mammals. In the C-terminus, Xinalphas and Xinbetas are more divergent relative to each other but each isoform from mammals shows a high degree of within-isoform sequence identity. This suggests different but conserved functions for mammalian Xinalpha and Xinbeta. Interestingly, the origin of Xin ca. 550 million years ago coincides with the genesis of heart chambers with complete endothelial and myocardial layers. We postulate that the emergence of the Xin paralogs and their functional differentiation may have played a key role in the evolutionary development of the heart.     

The LIM domains of WLIM1 define a new class of actin bundling modules

Actin filament bundling, i.e. the formation of actin cables, is an important process that relies on proteins able to directly bind and cross-link subunits of adjacent actin filaments. Animal cysteine-rich proteins and their plant counterparts are two LIM domain-containing proteins that were recently suggested to define a new family of actin cytoskeleton regulators involved in actin filament bundling. We here identified the LIM domains as responsible for F-actin binding and bundling activities of the tobacco WLIM1. The deletion of one of the two LIM domains reduced significantly, but did not entirely abolish, the ability of WLIM1 to bind actin filaments. Individual LIM domains were found to interact directly with actin filaments, although with a reduced affinity compared with the native protein. Variants lacking the C-terminal or the inter-LIM domain were only weakly affected in their F-actin stabilizing and bundling activities and trigger the formation of thick cables containing tightly packed actin filaments as does the native protein. In contrast, the deletion of one of the two LIM domains negatively impacted both activities and resulted in the formation of thinner and wavier cables. In conclusion, we demonstrate that the LIM domains of WLIM1 are new autonomous actin binding and bundling modules that cooperate to confer WLIM1 high actin binding and bundling activities.     

Loss of mXinalpha, an intercalated disk protein, results in cardiac hypertrophy and cardiomyopathy with conduction defects

The intercalated disk protein Xin was originally discovered in chicken striated muscle and implicated in cardiac morphogenesis. In the mouse, there are two homologous genes, mXinalpha and mXinbeta. The human homolog of mXinalpha, Cmya1, maps to chromosomal region 3p21.2-21.3, near a dilated cardiomyopathy with conduction defect-2 locus. Here we report that mXinalpha-null mouse hearts are hypertrophied and exhibit fibrosis, indicative of cardiomyopathy. A significant upregulation of mXinbeta likely provides partial compensation and accounts for the viability of the mXinalpha-null mice. Ultrastructural studies of mXinalpha-null mouse hearts reveal intercalated disk disruption and myofilament disarray. In mXinalpha-null mice, there is a significant decrease in the expression level of p120-catenin, beta-catenin, N-cadherin, and desmoplakin, which could compromise the integrity of the intercalated disks and functionally weaken adhesion, leading to cardiac defects. Additionally, altered localization and decreased expression of connexin 43 are observed in the mXinalpha-null mouse heart, which, together with previously observed abnormal electrophysiological properties of mXinalpha-deficient mouse ventricular myocytes, could potentially lead to conduction defects. Indeed, ECG recordings on isolated, perfused hearts (Langendorff preparations) show a significantly prolonged QT interval in mXinalpha-deficient hearts. Thus mXinalpha functions in regulating the hypertrophic response and maintaining the structural integrity of the intercalated disk in normal mice, likely through its association with adherens junctional components and actin cytoskeleton. The mXinalpha-knockout mouse line provides a novel model of cardiac hypertrophy and cardiomyopathy with conduction defects.     

Modulation of actin-bundling activity of 55-kDa protein by multiple isoforms of tropomyosin

Cultured rat cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J.J.-C. (1983) J. Biol. Chem. 258, 6636-6644). To explore the roles of the multiple tropomyosin isoforms in the microfilament organization of cultured cells, we have examined effects of tropomyosins on the bundling activity of the 55-kDa protein recently purified from HeLa cells (Yamashiro-Matsumura, S., and Matsumura, F. (1985) J. Biol. Chem. 260, 5087-5097). Maximum bundling of F-actin was observed at a molar ratio of 55-kDa protein to actin higher than 1:8. None of the isoforms of cultured rat cell tropomyosin significantly altered the F-actin-bundling activity of 55-kDa protein at this ratio, whereas skeletal muscle tropomyosin inhibited the bundling activity to about 50%. Also, cultured cell tropomyosins did not inhibit binding of 55-kDa protein to actin, whereas skeletal muscle tropomyosin inhibited it by 50%. The effect of 55-kDa protein on the binding of tropomyosin to actin varied with the isoform type of tropomyosin. Most (80%) of the tropomyosins with low Mr values (Mr 32,400 or 32,000) were caused to dissociate from actin by 55-kDa protein, but only 20% of tropomyosins with high Mr values (Mr 40,000 or 36,500) was dissociated from actin in these conditions. Immunofluorescence has shown that, while tropomyosin was localized in stress fibers, 55-kDa protein was found in microspikes as well as stress fibers, both of which are known to contain bundles of microfilaments. Therefore, we suggest that 55-kDa protein together with the multiple tropomyosin isoforms may regulate the formation of two types of actin-filament bundles, bundles containing tropomyosin and those without tropomyosin.     

Myosin binding protein C interaction with actin: characterization and mapping of the binding site

Myosin binding protein C (MyBPC) is a multidomain protein associated with the thick filaments of striated muscle. Although both structural and regulatory roles have been proposed for MyBPC, its interactions with other sarcomeric proteins remain obscure. The current study was designed to examine the actin-binding properties of MyBPC and to define MyBPC domain regions involved in actin interaction. Here, we have expressed full-length mouse cardiac MyBPC (cMyBPC) in a baculovirus system and shown that purified cMyBPC binds actin filaments with an affinity of 4.3 ± 1.1 μM and a 1:1 molar ratio with regard to an actin protomer. The actin binding by cMyBPC is independent of protein phosphorylation status and is not significantly affected by the presence of tropomyosin and troponin on the actin filament. In addition, cMyBPC-actin interaction is not modulated by calmodulin. To determine the region of cMyBPC that is responsible for its interaction with actin, we have expressed and characterized five recombinant proteins encoding fragments of the cMyBPC sequence. Recombinant N-terminal fragments such as C0-C1, C0-C4, and C0-C5 cosediment with actin in a linear, nonsaturable manner. At the same time, MyBPC fragments lacking either the C0-C1 or C0-C4 region bind F-actin with essentially the same properties as full-length protein. Together, our results indicate that cMyBPC interacts with actin via a single, moderate affinity site localized to the C-terminal region of the protein. In contrast, certain basic regions of the N-terminal domains of MyBPC may act as small polycations and therefore bind actin via nonspecific electrostatic interactions.     

Identification of a Novel Actin-Binding Domain within the Rho Guanine Nucleotide Exchange Factor TEM4

Spatio-temporal activation of Rho GTPases is essential for their function in a variety of biological processes and is achieved in part by regulating the localization of their activators, the Rho guanine nucleotide exchange factors (RhoGEFs). In this study, we provide the first characterization of the full-length protein encoded by RhoGEF TEM4 and delineate its domain structure, catalytic activity, and subcellular localization. First, we determined that TEM4 can stimulate guanine nucleotide exchange on RhoA and the related RhoB and RhoC isoforms. Second, we determined that TEM4, like other Dbl RhoGEFs, contains a functional pleckstrin homology (PH) domain immediately C-terminal to the catalytic Dbl homology (DH) domain. Third, using immunofluorescence analysis, we showed that TEM4 localizes to the actin cytoskeleton through sequences in the N-terminus of TEM4 independently of the DH/PH domains. Using site-directed mutagenesis and deletion analysis, we identified a minimal region between residues 81 and 135 that binds directly to F-actin and has an ～90-fold higher affinity for ATP-loaded F-actin. Finally, we demonstrated that a single point mutation (R130D) within full-length TEM4 abolishes actin binding and localization of TEM4 to the actin cytoskeleton, as well as dampens the in vivo activity of TEM4 towards RhoC. Taken together, our data demonstrate that TEM4 contains a novel actin binding domain and binding to actin is essential for TEM4 subcellular localization and activity. The unique subcellular localization of TEM4 suggests a spatially-restricted activity and expands the diversity of mechanisms by which RhoGEF function can be regulated.     

Domain analysis of cortexillin I: actin-bundling, PIP(2)-binding and the rescue of cytokinesis

Cortexillins are actin-bundling proteins that form a parallel two-stranded coiled-coil rod. Actin-binding domains of the alpha-actinin/spectrin type are located N-terminal to the rod and unique sequence elements are found in the C-terminal region. Domain analysis in vitro revealed that the N-terminal domains are not responsible for the strong actin-filament bundling activity of cortexillin I. The strongest activity resides in the C-terminal region. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) suppresses this bundling activity by binding to a C-terminal nonapeptide sequence. These data define a new PIP(2)-regulated actin-bundling site. In vivo the PIP(2)-binding motif enhances localization of a C-terminal cortexillin I fragment to the cell cortex and improves the rescue of cytokinesis. This motif is not required, however, for translocation to the cleavage furrow. A model is presented proposing that cortexillin translocation is based on a mitotic cycle of polar actin polymerization and midzone depolymerization.     

Molecular and biochemical characterization of a novel actin bundling protein in Acanthamoeba

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 microM of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.     

Phosphoinositide binding inhibits alpha-actinin bundling activity

alpha-Actinin is an abundant actin-bundling and adhesion protein that directly links actin filaments to integrin receptors. Previously, in platelet-derived growth factor-treated fibroblasts, we demonstrated that phosphoinositides bind to alpha-actinin, regulating its localization (Greenwood, J. A., Theibert, A. B., Prestwich, G. D., and Murphy-Ullrich, J. E. (2000) J. Cell Biol. 150, 627- 642). In this study, phosphoinositide binding and regulation of alpha-actinin function is further characterized. Phosphoinositide binding specificity, determined using a protein-lipid overlay procedure, suggests that alpha-actinin interacts with phosphates on the 4th and 5th position of the inositol head group. Binding assays and mutational analyses demonstrate that phosphoinositides bind to the calponin homology domain 2 of alpha-actinin. Phosphoinositide binding inhibited the bundling activity of alpha-actinin by blocking the interaction of the actin-binding domain with actin filaments. Consistent with these results, excessive bundling of actin filaments was observed in fibroblasts expressing an alpha-actinin mutant with decreased phosphoinositide affinity. We conclude that the interaction of alpha-actinin with phosphoinositides regulates actin stress fibers in the cell by controlling the extent to which microfilaments are bundled.     

Membrane localization of adenomatous polyposis coli protein at cellular protrusions: targeting sequences and regulation by beta-catenin

Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo- (binds KAP3A, B56alpha, and ASEF) and beta-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of beta-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of beta-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of beta-catenin reduced APC at membrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with beta-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only beta-catenin stimulated APC anchorage at the membrane. Our findings identify beta-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.     

F-actin and myosin II binding domains in supervillin

Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids approximately 280-342, approximately 344-422, and approximately 700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains.     

Beta-catenin N- and C-terminal tails modulate the coordinated binding of adherens junction proteins to beta-catenin

beta-Catenin plays a central role in the establishment and regulation of adherens junctions because it interacts with E-cadherin and, through alpha-catenin, with the actin cytoskeleton. beta-Catenin is composed of three domains: a central armadillo repeat domain and two N- and C-terminal tails. The C-tail interacts with the armadillo domain and limits its ability to bind E-cadherin and other cofactors. The two beta-catenin tails are mutually inter-regulated because the C-tail is also necessary for binding of the N-tail to the armadillo domain. Moreover, the N-tail restricts the interaction of the C-tail with the central domain. Depletion of either of the two tails has consequences for the binding of factors at the other end: deletion of the C-tail increases alpha-catenin binding, whereas deletion of the N-tail blocks E-cadherin interaction to the armadillo repeats. As an effect of the interconnection of the tails, the association of alpha-catenin and E-cadherin to beta-catenin is interdependent. Thus, binding of alpha-catenin to the N-tail, through conformational changes that affect the C-tail, facilitates the association of E-cadherin. These results indicate that different cofactors of beta-catenin bind coordinately to this protein and indicate how the two terminal ends of beta-catenin exquisitely modulate intermolecular binding within junctional complexes.     

Glucocorticoid down-regulation of fascin protein expression is required for the steroid-induced formation of tight junctions and cell-cell interactions in rat mammary epithelial tumor cells

Glucocorticoid hormones, which are physiological regulators of mammary epithelium development, induce the formation of tight junctions in rat Con8 mammary epithelial tumor cells. We have discovered that, as part of this process, the synthetic glucocorticoid dexamethasone strongly and reversibly down-regulated the expression of fascin, an actin-bundling protein that also interacts with the adherens junction component beta-catenin. Ectopic constitutive expression of full-length mouse fascin containing a Myc epitope tag (Myc-fascin) in Con8 cells inhibited the dexamethasone stimulation of transepithelial electrical resistance, disrupted the induced localization of the tight junction protein occludin and the adherens junction protein beta-catenin to the cell periphery, and prevented the rearrangement of the actin cytoskeleton. Ectopic expression of either the carboxyl-terminal 213 amino acids of fascin, which includes the actin and beta-catenin-binding sites, or the amino-terminal 313 amino acids of fascin failed to disrupt the glucocorticoid induction of tight junction formation. Mammary tumor cells expressing the full-length Myc-fascin remained generally glucocorticoid responsive and displayed no changes in the levels or protein-protein interactions of junctional proteins or the amount of cytoskeletal associated actin filaments. However, a cell aggregation assay demonstrated that the expression of Myc-fascin abrogated the dexamethasone induction of cell-cell adhesion. Our results implicate the down-regulation of fascin as a key intermediate step that directly links glucocorticoid receptor signaling to the coordinate control of junctional complex formation and cell-cell interactions in mammary tumor epithelial cells.     

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin.

Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. We have isolated here a GSK-3beta-interacting protein from a rat brain cDNA library using a yeast two-hybrid method. This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and dishevelled (Dsh) homologous domains in its N- and C-terminal regions, respectively. The predicted amino acid sequence of this GSK-3beta-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, we termed this protein rAxin (rat Axin). rAxin interacted directly with, and was phosphorylated by, GSK-3beta. rAxin also interacted directly with the armadillo repeats of beta-catenin. The binding site of rAxin for GSK-3beta was distinct from the beta-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promoted GSK-3beta-dependent phosphorylation of beta-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3beta and beta-catenin and mediating the signal from GSK-3beta to beta-catenin.     

Microtubule bundling by tau proteins in vivo: analysis of functional domains.

Tau varies both in the N-terminal region (three types) and in the C-terminal repeated microtubule binding domain (two types), generating six isoforms through alternative splicing. To understand the differences between the isoforms and to determine which domains are important for microtubule bundling, we performed transfection studies on fibroblasts using tau isoforms and deletion mutants to quantify their ability to bundle microtubules. By comparing the isoforms, we found that a longer N-terminal region induced microtubule bundling more efficiently, but changes in the microtubule binding domain did not. Mutants lacking the proline rich region or the repeated domain did not bind to microtubules. Although all the other mutants could bind to and bundle microtubules, deletion in the N-terminal neutral region or the first half of the C-terminal tail caused a significant decrease in microtubule bundling, indicating the importance of these regions in microtubule bundling.     

Properties of long myosin light chain kinase binding to F-actin in vitro and in vivo

Short and long myosin light chain kinases (MLCKs) are Ca(2+)/calmodulin-dependent enzymes that phosphorylate the regulatory light chain of myosin II in thick filaments but bind with high affinity to actin thin filaments. Three repeats of a motif made up of the sequence DFRXXL at the N terminus of short MLCK are necessary for actin binding (Smith, L., Su, X., Lin, P., Zhi, G., and Stull, J. T. (1999) J. Biol. Chem. 274, 29433-29438). The long MLCK has two additional DFRXXL motifs and six Ig-like modules in an N-terminal extension, which may confer unique binding properties for cellular localization. Two peptides containing either five or three DFRXXL motifs bound to F-actin and smooth muscle myofilaments with maximal binding stoichiometries consistent with each motif binding to an actin monomer in the filaments. Both peptides cross-linked F-actin and bound to stress fibers in cells. Long MLCK with an internal deletion of the five DFRXXL motifs and the unique NH(2)-terminal fragment containing six Ig-like motifs showed weak binding. Cell fractionation and extractions with MgCl(2) indicate that the long MLCK has a greater affinity for actin-containing filaments than short MLCK in vitro and in vivo. Whereas DFRXXL motifs are necessary and sufficient for short MLCK binding to actin-containing filaments, the DFRXXL motifs and the N-terminal extension of long MLCK confer high affinity binding to stress fibers in cells.     

The C-terminal aqueous-exposed domain of the 45 kDa subunit of the particulate methane monooxygenase in Methylococcus capsulatus (Bath) is a Cu(I) sponge

The crystal structure of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) has been reported recently [Lieberman, R. L., and Rosenzweig, A. C. (2005) Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane, Nature 434, 177-182]. Subsequent work has shown that the preparation on which the X-ray analysis is based might be missing many of the important metal cofactors, including the putative trinuclear copper cluster at the active site as well as ca. 10 copper ions (E-clusters) that have been proposed to serve as a buffer of reducing equivalents to re-reduce the copper atoms at the active site following the catalytic chemistry [Chan, S. I., Wang, V. C.-C., Lai, J. C.-H., Yu, S. S.-F., Chen, P. P.-Y., Chen, K. H.-C., Chen, C.-L., and Chan, M. K. (2007) Redox potentiometry studies of particulate methane monooxygenase: Support for a trinuclear copper cluster active site, Angew. Chem., Int. Ed. 46, 1992-1994]. Since the aqueous-exposed domains of the 45 kDa subunit (PmoB) have been suggested to be the putative binding domains for the E-cluster copper ions, we have cloned and overexpressed in Escherichia coli the two aqueous-exposed subdomains toward the N- and C-termini of the subunit: the N-terminal subdomain (residues 54-178) and the C-terminal subdomain (residues 257-394 and 282-414). The recombinant C-terminal water-exposed subdomain is shown to behave like a Cu(I) sponge, taking up to ca. 10 Cu(I) ions cooperatively when cupric ions are added to the protein fragment in the presence of dithiothreitol or ascorbate. In addition, circular dichroism measurements reveal that the C-terminal subdomain folds into a beta-sheet structure in the presence of Cu(I). The propensity for the C-terminal subdomain to bind Cu(I) is consistent with the high redox potential(s) determined for the E-cluster copper ions in the pMMO. These properties of the E-clusters are in accordance with the function proposed for these copper ions in the turnover cycle of the enzyme.     

Glycine-rich region regulates cysteine-rich protein 1 binding to actin cytoskeleton

Cysteine-rich protein 1 (CRP1) has a unique structure with two well separated LIM domains, each followed by a glycine-rich region. Although CRP1 has been shown to interact with actin-binding proteins and actin filaments, the mechanism regulating localization to the actin cytoskeleton in cells is not clear. Experiments using truncated forms showed that the first LIM domain and glycine-rich region are necessary for CRP1 bundling of actin filaments and localization to the actin cytoskeleton. Furthermore, domain swapping experiments replacing the first glycine-rich region with the second resulted in the loss of CRP1 bundling activity and localization to the actin cytoskeleton, identifying seven critical amino acid residues. These results highlight the importance of the first glycine-rich region for CRP1 bundling activity and localization to the actin cytoskeleton. In addition, this work identifies the first LIM domain and glycine-rich region as a distinct actin filament bundling module.     

The carboxy-terminal third of dystrophin enhances actin binding activity

Dystrophin is an actin binding protein that is thought to stabilize the cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene-actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule [from N-terminal actin binding domain (ABD) 1 through ABD2] bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays than full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction by restricting actin rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin.