Mathematical modeling of the dynamic mechanical behavior of neighboring sarcomeres in actin stress fibers.

Actin stress fibers (SFs) in live cells consist of series of dynamic individual sarcomeric units. Within a group of consecutive SF sarcomeres, individual sarcomeres can spontaneously shorten or lengthen without changing the overall length of this group, but the underlying mechanism is unclear. We used a computational model to test our hypothesis that this dynamic behavior is inherent to the heterogeneous mechanical properties of the sarcomeres and the cytoplasmic viscosity. Each sarcomere was modeled as a discrete element consisting of an elastic spring, a viscous dashpot and an active contractile unit all connected in parallel, and experiences forces as a result of actin filament elastic stiffness, myosin II contractility, internal viscoelasticity, or cytoplasmic drag. When all four types of forces are considered, the simulated dynamic behavior closely resembles the experimental observations, which include a low-frequency fluctuation in individual sarcomere length and compensatory lengthening and shortening of adjacent sarcomeres. Our results suggest that heterogeneous stiffness and viscoelasticity of actin fibers, heterogeneous myosin II contractility, and the cytoplasmic drag are sufficient to cause spontaneous fluctuations in SF sarcomere length. Our results shed new light to the dynamic behavior of SF and help design experiments to further our understanding of SF dynamics.

LIM proteins in actin cytoskeleton mechanoresponse.

The actin cytoskeleton assembles into branched networks or bundles to generate mechanical force for critical cellular processes such as establishment of polarity, adhesion, and migration. Stress fibers (SFs) are contractile actomyosin structures that physically couple to the extracellular matrix through integrin-based focal adhesions (FAs), thereby transmitting force into and across the cell. Recently, LIN-11, Isl1, and MEC-3 (LIM) domain proteins have been implicated in mediating this cytoskeletal mechanotransduction. Among the more well-studied LIM domain adapter proteins is zyxin, a dynamic component of both FAs and SFs. Here we discuss recent research detailing the mechanisms by which SFs adjust their structure and composition to balance mechanical forces and suggest ways that zyxin and other LIM domain proteins mediate mechanoresponse.

Targeting of zyxin to sites of actin membrane interaction and to the nucleus.

The localization of proteins to particular intracellular compartments often regulates their functions. Zyxin is a LIM protein found prominently at sites of cell adhesion, faintly in leading lamellipodia, and transiently in cell nuclei. Here we have performed a domain analysis to identify regions in zyxin that are responsible for targeting it to different subcellular locations. The N-terminal proline-rich region of zyxin, which harbors binding sites for alpha-actinin and members of the Ena/VASP family, concentrates in lamellipodial extensions and weakly in focal adhesions. The LIM region of zyxin displays robust targeting to focal adhesions. When overexpressed in cells, the LIM region of zyxin causes displacement of endogenous zyxin from focal adhesions. Upon mislocalization of full-length zyxin, at least one member of the Ena/VASP family is also displaced, and the organization of the actin cytoskeleton is perturbed. Zyxin also has the capacity to shuttle between the nucleus and focal adhesion sites. When nuclear export is inhibited, zyxin accumulates in cell nuclei. The nuclear accumulation of zyxin occurs asynchronously with approximately half of the cells exhibiting nuclear localization of zyxin within 2.3 h of initiating leptomycin B treatment. Our results provide insight into the functions of different zyxin domains.

Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy.

Although cytoskeletal mutations are known causes of genetically based forms of dilated cardiomyopathy, the pathways that link these defects with cardiomyopathy are unclear. Here we report that the alpha-actinin-associated LIM protein (ALP; Alp in mice) has an essential role in the embryonic development of the right ventricular (RV) chamber during its exposure to high biomechanical workloads in utero. Disruption of the gene encoding Alp (Alp) is associated with RV chamber dilation and dysfunction, directly implicating alpha-actinin-associated proteins in the onset of cardiomyopathy. In vitro assays showed that Alp directly enhances the capacity of alpha-actinin to cross-link actin filaments, indicating that the loss of Alp function contributes to destabilization of actin anchorage sites in cardiac muscle. Alp also colocalizes at the intercalated disc with alpha-actinin and gamma-catenin, the latter being a known disease gene for human RV dysplasia. Taken together, these studies point to a novel developmental pathway for RV dilated cardiomyopathy via instability of alpha-actinin complexes.

Identification of a CArG box-dependent enhancer within the cysteine-rich protein 1 gene that directs expression in arterial but not venous or visceral smooth muscle cells.

Smooth muscle cells (SMCs) are heterogeneous with respect to their contractile, synthetic, and proliferative properties, though the regulatory factors responsible for their phenotypic diversity remain largely unknown. To further our understanding of smooth muscle gene regulation, we characterized the cis-regulatory elements of the murine cysteine-rich protein 1 gene (CRP1/Csrp1). CRP1 is expressed in all muscle cell types during embryogenesis and predominates in vascular and visceral SMCs in the adult. We identified a 5-kb enhancer within the CRP1 gene that is sufficient to drive expression in arterial but not venous or visceral SMCs in transgenic mice. This enhancer also exhibits region-specific activity in the outflow tract of the heart and the somites. Within the 5-kb CRP1 enhancer, we found a single CArG box that binds serum response factor (SRF), and by mutational analysis, demonstrate that the activity of the enhancer is dependent on this CArG element. Our findings provide further evidence for the existence of distinct regulatory programs within SMCs and suggest a role for SRF in the activation of the CRP1 gene.

Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm.

Mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor initiates most hereditary and sporadic colon carcinomas. Although APC protein is located in both the cytoplasm and the nucleus, the protein domains required to maintain a predominantly cytoplasmic localization are unknown. Here, we demonstrate that nuclear export of APC is mediated by two intrinsic, leucine-rich, nuclear export signals (NESs) located near the amino terminus. Each NES was able to induce the nuclear export of a fused carrier protein. Both APC NESs were independently able to interact with the Crm1 nuclear export factor and substitute for the HIV-1 Rev NES to mediate nuclear mRNA export. Both APC NESs functioned within the context of APC sequence: an amino-terminal APC peptide containing both NESs interacted with Crm1 and showed nuclear export in a heterokaryon nucleocytoplasmic shuttling assay. Also, mutation of both APC NESs resulted in the nuclear accumulation of the full-length, approximately 320-kDa APC protein, further establishing that the two intrinsic APC NESs are necessary for APC protein nuclear export. Moreover, endogenous APC accumulated in the nucleus of cells treated with the Crm1-specific nuclear export inhibitor leptomycin B. Together, these data indicate that APC is a nucleocytoplasmic shuttle protein whose predominantly cytoplasmic localization requires NES function and suggests that APC may be important for signaling between the nuclear and cytoplasmic compartments of epithelial cells.

Fine mapping of the alpha-actinin binding site within cysteine-rich protein.

The cysteine-rich proteins (CRPs) are a family of highly conserved LIM (an acronym derived from the three gene products lin-11, isl-1 and mec-3) domain proteins that have been implicated in muscle differentiation. All CRP family members characterized so far have been shown to interact with the filamentous actin cross-linker alpha-actinin. The region of CRP required for this interaction has previously been broadly mapped to the molecule's N-terminal half. Here we report that the alpha-actinin-binding region of CRP, which we have mapped by using a combination of blot overlay and Western immunoblot techniques, is confined to an 18-residue sequence occurring within the protein's N-terminal glycine-rich repeat. A site-directed mutagenesis analysis of the binding region has revealed the critical importance of a single lysine residue (lysine 65 in human CRP1). Alterations at this site lead to a 10-fold decrease in alpha-actinin binding in comparison with wild-type CRP. The critical lysine residue localizes within a short alpha-helix, raising the possibility that mutagenesis-induced alterations in alpha-actinin-binding capacity might be attributed to the disruption of a key structural element.

Characterization of the interaction between zyxin and members of the Ena/vasodilator-stimulated phosphoprotein family of proteins.

Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.

Molecular dissection of zyxin function reveals its involvement in cell motility.

Spatially controlled actin filament assembly is critical for numerous processes, including the vectorial cell migration required for wound healing, cell- mediated immunity, and embryogenesis. One protein implicated in the regulation of actin assembly is zyxin, a protein concentrated at sites where the fast growing ends of actin filaments are enriched. To evaluate the role of zyxin in vivo, we developed a specific peptide inhibitor of zyxin function that blocks its interaction with alpha-actinin and displaces it from its normal subcellular location. Mislocalization of zyxin perturbs cell migration and spreading, and affects the behavior of the cell edge, a structure maintained by assembly of actin at sites proximal to the plasma membrane. These results support a role for zyxin in cell motility, and demonstrate that the correct positioning of zyxin within the cell is critical for its physiological function. Interestingly, the mislocalization of zyxin in the peptide-injected cells is accompanied by disturbances in the distribution of Ena/VASP family members, proteins that have a well-established role in promoting actin assembly. In concert with previous work, our findings suggest that zyxin promotes the spatially restricted assembly of protein complexes necessary for cell motility.

Purification and characterization of an alpha-actinin-binding PDZ-LIM protein that is up-regulated during muscle differentiation.

alpha-Actinin is required for the organization and function of the contractile machinery of muscle. In order to understand more precisely the molecular mechanisms by which alpha-actinin might contribute to the formation and maintenance of the contractile apparatus within muscle cells, we performed a screen to identify novel alpha-actinin binding partners present in chicken smooth muscle cells. In this paper, we report the identification, purification, and characterization of a 36-kDa smooth muscle protein (p36) that interacts with alpha-actinin. Using a variety of in vitro binding assays, we demonstrate that the association between alpha-actinin and p36 is direct, specific, and saturable and exhibits a moderate affinity. Furthermore, native co-immunoprecipitation reveals that the two proteins are complexed in vivo. p36 is expressed in cardiac muscle and tissues enriched in smooth muscle. Interestingly, in skeletal muscle, a closely related protein of 40 kDa (p40) is detected. The expression of p36 and p40 is dramatically up-regulated during smooth and skeletal muscle differentiation, respectively, and p40 colocalizes with alpha-actinin at the Z-lines of differentiated myotubes. We have established the relationship between p36 and p40 by molecular cloning of cDNAs that encode both proteins and have determined that they are the products of a single gene. Both proteins display an identical N-terminal PDZ domain and an identical C-terminal LIM domain; an internal 63-amino acid sequence present in p36 is replaced by a unique 111-amino acid sequence in p40. Analysis of the sequences of p36 and p40 suggest that they are the avian forms of the actinin-associated LIM proteins (ALPs) recently described in rat (Xia, H., Winokur, S. T., Kuo, W.-L., Altherr, M. R., and Bredt, D. S. (1997) J. Cell Biol. 139, 507-515). The expression of the human ALP gene has been postulated to be affected by mutations that cause facioscapulohumeral muscular dystrophy; thus, the characterization of ALP function may ultimately provide insight into the mechanism of this disease.

Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis.

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.

Solution structure of the chicken cysteine-rich protein, CRP1, a double-LIM protein implicated in muscle differentiation.

The mechanism by which the contractile machinery of muscle is assembled and maintained is not well-understood. Members of the cysteine-rich protein (CRP) family have been implicated in these processes. Three vertebrate CRPs (CRP1-3) that exhibit developmentally regulated muscle-specific expression have been identified. All three proteins are associated with the actin cytoskeleton, and one has been shown to be required for striated muscle structure and function. The vertebrate CRPs identified to date display a similar molecular architecture; each protein is comprised of two tandemly arrayed LIM domains, protein-binding motifs found in a number of proteins with roles in cell differentiation. Each LIM domain coordinates two Zn(II) ions that are bound independently in CCHC (C=Cys, H=His) and CCCC modules. Here we describe the solution structure of chicken CRP1 determined by homonuclear and 1H-15N heteronuclear magnetic resonance spectroscopy. Comparison of the structures of the two LIM domains of CRP1 reveals a high degree of similarity in their tertiary folds. In addition, the two component LIM domains represent two completely independent folding units and exhibit no apparent interactions with each other. The structural independence and spatial separation of the two LIM domains of CRP1 are compatible with an adapter or linker role for the protein.

Quantitative measurement of proteins by western blotting with Cy5-coupled secondary antibodies.

The concentration of proteins in cells is an important parameter that determines how a protein will interact with other proteins or pharmacological agents. Recent developments in Western blotting techniques have now made this a method of choice to measure protein concentration in complex solutions such as total cell extracts. We show that detection of Cy5-coupled secondary antibodies by PhosphorImager analysis produces signals that approach linearity with respect to protein concentration over a 20-fold range. We used this technique to estimate cellular levels of zyxin, which is an important protein component of the actin cytoskeleton in mammalian cells. By producing specific protein standards based on sequences that are available from public databases, it is now possible to estimate the concentration of almost any protein by this technique.

The LIM protein, CRP1, is a smooth muscle marker.

LIM domains are double zinc-finger motifs found in many proteins that play central roles in cell differentiation. Members of the cysteine-rich protein (CRP) family display two LIM domains and are implicated in muscle development. Here we describe the characterization of one member of this family, CRP1, in the mouse. We have isolated and sequenced murine cDNAs that encode CRP1. We have determined by Northern analysis and in situ hybridization that CRP1 expression is developmentally regulated in the embryonic mouse and displays organ specific regulation in adults. The gene encoding CRP1 is expressed in the smooth muscle cells (SMCs) of the dorsal aorta at E9.5, thus illustrating that CRP1 is an early marker for SMC differentiation at that site. As development proceeds, CRP1 transcripts are observed throughout the SMC lineage, with minimal, transient expression detected in skeletal and cardiac muscle. Interestingly, although several markers of mature smooth muscle are already expressed, CRP1 expression in the bladder is not upregulated until the onset of bladder expansion at embryonic day 16.5, at which time its expression becomes very prominent. CRP1 expression persists into adulthood with prominent expression observed in both vascular and visceral smooth muscle. The results reported here define CRP1 as a general marker of smooth muscle lineages.

A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans.

We describe here the molecular and functional characterization of the Caenorhabditis elegans unc-97 gene, whose gene product constitutes a novel component of muscular adherens junctions. UNC-97 and homologues from several other species define the PINCH family, a family of LIM proteins whose modular composition of five LIM domains implicates them as potential adapter molecules. unc-97 expression is restricted to tissue types that attach to the hypodermis, specifically body wall muscles, vulval muscles, and mechanosensory neurons. In body wall muscles, the UNC-97 protein colocalizes with the beta-integrin PAT-3 to the focal adhesion-like attachment sites of muscles. Partial and complete loss-of-function studies demonstrate that UNC-97 affects the structural integrity of the integrin containing muscle adherens junctions and contributes to the mechanosensory functions of touch neurons. The expression of a Drosophila homologue of unc-97 in two integrin containing cell types, muscles, and muscle-attached epidermal cells, suggests that unc-97 function in adherens junction assembly and stability has been conserved across phylogeny. In addition to its localization to adherens junctions UNC-97 can also be detected in the nucleus, suggesting multiple functions for this LIM domain protein.

LIM domain-containing protein trip6 can act as a coactivator for the v-Rel transcription factor.

The retroviral oncoprotein v-Rel is a transcriptional activator in the Rel/NF-kappaB family of eukaryotic transcription factors. v-Rel malignantly transforms a variety of cell types in vitro and in vivo, and its transforming activity is dependent on the ability of v-Rel to bind to DNA and activate transcription. In this report, we used the yeast two-hybrid assay to identify proteins that interact with C-terminal sequences of v-Rel that are needed for transcriptional activation and transformation. One protein, Trip6, that we identified in this screen was previously identified as a thyroid hormone receptor-interacting protein. Trip6 is a member of a subfamily of LIM domain-containing proteins that are thought to transport intracellular signals from the cell surface to the nucleus. By several criteria, we show that sequences from Trip6, which include the LIM domains, behave as a coactivator for transcriptional activation by v-Rel. That is, a GAL4-Trip6 fusion protein can activate transcription in yeast and chicken cells, Trip6 can enable C-terminal sequences of v-Rel to activate transcription in yeast, and Trip6 can enhance activation by v-Rel from a kappaB site reporter plasmid in yeast. Although full-length Trip6 localizes to adhesion plaques, deletion of N-terminal sequences allows human Trip6 to enter the nucleus of chicken cells. Lastly, Northern blotting shows that Trip6 mRNA is expressed in many human tissues. Coexpression of Trip6 does not affect the transforming activity of v-Rel. Taken together, our results indicate that Trip6 may be a protein that is important for the ability of v-Rel to activate transcription and transform cells, and may represent a potential target for blocking Rel-mediated oncogenesis and transcriptional activation.

Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains.

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

The human TRIP6 gene encodes a LIM domain protein and maps to chromosome 7q22, a region associated with tumorigenesis.

The thyroid receptor interacting protein-6 (TRIP6) was first identified as a ligand-dependent binding partner for the thyroid hormone receptor in a yeast two-hybrid screen. A partial TRIP6 cDNA clone that was isolated in the initial screen encodes two copies of the LIM domain. The LIM domain is a double zinc-finger structure that mediates protein-protein interactions. Here we report the complete amino acid sequence of human TRIP6. The TRIP6 protein displays a proline-rich N-terminal region linked to three tandemly arrayed C-terminal LIM domains. The global molecular architecture and sequence of TRIP6 place it in the same family as the adhesion plaque protein, zyxin, and the lipoma preferred partner (LPP). Zyxin and LPP are implicated in cellular signaling and tumorigenesis, respectively. By radiation hybrid mapping, the human TRIP6 gene was assigned to a segment of chromosome 7q22 that is commonly deleted in malignant myeloid diseases and uterine leiomyoma.