Visualization of myosin in living cells.

Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasingly more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.

Banding and polarity of actin filaments in interphase and cleaving cells.

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.

Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.

Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models.

To determine if a living cell is necessary for the incorporation of actin, alpha-actinin, and tropomyosin into the cytoskeleton, we have exposed cell models to fluorescently labeled contractile proteins. In this in vitro system, lissamine rhodamine-labeled actin bound to attachment plaques, ruffles, cleavage furrows and stress fibers, and the binding could not be blocked by prior exposure to unlabeled actin. Fluorescently labeled alpha-actinin also bound to ruffles, attachment plaques, cleavage furrows, and stress fibers. The periodicity of fluorescent alpha-actinin along stress fibers was wider in gerbil fibroma cells than it was in PtK2 cells. The fluorescent alpha-actinin binding in cell models could not be blocked by the prior addition of unlabeled alpha-actinin suggesting that alpha-actinin was binding to itself. While there was only slight binding of fluorescent tropomyosin to the cytoskeleton of interphase cells, there was stronger binding in furrow regions of models of dividing cells. The binding of fluorescently labeled tropomyosin could be blocked by prior exposure of the cell models to unlabeled tropomyosin. If unlabeled actin was permitted to polymerize in the stress fibers in cell models, fluorescently labeled tropomyosin stained the fibers. In contrast to the labeled contractile proteins, fluorescently labeled ovalbumin and BSA did not stain any elements of the cytoskeleton. Our results are discussed in terms of the structure and assembly of stress fibers and cleavage furrows.

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.

Differences in the stress fibers between fibroblasts and epithelial cells.

In the stress fibers of two types of nonmuscle cells, epithelia (PtK2, bovine lens) and fibroblasts (Gerbil fibroma, WI-38, primary human) the spacing between sites of alpha-actinin localization differs by a factor of about 1.6 as determined by indirect immunofluorescence and ultrastructural localization with peroxidase-labeled antibody. Both methods reveal striations along the stress fibers with a center-to-center spacing in the range of 0.9 mum in epithelial cells and 1.5 mum in fibroblasts. Periodic densities spaced at comparable distances are seen in PtK2 and in gerbil fibroma cells when they are treated with tannic acid and examined in the electron microscope. In such cells, densities are found not only along stress fibers but also at cell-cell junctions, attachment plaques, and foci from which stress fibers radiate. These latter three sites all stain with alpha-actinin antibody on the light and electron microscope level. Stress fibers in the two cell types also vary in the periodicity produced by indirect immunofluorescence with tropomyosin antibodies. As is the case for alpha-actinin, the tropomyosin center-to-center banding is approximately 1.6 times as long in gerbil fibroma cells (1.7 mum) as it is in PtK2 cells (1.0 mum). These results suggest that the densities seen in the electron microscope are sites of alpha-actinin localization and that the proteins in stress fibers have an arrangement similar to that in striated muscle. We propose a sarcomeric model of stress fiber structure based on light and electron microscopic findings.

Costameres are sites of force transmission to the substratum in adult rat cardiomyocytes.

Costameres, the vinculin-rich, sub-membranous transverse ribs found in many skeletal and cardiac muscle cells (Pardo, J. V., J. D. Siciliano, and S. W. Craig. 1983. Proc. Natl. Acad. Sci. USA. 80:363-367.) are thought to anchor the Z-lines of the myofibrils to the sarcolemma. In addition, it has been postulated that costameres provide mechanical linkage between the cells' internal contractile machinery and the extracellular matrix, but direct evidence for this supposition has been lacking. By combining the flexible silicone rubber substratum technique (Harris, A. K., P. Wild, and D. Stopak. 1980. Science (Wash. DC). 208:177-179.) with the microinjection of fluorescently labeled vinculin and alpha-actinin, we have been able to correlate the distribution of costameres in adult rat cardiac myocytes with the pattern of forces these cells exert on the flexible substratum. In addition, we used interference reflection microscopy to identify areas of the cells which are in close contact to the underlying substratum. Our results indicate that, in older cell cultures, costameres can transmit forces to the extracellular environment. We base this conclusion on the following observations: (a) adult rat heart cells, cultured on the silicone rubber substratum for 8 or more days, produce pleat-like wrinkles during contraction, which diminish or disappear during relaxation; (b) the pleat-like wrinkles form between adjacent alpha-actinin-positive Z-lines; (c) the presence of pleat-like wrinkles is always associated with a periodic, "costameric" distribution of vinculin in the areas where the pleats form; and (d) a banded or periodic pattern of dark gray or close contacts (as determined by interference reflection microscopy) has been observed in many cells which have been in culture for eight or more days, and these close contacts contain vinculin. A surprising finding is that vinculin can be found in a costameric pattern in cells which are contracting, but not producing pleat-like wrinkles in the substratum. This suggests that additional proteins or posttranslational modifications of known costamere proteins are necessary to form a continuous linkage between the myofibrils and the extracellular matrix. These results confirm the hypothesis that costameres mechanically link the myofibrils to the extracellular matrix. We put forth the hypothesis that costameres are composite structures, made up of many protein components; some of these components function primarily to anchor myofibrils to the sarcolemma, while others form transmembrane linkages to the extracellular matrix.

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin.

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.

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.

Aberrant postendocytotic fate of a 34-kDa molecular mass growth factor from human trophoblasts.

A 34-kDa growth factor expressed by trophoblasts and certain carcinomas binds to target fibroblastic cells through specific high-affinity receptors. Here we report studies on the cellular routing behavior of the receptor-bound 34-kDa protein. Internalization was visualized by using lissamine rhodamine-conjugated 34-kDa protein and was quantified by analyzing the acid dissociability of cell-bound radioiodinated protein after incubation at 37 degrees C. The protein was found to be rapidly internalized in a temperature-sensitive manner. However, in contrast with other protein ligands, the 34-kDa protein was not rapidly degraded. The extent of ligand degradation was small as quantified by gel filtration analysis. Studies on the receptor showed that there was an atypical up-regulation, i.e., increase in surface receptors in response to ligand binding at 37 degrees C. The up-regulation was partially blocked by cycloheximide, an inhibitor of protein biosynthesis, but not by known inhibitors of receptor recycling such as monensin, chloroquine, and methylamine, suggesting that enhanced receptor biosynthesis may be responsible for the process. These studies indicate that the cellular routing and receptor regulatory characteristics of the internalized 34-kDa growth factor are different from those of most growth factor ligands and imply the involvement of receptor up-regulation in signal transduction.

Intermediate filament aggregation: effect on cell topology, cell spreading and surface transport.

When chick fibroblasts are treated with cytochalasin-B and then transferred to medium containing colcemid, they become very flat and acquire corrugations on the apical cell surface. Bundles of intermediate filaments are found beneath the cell membrane in the troughs of the corrugations. When subcultured in colcemid, the cells retain the bundles of intermediate filaments and corrugations, and spread out both on untreated surfaces and on surfaces coated with gold particles. Gold particles are transported over the apical surface and phagocytosed by the cell. There is no accumulation of gold particles on the membrane under which the cables of intermediate filaments lie, and phagocytosed gold is excluded from the area occupied by the cables. These experiments indicate that a cell does not need microtubules and (a normal distribution of) intermediate filaments in order to spread, or to pick up, transport and phagocytose particulate material. The experimental aggregation of intermediate filaments does, however, alter the cell topology and the way in which particles are transported over the surface and ingested.

Differential response of three types of actin filament bundles to depletion of cellular ATP levels.

The effect of low levels of ATP on actin filament bundles in PtK2 cells was investigated by using 2-deoxyglucose, together with either sodium cyanide, sodium azide, or 2,4-dinitrophenol. Three actin filament systems were examined: stress fibers, cleavage rings, and dimethyl-sulfoxide (DMSO)-induced actin bundles in the nucleus. Of the three, only stress fibers disassembled when the ATP production was inhibited. The disassembly progressed slowly with the cells losing all stress fibers after about 90 min, but remaining in flat interconnected sheets. Mitotic cells that had progressed as far as metaphase when inhibitors were added, assembled cleavage rings. The process of cytokinesis took place in these cells but at a rate 5 to 10 times slower than normal, and disassembly of the cleavage ring was inhibited after the completion of cytokinesis. DMSO-induced nuclear actin bundles did not disassemble in cells depleted of ATP even when DMSO was eliminated from the medium. The peripheral aggregates of contractile proteins present in these cells became redistributed, however, and the cells flattened in the low ATP environment when DMSO was removed. Nuclear actin bundles did not form in DMSO-treated cells if the ATP inhibitors were present for as little as 5 min prior to DMSO exposure. Thus, the three types of actin filament bundles are affected in different ways by low intracellular levels of ATP. Stress fibers are most sensitive and cleavage rings, the least.

Differential response of stress fibers and myofibrils to gelsolin.

The actin-severing activity of human platelet gelsolin was analyzed on embryonic skeletal and cardiac myofibrils, and on stress fibers in non-muscle cells. These subcellular structures, although in all three cell types composed of contractile proteins arranged in sarcomeric units, were found to respond differently to gelsolin. The myofibrils in permeabilized myotubes or cardiac cells, as well as in living, microinjected muscle cells proved resistant to a wide concentration range of gelsolin. The same was found for the "mini-sarcomeres" which are seen in developing muscle cells. In contrast, stress fibers in microinjected fibroblasts or epithelial cells, as well as in permeabilized cells, were broken down rapidly by the platelet gelsolin. We conclude from these results that the mini-sarcomeres in embryonic myotubes and cardiac myocytes are not identical with stress fibers.

Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.

Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.

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.

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.

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.

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.

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.