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1.
FORMATION OF ARROWHEAD COMPLEXES WITH HEAVY MEROMYOSIN IN A VARIETY OF CELL TYPES 总被引:157,自引:112,他引:45
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Heavy meromyosin (HMM) forms characteristic arrowhead complexes with actin filaments in situ. These complexes are readily visualized in sectioned muscle. Following HMM treatment similar complexes appear in sectioned fibroblasts, chondrogenic cells, nerve cells, and several types of epithelial cells. Thin filaments freshly isolated from chondrogenic cells also bind HMM and form arrowhead structures in negatively stained preparations. HMM-filament complexes are prominent in the cortex of a variety of normal metaphase and Colcemid-arrested metaphase cells. There is no detectable binding of HMM with other cellular components such as microtubules, 100-A filaments, tonofilaments, membranes, nuclei, or collagen fibrils. The significance of HMM-filament binding is discussed in view of the finding that arrowhead complexes form in types of cells not usually thought to contain actin filaments. 相似文献
2.
An actin-like component in spermatocytes of a crane fly (Nephrotoma suturalis Loew) 总被引:15,自引:0,他引:15
Decorated actin-like filaments were seen in spindles after crane fly spermatocytes were glycerinated and then treated with rabbit skeletal muscle heavy meromyosin (HMM). Both ATP and pyrophosphate inhibited the HMM reaction. In prometaphase, metaphase, and mid-anaphase cells, actin-like filaments were seen near regions where chromosomal spindle fibres are seen in living cells, and were oriented in the pole-to-pole direction. In the interzone of anaphase cells, actin-like filaments were not oriented in a preferential direction when they were not associated with the microtubules attached to the sex chromosomes. No filaments were seen in glycerinated spindles not treated with HMM. We discuss reasons why filaments might not be seen without prior HMM treatment, and we discuss the possible role of the actin-like filaments in the spindles. — Spindle microtubules often were not seen in cells treated with HMM. This depended on the stage of division: in prometaphase no microtubules were seen; in metaphase microtubules were seen, in apparently normal numbers; in mid-anaphase, microtubules between the autosomes and the poles were seen in reduced numbers, those associated with the equatorial sex-chromosomes were seen in apparently normal numbers, while those between the separating autosomal half-bivalents were not seen. Microtubules were not seen in glycerinated spindles not treated with HMM, suggesting that HMM in some way affects microtubule stability. The question of microtubule stability is briefly discussed. 相似文献
3.
Several and various types of cells contain fine cytoplasmic filaments closely resembling the myofilaments of muscle cells (2, 18, 23, 24). In many of these cells and especially when cultured, it has been demonstrated that some of these filaments react with heavy meromyosin (HMM) in the same way as do the actin filaments of muscle cells (3, 6 7). This suggests that these filaments may be actinoid and form part of a contractile system. As fine intracytoplasmic filaments do occur in lymphatic endothelial cells (2, 14), we undertook an electron microscope investigation of their fine structure and their reaction on incubation with HMM and EDTA. We postulated that lymphatic endothelial cells possess a contractile filamentous system to which these filaments belong. 相似文献
4.
Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca2+-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg2+-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg2+-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi. 相似文献
5.
Ultrastructurally, myoepithelial cells were shown to contain numerous fine filaments in their cytoplasm and resembled smooth muscle cells. The myoepithelial cell of the salivary gland has been considered to play an important role in the secretion of saliva. The present study showed that all the thin filaments (actin filaments) in the myoepithelial cell of the human parotid gland bound heavy meromyosin (HMM) and formed characteristic arrowhead structures. These filaments ran in two opposite directions with the poles at different ends. On the other hand, there was no binding of HMM with thicker filaments (10-nm filaments), plasma membrane, nuclear membrane, collagen fibrils, basement membrane or other cytoplasmic organelles. The present results strongly suggest that myoepithelial cells possess a contractile function parallel to the long axis of the cell for supporting the secretion of saliva in the parotid gland. 相似文献
6.
Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin 总被引:6,自引:0,他引:6
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D H Schwyter S J Kron Y Y Toyoshima J A Spudich E Reisler 《The Journal of cell biology》1990,111(2):465-470
Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule. 相似文献
7.
Actin-like filaments in bone cells of cultured mouse calvaria as demonstrated by binding to heavy meromyosin 总被引:4,自引:4,他引:0
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《The Journal of cell biology》1975,66(2):445-451
A variety of intracellular filaments (50-70 A in diameter) found in bone cells was shown to bind specifically to HMM. Because of this property, these filaments are probably biochemically similar to muscle actin. in osteoblasts and osteocytes, these reactive filaments were oriented in bundles parallel to the plasma membrane and filling the cell processes. In the osteoclast the filaments along the cell membrane were not so highly organized. In the clear zone, the quiescent part of the cell adjacent to the motile ruffled border, organized filament bundles were oriented perpendicular to the cell membrane and terminated in short processes at the bone surface. These filaments were also reactive with HMM. The possible significance of the filaments with respect to the physiology of bone cells is discussed. 相似文献
8.
Banding and polarity of actin filaments in interphase and cleaving cells 总被引:26,自引:19,他引:7
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《The Journal of cell biology》1980,86(2):568-575
Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments. 相似文献
9.
Actin-like filaments in the myoid cell of the testis 总被引:1,自引:0,他引:1
Yoshiro Toyama M.Sc. 《Cell and tissue research》1977,177(2):221-226
Microfilaments in the myoid cells of the peritubular tissue in the mouse, swine and human testis bind heavy meromyosin (HMM) and form arrowhead complexes. The periodicity of the arrowhead complexes is about 35 nm. Individual filaments show arrowheads that point in the same direction. Opposing polarity of the HMM-bound filaments is also observed. The microfilaments do not bind HMM in the presence of 10 mM ATP. After treatment with the contraction medium of Hoffmann-Berling, the filaments appear to be undulated. These observations indicate that the microfilaments in the myoid cell are actin-like in nature. A small number of thicker filaments (about 10 nm in diameter) which do not bind HMM is also observed in the cell. Microfibrils which have been reported around the human myoid cell are also found in the swine. 相似文献
10.
TAKASHI OBINATA IZUMI HAYASHI DONALD A. FISCHMAN 《Development, growth & differentiation》1974,16(2):105-122
A method is presented for the release of “native” thin filaments from 13-day old embryonic chick muscle without tryptic digestion or desoxycholate (DOC) solubilization of Z bands. The isolated filaments were 50–60 Å in diameter, of variable length, and formed “arrowhead-like” complexes with heavy meromyosin (HMM). In addition, the filaments interacted with purified myosin to form actomyosin as effectively as action extracted from an acetone powder of muscle. The Mg++-dependent ATPase activity and extent of superprecipitation of the synthetic actomyosin required a low concentration of Ca++, strongly suggesting the presence of troponin and tropomyosin on the thin filaments isolated from muscle at this stage of embryogenesis. The native thin filaments were more sensitive to trypsin than synthetic F-actin prepared from an acetone powder based on measurements of flow birefrengence, viscosity and the ability to activate myosin ATPase. 相似文献
11.
HEAVY meromyosin (HMM) fragments of myosin from striated muscle specifically bind with actin filaments to form complexes that are readily observed by electron microscopy1 in both negatively-stained preparations and sectioned material. The composite or “decorated filaments” appear like a line of arrowheads. The existence of such decorated filaments in cells or some cell fraction after treatment with HMM indicates that actin is present. Ishikawa et al.2 used this to demonstrate actin in a number of cultured cell types. More recently, other workers have similarly demonstrated actin filaments in slime mould3, amoebae4,5, blood platelets6, microvilli7, macrophages8 and, less convincingly, in sperm tails9 and the mitotic spindle10. We prove here that filaments from the cortical region of mouse neuroblastoma cells bind HMM and therefore contain actin. 相似文献
12.
To study the orientation and dynamics of myosin, we measured fluorescence polarization of single molecules and ensembles of myosin decorating actin filaments. Engineered chicken gizzard regulatory light chain (RLC), labeled with bisiodoacetamidorhodamine at cysteine residues 100 and 108 or 104 and 115, was exchanged for endogenous RLC in rabbit skeletal muscle HMM or S1. AEDANS-labeled actin, fully decorated with labeled myosin fragment or a ratio of approximately 1:1000 labeled:unlabeled myosin fragment, was adhered to a quartz slide. Eight polarized fluorescence intensities were combined with the actin orientation from the AEDANS fluorescence to determine the axial angle (relative to actin), the azimuthal angle (around actin), and RLC mobility on the <10 ms timescale. Order parameters of the orientation distributions from heavily labeled filaments agree well with comparable measurements in muscle fibers, verifying the technique. Experiments with HMM provide sufficient angular resolution to detect two orientations corresponding to the two heads in rigor. Experiments with S1 show a single orientation intermediate to the two seen for HMM. The angles measured for HMM are consistent with heads bound on adjacent actin monomers of a filament, under strain, similar to predictions based on ensemble measurements made on muscle fibers with electron microscopy and spectroscopic experiments. 相似文献
13.
Evidence for an interaction between the cell surface and intermediate filaments in cultured fibroblasts 总被引:6,自引:0,他引:6
Intermediate filaments (IF) were found in close proximity to the plasma membrane in substrate attached baby hamster kidney cells (BHK-21) and chick embryo fibroblasts (CEF) as well as cells removed from their substrate in the absence of trypsin. However, in cells removed with trypsin, it appeared that IF had retracted away from the membrane. In cells with abundant extracellular matrix (ECM), colchicine induced massive cables of IF, which appeared to interact with specialized areas of the inner plasma membrane. In cells lysed to extract most microfilaments and cytoplasmic constituents, the intact IF network which remained was closely associated with the ECM. From these ultrastructural observations it was concluded that IF interact in some way with a "cell membrane complex" defined as comprising the plasma membrane and molecules attached to its inner and outer surfaces. In order to investigate the possibility that components of the membrane complex may co-isolate with IF, native intermediate filaments (NIF) were prepared. In addition to the structural subunits and other associated polypeptides, a approximately 220 kd species which reacted specifically with antibodies directed against the ECM protein fibronectin (FN) was observed; 220 kd was still present after NIF were isolated under pH conditions where FN is more soluble, suggesting that its presence was not simply due to the coprecipitation of two insoluble proteins. Immunofluorescence and immunogold localization confirmed that FN is a component of the cell membrane complex with which IF appeared to interact. 相似文献
14.
Jack Rosenbluth 《The Journal of cell biology》1971,48(1):174-188
Segments of the lower small intestine of the toad Bufo marinus were excised and soaked for approximately 2 hr in Ringer's solution (pH 7.4 or 7.8) containing crystalline trypsin and then fixed for electron microscopy at approximately the same pH. Thin sections of the tunica muscularis of these specimens show smooth muscle cells ranging in appearance from severely damaged at one extreme to apparently unaffected at the other. Among these are cells at intermediate stages, including some which exhibit large and conspicuous populations of thick filaments closely resembling artificially prepared aggregates of smooth muscle myosin. The thick filaments have the form of tactoids ~ 250–300 A in diameter in their middle regions and are ~ 0.5–1.0 µ in length. In some preparations they also display an axial periodicity approximating 143 A. They are usually randomly oriented and segregated from the thin filaments, which tend to form closely packed, virtually crystalline bundles at the periphery of these cells. "Dense bodies" are absent from cells showing these changes. The simplest interpretation of these data is that smooth muscle myosin normally exists among the actin filaments in a relatively disaggregated state and that trypsin induces aggregation by altering the conformation of the myosin molecule. Alternatively, trypsin may act indirectly through an effect on some other smooth muscle protein which normally forms a stable complex with relatively disaggregated myosin. 相似文献
15.
Control of stimulus-secretion coupling in adrenal medullary chromaffin cells by microfilament-specific macromolecules 总被引:2,自引:0,他引:2
J E Friedman P I Lelkes K Rosenheck A Oplatka 《The Journal of biological chemistry》1986,261(13):5745-5750
We have incorporated the myosin fragment heavy meromyosin (HMM), which is known to interact mechanochemically and enzymatically with actin filaments, into intact chromaffin cells of the bovine adrenal medulla, in order to study the possible involvement of actin and myosin in stimulus-secretion coupling. HMM was found to stimulate secretion of catecholamines, to cause depolarization of the plasma membrane, and to enhance 22Na+ uptake. HMM-stimulated catecholamine secretion was dependent on the presence of extracellular Na+. The Na+ uptake caused by HMM was inhibited by 10 microM amiloride. Acetylcholine-stimulated catecholamine secretion and 22Na+ uptake were both enhanced by HMM incorporation. A Na+/H+ antiporter, activated by the interaction of HMM with the cells' microfilaments, seems to be involved in HMM action and could possibly also be a component of stimulus-secretion coupling in chromaffin cells, induced by regular agonists. 相似文献
16.
Actin in erythrocyte ghosts and its association with spectrin. Evidence for a nonfilamentous form of these two molecules in situ 总被引:47,自引:28,他引:19
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《The Journal of cell biology》1975,66(3):508-520
Actin was isolated from erythrocyte ghosts. It is identical to muscle actin in its molecular weight, net charge, ability to polymerize into filaments with the double helical morphology, and its decoration with heavy meromyosin (HMM). when erythrocyte ghosts are incubated in 0.1 mM EDTA, actin and spectrin are solubilized. Spectrin has a larger molecular weight than muscle myosin. When salt is added to the EDTA extract, a branching filamentous polymer is formed. However, when muscle actin and the EDTA extract are mixed together in the presence of salt, the viscosity achieved is less than the viscosity of the solution if spectrin is omitted. Thus, spectrin seems to inhibit the polymerization of actin. If the actin is already polymerized, the addition of spectrin increases the viscosity of the solution, presumably by cross-linking the actin filaments. The addition of HMM of trypsin to erythrocyte ghosts results in filament formation in situ. These agents apparently act by detaching erythrocyte actin from spectrin, thereby allowing the polmerization of one or both proteins to occur. Since filaments are not present in untreated erythrocyte ghosts, we conclude that erythrocyte actin and spectrin associate to form an anastomosing network beneath the erythrocyte membrane. This network presumably functions in restricting the lateral movement of membrane-penetrating particles. 相似文献
17.
Summary The cytochemistry and ultrastructure of intracytoplasmic filaments of pulmonary lymphatic endothelial cells of neonatal rabbits were studied by comparison with myofilaments of the peribronchial and pulmonary vascular smooth muscle cells. Two types of endothelial filaments were observed: thin filaments (diameter: 50 Å) which lie close to the abluminal cell membrane; and thick filaments (diameter: 90 Å) which are dispersed throughout the cell cytoplasm.Following heavy meromyosin (HMM) treatment, characteristic arrowhead complexes formed in the thin lymphatic endothelial filaments as well as in the actin filaments of the smooth muscle cells. There was no detectable reaction of HMM with the thick filaments.After incubation with EDTA, the thin filaments were labile, and the thick filaments became the major filamentous component in the endothelial cells. In smooth muscle cells, the actin myofilaments were also labile while the 100 Å filaments were stable.These observations support the hypothesis that the actin-like thin endothelial lymphatic filaments form part of a contractile system, while the thick filaments constitute a plastic cell skeleton. The significance of the contractile system in lymphatic endothelial cells might lie in a mechanism for the active regulation of the endothelial intercellular junctions and gaps and hence the permeability of the lymphatic endothelial cell lining.This study was supported by The Council for Tobacco Research—U.S.A. The authors thank Professor Robert C. Rosan, M.D. (Saint-Louis University—U.S.A.) for expert advice. R. Renwart, B. Emanuel and R. Jullet for technical, G. Pison and St. Ons for photographical and N. Tyberghien for secretarial assistance. 相似文献
18.
Previous cross-linking studies [Kim E, Bobkova E, Hegyi G, Muhlrad A & Reisler E (2002) Biochemistry 41, 86-93] have shown that site-specific cross-linking among F-actin monomers inhibits the motion and force generation of actomyosin. However, it does not change the steady-state ATPase parameters of actomyosin. These apparently contradictory findings have been attributed to the uncoupling of force generation from other processes of actomyosin interaction as a consequence of reduced flexibility at the interface between actin subdomains-1 and -2. In this study, we use EPR spectroscopy to investigate the effects of cross-linking constituent monomers upon the molecular dynamics of the F-actin complex. We show that cross-linking reduces the rotational mobility of an attached probe. It is consistent with the filaments becoming more rigid. Addition of heavy meromyosin (HMM) to the cross-linked filaments further restricts the rotational mobility of the probe. The effect of HMM on the actin filaments is highly cooperative: even a 1 : 10 molar ratio of HMM to actin strongly restricts the dynamics of the filaments. More interesting results are obtained when nucleotides are also added. In the presence of HMM and ADP, similar strongly reduced mobility of the probe was found than in a rigor state. In the presence of adenosine 5'[betagamma-imido] triphosphate (AMPPNP), a nonhydrolyzable analogue of ATP, weak binding of HMM to either cross-linked or native F-actin increases probe mobility. By contrast, weak binding by the HMM/ADP/AlF4 complex has different effects upon the two systems. This protein-nucleotide complex increases probe mobility in native actin filaments, as does HMM + AMPPNP. However, its addition to cross-linked filaments leaves probe mobility as constrained as in the rigor state. These findings suggest that the dynamic change upon weak binding by HMM/ADP/AlF4 which is inhibited by cross-linking is essential to the proper mechanical behaviour of the filaments during movement. 相似文献
19.
K Takehana M Abe M Yamaguchi T Uchida M Inagaki K Yamamoto J Masty A Winnard H Ueda H Miyata 《Acta anatomica》1992,143(4):294-300
Cytoplasmic filaments of the endothelial cells of sheathed capillaries in the pig spleen were identified and their ultrastructure was studied. Two types of cytoplasmic filaments were found: intermediate filaments (diameter: 10 nm) which filled most of the interior of the cells, and thin filaments (diameter: 5 nm) which were located just beneath the cell membrane and filled the lateral cytoplasmic processes. In immunocytochemical preparations, the intermediate filaments were positive for vimentin and desmin, and were negative for keratin. Staining of the thin filaments with heavy meromyosin resulted in arrowhead formations. These observations suggest that the intermediate filaments maintain the cytoarchitecture, possibly protecting the cell from structural alterations induced by blood pressure changes. Concurrently, thin filaments may facilitate the passage of red blood cells and blood platelets through the interendothelial fenestrae of the sheathed endothelial cell to the reticular meshwork in the capillary sheath. 相似文献
20.
Intensity fluctuations of laser light scattering were utilized in order to follow enhancement of translational motion of the actin-heavy meromyosin (HMM) complex in extremely dilute solutions accompanied by the hydrolysis of MgATP. Such enhancement was anticipated on the basis of the idea that active streaming along actin filaments should be associated with their mechanochemical reactivity. Native tropomyosin was added in order to stabilize actin in its filamentous form, thus allowing the reduction of actin concentration below 50 micrograms/ml to enable free movement of neighboring filaments and yet give a reliable signal. Analysis of the data in terms of Doppler broadening led to an approximate evaluation of the average velocity of translation of the mobile filaments. This velocity was found to increase with increasing HMM concentration up to a maximum attained at a molar ratio HMM/actin of 1:2, and then decreased. Total intensity measurements indicate that the mobile scatterer is actually a complex of HMM with an isolated actin filament. HMM subfragment-1 was found to be ineffective. These results suggest that cooperation between the two myosin heads is necessary for efficient induction of active streaming along isolated actin filaments. 相似文献