Targeting of cardiac muscle titin fragments to the Z-bands and dense bodies of living muscle and non-muscle cells.

A 6.5-kb N-terminal region of embryonic chick cardiac titin, including the region previously reported as part of the protein zeugmatin, has been sequenced, further demonstrating that zeugmatin is part of the N-terminal region of titin, and not a separate Z-band protein. This Z-band region of cardiac titin, from both 7- and 19-day embryos as well as from adult animals, was found to contain six different small motifs, termed z-repeats [Gautel et al., 1996: J. Cell Sci. 109:2747-2754], of approximately 45 amino acids each sandwiched between flanking regions containing Ig domains. Fragments of Z-band titin, linked to GFP, were expressed in cultured cardiomyocytes to determine which regions were responsible for Z-band targeting. Transfections of primary cultures of embryonic chick cardiomyocytes demonstrated that the z-repeats play the major role in targeting titin fragments to the Z-band. Similar transfections of skeletal myotubes and non-muscle cells lead to the localization of these cardiac z-repeats in the Z-bands of the myofibrils and the dense bodies of the stress fibers. Over-expression of these z-repeat constructs in either muscle or non-muscle cells lead to the loss of the myofibrils or stress fibers, respectively. The transfection experiments also indicated that small domains of a protein, 40 to 50 amino acids, can be studied for their localization properties in living cells if a suitable linker is placed between these small domains and the much larger 28 kDa GFP protein.

Use of green fluorescent proteins linked to cytoskeletal proteins to analyze myofibrillogenesis in living cells.

Once the appropriate site has been selected for the attachment of GFP to the sarcomeric protein, it is quite remarkable that the large size of the GFP molecule does not appear to interfere with the localization of the fluorescent sarcomeric proteins into the sarcomeric regions of the myofibrils. A similar approach using truncated parts of sarcomeric proteins linked to GFP should allow studies of the targeting properties of other sarcomeric domains for localization and assembly studies.

Dpc4 transcriptional activation and dysfunction in cancer cells.

Dpc4 (Smad4) is implicated in mediation of signals from transforming growth factor (TGF) beta and related ligands, and wild-type Dpc4 mediates TGF-beta-stimulated gene transcription at specific DNA sequences bound by Dpc4 [Smad binding element (SBE)]. We characterized panels of DPC4 tumor mutations and cancer cell lines. Amino acid substitutions within the NH2-terminal third of Dpc4 weakened or ablated SBE-mediated gene regulation by a disruption of DNA binding. An interaction of the COOH-terminal end with the DNA-binding domain of Dpc4 was evident but was not required to explain the functional impairment produced by NH2-terminal DPC4 mutations. Both substitution and truncation mutations of the COOH-terminal half of DPC4 lacked the ability to regulate transcription while retaining the sequence-specific DNA-binding function, but through differing mechanisms. A modular domain to redistribute Dpc4 to the nuclear compartment allowed SBE-mediated transcriptional activation in a cell line having a TGF-1 receptor defect and was sufficient to restore SBE-mediated transactivation ability to COOH-terminal DPC4 missense mutants. Cells harboring DPC4 alterations had a universal impairment of the TGF-beta-stimulated SBE transcriptional response. These studies identify the loss of SBE-mediated gene regulation as a uniform outcome of the selection for DPC4 alterations during tumorigenesis. They raise the possibility of restoration of some Dpc4-associated transcriptional events in cancer cells through the targeted redistribution of wild-type and some missense mutant forms of Dpc4 to the nucleus.

Sites of monomeric actin incorporation in living PtK2 and REF-52 cells.

The purpose of this study was to analyze where monomeric actin first becomes incorporated into the sarcomeric units of the stress fibers. We microinjected fluorescently labeled actin monomers into two cell lines that differ in the sarcomeric spacings of alpha-actinin and nonmuscle myosin II along their stress fibers: REF-52, a fibroblast cell line, and PtK2, an epithelial cell line. The cells were fixed at selected times after microinjection (30 s and longer) and then stained with an alpha-actinin antibody. Localization of the labeled actin and alpha-actinin antibody were recorded with low level light cameras. In both cell types, the initial sites of incorporation were in focal contacts, lamellipodia and in punctate regions of the stress fibers that corresponded to the alpha-actinin rich dense bodies. The adherent junctions between the epithelial PtK2 cells were also initial sites of incorporation. At longer times of incorporation, the actin fluorescence extended along the stress fibers and became almost uniform. We saw no difference in the pattern of incorporation in peripheral and perinuclear regions of the stress fibers. We propose that rapid incorporation of monomeric actin occurs at the cellular sites where the barbed ends of actin filaments are concentrated: at the edges of lamellipodia, the adherens junctions, the attachment plaques and in the dense bodies that mark out the sarcomeric subunits of the stress fibers.

Myofibrillogenesis visualized in living embryonic cardiomyocytes.

Myofibril formation was visualized in cultured live cardiomyocytes that were transfected with plasmids expressing green fluorescent protein (GFP) linked to the Z-band protein, alpha-actinin. The expression of this fluorescent protein provided an in vivo label for structures containing alpha-actinin. The GFP-alpha-actinin fusion protein was incorporated into Z-bands, intercalated discs, and attachment plaques, as well as into the punctate aggregates, or Z-bodies, that are thought to be the precursors of Z-bands. Observations of live cells over several days in culture permitted us to test aspects of several theories of myofibril assembly that had been proposed previously based on the study of fixed cells. Fine fibrils, called premyofibrils, that formed de novo at the spreading edges of cardiomyocytes, contained punctate concentrations of alpha-actinin, termed Z-bodies. The punctate Z-bodies grew and aligned with Z-bodies in adjacent fibrils. With increasing time, adjacent fibrils and Z-bodies appeared to fuse and form mature myofibrils and Z-bands in cytoplasmic regions where the linear arrays of Z-bodies had been. These new myofibrils became aligned with existing myofibrils at their Z-bands to form myofibrils that spanned the length of the spread cell. These results are consistent with a model that postulates that the fibrils that form de novo near the cell membrane are premyofibrils-i.e., the precursors of mature myofibrils.

An N-terminal fragment of titin coupled to green fluorescent protein localizes to the Z-bands in living muscle cells: overexpression leads to myofibril disassembly.

Cultures of nonmuscle cells, skeletal myotubes, and cardiomyocytes were transfected with a fusion construct (Z1.1GFP) consisting of a 1.1-kb cDNA (Z1.1) fragment from the Z-band region of titin linked to the cDNA for green fluorescent protein (GFP). The Z1.1 cDNA encodes only 362 amino acids of the approximately 2000 amino acids that make up the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets the alpha-actinin-rich Z-bands of contracting myofibrils in vivo. This fluorescent fusion protein also localizes in the nascent and premyofibrils at the edges of spreading cardiomyocytes. Similarly, in transfected nonmuscle cells, the Z1.1GFP fusion protein localizes to the alpha-actinin-containing dense bodies of the stress fibers in vivo. A dominant negative phenotype was also observed in living cells expressing high levels of this Z1.1GFP fusion protein, with myofibril disassembly occurring as titin-GFP fragments accumulated. These data indicate that the Z-band region of titin plays an important role in maintaining and organizing the structure of the myofibril. The Z1.1 cDNA was derived from a chicken cardiac lambda gt11 expression library, screened with a zeugmatin antibody. Recent work has suggested that zeugmatin is actually part of the N-terminal region of the 81-kb titin cDNA. A reverse transcriptase polymerase chain reaction using a primer from the distal end (5' end) of the Z1.1 zeugmatin cDNA and a primer from the nearest known proximal (3' end) chicken titin (also called connectin) cDNA resulted in a predicted 0.3-kb polymerase chain reaction product linking the two known chicken titin cDNAs to each other. The linking region had a 79% identity at the amino acid level to human cardiac titin. This result and a Southern blot analysis of chicken genomic DNA hybridized with Z1.1 add further support to our original suggestion that zeugmatin is a proteolytic fragment from the N-terminal region of titin.

Zeugmatin is part of the Z-band targeting region of titin.

Originally, zeugmatin was identified as a 600-800 kD muscle specific protein in Z-bands of cardiac and skeletal muscles by Maher et al. (1985). In this presentation we review our work on myofibrillogenesis and present evidence that zeugmatin is actually part of the Z-Band region of titin and that this region of titin plays an important role in the assembly of the Z-bands and myofibrils. Rhee et al. (1994) reported that during myofibrillogenesis, zeugmatin antibody localization is detected in fully formed Z-bands in the mature myofibrils, in the Z-bodies of the nascent myofibrils, but not in the Z-bodies of the premyofibrils. These observations lead to the suggestion that zeugmatin might be responsible for the fusion of the Z-bodies to form the solid Z-bands of the mature myofibrils (Rhee et al. 1994). As part of a study to test aspects of this model of myofibrillogenesis, we isolated a 1.8 kb cDNA from a chicken cardiac expression library using an anti-zeugmatin antibody (Turnacioglu et al., 1996). We found this chicken cDNA to be 60% identical at the amino acid level to a segment of the Z-band region of human cardiac titin (connectin) sequenced by Labeit and Kolmerer (1995). This homology along with Western blot analysis with purified titin, suggested that zeugmatin is in fact part of the N-terminal region of chicken titin. When expressed in non-muscle cells, Z1.1 product colocalized with the alpha-actinin in stress fiber dense bodies and focal adhesions. Cultures of non-muscle cells, skeletal myotubes and cardiomyocytes were also transfected with a fusion construct (Z1.1GFP) consisting of the Z1.1 kb cDNA linked to the cDNA for green fluorescent protein (GFP). The Z1.1 kb cDNA encodes only 362 of the approximately 2,000 amino acids which comprise the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets in vivo to the alpha-actinin rich Z-bands of contracting myofibrils. A dominant negative phenotype was observed in living cells expressing high levels of this Z1.1GFP fusion protein with inhibition of myofibrillogenesis as well as the disassembly of preexisting myofibrils in these cells. These data indicate that the Z-band region of titin (connectin) plays an important role in organizing and maintaining the structure of the myofibril.

Partial characterization of zeugmatin indicates that it is part of the Z-band region of titin.

Zeugmatin is a muscle specific protein discovered by Maher et al. [1985: J. Cell Biol. 101:1871-1883] to be in Z-Bands of muscle and in the dense bodies of smooth muscle. Maher et al. [1985] generated a zeugmatin specific monoclonal antibody, McAb20, and then used immunoaffinity chromatography to isolate a 600-800 kD protein. During myofibrillogenesis of embryonic cardiac muscle, zeugmatin is detected in fully formed Z-bands in the mature myofibrils but not in the Z-bodies of premyofibrils [Rhee et al., 1994: Cell Motil. Cytoskeleton 28:1-24]. Rhee et al. [1994] have postulated that zeugmatin may be responsible for the fusion of the alpha-actinin containing Z-bodies to form the solid Z-Bands of the mature myofibrils. The current studies were undertaken to characterize the properties of zeugmatin. The McAb20 was used to probe a chicken heart lamba gt11 expression library, and three unique positive clones of 1.1, 1.4, and 1.7 kB were isolated. These were inserted into pcDNA3, sequenced, and assembled into a 1.8 kB ORF. A 60% identity with N-terminal region of the human cardiac titin sequence was revealed at the amino acid level. This region of the 1.8 kB zeugmatin sequence is located entirely in the Z-band region of the human cardiac titin molecule. The 1.1 kB clone of zeugmatin was subcloned into pTrcHisC and expressed in bacteria. Bacterial lysates were prepared and run over nickel columns to isolate a 46 kD fusion protein. This fusion protein formed a complex with purified alpha-actinin that could be immunoprecipitated with the zeugmatin specific antibody, McAb 20. The 1.1 kB sequence was transfected into non-muscle cell lines, PtK2 and REF. Twenty-four hours after transfection, the 46 kD zeugmatin peptide, not present in control non-muscle cells, was localized in focal adhesions and in a punctate pattern along the stress fibers. Double immunofluorescence staining revealed that zeugmatin colocalized with the alpha-actinin in the dense bodies and focal contacts of the stress fibers. At longer time points, as the transfected cells accumulated more truncated zeugmatin molecules, the cells lost adhesion plaques and stress fibers, and became detached from the substratum. Our results indicate the zeugmatin is part of the titin molecule that is located within the Z-band and that this section of the titin molecule anchors the actin crosslinking alpha-actinin molecules.