Does Nebulin Make Tropomyosin Less Dynamic in Mature Myofibrils in Cross-Striated Myocytes?

Myofibrils in vertebrate cardiac and skeletal muscles are characterized by groups of proteins arranged in contractile units or sarcomeres, which consist of four major components - thin filaments, thick filaments, titin and Z-bands. The thin actin/tropomyosin-containing filaments are embedded in the Z-bands and interdigitate with the myosin-containing thick filaments aligned in A-bands. Titin is attached to the Z-band and extends upto the middle of the A-Band. In this mini review, we have addressed the mechanism of myofibril assembly as well as the dynamics and maintenance of the myofibrils in cardiac and skeletal muscle cells. Evidence from our research as well as from other laboratories favors the premyofibril model of myofibrillogenesis. This three-step model (premyofibril to nascent myofibril to mature myofibril) not only provides a reasonable mechanism for sequential interaction of various proteins during assembly of myofibrils, but also suggests why the dynamics of a thin filament protein like tropomyosin is higher in cardiac muscle than in skeletal muscles. The dynamics of tropomyosin not only varies in different muscle types (cardiac vs. skeletal), but also varies during myofibrillogenesis, for example, premyofibril versus mature myofibrils in skeletal muscle. One of the major differences in protein composition between cardiac and skeletal muscle is nebulin localized along the thin filaments (two nebulins/thin filament) of mature myofibrils in skeletal muscle cells, but which is expressed in a minimal quantity (one nebulin/50 actin filaments) in ventricular cardiomyocytes. Interestingly, nebulin is not associated with premyofibrils in skeletal muscle. Our FRAP(Fluorescence Recovery After Photobleaching) results suggest that tropomyosin is more dynamic in premyofibrils than in mature myofibrils in skeletal muscle, and also, the dynamics of tropomyosin in mature myofibrils is significantly higher in cardiac muscle compared to skeletal muscle. Our working hypothesis is that the association of nebulin in mature myofibrils renders tropomyosin less dynamic in skeletal muscle.

Potential role of the EPEC translocated intimin receptor (Tir) in host apoptotic events.

Apoptosis, or programmed cell death, is a well-ordered process that allows damaged or diseased cells to be removed from an organism without severe inflammatory reactions. Multiple factors, including microbial infection, can induce programmed death and trigger reactions in both host and microbial cellular pathways. Whereas an ultimate outcome is host cell death, these apoptotic triggering mechanisms may also facilitate microbial spread and prolong infection. To gain a better understanding of the complex events of host cell response to microbial infection, we investigated the molecular role of the microorganism Enteropathogenic Escherichia coli (EPEC) in programmed cell death. We report that wild type strain of EPEC, E2348/69, induced apoptosis in cultured PtK2 and Caco-2 cells, and in contrast, infections by the intracellularly localized Listeria monocytogenes did not. Fractionation and concentration of EPEC-secreted proteins demonstrated that soluble protein factors expressed by the bacteria were capable of inducing the apoptotic events in the absence of organism attachment, suggesting adherence is not required to induce host cell death. Among the known EPEC proteins secreted via the Type III secretion (TTS) system, we identified the translocated intimin receptor (Tir) in the apoptosis-inducing protein sample. In addition, host cell ectopic expression of an EPEC GFP-Tir showed mitochondrial localization of the protein and produced apoptotic effects in transfected cells. Taken together, these results suggest a potential EPEC Tir-mediated role in the apoptotic signaling cascade of infected host cells.

Fishing out proteins that bind to titin.

Another giant protein has been detected in cross-striated muscle cells. Given the name obscurin, it was discovered in a yeast two-hybrid screen in which the bait was a small region of titin that is localized near the Z-band. Obscurin is about 720 kD, similar in molecular weight to nebulin, but present at about one tenth the level (Young et al., 2001). Like titin, obscurin contains multiple immunoglobulin-like domains linked in tandem, but in contrast to titin it contains just two fibronectin-like domains. It also contains sequences that suggest obscurin may have roles in signal transduction. During embryonic development, its localization changes from the Z-band to the M-band. With these intriguing properties, obscurin may not remain obscure for long.

Expression of green or red fluorescent protein (GFP or DsRed) linked proteins in nonmuscle and muscle cells.

The introduction of the green fluorescent protein (GFP) plasmids that allow proteins and peptides to be expressed with a fluorescent tag has had a major impact on the field of cell biology. It has enabled the dynamics of a wide variety of proteins to be analyzed that could not otherwise be detected in live cells. Transient transfections of muscle and nonmuscle cells with plasmids encoding various cytoskeletal proteins ligated to green fluorescent protein or Ds red protein allow changes in the cytoskeletal network to be studied in the same cell for time periods up to several days. With this approach, proteins that could not be purified and directly labeled with fluorescent dyes and microinjected into cells can now be expressed and visualized in a wide variety of cells. Procedures are presented for transfection of the nonmuscle cell, PtK2, and primary cultures of embryonic chick myocytes, and for studying the live transfected cells.

Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins.

When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli.

Assembly of myofibrils in cardiac muscle cells.

How do myofibrils assemble in cardiac muscle cells? When does titin first assemble into myofibrils? What is the role of titin in the formation of myofibrils in cardiac muscle cells? This chapter reviews when titin is first detected in cultured cardiomyocytes that have been freshly isolated from embryonic avian hearts. Our results support a model for myofibrillogenesis that involves three stages of assembly: premyofibrils, nascent myofibrils and mature myofibrils. Titin and muscle thick filaments were first detected associated with the nascent myofibrils. The Z-band targeting site for titin is localized in the N-terminus of titin. This region of titin binds alpha-actinin and less avidly vinculin. Thus the N-terminus of titin via its binding to alpha-actinin, and vinculin could also help mediate the costameric attachment of the Z-bands of mature myofibrils to the nearest cell surfaces.

Assembly of cytoskeletal proteins into cleavage furrows of tissue culture cells.

We review results obtained after fluorescent actin and myosin II probes were microinjected into interphase and prophase PtK2 and LLC-PK tissue culture cells to follow the changing distribution of these cytoskeletal proteins in the live cells during division. The fluorescent probes first begin to assemble into the future furrow region during mid-anaphase before any sign of initial contractions. The total concentrations of F-actin and myosin in the cleavage furrow begin to decrease a few minutes after the onset of furrow contraction. The cell's shape and the position of its mitotic spindle affect the deposition of cytoskeletal proteins in the forming cleavage furrow. In cells with two spindles, contractile proteins were recruited not only to the cortex bordering the former metaphase plates but also to the cortex midway between each pair of adjacent non-daughter poles or centrosomes. The furrowing between adjacent poles seen in these cultured cells are similar to the furrows observed by Rappaport [(1961) J Exp Zool 148:81-89] when echinoderm eggs were manipulated into a torus shape so that the poles of two mitotic spindles were adjacent to one another. These observations on injected tissue culture cells suggest that vertebrate cells share common mechanisms for the establishment of the cleavage furrow with echinoderm cells.

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.

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.

Unusual cleavage furrows in vertebrate tissue culture cells: insights into the mechanisms of cytokinesis.

In cultures of the epithelial cell lines, PtK2 and LLC-PK, some cells assume unusually large flattened morphologies and, during cell division, produce unusual cleavage furrows. We have microinjected some of these large cells with fluorescent actin and myosin probes to determine how the cell's shape and the position of its mitotic spindle affect the deposition of actin and myosin in the forming cleavage furrow. In cells with two spindles, contractile proteins were recruited not only to the cortex bordering the former metaphase plates but also to the cortex midway between each pair of adjacent nondaughter poles or centrosomes. The furrowing between adjacent poles seen in these cultured epithelial cells conformed to the furrows seen when echinoderm eggs were manipulated into a torus shape so that the poles of two mitotic spindles were adjacent to one another [Rappaport, 1961]. The recruitment of contractile proteins and the formation of furrows between adjacent centrosomes was a function of the distances between them. When adjacent centrosomes were positioned too close together neither contractile protein recruitment nor furrow formation occurred. If a normal-sized spindle was present in a very large cell, fibers of contractile protein assembled in the cortex above the former metaphase plate but they did not extend to the cell periphery, resulting in an inhibition of cytokinesis. In all injected cells, the recruitment of actin and myosin to the cell surfaces could first be detected at mid-anaphase before there was any visible sign of furrowing. Our results suggest that vertebrate cells share common mechanisms for the establishment of the cleavage furrow with echinoderm cells. The results are consistent with a model in which (1) the positions of the centrosomes and their linearly connected microtubules determine the position for the assembly of the cleavage furrow, and (2) the signal arrives at the surface within a few minutes after the initiation of anaphase. We speculate that an interaction of the ends of microtubules from adjacent centrosomes with the cell surface promotes a clustering of integral membrane protein(s) that interact with and target contractile proteins to a position midway between centrosomes where furrowing occurs.

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.