Control of filament length by the regulatory light chains in skeletal and cardiac myosins

The possible role of the regulatory light chains (LC2) in in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by formation of minifilaments and synthetic thick filaments. After LC2 was removed, the resulting myosin preparations exhibited little aggregation in 0.5 M KCl and 0.05 M potassium phosphate (pH 6.5). Minifilaments migrated as a single, hypersharp peak during sedimentation velocity, but electron microscopic analysis revealed a more destabilized structure for LC2-deficient minifilaments. Thick filaments were formed in buffers containing 0.15 M KCl and the following: 20 mM imidazole; 20 mM imidazole, 5 mM ATP; or 20 mM imidazole, 5 mM ATP, and 5 mM MgCl2, all at pH 7.0. Skeletal and cardiac myosin filaments formed in imidazole buffer alone were bipolar, tapered at both ends, and about 1.6 micron long. Removal of LC2 resulted in the formation of shorter thick filaments (1.2 micron long). This effect could be reversed by reassociation with LC2. Inclusion of ATP in the buffer disrupted the filament structure, resulting in irregular, short filaments (less than 0.6 micron); addition of both ATP and MgCl2 largely reversed the effects of ATP alone. In cardiac myosin filaments, the bare zone diameter increased from 16 nm as measured in control and LC2-recombined samples to 20 nm in LC2-deficient myosin assemblies. These results implicate LC2 in an active role in controlling synthetic thick filament length in both skeletal and cardiac muscles.     

Self-assembly of myosin in vitro caused by rapid dilution. Effects of hydrogen ion, potassium chloride, and protein concentrations.

The in vitro assembly of rabbit skeletal myosin was studied by flow birefringence. Filaments were obtained from a solution of myosin in 0.5 M KCl by rapid dilution to lower ionic strength. In most cases, the filament length as determined from extinction angle measurements increased or decreased gradually for about 1 h after dilution, depending on pH, KCl concentration and the previous history. The filament length (l) immediately after dilution also showed a marked dependence on pH, KCl concentration and protein concentration (c) at the moment of assembly. The general characteristics obtained from our limited study (0.04-6.0 mg/ml) show three distinctive modes of effect of the protein concentration on the filament length: d logl/d log c is positive (0.1-1) at small c, negative (from -1 to -0.2) at intermediate c, and zero or slightly positive (0.0-0.3) at large c. Lowering of the KCl concentration (75-250 mM) as well as increase of the hydrogen ion concentration (pH 6-8) influenced the filament length in qualitatively the same manner as increase of the protein concentration. A model of the assembly reaction of myosin in which the polarity of filaments is crucial was constructed and shown to give qualitatively the experimental dependence of the filament length on the protein concentration.     

LC2 involvement in the assembly of skeletal myosin filaments

The assembly of LC2-deficient myosin was studied under conditions where control and LC2-reassociated myosin assemble around the native length of about 1.5 microns. The aim of this work was to determine how loss of LC2 affects the assembly characteristics. The findings of this study can be summarized as follows: (a) LC2-deficient myosin assembles into two populations of filaments, one around 0.5 micron in length and the other around 1 micron in length. This suggests that loss of the LC2 perturbs the length-determining mechanism. (b) The population of filaments around 0.5 micron has a diameter around 14 nm and that around 1 micron a diameter around 22 nm. Neither diameter corresponds to the 18 nm obtained with the control and LC2-reassociated myosins, suggesting that the presence of LC2 may have a role in regulating the side-to-side assembly of the myosin rods. (c) Filaments assembled from LC2-deficient myosin tend to aggregate side-by-side, but not those assembled from control and LC2-reassociated myosin. (d) The presence of MgATP has no effect on the length distribution of LC2-deficient myosin filaments in contrast to the sharpening of the distribution observed with control and reassociated myosin.     

The aggregation characteristics of column-purified rabbit skeletal myosin in the presence and absence of C-protein at pH 7.0.

The aggregation properties of column-purified rabbit skeletal myosin at pH 7.0 were investigated as functions of ionic strength, protein concentration, and time. Filaments prepared by dialysis exhibited the same average length and population distribution at 0.10 and 0.15 M KCl at protein concentrations greater than 0.10 mg/ml; similar results were obtained at .0.20 M KCl, although average filament length was approximately 0.5 micrometer shorter. Once formed, these length distributions remained virtually unchanged over an 8-d period. At and below 0.10 mg/ml, average filament length decreased as a function of protein concentration; filaments prepared from an initial concentration of 0.02 mg/ml were half the length of those prepared at 0.2 mg/ml. Filaments prepared by dilution exhibited a sharp increase in average length as the time-course increased up to 40 s, then altered only slightly over a further period of 4 min. Addition of C-protein in a molar ratio of 1-3.3 myosin molecules affected most of these results. Average filament length was affected neither by ionic strength nor by initial protein concentration down to 0.04 mg/ml or over an 8-d period. Filaments formed by dilution in the presence of C-protein exhibited a constant average length and hypersharp length distribution over variable time courses up to 7 min. It is possible that C-protein acts to stabilize the antiparallel intermediate during filamentogenesis, and may also affect subunit addition to this nucleus.     

Segregated assembly of muscle myosin expressed in nonmuscle cells.

Skeletal muscle myosin cDNAs were expressed in a simian kidney cell line (COS) and a mouse myogenic cell line to investigate the mechanisms controlling early stages of myosin filament assembly. An embryonic chicken muscle myosin heavy chain (MHC) cDNA was linked to constitutive promoters from adenovirus or SV40 and transiently expressed in COS cells. These cells accumulate hybrid myosin molecules composed of muscle MHCs and endogenous, nonmuscle, myosin light chains. The muscle myosin is found associated with a Triton insoluble fraction from extracts of the COS cells by immunoprecipitation and is detected in 2.4 +/- 0.8-micron-long filamentous structures distributed throughout the cytoplasm by immunofluorescence microscopy. These structures are shown by immunoelectron microscopy to correspond to loosely organized bundles of 12-16-nm-diameter myosin filaments. The muscle and nonmuscle MHCs are segregated in the transfected cells; the endogenous nonmuscle myosin displays a normal distribution pattern along stress fibers and does not colocalize with the muscle myosin filament bundles. A similar assembly pattern and distribution are observed for expression of the muscle MHC in a myogenic cell line. The myosin assembles into filament bundles, 1.5 +/- 0.6 micron in length, that are distributed throughout the cytoplasm of the undifferentiated myoblasts and segregated from the endogenous nonmuscle myosin. In both cell lines, formation of the myosin filament bundles is dependent on the accumulation of the protein. In contrast to these results, the expression of a truncated MHC that lacks much of the rod domain produces an assembly deficient molecule. The truncated MHC is diffusely distributed throughout the cytoplasm and not associated with cellular stress fibers. These results establish that the information necessary for the segregation of myosin isotypes into distinct cellular structures is contained within the primary structure of the MHC and that other factors are not required to establish this distribution.     

Modification of gizzard myosin and reconstituted actomyosin with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at low and high ionic strength.

Treatment of reconstituted gizzard actomyosin at 0.15 M or 0.6 M KCl with the fluorescent adenine analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, resulted in a significant decrease in the labeling of the myosin from actomyosin compared to that of myosin alone. Actin protected partially the K(+)-ATPase activity of myosin from modified actomyosin. The reagent was able to detect changes in the conformation of myosin as the distribution of the label in the heavy and light chains of myosin and actin was different at 0.15 M and 0.6 M KCl. The 6S and 10S transition, unique to smooth muscle myosin, can be monitored with the aid of this reagent.     

Binding of connectin to myosin filaments

Binding of native connectin (2,100 kDa fragment of alpha-connectin) to myosin filaments was investigated using a sedimentation technique and densitometric estimations of the separated proteins. In the presence of 60 mM KCl and 5 mM phosphate buffer, pH 7.0, as much as 1.5 mol of connectin was bound to 1 mol of myosin, suggesting that some 150 connectin filaments bound to a single myosin filament of approximately 0.5 micron in length. This value was much more than the ratio found in muscle (12:1). It appeared that C protein did not affect the binding of connectin to myosin filaments.     

Adenosine triphosphatase activity and "thick filament" formation of chicken gizzard myosin in low salt media.

The ATPase activity of chicken gizzard myosin was studied by varying the KCl concentration in the reaction medium. The following was thus found: (a) A sharp depression of the activity occurred when the KCl concentration was reduced to less than 0.3 M, showing the minimum activity around 0.15 M KCl. (b) The activity depression was removed by addition of urea or bay papain-digestion, but not by addition of p-chloromercuribenzoate. (c) In the KCl concentration where the activity depression occurred, the ATPase reaction proceeded in two distinct phases; the activity was relatively high in the early phase of the reaction and declined into the later phase where the steady state reaction took place. (d) In the KCl concentrations higher than that particular concentration or in the presence of urea, the ATPase reaction proceeded in one phase. (e) The temperature dependence of the ATPase activity in the early phase was of an ordinary magnitude being approximately equal to that of the ATPase activity in 0.6 M KCl. In contrast, the temperature dependence of the activity in the later phase was unusually small. Gizzard myosin in various concentrations of KCl was also examined by measuring the turbidity and the light-scattering intensity, and by observation under an electron microscope. The following was thus found: (a) In the KCl concentration where the activity depression occurred, there was a stagnation in the turbidity decrease as the KCl concentration was gradually increased and also the formation of "thick filaments," each of which was approximately 0.6-0.9 micron in length and 20-30 nm in diameter with no central "bare zone." (b) Addition of ATP induced dissociation of the thick filaments, and the dissociation occurred during the early phase of the ATPaseeaction. (c) Moreover, the temperature dependence of the ATP-induced dissociation rate was approximately equal to that of the ATPase activity in the early phase. On the basis of the findings mentioned above, it is concluded that the activity depression results from the ATP-induced dissociation of myosin filaments. Moreover, since high concentrations of KCl or urea also caused dissociation of myosin filaments and yet did not produce the activity depression, it was strongly suggested that gizzard myosin in the ATP-dissociated form must be different from that in the urea- or KCl-dissociated form, probably in the physical state of some myosin aggregates which were not detectable by the physical methods we used. As a side-observation, gizzard myosin filaments formed in the presence of ADP were found to be unusually long (longer than 2 micron), and they looked very similar to the particular filaments of skeletal myosin that were reported, by Moos, to be formed in the absence of the C protein.     

Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments

In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAMP. To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KCl, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that approximately 80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KCl (less than 10 mM) the Dictyostelium myosin was also soluble at 100,000 g. But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCl, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 mol Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of approximately 0.43 micron. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of approximately 5 micrograms/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCl, magnesium, ATP, or H+ strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli.     

Orchestrating nonmuscle myosin II filament assembly at the onset of cytokinesis

Contractile forces in the actomyosin cortex are required for cellular morphogenesis. This includes the invagination of the cell membrane during division, where filaments of nonmuscle myosin II (NMII) are responsible for generating contractile forces in the cortex. However, how NMII heterohexamers form filaments in vivo is not well understood. To quantify NMII filament assembly dynamics, we imaged the cortex of Caenorhabditis elegans embryos at high spatial resolution around the time of the first division. We show that during the assembly of the cytokinetic ring, the number of NMII filaments in the cortex increases and more NMII motors are assembled into each filament. These dynamics are influenced by two proteins in the RhoA GTPase pathway, the RhoA-dependent kinase LET-502 and the myosin phosphatase MEL-11. We find that these two proteins differentially regulate NMII activity at the anterior and at the division site. We show that the coordinated action of these regulators generates a gradient of free NMII in the cytoplasm driving a net diffusive flux of NMII motors toward the cytokinetic ring. Our work highlights how NMII filament assembly and disassembly dynamics are orchestrated over space and time to facilitate the up-regulation of cortical contractility during cytokinesis.     

Substructure and accessory proteins in scallop myosin filaments

Native myosin filaments from scallop striated muscle fray into subfilaments of approximately 100-A diameter when exposed to solutions of low ionic strength. The number of subfilaments appears to be five to seven (close to the sevenfold rotational symmetry of the native filament), and the subfilaments probably coil around one another. Synthetic filaments assembled from purified scallop myosin at roughly physiological ionic strength have diameters similar to those of native filaments, but are much longer. They too can be frayed into subfilaments at low ionic strength. Synthetic filaments share what may be an important regulatory property with native filaments: an order-disorder transition in the helical arrangement of myosin cross-bridges that is induced on activation by calcium, removal of nucleotide, or modification of a myosin head sulfhydryl. Some native filaments from scallop striated muscle carry short "end filaments" protruding from their tips, comparable to the structures associated with vertebrate striated muscle myosin filaments. Gell electrophoresis of scallop muscle homogenates reveals the presence of high molecular weight proteins that may include the invertebrate counterpart of titin, a component of the vertebrate end filament. Although the myosin molecule itself may contain much of the information required to direct its assembly, other factors acting in vivo, including interactions with accessory proteins, probably contribute to the assembly of a precisely defined thick filament during myofibrillogenesis.     

Interactions of muscle beta-connectin with myosin, actin, and actomyosin at low ionic strengths

The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15 M at pH 7.0 at 25 degrees C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of alpha-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils. The Mg2+-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1 M. The extent of activation by connectin was smaller than by alpha-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.     

In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.     

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.     

Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro

We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin- dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.     

Purealin, a novel stabilizer of smooth muscle myosin filaments that modulates ATPase activity of dephosphorylated myosin

Effects of purealin isolated from a sea sponge, Psammaplysilla purea, on the enzymatic and physiochemical properties of chicken gizzard myosin were studied. At 0.15 M KCl, 40 microM purealin increased the Ca2+- and Mg2+-ATPase activity of dephosphorylated gizzard myosin to 2.5- and 3-fold, respectively, but decreased the K+-EDTA-ATPase activity of the myosin to 0.25-fold. In contrast, purealin had little effect on the ATPase activities of phosphorylated gizzard myosin. The ATP-induced decrease in light scattering of dephosphorylated gizzard myosin at 0.15 M KCl was lessened by 40 microM purealin. Electron microscopic observations indicated that thick filaments of dephosphorylated myosin were disassembled immediately by addition of 1 mM ATP at 0.15 M KCl, although they were preserved by purealin for a long time even after addition of ATP. Upon ultracentrifugation, dephosphorylated myosin sedimented as two components, the 10 S species and myosin filaments, in the solution containing 0.18 M KCl and 1 mM Mg X ATP in the presence of 60 microM purealin. These results suggest that purealin modulates the ATPase activities of dephosphorylated gizzard myosin by enhancing the stability of myosin filaments against the disassembling action of ATP.     

Native bare zone assemblage nucleates myosin filament assembly

Native myosin filaments from rabbit psoas muscle are always 1·5 μm long. The regulated assembly of these filaments is generally considered to occur by an initial antiparallel and subsequent parallel aggregation of identical myosin subunits. In this schema myosin filament length is controlled by either a self-assembly or a Vernier process. We present evidence which refines these ideas. Namely, that the intact myosin bare zone assemblage nucleates myosin filament assembly. This suggestion is based on the following experimental evidence. (1) A native bare zone assemblage about 0·3 μm long can be formed by dialysis of native myosin filaments to either a pH 8 or a 0·2 m-KCl solution. (2) Upon dialysis back to 0·1 m-KCl, bare zone assemblages and distal myosin molecules recombine to form 1·5 μm long bipolar filaments. (3) The bare zone assemblage can be separated from the distal myosin molecules by column chromatography in 0·2 m-KCl. Upon dialysis of the fractionated subsets back to 0.1 m-KCl, the bare zone assemblage retains its length of about 0·3 μm. However, the distal molecules reassemble to form filaments about 5 μm long. (4) Filaments are formed from mixes of the isolated subsets. The lengths of these filaments vary with the amount of distal myosin present. (5) When native filaments, isolated bare zone assemblages or distal myosin molecules are moved sequentially to 0·6 m-KCl and then to 0·1 m-KCl. the final filament lengths are all about 5 μm. The capacity of the bare zone assemblage to nucleate filament assembly may be due to the bare zone myosin molecules, the associated M band components or both.     

Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial regulation of myosin II filament disassembly

Studies in Dictyostelium discoideum have established that the cycle of myosin II bipolar filament assembly and disassembly controls the temporal and spatial localization of myosin II during critical cellular processes, such as cytokinesis and cell locomotion. Myosin heavy chain kinase A (MHCK A) is a key enzyme regulating myosin II filament disassembly through myosin heavy chain phosphorylation in Dictyostelium. Under various cellular conditions, MHCK A is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. MHCK A possesses an aminoterminal coiled-coil domain that participates in the oligomerization, cellular localization, and actin binding activities of the kinase. In the current study, we show that the interaction between the coiled-coil domain of MHCK A and filamentous actin leads to an approximately 40-fold increase in the initial rate of kinase catalytic activity. Actin-mediated activation of MHCK A involves increased rates of kinase autophosphorylation and requires the presence of the coiled-coil domain. Structure-function analyses revealed that the coiled-coil domain alone binds to actin filaments (apparent K(D) = 0.9 microm) and thus mediates the direct interaction with F-actin required for MHCK A activation. Collectively, these results indicate that MHCK A recruitment to actin-rich sites could lead to localized activation of the kinase via direct interaction with actin filaments, and thus this mode of kinase regulation may represent an important mechanism by which the cell achieves localized disassembly of myosin II filaments required for specific changes in cell shape.     

Myosin and actin from Escherichia coli K12 C600.

Myosin-like protein and actin-like protein from E. coli formed filaments very similar in structure to those of myosin and actin from skeletal muscle. At 0.2 M KCl, a large number of "thick filaments" of uniform size (about 0.6-0.7 micron long and about 20 nm wide) was present. These thick filaments aggregated as the KCl concentration decreased to less than 0.2 M. Filaments of actin-like protein were decorated with muscle heavy meromyosin, showing "arrowheads". The arrowhead structure disappeared in the presence of ATP. A mixture of E. coli myosin-like protein and rabbit skeletal actin exhibited a gelation phenomenon on the additon of ATP. The phenomenon was reversible and showed ATP specificity. However, the gelation phenomenon was not observed with the mixture of E. coli actin-like protein and E. coli myosin-like protein. These results provide compelling evidence that the E. coli myosin-like protein and actin-like protein we isolated are essentially identical to myosin and actin, respectively.     

Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly

Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994, J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, alpha-actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (approximately 39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity. We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled.