Immunochemical analysis of the supramolecular structure of myosin in contractile cytoskeletons of Dictyostelium amoebae

A collection of monoclonal antibodies against Dictyostelium myosin was screened to identify an antibody that could distinguish monomeric from polymeric myosin. An antibody was found that reacted only with monomeric myosin, provided that the antigen-antibody reaction was carried out in solution. This antibody was used in competition radioimmunoassays to probe the supramolecular structure of myosin in Triton-extracted cell models, or cytoskeletons, of Dictyostelium amoebae. The competition assay showed that, as isolated, cytoskeletal myosin was entirely filamentous, but could be converted to monomeric form by increasing the ionic strength of the surrounding buffer. As monomer, it remained associated with the cytoskeleton and could be cycled back to filament form by a second change of buffer. The ability of cytoskeletons to carry out ATP-dependent contraction was examined as a function of the assembly state of myosin. The results suggested that filamentous myosin is responsible for contraction of the cortical filament matrix.     

Organization of stress fibers in cultured fibroblasts after extraction of actin with bovine brain gelsolin-like protein

Mouse and quail embryo fibroblasts were extracted with Triton X-100 and the resulting cytoskeletons were treated with gelsolin-like actin-capping protein (the 90-kDa protein-actin complex isolated from bovine brain). Staining of cells with rhodamine-conjugated phalloin or an antibody to actin did not reveal any actin-containing structures after treatment with the 90-kDa protein-actin complex. Extraction of actin was confirmed by SDS-gel electrophoresis. Immunofluorescence microscopy showed that vinculin and α-actinin were released from the cytoskeletons together with actin. However, myosin remained associated with the cytoskeleton after treatment with the 90-kDa protein-actin complex. The distribution of myosin in treated cells showed no significant difference from that in control cells: in both cases myosin was localized mainly in the stress fibers. Double-fluorescence staining showed the absence of actin in myosin-containing stress fibers of treated cells. The ultrastructural organization of actin-depleted stress fibers was studied by transmission electron microscopy of platinum replicas. On electron micrographs these fibers appeared as bundles of filaments containing clusters of globular material. It is concluded that myosin localization in stress fibers does not depend on actin.     

Non-sarcomeric mode of myosin II organization in the fibroblast lamellum

The organization of myosin in the fibroblast lamellum was studied by correlative fluorescence and electron microscopy after a novel procedure to reveal its underlying morphology. An X-rhodamine analog of conventional smooth muscle myosin (myosin II) that colocalized after microinjection with endogenous myosin was used to trace myosin distribution in living fibroblasts. Then, the same cells were examined by EM of platinum replicas. To visualize the structural arrangement of myosin, other cytoskeletal fibrillar structures had to be removed: microtubules were depolymerized by nocodazole treatment of the living cells before injection of myosin; continued nocodazole treatment also induced the intermediate filaments to concentrate near the nucleus, thus removing them from the lamellar region; actin filaments were removed after lysis of the cells by incubation of the cytoskeletons with recombinant gelsolin. Possible changes in myosin organization caused by this treatment were examined by fluorescence microscopy. No significant differences in myosin distribution patterns between nocodazole-treated and control cells were observed. Cell lysis and depletion of actin also did not induce reorganization of myosin as was shown by direct comparison of myosin distribution in the same cells in the living state and after gelsolin treatment. EM of the well-spread, peripheral regions of actin-depleted cytoskeletons revealed a network of bipolar myosin mini-filaments, contracting each other at their terminal, globular regions. The morphology of this network corresponded well to the myosin distribution observed by fluorescence microscopy. A novel mechanism of cell contraction by folding of the myosin filament network is proposed.     

Stopped-flow measurement of cytoskeletal contraction: Dictyostelium myosin II is specifically required for contraction of amoeba cytoskeletons

Cytoskeletons provide valuable information on the composition and organization of the cell's contractile machinery, and in many cases these cell models retain the ability to contract. To quantitate contraction rates, we developed a novel stopped-flow assay permitting simultaneous analysis of thousands of Dictyostelium cytoskeletons within milliseconds of mixing with Mg-ATP. Cytoskeletons were placed in one syringe of the stopped flow apparatus and the appropriate buffer was placed in the second syringe. Mixing with Mg-ATP caused an immediate increase in the absorbance at 310 nm. Rapid fixation of the cytoskeletons during the reaction confirmed that this change in absorbance was highly correlated with contraction of the cytoskeletons. This spectroscopic change was used to measure the effects of temperature, pH, ionic strength, and nucleotides on contraction rate. Treatment with high salt and ATP removed most of the myosin, some actin, and small amounts of minor proteins. These extracted cytoskeletons lost the ability to contract, but after the addition of purified Dictyostelium myosin they regained full function. In contrast, rabbit skeletal muscle myosin was unable to restore contractility, even though it bound to the extracted cytoskeletons. Cytoskeletons prepared from a myosin-null mutant did not contract. Upon the addition of purified ameba myosin, however, they became contractile. These results suggest that filamentous Dictyostelium myosin II is essential for contraction, and that the actin cytoskeleton and associated proteins retain their functional organization in the absence of myosin.     

Actomyosin organization during cytokinesis: reversible translocation and differential redistribution in Dictyostelium

Synchronized cultures of Dictyostelium discoideum were used to study organizational changes of the cytoskeleton during mitotic cell division. The agar-overlay technique (Yumura et al.: J. Cell Biol. 99:894-899, 1984) was employed for immunofluorescence localization and video microscopic observation of living mitotic cells. The mitotic phase was defined by changes in chromosome configuration by using a double stain with the fluorescent dye DAPI. This study showed that the actin- and myosin-containing cytoskeleton was reversibly redistributed between the cortical ectoplasm and the endoplasm during prophase and telophase. Both actin and myosin filaments were dissociated from the cell cortex in prophase. Most of the actin and myosin was filamentous and remained in the endoplasm until telophase. Saltatory movements of organelles stopped suddenly, coincident with the breakdown of the cytoplasmic microtubule network. This change in the microtubule system was temporally coupled with the disappearance of actomyosin from the cortex. At the same time, the local vibrating movement of particles almost stopped, suggesting that the viscoelastic nature of the endoplasm was altered. In the late anaphase, actin and myosin relocalized to the cortical ectoplasm. Early in this phase, myosin filaments were localized specifically at the anticipated cleavage furrow region of the cleavage furrow, whereas actin filaments were redistributed more uniformly in the cell cortex, with an extremely large accumulation in the polar pseudopods. Subsequently the actin formed an orderly parallel array of cables along with myosin filaments in the contractile ring. The spatial segregation of actin and myosin in late anaphase was clearly demonstrated by multipolar cell division of artificially induced giant cells. Actin was relocalized in both the polar and the proximal constricting regions whereas myosin was only localized in the center of each pair of daughter microtubule networks where the cleavage furrow was formed. This study demonstrates that actin and myosin are reorganized by a temporally coordinated but spatially different mechanism during cytokinesis of Dictyostelium.     

The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages

A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and bifurcates to form 0.2-0.5 micron thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment 1 uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and approximately 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAase I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 +/- 47 nm and 118 +/- 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130 degrees angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of cell cytoskeletons and of an actin gel made with actin- binding protein with anti-actin-binding protein IgG and anti-IgG-coated gold beads resulted in the deposition of clusters of gold at points where filaments intersect and at the ends of filaments that may have been in contact with the membrane before its removal with detergent. In the actin gel made with actin-binding protein, 75% of actin-fiber intersections labeled, and the filament spacing between intersections is consistent with that predicted on theoretical grounds if each added actin-binding protein molecule cross-links two filaments to form an intersection in the gel.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of native myosin filaments from muscle

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.     

Isolation, electron microscopy and 3D reconstruction of invertebrate muscle myofilaments

Understanding the molecular mechanism of muscle contraction and its regulation has been greatly influenced and aided by studies of myofilament structure in invertebrate muscles. Invertebrates are easily obtained and cover a broad spectrum of species and functional specializations. The thick (myosin-containing) filaments from some invertebrates are especially stable and simple in structure and thus much more amenable to structural analysis than those of vertebrates. Comparative studies of invertebrate filaments by electron microscopy and image processing have provided important generalizations of muscle molecular structure and function. This article reviews methods for preparing thick and thin filaments from invertebrate muscle, for imaging filaments by electron microscopy, and for determining their three dimensional structure by image processing. It also highlights some of the key insights into filament function that have come from these studies.     

Actin depolymerization drives actomyosin ring contraction during budding yeast cytokinesis

Actin filaments and myosin II are evolutionarily conserved force-generating components of the contractile ring during cytokinesis. Here we show that in budding yeast, actin filament depolymerization plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuate actomyosin ring constriction. Deletion of myosin II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin II motor activity. Model simulations based on experimental measurements support the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time regardless of the initial ring size, as originally reported for C. elegans embryonic cells. This prediction was validated in yeast cells of different sizes due to different ploidies.     

Expression in Escherichia coli of a functional Dictyostelium myosin tail fragment

The amino acid sequence of the myosin tail determines the specific manner in which myosin molecules are packed into the myosin filament, but the details of the molecular interactions are not known. Expression of genetically engineered myosin tail fragments would enable a study of the sequences important for myosin filament formation and its regulation. We report here the expression in Escherichia coli of a 1.5- kb fragment of the Dictyostelium myosin heavy chain gene coding for a 58-kD fragment of the myosin tail. The expressed protein (DdLMM-58) was purified to homogeneity from the soluble fraction of E. coli extracts. The expressed protein was found to be functional by the following criteria: (a) it appears in the electron microscope as a 74-nm-long rod, the predicted length for an alpha-helical coiled coil of 500 amino acids; (b) it assembles into filamentous structures that show the typical axial periodicity of 14 nm found in muscle myosin native filaments; (c) its assembly into filaments shows the same ionic strength dependence as Dictyostelium myosin; (d) it serves as a substrate for the Dictyostelium myosin heavy chain kinase which phosphorylates myosin in response to chemotactic signaling; (e) in its phosphorylated form it has the same phosphoamino acids and similar phosphopeptide maps to those of phosphorylated Dictyostelium myosin heavy chain; (f) it competes with myosin for the heavy chain kinase. Thus, all the information required for filament formation and phosphorylation is contained within this expressed protein.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Multiple forms of cardiac myosin-binding protein C exist and can regulate thick filament stability

Although absence or abnormality of cardiac myosin binding protein C (cMyBP-C) produces serious structural and functional abnormalities of the heart, function of the protein itself is not clearly understood, and the cause of the abnormalities, unidentified. Here we report that a major function of cMyBP-C may be regulating the stability of the myosin-containing contractile filaments through phosphorylation of cMyBP-C. Antibodies were raised against three different regions of cMyBP-C to detect changes in structure within the molecule, and loss of myosin heavy chain was used to monitor degradation of the thick filament. Results from Western blotting and polyacrylamide gel electrophoresis indicate that cMyBP-C can exist in two different forms that produce, respectively, stable and unstable thick filaments. The stable form has well-ordered myosin heads and requires phosphorylation of the cMyBP-C. The unstable form has disordered myosin heads. In tissue with intact cardiac cells, the unstable unphosphorylated cMyBP-C is more easily proteolyzed, causing thick filaments first to release cMyBP-C and/or its proteolytic peptides and then myosin. Filaments deficient in cMyBP-C are fragmented by shear force well tolerated by the stable form. We hypothesize that modulation of filament stability can be coupled at the molecular level with the strength of contraction by the sensitivity of each to the concentration of calcium ions.     

Immunoelectron microscopic studies of the ultrastructure of myosin filaments in Dictyostelium discoideum

We have examined the characteristics of myosin in situ in Dictyostelium amoebae. By an improved immunofluorescence method, we previously found rod-like structures that contain myosin, which we call "myosin rods", in amoebae (Yumura. S., and Fukui, Y. (1985) Nature, 314: 194-196). Although we prepared samples for electron microscopy using conventional chemical fixation to clarify the ultrastructure of the myosin rods, we could not find any filamentous structures similar to myosin thick filaments. Therefore, we examined the effects of chemical fixatives on the myosin rods in situ by immunofluorescence staining. When cells were fixed in more than 0.05% glutaraldehyde or more than 1% osmium tetroxide at 4 degrees C, the myosin rods disappeared. These effects did not result from loss of the antigenicity, because a monoclonal myosin-specific antibody was able to react with synthetic myosin filaments treated with 0.5% glutaraldehyde or 2% osmium tetroxide. Cells fixed by the procedure used for immunofluorescence staining were post-fixed with permissible concentrations of chemical fixatives and prepared for examination by transmission electron microscopy. We found discrete filaments of about 12 nm thickness between the microfilaments. These filaments were shown to contain myosin by immunoelectron microscopy with an immunogold probe. These filaments were thinner than synthetic myosin thick filaments formed in vitro in the presence of 10 mM MgCl2, but they were similar to those formed in the presence of 2 mM MgCl2, or under nearly physiological ionic conditions. The images after immunofluorescence and immunogold labeling both suggested that these 12-nm-thick filaments in Dictyostelium amoebae were myosin filaments in situ.     

Contraction of Dictyostelium ghosts reconstituted with myosin II.

Cytoskeletons, or 'Triton ghosts,' that contained mainly actin and myosin II were prepared from Dictyostelium discoideum amoebae by extraction with Triton X-100. The Triton ghosts contracted immediately upon addition of ATP. However, under high-salt conditions in the presence of ATP, they did not contract but released myosin II. Almost all of the applied myosin II became associated with ghosts when myosin-free Triton ghosts, prepared in this way, were incubated with purified actin and then with myosin II from Dictyostelium. Immunofluorescence microscopy demonstrated that the associated myosin was localized in the cortical actin layer of the ghosts. Furthermore, the ghosts reconstituted with purified myosin resumed ATP-dependent contraction. Skeletal muscle myosin could also restore contractility to ghosts from which myosin had been extracted. The amount of myosin II necessary for the contraction of the ghosts was calculated by two methods. Less than 10% of the myosin II in intact cells was necessary for the contraction. These results show that myosin II is responsible for the contraction of the Dictyostelium cytoskeleton.     

Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts

The authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin-like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin-free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde. Electron microscopy of platinum replicas of these preparations revealed dumbbell-shaped structures of approximately 0.28 micron in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment 1 and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin-free cytoskeletons were often organized in chains and lattices formed by end-to-end contacts of individual filaments at their head-containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.     

Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state

Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electron microscopy to study the effects of blebbistatin on the organization of the myosin heads on muscle thick filaments. Loss of ADP and Pi from the heads causes thick filaments to lose their helical ordering. In the presence of 100 μM blebbistatin, disordering was at least 10 times slower. In the M·ADP state, myosin heads are also disordered. When blebbistatin was added to M·ADP thick filaments, helical ordering was restored. However, blebbistatin did not improve the order of thick filaments lacking bound nucleotide. Addition of calcium to relaxed muscle homogenates induced thick-thin filament interaction and filament sliding. In the presence of blebbistatin, filament interaction was inhibited. These structural observations support the conclusion, based on biochemical studies, that blebbistatin inhibits myosin ATPase and actin interaction by stabilizing the closed switch 2 structure of the myosin head. These properties make blebbistatin a useful tool in structural and functional studies of cell motility and muscle contraction.     

Improved Procedures for Electron Microscopic Visualization of the Cytoskeleton of Cultured Cells

We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.     

Myosin II redistribution during rear retraction and the role of filament assembly and disassembly

The literature to date suggests a role for myosin II in rear retraction, including evidence that myosin undergoes a characteristic 'C'-to-spot redistribution at the cell posterior which is associated with retraction. Here we investigate the mechanism of both retraction and the'C'-to-spot using Dictyostelium cells containing mutant forms of myosin that affect its polymerization. 3 x Asp-myosin forms few if any filaments. When 3 x Asp cells are added to a wild-type mound, the mutant cells move directionally, but rear retraction is markedly delayed,demonstrating that myosin II filaments are essential for efficient retraction. In addition, using a GFP-tagged 3 x Asp-myosin, we observed a posterior spot pattern associated with retraction,but no cortical 'C' pattern preceding it. This suggests that filamentous myosin is required to produce the 'C', and that its failure to form results in defective rear retraction. In contrast, an alternate mutant myosin that forms filaments constitutively, 3 x Ala-myosin, forms 'Cs' and then spot patterns at the posterior, but in the interim the spots do not disintegrate. This suggests that spot dissolution occurs by filament depolymerization. In summary our data demonstrate a role for myosin II and the 'C'-to-spot in efficient rear retraction, and define filament assembly as critical for formation of the 'C' and filament disassembly as critical for dissolution of the spot.     

Multiple mechanisms for accumulation of myosin II filaments at the equator during cytokinesis

Total internal reflection fluorescence microscopy revealed how individual bipolar myosin II filaments accumulate at the equatorial region in dividing Dictyostelium cells. Direct observation of individual filaments in live cells provided us with much convincing information. Myosin II filaments accumulated at the equatorial region by at least two independent mechanisms: (i) cortical flow, which is driven by myosin II motor activities and (ii) de novo association to the equatorial cortex. These two mechanisms were mutually redundant. At the same time, myosin II filaments underwent rapid turnover, repeating their association and dissociation with the actin cortex. Examination of the lifetime of mutant myosin filaments in the cortex revealed that the turnover mainly depended on heavy chain phosphorylation and that myosin motor activity accelerated the turnover. Double mutant myosin II deficient in both motor and phosphorylation still accumulated at the equatorial region, although they displayed no cortical flow and considerably slow turnover. Under this condition, the filaments stayed for a significantly longer time at the equatorial region than at the polar regions, indicating that there are still other mechanisms for myosin II accumulation such as binding partners or stabilizing activity of filaments in the equatorial cortex.     

Human platelet myosin. II. In vitro assembly and structure of myosin filaments

We have used electron microscopy and solubility measurements to investigate the assembly and structure of purified human platelet myosin and myosin rod into filaments. In buffers with ionic strengths of less than 0.3 M, platelet myosin forms filaments which are remarkable for their small size, being only 320 nm long and 10-11 nm wide in the center of the bare zone. The dimensions of these filaments are not affected greatly by variation of the pH between 7 and 8, variation of the ionic strength between 0.05 and 0.2 M, the presence or absence of 1 mM Mg++ or ATP, or variation of the myosin concentration between 0.05 and 0.7 mg/ml. In 1 mM Ca++ and at pH 6.5 the filaments grow slightly larger. More than 90% of purified platelet myosin molecules assemble into filaments in 0.1 M KC1 at pH 7. Purified preparations of the tail fragment of platelet myosin also form filaments. These filaments are slightly larger than myosin filaments formed under the same conditions, indicating that the size of the myosin filaments may be influenced by some interaction between the head and tail portions of myosin molecules. Calculations based on the size and shape of the myosin filaments, the dimensions of the myosin molecule and analysis of the bare zone reveal that the synthetic platelet myosin filaments consists of 28 myosin molecules arranged in a bipolar array with the heads of two myosin molecules projecting from the backbone of the filament at 14-15 nm intervals. The heads appear to be loosely attached to the backbone by a flexible portion of the myosin tail. Given the concentration of myosin in platelets and the number of myosin molecules per filament, very few of these thin myosin filaments should be present in a thin section of a platelet, even if all of the myosin molecules are aggregated into filaments.