Models of contractile units and their assembly in smooth muscle

It is believed that the contractile filaments in smooth muscle are organized into arrays of contractile units (similar to the sarcomeric structure in striated muscle), and that such an organization is crucial for transforming the mechanical activities of actomyosin interaction into cell shortening and force generation. Details of the filament organization, however, are still poorly understood. Several models of contractile filament architecture are discussed here. To account for the linear relationship observed between the force generated by a smooth muscle and the muscle length at the plateau of an isotonic contraction, a model of contractile unit is proposed. The model consists of 2 dense bodies with actin (thin) filaments attached, and a myosin (thick) filament lying between the parallel thin filaments. In addition, the thick filament is assumed to span the whole contractile unit length, from dense body to dense body, so that when the contractile unit shortens, the amount of overlap between the thick and thin filaments (i.e., the distance between the dense bodies) decreases in exact proportion to the amount of shortening. Assembly of the contractile units into functional contractile apparatus is assumed to involve a group of cells that form a mechanical syncytium. The contractile apparatus is assumed malleable in that the number of contractile units in series and in parallel can be altered to accommodate strains on the muscle and to maintain the muscle's optimal mechanical function.     

Structural and functional reconstitution of thin filaments in the contractile apparatus of cardiac muscle.

The muscle contractile apparatus has a highly ordered liquid crystalline structure. The molecular mechanism underlying the formation of this apparatus remains, however, to be elucidated. Selective removal and reconstitution of the components are useful means of examining this mechanism. In addition, this approach is a powerful technique for examining the structure and function of a specific component of the contractile system. In this study we have achieved the structural and functional reconstitution of thin filaments in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with gelsolin. Under these conditions no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached 135 +/- 64% of the original level. The augmentation of tension was attributable to the elongation of reconstituted filaments. As another possibility for augmented tension generation, we suggest the presence of an inhibitory system that was not reconstituted. In any case, the thin filaments of the cardiac contractile apparatus are considered to be assembled so as not to develop the highest degree of tension. Incorporation of the tropomyosin-troponin complex fully restored Ca2+ sensitivity without affecting maximum tension. The present results indicate that a muscle contractile apparatus with a higher order structure and function can be constructed by the self-assembly of constituent proteins.     

The control of anthozoan cilia by the basal apparatus

The ciliary basal apparatus in the pharynx of the sea anemone, Calliactis parasitica (Couch), is composed of two centrioles, a single striated rootlet at least 20 microns long, and a basal foot, to the tip of which is attached a bundle of microtubules leading to the rootlet. When the basal apparatus is sectioned in the plane of the ciliary power-stroke, the distal centriole, with which the cilium base is continuous, is rarely found to be erect. The orientation of the distal centriole is determined by bending in the basal apparatus. Bending occurs only in the plane of the ciliary power-stroke towards the side from which the basal foot projects, and it is closely correlated with membrane buckling in the belt desmosome region of the cell apex. Associated with the belt desmosome, but not directly with the basal apparatus, are bundles of filaments. These filaments are of two size classes, 5-6 and 10 nm in diameter. A model is presented in which the 5-6 nm filaments form the basis of a contractile system which mediates membrane buckling in the region of the belt desmosome. This action effectively shortens the cell apex and thus forces the apparatus to bend. The precise reorientation of the distal centriole is a result of the mechanical properties of the basal apparatus.     

Influence of dispersion in myosin filament orientation and anisotropic filament contractions in smooth muscle

A new constitutive model for the biomechanical behaviour of smooth muscle tissue is proposed. The active muscle contraction is accomplished by the relative sliding between actin and myosin filaments, comprising contractile units in the smooth muscle cells. The orientation of the myosin filaments, and thereby the contractile units, are taken to exhibit a statistical dispersion around a preferred direction. The number of activated cross-bridges between the actin and myosin filaments governs the contractile force generated by the muscle and also the contraction speed. A strain-energy function is used to describe the mechanical behaviour of the smooth muscle tissue. Besides the active contractile apparatus, the mechanical model also incorporates a passive elastic part. The constitutive model was compared to histological and isometric tensile test results for smooth muscle tissue from swine carotid artery. In order to be able to predict the active stress at different muscle lengths, a filament dispersion significantly larger than the one observed experimentally was required. Furthermore, a comparison of the predicted active stress for a case of uniaxially oriented myosin filaments and a case of filaments with a dispersion based on the experimental histological data shows that the difference in generated stress is noticeable but limited. Thus, the results suggest that myosin filament dispersion alone cannot explain the increase in active muscle stress with increasing muscle stretch.     

Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.     

Response of the cortex to the mitotic apparatus during polar body formation in the starfish oocyte of Asterina pectinifera

In order to understand the mechanism of unequal division, polar body formation was investigated using the oocytes of the starfish, Asterina pectinifera. Cortical actin filaments were quantitatively measured after staining the maturing oocytes with fluorescently labeled phalloidin using a computer and image-processing software. Before polar body formation, at first the actin filaments at the animal pole decreased and subsequently the animal pole bulged. On the other hand, actin filaments surrounding the animal pole increased gradually and made a cleavage furrow around the animal pole as the bulge grew. Then the furrow ingressed and finally a polar body formed. When the surface force was calculated according to the cell shape, the surface force decreased at the animal pole but the force at the contractile ring increased. When by micromanipulation the mitotic apparatus was detached and translocated to the cortex other than the animal pole, polar body formation occurred all over the cortex of the oocyte, which indicates that the response of the whole cortex to the mitotic apparatus is equal. These results indicate that the decrease in the actin filaments and surface force near the centrosome of the mitotic apparatus as well as the increase in the actin filaments and surface force at some distance of the centrosome is important for cytokinesis.     

Identification and characterization of Myosin from rat testicular peritubular myoid cells

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth muscle-alpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMC-myosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 4 degrees C, but did at 20 degrees C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1.     

Arrangement of actin filaments and myosin-like filaments in the contractile ring and of actin-like filaments in the mitotic spindle of dividing HeLa cells

We used a glutaraldehyde-tannic acid-saponin fixative to improve the preservation of actin filaments in dividing HeLa cells during preparation for thin sectioning. The contractile ring in the cleavage furrow is composed of a parallel array of actin filaments that circle the equator. We show that many of these actin filaments are arranged in small bundles. These bundles consist of about 25 filaments throughout cytokinesis. For comparison, filopodia on these cells have about 23 actin filaments packed at a higher density than the filaments in the contractile ring bundles. Some of the contractile ring actin filaments appear to radiate out from electron-dense sites on the plasma membrane. The contractile ring also has a large number of short filaments 13 nm in diameter that closely resemble filaments formed from purified human cytoplasmic myosin. These thick filaments are aligned circumferentially and interdigitate with the actin filaments, as expected for a sliding filament mechanism of tension generation. There are no long actin filaments in the mitotic spindle, but there are a large number (400 to 1000 per μm ³) of very short filaments identical in appearance to actin filaments in other parts of these cells. These short filaments may account for the reported staining of the mitotic spindle with fluorescent antibodies to actin and with fluorescent myosin fragments.     

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) ^1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency ³. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction ⁴. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues ⁵. This later change increases muscle stiffness ⁶. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.     

Cardiac Fine Structure in Selected Arthropods and Molluscs

The ultrastructure of the single-chambered hearts of selectedarthropods is compared with that of the multi-chambered heartsof three molluscs. I used the following four systems to makethe comparison: (1) contractile apparatus, (2) sarcoplasmicreticulum and surface invaginations, (3) cell to cell junctions,and (4) nerves. The contractile apparatus is composed of thinand thick filaments. While the thin filaments have the samediameter, the diameter of the thick filaments differs from oneheart to another. Evidence is presented to indicate that thisis due to varying amounts of paramyosin in the thick filaments.The arthropod cardiac cells have an extensive system of sarcoplasmicreticulum, the terminal vesicles of which are coupled to theplasmalemma and to the invaginations of the plasmalemma, theT-system. The molluscan cardiac cells lack a typical T-system,which is presumably due to their small cell size (about 10 µm).They possess, however, an elaborate system of sarcoplasmic reticulumwhich extends from just under the plasmalemma to the middleof the cell. In addition to elaborate sarcoplasmic reticulum,the heart of the whelk (Busycon canaliculatum) possess manysmall invaginations of the plasmalemma, called sarcolemmic tubules.These invaginations of the cell surface are not found in thehearts of the few bivalves examined. All arthropod and molluscanhearts have intercalated discs which can be seen in the lightmicroscope. Two types of junctions can be distinguished in theelectron microscope. The mechanical junction is at the levelof the terminal sarcomere where the thin filaments are embeddedin the cell wall and dense granular material appears to causethe two adjacent cells to adhere to each other. The electricaljunction is found along the lateral borders of cells of boththe molluscan and arthropod hearts. Finally, while nerves appearto be absent in the myogenic moth heart, they are abundant inthe myogenic cockroach heart and in the neurogenic lobster heart.Furthermore, two types of nerves appear very prominently inthe myogenic molluscan hearts.     

Isolation and characterization of actin and myosin from B-lymphocytic guinea pig leukemia cells.

Actin and myosin have been isolated from a guinea pig B cell leukemia line, L2C. The m.w. and amino acid compositions of these proteins are similar to actin and myosin from other nonmuscle cell types. L2C actin polymerizes to form filaments and activates the ATPase activity of skeletal muscle myosin. Actin in crude lymphocyte extracts does not polymerize as well as predicted from the critical concentration of purified lymphocyte actin suggesting that other factors in lymphocyte extracts regulate actin polymerization. Lymphocyte myosin polymerizes to form synthetic filaments at low ionic strength. Lymphocyte myosin binds to actin, but its ATPase activity is not activated by actin. Possible mechanisms for regulation of the lymphocyte contractile apparatus and its importance in a number of lymphocyte functions are discussed.     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Fine structure of the nephridial muscle layer cells of Phascolosoma granulatum (Sipuncula)

The nephridial muscle layer of Phascolosoma granulatum consists of a network of longitudinal and circular cells separated by connective tissue matrix. The muscle fibers are densely packed with thick and thin myofilaments, among which are scattered cytoplasmic dense bodies. The nucleus and noncontractile cytoplasmic organelles occupy a lateral projection from the contractile portion of the fiber. Cytoplasmic dense bodies are the result of a clustering of an indeterminate number of the thin actin filaments that fill the cytoplasm between thick filaments. Attached to the cytoplasmic face of the cell membrane are membrane-associated electron-dense plaques. These sites are linked to the contractile myofilaments by narrow filamentous bridges. Extracellular narrow filaments extend from these plaques to collagen fibers of the connective tissue matrix. Differences in length of the dense plaques may be related to differences in thick myofilament diameter in three types of muscle fiber, types A, B and C, statistically distinguished by mean fiber size differences. The plaques may serve as connecting links for the transmission of tension from contractile units to the connective tissue of the muscle layer. © 1993 Wiley-Liss, Inc.     

The organization and fine structure of the muscles of the scolex of the cysticercoid of Hymenolepis microstoma

The organization and fine structure of the muscles of the scolex of the cysticercoid of Hymenolepis microstoma are described. The contractile apparatus consists of thick (175–325 Å diameter × 1.4 μm) and thin (60–80 Å diameter × 1 μm) filaments. The thick filaments are occasionally attached to the thin filaments by cross bridges. The thin filaments are attached to the dense bodies or to a dense zone at the sarcolemma at muscle insertions. In contracted muscle the thick filaments appear as quasi-hexagonal arrays or in lines. Each thick filament is surrounded by an orbit of up to 12 thin filaments, which in turn may be shared by adjacent thick filaments. Thin filaments may be present in quasi-rectangular or hexagonal groupings indicating some low order degree of actin lattice. The fusiform dense bodies (1,500 Å × 900 Å), consisting of up to 25 discrete substructures, are distributed uniformly throughout the myofiber and/or attached to the sarcolemma at attachment plaques. The sarcoplasmic reticulum, consisting of a presumed anastomosing network of tubules is structurally connected to the sarcolemma by periodic deposits of electron opaque material. Sarcoplasmic extensions of the myofiber(s) contain the nucleus, Golgi complexes, rough endoplasmic reticulum, ribosomes, β-glycogen, mitochondria and membrane bound electron dense structures. Upon activation of the metacestode, groups of α-glycogen and enlargement of the rough endoplasmic reticulum were observed. Microtubules which were conspicuously absent from the sarcoplasm of the unactivated worms appeared adjacent to the myofibers in activated worms.     

Physiological and cytochemical studies of Ca in the primary muscle of the trunk of Sagitta setosa (chaetognath)

The movements of Sagitta are conditioned by the presence of Ca(2+) in the external medium. When this ion is removed from artificial sea water, animals do not move. They swim again when Ca(2+) is present. Among the problems raised by this observation, we have studied the role of Ca(2+) in the contraction of the primary musculature. Physiological experiments show the central importance of the extracellular Ca(2+) and of its translocation through the membrane during the initiation of the contraction. Cytochemical data correlate these observations. They show that Ca(2+) is localized mainly at the level of the plasma membrane, its invaginations and in the poorly developed SR (less than 2% of cell). Like SR, mitochondria accumulate Ca(2+) but do not seem to participate in the regulation of these Ca movements except in abnormal situations. La(3+) blocks the entry of extracellular Ca(2+) and attaches to the membranes; this fixation is not the same on the plasma membrane and in its invaginations. The contractile apparatus of Sagitta primary musculature show remarkable specializations (Duvert and Savineau, 1986). It is composed of ribbon-shaped myofibrils of regular thickness surrounded by external membranes implicated in the fixation and the translocation of a pool of Ca(2+) necessary for initiating contraction. The poorly developed SR and the mitochondria probably modulate the functioning of the two types of fibres (A and B).     

Microtubles and actin filaments in teleost visual cone elongation and contraction

Teleost retinal cones contract in light and elongate in darkness. This paper describes the disposition of microtubules and cytoplasmic filaments in cone cells of 2 species of fish (Haemulon sciurus and Lutjanus griseus). In Haemulon, the neck-like “myoid” region of the cone changes in length from 5 μ to 75 μ. Maximal observed rates of elongation and contraction are comparable to that of chromosome movement in mitosis (2–3 μ/min). Microtubules presumably participate in cone elongation, since numerous longitudinal microtubules are present in the myoid region, and colchicine blocks dark-induced elongation. Myoid shortening, on the other hand, appears to be an active contractile process. Disruption of microtubules in dark-adapted cones does not produce myoid shortening in the absence of light, and light-induced myoid shortening is blocked by cytochalasin-B. Cone cells possess longitudinally-oriented thin filaments which bind myosin subfragment-1 to form arrowhead complexes typical of muscle actin. Myoid thin filaments are clearly observed in negatively stained preparations of isolated cones which have been disrupted with detergent after attachment to grids. These myoid filaments are not, however, generally preserved by conventional fixation, though bundles of thin filaments are preserved in other regions of the cell. Thus, actin filaments are poorly retained by fixation in precisely the region of the cone cell where contraction occurs. Cone cells also possess longitudinally-oriented thick filaments 130–160Å in diameter. That these thick filaments may be myosin is suggested by the presence of side-arms with approximately 150 Å periodicity. The linear organization of the contractile apparatus of the retinal cone cell makes this cell a promising model for morphological characterization of the disposition of actin and myosin filaments during contraction in a nonmuscle cell.     

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

Summary In the primitive red algaCyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study.C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules ofC. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against aC. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin immature contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments.Abbreviations ORF open reading frame - IPTG isopropyl--D(–)-thiogalactopyranoside - SDS-PAGE sodium dodecyl sulphate-poly-acrylamide gel electrophoresis - DAPI 4,6-diamidino-2-phenylindole