Heat-induced Gelation of Myosin from Leg and Breast Muscles of Chicken

Heat-induced gelation of myosin from leg and breast muscles of chicken was studied in 0.6 m KC1. Gel strength of breast myosin was higher than that of leg myosin between pH 5.2 and 6.0. Turbidity of breast myosin increased below pH 6.0 but that of leg myosin did not increase at pH 5.7. Turbidity of leg myosin was higher than that of breast myosin below pH 5.6. Viscosity of breast myosin increased between pH 5.5 and 6.0 as the pH decreases, although that of leg myosin decreased. The breast myosin assembled to form long filaments at pH 5.7, but leg myosin failed to form long filaments. At pH 5.4, breast myosin filaments became longer and leg myosin assembled into filaments though they were shorter than breast myosin filaments. The strength of heat-induced gel formed from the filamentous leg and breast myosins at acidic region was not influenced by F-actin. These results indicate that the strength of heat-induced gel of both myosins is closely related to their morphological properties.     

Activation of myosin Va function by melanophilin, a specific docking partner of myosin Va

It is known that melanophilin is a myosin Va-targeting molecule that links myosin Va and the cargo vesicles in cells. Here we found that melanophilin directly activates the actin-activated ATPase activity of myosin Va and thus its motor activity. The actin-activated ATPase activity of the melanocyte-type myosin Va having exon-F was significantly activated by melanophilin by 4-fold. Although Rab27a binds to myosin Va/melanophilin complex, it did not affect the melanophilin-induced activation of myosin Va. Deletion of the C-terminal actin binding domain and N-terminal Rab binding domain of melanophilin resulted in no change in the activation of the ATPase by melanophilin, indicating that the myosin Va binding domain (MBD) is sufficient for the activation of myosin Va. Among MBDs, the interaction of MBD-2 with exon-F of myosin Va is critical for the binding of myosin Va and melanophilin, whereas MBD-1 interacting with the globular tail of myosin Va plays a more significant role in the activation of myosin Va ATPase activity. This is the first demonstration that the binding of the cargo molecule directly activates myosin motor activity. The present finding raises the idea that myosin motors are switched upon their binding to the cargo molecules, thus avoiding the waste of ATP consumption.     

Changes in myosin isozymes during development of chicken breast muscle

The patterns of myosin isozymes in embryonic and adult chicken pectoralis muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light chains and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, the predominant isozyme component in embryonic pectoralis myosin could be clearly distinguished from adult myosin isozymes. SDS-polyacrylamide gel electrophoresis indicated that the light chain composition of embryonic myosin was also different from that of adult myosin. The pattern of peptide fragments produced by myosin digestion with a-chymotrypsin differed significantly between embryonic and adult skeletal myosin. These results suggest that myosin in the embryonic pectoralis muscle is different in both light and heavy chain composition from myosin in the same adult tissue.     

M-RIP targets myosin phosphatase to stress fibers to regulate myosin light chain phosphorylation in vascular smooth muscle cells

Vascular smooth muscle cell contraction and relaxation are directly related to the phosphorylation state of the regulatory myosin light chain. Myosin light chains are dephosphorylated by myosin phosphatase, leading to vascular smooth muscle relaxation. Myosin phosphatase is localized not only at actin-myosin stress fibers where it dephosphorylates myosin light chains, but also in the cytoplasm and at the cell membrane. The mechanisms by which myosin phosphatase is targeted to these loci are incompletely understood. We recently identified myosin phosphatase-Rho interacting protein as a member of the myosin phosphatase complex that directly binds both the myosin binding subunit of myosin phosphatase and RhoA and is localized to actin-myosin stress fibers. We hypothesized that myosin phosphatase-Rho interacting protein targets myosin phosphatase to the contractile apparatus to dephosphorylate myosin light chains. We used RNA interference to silence the expression of myosin phosphatase-Rho interacting protein in human vascular smooth muscle cells. Myosin phosphatase-Rho interacting protein silencing reduced the localization of the myosin binding subunit to stress fibers. This reduction in stress fiber myosin phosphatase-Rho interacting protein and myosin binding subunit increased basal and lysophosphatidic acid-stimulated myosin light chain phosphorylation. Neither cellular myosin phosphatase, myosin light chain kinase, nor RhoA activities were changed by myosin phosphatase-Rho interacting protein silencing. Furthermore, myosin phosphatase-Rho interacting protein silencing resulted in marked phenotypic changes in vascular smooth muscle cells, including increased numbers of stress fibers, increased cell area, and reduced stress fiber inhibition in response to a Rho-kinase inhibitor. These data support the importance of myosin phosphatase-Rho interacting protein-dependent targeting of myosin phosphatase to stress fibers for regulating myosin light chain phosphorylation state and morphology in human vascular smooth muscle cells.     

BIG1 is a binding partner of myosin IXb and regulates its Rho-GTPase activating protein activity

Myosin IXb, a member of the myosin superfamily, is a molecular motor that possesses a GTPase activating protein (GAP) for Rho. Through the yeast two-hybrid screening using the tail domain of myosin IXb as bait we found BIG1, a guanine nucleotide exchange factor for ADP-ribosylation factor (Arf1), as a potential binding partner for myosin IXb. The interaction between myosin IXb and BIG1 was demonstrated by co-immunoprecipitation of endogenous myosin IXb and BIG1 with anti-BIG1 antibodies in normal rat kidney cells. Using the isolated proteins, it was demonstrated that myosin IXb and BIG1 directly bind to each other. Various truncation mutants of the myosin IXb tail domain were produced, and it was revealed that the binding region of myosin IXb to BIG1 is the zinc finger/GAP domain. Interestingly, the GAP activity of myosin IXb was significantly inhibited by the addition of BIG1 with IC(50) of 0.06 microm. The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with the concentration similar to the inhibition of the GAP activity. Likewise, RhoA inhibited the BIG1 binding of myosin IXb. These results suggest that BIG1 and RhoA compete with each other for the binding to myosin IXb, thus resulting in the inhibition of the GAP activity by BIG1. The present study identified BIG1, the Arf guanine nucleotide exchange factor, as a new binding partner for myosin IXb, which inhibited the GAP activity of myosin IXb. The findings raise a concept that the myosin transports the signaling molecule as a cargo that functions as a regulator for the myosin molecule.     

Isolation of a cDNA encoding the motor domain of nonmuscle myosin which is specifically expressed in the mantle pallial cell layer of scallop (Patinopecten yessoensis)

It has been reported that catch and striated muscle myosin heavy chains of scallop are generated through alternative splicing from a single gene [Nyitray et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12686-12690]. They suggested that the catch muscle type myosin was expressed in various tissues of scallop, including the gonad, heart, foot, and mantle. However, there have been no reports of the primary structure of myosin from tissues other than the adductor muscles. In this study, we isolated a cDNA encoding the motor domain of myosin from the mantle tissue of scallop (Patinopecten yessoensis), and determined its nucleotide sequence. Sequence analysis revealed that mantle myosin exhibited 65% identity with Drosophila non muscle myosin, 60% with chicken gizzard smooth muscle myosin, and 44% with scallop striated muscle myosin. The mantle myosin has inserted sequences in the 27 kDa domain of the head region, and has a longer loop 1 structure than those of scallop striated and catch muscle myosins. Phylogenetic analysis suggested that the mantle myosin is classified as a smooth/nonmuscle type myosin. Western blot analysis with antibodies produced against the N-terminal region of the mantle myosin revealed that this myosin was specifically expressed in the mantle pallial cell layer consisting of nonmuscle cells. Our results show that mantle myosin is classified as a nonmuscle type myosin in scallop.     

The influence of trace amount of calponin on the smooth muscle myosins in different states

Calponin (CaP), a thin filament-associated protein, plays an important role in the regulation of smooth muscle contractility. It has been known that CaP inhibits the actin-activated myosin MgATPase activity via binding to F-actin, and stimulates myosin MgATPase activity via binding to myosin. Our recent study revealed a new phenomenon that trace amount of CaP (TAC) could influence the function of different states of myosin. Our data showed that in the absence of actin, CaP, even in the concentration of 0.0001 microM, significantly increased the precipitations of 1 microM unphosphorylated myosin, Ca(2+)-CaM dependently, and independently phosphorylated myosin by MLCK, and stimulated the MgATPase activities of these myosins slightly but significantly. However, no obvious change of precipitation of myosin phosphorylated by PKA was observed, indicating the relative selective effect of TAC. In the presence of actin, myosin, and TAC, the increase of myosin precipitation was abolished, and no obvious changes of actin precipitations and actin-activated myosin MgATPase activities were observed implicating the highly efficiency of TAC on myosin being present in the absence of actin. Although we cannot give conclusive comments to our results, we propose that the high efficiency of TAC-myosin interaction is present in the regulation of the function of myosin when actin is dissociated from myosin, even if CaP/myosin ratio is very low; this high efficient interaction between TAC and myosin can be abolished by actin. However, why and how TAC can possess such a high efficiency to influence myosin and how the physiological significance of the high efficiency of TAC is in regulating the interaction between myosin and actin remain to be investigated.     

Subcellular compartmentalization of myosin isoforms in embryonic chick heart ventricle myocytes during cytokinesis

Embryonic chick heart ventricle myocytes retain the ability to alternate between proliferation and functional differentiation. A cytoplasmic isoform of myosin is present in cleavage furrows of various nonmuscle cells during cytokinesis, whereas one or more of the cardiac myosin isoforms are localized in sarcomeres of beating cardiomyocytes. Antibodies were employed to reveal the subcellular localizations of cytoplasmic and cardiac myosin isoforms in embryonic chick ventricle cardiomyocytes during cytokinesis. Monoclonal anticytoplasmic myosin antibodies were prepared against myosin purified from brains of 1-day-posthatched chickens and shown to react with chick brain myosin heavy chain by Western blots and/or ELISA tests. One monoclonal antibrain myosin antibody also cross-reacted with chick cardiac myosin but not with skeletal or smooth muscle myosins. Two antichick cardiac myosin monoclonal antibodies and one antichick skeletal myosin polyclonal antibody that cross-reacts with cardiac myosin were employed to identify cardiac sarcomeric myosin. Cells were isolated from day 8 embryonic chick heart ventricles, enriched for myocytes, grown in vitro for 3 days, and then examined by immunofluorescence techniques. Monoclonal antibodies against cytoplasmic myosin preferentially localized in the cleavage furrows of both cardiofibroblasts and cardiomyocytes in all stages of cytokinesis. In contrast, antibodies that recognize cardiac myosin were distributed throughout cardiomyocytes during early stages of cytokinesis, but became progressively excluded from the furrow area during middle and late stages of cytokinesis. These data suggest that in cells that contain both cytoplasmic and sarcomeric myosin isoforms, only cytoplasmic myosin isoforms are mobilized to from the contractile ring for cytokinesis.     

Monoclonal antibodies localize changes on myosin heavy chain isozymes during avian myogenesis

Monoclonal antibodies were used to identify and localize by immunoelectron microscopy epitopes on myosin isozymes. An antibody that reacts with an amino-terminal fragment of the myosin heavy chain maps on the myosin head 140 A distal to the head-rod junction. It identifies an epitope that is shared on adult and embryonic myosin, and detects two transitions in myosin expression during avian pectoralis myogenesis. Another antibody maps to the carboxyl terminus of the myosin rod. It is specific for an adult fast myosin epitope that is not detected in early developing pectoralis muscle. In contrast, an epitope that is present throughout development is identified by an antibody that reacts with a myosin light chain. This light chain epitope is localized at the head-rod junction. These results demonstrate structural changes in widely separated regions of the myosin molecule accompanying the sequential expression of developmental myosin isozymes.     

Properties of porcine platelet myosin. I. Similarity between vertebrate smooth muscle and nonmuscle myosins in their binding properties with F-actin

The mechanism of the ATPase [EC 3.6.1.3] reaction of porcine platelet myosin and the binding properties of platelet myosin with rabbit skeletal muscle F-actin were investigated. The kinetic properties of the platelet myosin ATPase reaction, that is, the rate, the extent of fluorescence enhancement of myosin, the size of the initial P1 burst of myosin, and the amount of nucleotides bound to myosin during the ATPase reaction, were very similar to those found for other myosins. Strong binding of platelet myosin with rabbit skeletal muscle F-actin, as found for smooth muscle myosin, was suggested by the following results. The rate of the ATP-induced dissociation of hybrid actomyosin, reconstituted from platelet myosin and skeletal muscle F-actin, was very slow. The amount of ATP necessary for complete dissociation of hybrid actomyosin was 2 mol/mol of myosin, although skeletal muscle actomyosin is known to dissociate completely upon addition of 1 mol ATP per mol of myosin. Unlike skeletal muscle myosin, the EDTA(K+)-ATPase activity of platelet myosin was inhibited by skeletal muscle F-actin. These observations indicate that ATP hydrolysis by vertebrate nonmuscle myosin follows the same mechanism as with other myosins and that the binding properties of nonmuscle myosin with F-actin are similar to those of smooth muscle myosin but not to those of skeletal muscle myosin.     

Macrophage myosin. Regulation of actin-activated ATPase, activity by phosphorylation of the 20,000-dalton light chain.

Myosin was purified from rabbit alveolar macrophages in a form that could not be activated by actin. This myosin could be phosphorylated by an endogenous myosin light chain kinase, up to 2 mol of phosphate being incorporated/mol of myosin. The site phosphorylated was located on the 20,000-dalton myosin light chain. Phosphorylation of macrophage myosin was found to be necessary for actin activation of myosin ATPase activity. Moreover, the actin-activated ATPase activity was found to vary directly with the extent of myosin phosphorylation, maximal phosphorylation (2 mol of Pi/mol of myosin) resulting in an actin-activated MgATPase activity of approximately 200 nmol of Pi/mg of myosin/min at 37 degrees C. These results establish that phosphyoyration of the 20,000-dalton light chain of myosin is sufficient to regulate the actin-activated ATPase activity of macrophage myosin.     

Functional expression of a chimeric myosin-containing motor domain of Chara myosin and neck and tail domains of Dictyostelium myosin II

We succeeded in expressing a chimeric myosin that comprises the motor domain of characean algal myosin, (the fastest known motor protein), and the neck and tail domains of Dictyostelium myosin II. Although the chimeric myosin showed an ATPase activity comparable to that of muscle myosin (15 times higher than that of the wild-type Dictyostelium myosin II), the motile activity of the chimera was only 1.3 times higher than that of the wild-type. However, this is the first chimeric myosin that showed motile activity faster than at least one of the parent myosins. It was suggested, therefore, that the motor domain of Chara myosin has the potential for performing fast sliding movement.     

Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis

In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3–activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.     

Importance of the converter region for the motility of myosin as revealed by the studies on chimeric Chara myosins

A long alpha-helix in myosin head constitutes a lever arm together with light chains. It is known from X-ray crystallographic studies that the first three turns of this lever arm alpha-helix are inserted into the converter region of myosin. We previously showed that chimeric Chara myosin in which the motor domain of Chara myosin was connected to the lever arm alpha-helix of Dictyostelium myosin had motility far less than that expected for the motor domain of Chara myosin. Here, we replaced the inserted three turns of alpha-helix of Dictyostelium myosin with that of the Chara myosin and found that the replacement enhanced the motility 2.6-fold without changing the ATPase activity so much. The result clearly showed the importance of interaction between the converter region and the lever arm alpha-helix for the efficient motility of myosin.     

Regulation of Drosophila myosin ATPase activity by phosphorylation of myosin light chains--II. Flightless mfd- fly

1. The Ca2(+)-activated and Mg2+ actin-activated myosin ATPase activities of flightless mfd- mutant Drosophila flight muscle myosin were one-half and one-third of those of the wild-type fly muscle myosin, respectively. 2. In the two-dimensional gel electrophoresis, the spots corresponding to phosphorylated myosin light chains, Lfl and Ltl, were hardly detected in mfd- mutant myosin. 3. These results support not only the conclusion that phosphorylation of myosin light chains regulates Drosophila myosin ATPase activity but also the assumption that the phosphorylation of myosin light chains is directly involved in flight function of the Drosophila fly.     

Regulation of Dictyostelium myosin I and II

Dictyostelium expresses 12 different myosins, including seven single-headed myosins I and one conventional two-headed myosin II. In this review we focus on the signaling pathways that regulate Dictyostelium myosin I and myosin II. Activation of myosin I is catalyzed by a Cdc42/Rac-stimulated myosin I heavy chain kinase that is a member of the p21-activated kinase (PAK) family. Evidence that myosin I is linked to the Arp2/3 complex suggests that pathways that regulate myosin I may also influence actin filament assembly. Myosin II activity is stimulated by a cGMP-activated myosin light chain kinase and inhibited by myosin heavy chain kinases (MHCKs) that block bipolar filament assembly. Known MHCKs include MHCK A and MHCK B, which have a novel type of kinase catalytic domain joined to a WD repeat domain, and MHC-protein kinase C (PKC), which contains both diacylglycerol kinase and PKC-related protein kinase catalytic domains. A Dictyostelium PAK (PAKa) acts indirectly to promote myosin II filament formation, suggesting that the MHCKs may be indirectly regulated by Rac GTPases.     

Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle

The myosin isozymes present in the developing rat soleus muscle from 1 week to 6 weeks after birth were investigated using biochemical and immunological methods. Electrophoresis of native myosin reveals that adult slow myosin is present in the soleus as early as 1 week after birth. At this time, embryonic and neonatal myosin can also be demonstrated. Using an immunotransfer technique, the presence of slow myosin heavy chain can be demonstrated at all time points examined whereas neonatal myosin heavy chain diminishes in quantity between 2 and 3 weeks, and is undetectable in the adult soleus. Specific polyclonal antibodies were prepared to embryonic, neonatal, and adult fast and slow myosins. Immunocytochemistry reveals a cellular heterogeneity at all stages examined. Different combinations of myosin isozymes can be found in the soleus fibers depending on the stage of development; these results suggest therefore that myosin isozyme transitions are occurring. Approximately half the fibers contain embryonic and slow myosin at 1 week after birth; these fibers subsequently contain only slow myosin. A second group of fibers contains embryonic and neonatal myosin at 1 week and most of them subsequently accumulate adult fast myosin. A portion of this latter group begins to acquire slow myosin from 4 weeks of age. These data are interpreted to suggest that a preprogrammed sequence of myosin isozymes is embryonic----neonatal----adult fast. At any time during development of an individual fiber, induction of slow myosin accumulation and repression of other types can occur.     

The regulatory light chain is required for folding of smooth muscle myosin

Light chain phosphorylation causes the folded monomeric form of myosin to extend and assemble into filaments. This observation established the involvement of the 20-kDa regulatory light chain (LC20) in conformational transitions of smooth muscle myosin. To further assess the role of this subunit in the intramolecular folding of myosin, LC20 was removed from turkey gizzard myosin at elevated temperatures in the presence of EDTA through the use of an antibody affinity column. Metal-shadowed images showed that LC20-deficient myosin had a tendency to aggregate through the neck region. When MgATP was added to filaments formed from this myosin, less than 10% of the myosin was solubilized, indicating that myosin could not fold in the absence of light chain. Readdition of native regulatory light chain restored the myosin to its original solubility properties, thus establishing reversibility. Addition of foreign light chains from skeletal muscle myosin or a chymotryptic-cleaved gizzard light chain produced the same amount of monomeric myosin in high salt that was obtained by recombination with the homologous light chain. However, the ability of the hybrid myosins to assume the folded conformation was impaired, and only a partially folded species was obtained. Single-headed myosin, like rod and light chain-deficient myosin, remained filamentous in the presence of MgATP. These results are consistent with the hypothesis that the regulatory light chain in the neck region of myosin contributes to a binding site for the myosin tail.     

Targeting by myosin phosphatase-RhoA interacting protein mediates RhoA/ROCK regulation of myosin phosphatase

Vascular smooth muscle cell contractile state is the primary determinant of blood vessel tone. Vascular smooth muscle cell contractility is directly related to the phosphorylation of myosin light chains (MLCs), which in turn is tightly regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. Myosin phosphatase is the principal enzyme that dephosphorylates MLCs leading to relaxation. Myosin phosphatase is regulated by both vasoconstrictors that inhibit its activity to cause MLC phosphorylation and contraction, and vasodilators that activate its activity to cause MLC dephosphorylation and relaxation. The RhoA/ROCK pathway is activated by vasoconstrictors to inhibit myosin phosphatase activity. The mechanism by which RhoA and ROCK are localized to and interact with myosin light chain phosphatase (MLCP) is not well understood. We recently found a new member of the myosin phosphatase complex, myosin phosphatase-rho interacting protein, that directly binds to both RhoA and the myosin-binding subunit of myosin phosphatase in vitro, and targets myosin phosphatase to the actinomyosin contractile filament in smooth muscle cells. Because myosin phosphatase-rho interacting protein binds both RhoA and MLCP, we investigated whether myosin phosphatase-rho interacting protein was required for RhoA/ROCK-mediated myosin phosphatase regulation. Myosin phosphatase-rho interacting protein silencing prevented LPA-mediated myosin-binding subunit phosphorylation, and inhibition of myosin phosphatase activity. Myosin phosphatase-rho interacting protein did not regulate the activation of RhoA or ROCK in vascular smooth muscle cells. Silencing of M-RIP lead to loss of stress fiber-associated RhoA, suggesting that myosin phosphatase-rho interacting protein is a scaffold linking RhoA to regulate myosin phosphatase at the stress fiber.     

Susceptibility of Chara myosin to SH reagents

Cellular and intracellular motile events in plants are susceptible to SH reagents such as N-ethylmaleimide (NEM). It has long been believed that the target of the reagent is myosin. We compared the effect of NEM on the motile and ATPase activities of skeletal muscle myosin with that on plant myosin using characean algal myosin. It was found that the motile activity of myosin prepared from NEM-treated C. corallina decreased to a level accountable for the decrease in the velocity of cytoplasmic streaming but it was also found that Chara myosin was far less susceptible to NEM than skeletal muscle myosin.