Actomyosin Filaments Research Articles

Myosin light chain kinase: functional domains and structural motifs

Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca2+/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca2+/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca2+/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca2+/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca2+/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.

Actomyosin motor in the merozoite of the malaria parasite, Plasmodium falciparum: implications for red cell invasion

The genome of the malaria parasite, Plasmodium falciparum, contains a myosin gene sequence, which bears a close homology to one of the myosin genes found in another apicomplexan parasite, Toxoplasma gondii. A polyclonal antibody was generated against an expressed polypeptide of molecular mass 27,000, based on part of the deduced sequence of this myosin. The antibody reacted with the cognate antigen and with a component of the total parasite protein on immunoblots, but not with vertebrate striated or smooth muscle myosins. It did, however, recognise two components in the cellular protein of Toxoplasma gondii. The antibody was used to investigate stage-specificity of expression of the myosin (here designated Pf-myo1) in P. falciparum. The results showed that the protein is synthesised in mature schizonts and is present in merozoites, but vanishes after the parasite enters the red cell. Pf-myo1 was found to be largely, though not entirely, associated with the particulate parasite cell fraction and is thus presumably mainly membrane bound. It was not solubilised by media that would be expected to dissociate actomyosin or myosin filaments, or by non-ionic detergent. Immunofluorescence revealed that in the merozoite and mature schizont Pf-myo1 is predominantly located around the periphery of the cell. Immuno-gold electron microscopy also showed the presence of the myosin around almost the entire parasite periphery, and especially in the region surrounding the apical prominence. Labelling was concentrated under the plasma membrane but was not seen in the apical prominence itself. This suggests that Pf-myo1 is associated with the plasma membrane or with the outer membrane of the subplasmalemmal cisterna, which forms a lining to the plasma membrane, with a gap at the apical prominence. The results lead to a conjectural model of the invasion mechanism.

Three-dimensional image reconstruction of reconstituted smooth muscle thin filaments: effects of caldesmon

Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities. A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function. Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C. Helically arranged actin monomers and tropomyosin strands were observed in both cases. In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin. In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites. The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.

Binding of Myosin Light Chain Kinase to Cellular Actin-Myosin Filaments

Myosin light chain kinase binds to the actomyosin-containing filaments in smooth and nonmuscle cells. However, the region of the kinase necessary for this high affinity binding in vivo is not known, although it has been proposed that the N and C termini bind to actin and myosin in vitro, respectively. Truncated myosin light chain kinases containing the catalytic core and calmodulin-binding domain but lacking N (amino acids 1-655) and/or C (amino acids 1004-1147) termini were expressed in the baculovirus system and purified. All enzymes were catalytically active and Ca2+/calmodulin-dependent. The C-terminal truncated myosin light chain kinase bound to detergent-washed smooth muscle contractile proteins similar to recombinant full-length myosin light chain kinase or enzyme purified from smooth muscle. The apparent affinity of the full-length kinase was greater for the actomyosin-containing filaments with associated proteins than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. In contrast, truncations at the N terminus alone or at both N and C termini resulted in no significant binding. Similar effects were observed by two other assays: binding of fluorescently labeled myosin light chain kinases to actin-containing stress fibers in detergent-treated fibroblasts and localization of fluorescently labeled kinases after microinjection into primary smooth muscle cells in culture. The full-length and the C-terminal truncated myosin light chain kinases, but not myosin light chain kinases truncated at the N terminus or both N and C termini, associated with filaments in cells. Thus, the N terminus and not the C terminus of myosin light chain kinase is necessary for high affinity binding to actomyosin-containing filaments in smooth and nonmuscle cells.

Helical Processing Using PHOELIX

We describe here a set of procedures and algorithms for helical processing that we refer to as the PHOELIX package. The package was developed to provide a time-efficient and semiautomated method for determining a three-dimensional density map from a specimen with helical symmetry. The procedures which are part of PHOELIX are drawn from the original MRC helical processing suite with extensions principally developed using the SUPRIM image processing package. The package in its current form has been optimized for the processing of actomyosin filaments but has been modified and applied to other helical structures.

Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments.

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.

Relationship between expression of serendipity alpha and cellularisation of the Drosophila embryo as revealed by interspecific transformation.

A dramatic reorganization of the cytoskeleton underlies the cellularisation of the syncytial Drosophila embryo. Formation of a regular network of acto-myosin filaments, providing a structural framework, and possibly a contractile force as well, appears essential for the synchronous invagination of the plasma membrane between adjacent nuclei. The serendipity alpha (sry alpha) gene is required for this complete reorganization of the microfilaments at the onset of membrane invagination. We compare here the structure and expression of sry alpha between D. pseudoobscura, D. subobscura and D. melanogaster. Interspersion of evolutionarily highly conserved and divergent regions is observed in the protein. One such highly conserved region shows sequence similarities to a motif found in proteins of the ezrin-radixin-moesin (ERM) family. Four 7-13 bp motifs are conserved in the 5' promoter region; two of these are also found, and at the same position relative to the TATA box, in nullo, another zygotic gene recently shown to be involved in cellularisation. The compared patterns of expression of D. melanogaster sry alpha and nullo, and D. pseudoobscura sry alpha reveal a complex regulation of the spatiotemporal accumulation of their transcripts. The D. pseudoobscura sry alpha gene is able to rescue the cellularisation defects associated with a complete loss of sry alpha function in D. melanogaster embryos, even though species-specific aspects of its expression are maintained. Despite their functional homologies, the D. melanogaster and D. pseudoobscura sry alpha RNAs have different subcellular localisations, suggesting that this specific localization has no conserved role in targeting the sry alpha protein to the apical membranes.

Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape.

Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopical and biochemical studies of actomyosin from pre- and post-spawned hake

1. 1. The ultrastructure of actomyosin obtained from pre- and post-spawned hake was investigated. 2. 2. A characteristic arrowhead structure was found on actomyosin filaments obtained from post-spawned hake. 3. 3. The protein obtained from pre-spawned fish showed a loss of the filamentous structure. 4. 4. The loss of the filamentous structure in actomyosin from pre-spawned hake was related to a decreased affinity between myosin and actin.

Slow calcium waves accompany cytokinesis in medaka fish eggs.

Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell. We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at approximately 0.5 micron/s. The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first waves travel within the assembling contractile band, they would indicate local increases of free calcium to concentrations of about five to eight micromolar. This is the first report to visualize high free calcium within cleavage furrows. Moreover, this is also the first report to visualize slow (0.3-1.0 micron/s) as opposed to fast (10-100 microns/s) calcium waves. We suggest that these first waves are needed for furrow growth; that in part they further furrow growth by speeding actomyosin filament shortening, while such shortening in turn acts to mechanically release calcium and thus propagates these waves as well as furrow growth. We also suggest that the second waves act to induce the exocytosis which provides new furrow membrane.

Pleiotropic effect of okadaic acid on maturing mouse oocytes

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

冷凍すり身中のタンパク質の分散状態に及ぼす重合リン酸塩の影響

For clarifying what dispersion state the proteins assume in Alaska pollack frozen surimis, effects of polyphosphates and of pH, ionic strength and Mg2+ on dispersion state of proteins were examined by phase contrast microscopy and electron microscopy. Frozen surimi and carp myofibril were homogenized with various dispersion media, and the dispersion states of proteins in the resultant homogenates were compared with respect to the effects of the medium composition. The pH of the dispersion media was found to affect the dispersion state of proteins, while ionic strength of polyphosphates did not affect the dispersion significantly. The ionic strength of polyphosphates, low or high, was not found to increase the effect of polyphosphates to cause dissociation of actin and myosin, resulting in occurrence of myosin-poor actomyosin filaments and myosin free F-actin filaments. Based on the facts obtained, it was estimated that actin and myosin in the frozen surimi exist in the state of myofibrillar structure.

Oxygen Exchange Reaction during ATP Hydrolysis by Glycerinated Muscle Fibers, Myofibrils, and Synthetic Actomyosin Filaments1

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.

A note on elastic energy density functions for largely deformed compressible rubber solids

Active chirality is an universal property of macromolecules as well as cellular systems and processes, such as nucleic acid, proteins, bacterial flagella and its motions, actomyosin filaments and network, and the left–right (LR) asymmetric morphogenesis, etc. Despite of attempts to elucidate the relations among cellular chirality, spontaneous cellular rotation and extracellular matrix properties, many features of the chiral active matter that are in contact with elastic substrates are still not clear in terms of fundamental physical principles. For instance, when an active chiral droplet in contact with an elastic substrate, the underline interplay among surface elasticity, chirality, and surface tension is largely unknown. In this work, based on the Gurtin–Murdoch surface elasticity we developed an adhesive surface hydro-elasticity theory of moving line to model and simulate the spontaneous rotation of a chiral active soft matter droplet on soft elastic substrates to study the rich morphological, conformational, and dynamic behaviors of the droplet/substrate interaction. In this study, we have found that both surface tension and substrate elasticity significantly affect the spontaneous rotation of the active chiral droplet on the substrate from several crucial aspects: (1) the geometry and morphology of the rotating droplet; (2) the angular velocity and helical vertices of the rotating droplet; (3) the order parameter distribution inside the rotating droplet, (4) the dynamic contact area of the rotating droplet, and (5) the contact stress distribution in both the droplet as well as the elastic substrate.

かまぼこの調製過程の電子顕微鏡による解析

Changes of fine structures of fish muscle matters along the kamaboko preparation process were examined under electron microscope. In the minced meat (nama surimi), nearly intact myofibrillar structures were seen. The cross sections showed a hexagonal arrangement with six actin filaments surrounding each myosin filament. In the minced meat adjusted to 75-78% moisture, myofibrillar structures without any appreciable M-lines were found, while bundles of irregularly arranged filaments were observed. When the minced meat is ground with NaCl, disintegration of the myofibrillar structures was found to proceed with the length of grinding time. In the meat paste obtained by grinding the minced meat with NaCl for 15 minutes, only filaments of 7nm in diameter were observed, which was assigned as actomyosin filaments, based on its cross section structure's similar to that of actomyosin. There was a difference in fine structures between the kamabokos which were cooked with and without setting treatment. Dispersion profiles of protein were more uniform in the former than in the latter. In low protein concentration portions, network structures of single and bundled filaments were found regardless of setting treatment applied. However, filaments of setting treated kamabokos were thicker than those of non-setting kamabokos.

冷凍すり身中のタンパク質の分散状態の電子顕微鏡的研究

Dispersion profiles of proteins in “muen” (non-salt) and “kaen” (salt added) frozen surimi (fish mince) were examined under electron microscope. The cross sections of the muen surimis gave various profiles but no appreciable difference related with the jelly strength of the drived kamabokos was found. The dispersion profiles of the muen surimis were classfied roughly into following four types: (1) a profile demonstrating well reserved myofibrillar structures in which the M-lines and the Z-bands remain in nearly intact state, (2) a profile demonstrating moyfibrillar structures in which the M-lines are inappreciable and the vague Z-bands are shown, (3) a profile demonstrating parallel streams of fine filaments which are probably assingnable as actomyosin filaments, (4) a profile in which no uniform structure is appeciable. The dispersion profiles in the kaen surimis were similar to those found in kamabokos (cooked jellies) and showed dense or loose matrices of cross-linked filaments. Dilute homogenate suspensions of the surimis illustrated two types of filaments which were probably assignable as actomyosin filaments and as naked F-action filaments beside aggregates of myosin. The grade SA surimis and some grade C surimis demonstrated fine arrowheaded filaments of actomyosin and gave good jellies. Other grade C surimis demonstrated filaments of myosin-poor actomyosin filaments and gave poor jellies.

Presence of connectin-like protein in white blood cells and platelets.

Connectin is known to be an anchoring protein of actomyosin filaments in skeletal muscle. Attempts were made to extract this protein from white blood cells and platelets in order to investigate its properties. The purified connectin-like protein obtained had a rubbery appearance and was insoluble in water and immobile on SDS gel-electrophoresis. Amino acid composition and electron microscopic features of the protein were similar to those of skeletal muscles. FITC-labeled antibody to the protein showed a positive reaction to the membrane fractions of red cells, white cells and platelets. In moving cells fluorescence was observed not only in pseudopod, but also in whole cytoplasm. No changes in the fluorescence patterns were observed with respect to moving stages or cell types. The presence of the protein beneath the cell membrane was also confirmed electron micrographically.

Receptor capping in mouse T-lymphoma cells: a Ca2+ and calmodulin-stimulated ATP-dependent process.

The roles that Ca2+, calmodulin, and ATP play in the redistribution of concanavalin A (Con A) binding sites on the surface of mouse T-lymphoma cells were examined. Membranes of cells labeled with fluorescein-conjugated Con A (Fl-Con A) were made permeable ("skinned") to ions and proteins by incubation in a solution containing no added Ca2+, 7 mM EGTA, and ATP. The intracellular ionic and protein concentrations could then be varied, and the degree of Con A receptor capping monitored simultaneously. A graded increase (9.0 to 30%) was found in the number of capped cells with increasing Ca2+ concentration from 10(-6)-10(-4.9) M. Increasing concentrations of trifluoperazine, chlorpromazine, and promethazine (1.5 x 10(-6) to 1.0 x 10(-4) M) in cell suspensions containing 10(-4) M Ca2+ produced graded inhibition of capping in the same order that the drugs bind to calmodulin. Removal of extracellular Ca2+ dissociated (reversed) some of the caps into patches, thus reducing their number (12%). ATP was required for either capping or cap dissociation to occur. Addition of calmodulin (3.9 x 10(-8)-6.3 x 10(-7) M) to the cell suspension increased the Ca2+ sensitivity. These results provide direct evidence that capping of Con A receptors is a reversible process (i) regulated by intracellular Ca2+ concentrations, (ii) requiring ATP as an energy source, and (iii) susceptible to the influence of calmodulin. These findings are consistent with the hypothesis that the collection of surface receptor patches into cap structures is controlled by the interaction of actomyosin filaments, which in turn is regulated by a Ca2+-calmodulin-activated control system.

Actomyosin interactions with insulin-storage granules in vitro.

Interactions between actomyosin and insulin storage granules isolated from rat islets of Langerhans have been examined in a simple system in vitro, which allows comparison of the sedimentation of the granules in the presence of absence of actomyosin in various conditions. Actomyosin altered granule-sedimentation rates in a manner consistent with the binding of the granules of actomyosin filaments. This interaction was enhanced by addition of ATP (1.5 mM) but unaltered by addition of CaCl2, by calmodulin or by calmodulin in the presence of 10 microM-CaCl2. Addition of EGTA (0.1 mM), cyclic AMP (10 microM) of cytochalasin B (10 microgram/ml) were also without effects in these conditions. Pre-incubation of granules with phospholipase c did not affect granule-actomyosin interaction. Ultrastructural studies showed close contacts between the membranes of the granules and actomyosin filaments. The results indicate the possibility that actomyosin might provide the motile force for granule translocation during the insulin secretory process.

The Cytoskeleton of Intestinal Microvilli Contains Two Polypeptides Immunologically Related to Proteins of Striated Muscle

The microvilli of intestinal cells contain a highly organized network of microfilaments. The central core of the microvillus is occupied by a bundle of polarized actin filaments, originating at the tip of the microvillus (Mooseker and Tilney 1975) and extending into a network made of actomyosin and intermediate filaments, called the terminal web. The terminal web is oriented perpendicularly to the microvillar axis. The actin microfilaments in these bundles are linked to one another by cross filaments (Mooseker 1976). Laterally, these bundles are connected to the plasma membrane via bridge filaments (Murkerjee and Staehelin 1971; Mooseker 1976; Matsudaira and Burgess 1979).