To the heart of myofibril assembly

One of the most fascinating examples of cytoskeletal assembly is the myofibril, the contractile structure of striated (i.e. skeletal and cardiac) muscle. Myofibrils are composed of repeating contractile units known as sarcomeres, perhaps the most highly ordered macromolecular structures in eukaryotic cells. When skeletal and cardiac muscle cells differentiate, thousands of structural and regulatory molecules assemble into the semicrystalline sarcomeric contractile units. As a consequence of this precise assembly, many different classes of proteins function together to convert the molecular interactions of actin and myosin efficiently into the macroscopic movements of contractile activity.     

Laser ablation reveals the impact of Cdc15p on the stiffness of the contractile ring

The mechanics that govern the constriction of the contractile ring remain poorly understood yet are critical to understanding the forces that drive cytokinesis. We used laser ablation in fission yeast cells to unravel these mechanics focusing on the role of Cdc15p as a putative anchoring protein. Our work shows that the severed constricting contractile ring recoils to a finite point leaving a gap that can heal if less than ∼1 µm. Severed contractile rings in Cdc15p-depleted cells exhibit an exaggerated recoil, which suggests that the recoil is limited by the anchoring of the ring to the plasma membrane. Based on a physical model of the severed contractile ring, we propose that Cdc15p impacts the stiffness of the contractile ring more than the viscous drag.     

A model of contractile tubules showing how they contract in the heliozoan Echinosphaerium

In the large heliozoan Echinosphaerium, contractile tubules (formerly called X-bodies) are located between the axopodial membrane and the axonemal microtubules. When axopodial contraction occurs, the tubules have been thought to be transformed from a tubular to a granular form, as seen in ultra-thin sections. Our detailed morphological observations of the contractile tubules, however, have revealed that this so-called granulation of the contractile tubules is mediated by self-twisting and supercoiling during contraction. We also examined the localization of calcium during axopodial contraction using a potassium pyroantimonate assay. Ca-Sb deposits were detected on contractile tubules only during the twisting and coiling of this organelle. Our results indicate that axopodial contraction is enforced by the twisting and coiling of contractile tubules, which action probably is mediated by Ca2+ ions.     

Involvement of smooth and coated vesicles in the function of the contractile vacuole complex of some cryptophycean flagellates

The contractile vacuole complex of cryptophycean flagellates comprises the contractile vacuole, a pore and a vesicular spongiome. A minority of spongiome vesicles bear a 15-nm coat on the cytoplasmic surface of the membrane. The coat superficially resembles a clathrin coat. The majority of vesicles are smooth surfaced. Both types of vesicles are found at the same time. Smooth vesicles can be seen in profile suggesting vesicle-vesicle and vesicle-vacuole fusion. It is suggested that smooth vesicles are involved in the segregation of fluid from the cytoplasm and in filling the vacuole. Coated elements exist only as independent vesicles and as coated pits in the contractile vacuole membrane. There is no evidence of fusion of coated vesicles. It is suggested that coated vesicles function to retrieve specific membrane components from the contractile vacuole.     

Dissecting the role of Rho-mediated signaling in contractile ring formation

In anaphase, microtubules provide a specification signal for positioning of the contractile ring. However, the nature of the signal remains unknown. The small GTPase Rho is a potent regulator of cytokinesis, but the involvement of Rho in contractile ring formation is disputed. Here, we show that Rho serves as a microtubule-dependent signal that specifies the position of the contractile ring. We found that Rho translocates to the equatorial region before furrow ingression. The Rho-specific inhibitor C3 exoenzyme and small interfering RNA to the Rho GDP/GTP exchange factor ECT2 prevent this translocation and disrupt contractile ring formation, indicating that active Rho is required for contractile ring formation. ECT2 forms a complex with the GTPase-activating protein MgcRacGAP and the kinesinlike protein MKLP1 at the central spindle, and the localization of ECT2 at the central spindle depends on MgcRacGAP and MKLP1. In addition, we show that the bundled microtubules direct Rho-mediated signaling molecules to the furrowing site and regulate furrow formation. Our study provides strong evidence for the requirement of Rho-mediated signaling in contractile ring formation.     

The ionic composition of the contractile vacuole fluid of Paramecium mirrors ion transport across the plasma membrane

In vivo K+, Na+, Ca2+, Cl- and H+ activities in the cytosol and the contractile vacuole fluid, the overall cytosolic osmolarity, the fluid segregation rate per contractile vacuole and the membrane potential of the contractile vacuole complex of Paramecium multimicronucleatum were determined in cells adapted to 24 or 124 mosm l(-1) solutions containing as the monovalent cation(s): 1) 2 mmol l(-1) K+; 2) 2 mmol l(-1) Na+; 3) 1 mmol l(-1) K+ plus 1 mmol l(-1) Na+; or 4) 2 mmol l(-1) choline. In cells adapted to a given external osmolarity i) the fluid segregation rate was the same if adapted to either K+ or Na+, twice as high when adapted to solutions containing both K+ and Na+, and reduced by 50% or more in solutions containing only choline, ii) the fluid of the contractile vacuole was always hypertonic to the cytosol while the sum of the ionic activities measured in the fluid of the contractile vacuole was the same in cells adapted to either K+ or Na+, at least 25% higher in cells adapted to solutions containing both K+ and Na+, and was reduced by 55% or more in solutions containing only choline, iii) the cytosolic osmolarity was the same in cells adapted to K+ alone, to Na+ alone or to both K+ and Na+, whereas it was significantly lower in cells adapted to choline. At a given external osmolarity, a positive relationship between the osmotic gradient across the membrane of the contractile vacuole complex and the fluid segregation rate was observed. We conclude that both the plasma membrane and the membrane of the contractile vacuole complex play roles in fluid segregation. The presence of external Na+ moderated K+ uptake and caused the Ca2+ activity in the contractile vacuole fluid to rise dramatically. Thus, Ca2+ can be eliminated through the contractile vacuole complex when Na+ is present externally. The membrane potential of the contractile vacuole complex remained essentially the same regardless of the external ionic conditions and the ionic composition of the fluid of the contractile vacuole. Notwithstanding the large number of V-ATPases in the membrane of the decorated spongiome, the fluid of the contractile vacuole was found to be only mildly acidic, pH 6.4.     

Dependence of the elastic characteristics and contractile activity of the rat tail artery on the transmural difference of potentials

Change in the diffuse portion of the double electric layer at the blood-vascular wall border under the effect of thorium nitric oxide and heparin affects the tone and contractile ability of the rat tail artery: thorium diminishes the vessel rigidity and the amplitude of its contractile response, whereas heparin augments the rigidity as well as the amplitude of the response. Thorium nitric oxide also diminishes rigidity of the vessel connective tissue skeleton. The difference of potentials between the vessel lumen and its wall seems to be capable of affecting the contractile apparatus of the smooth muscle cells by means of activation of potential-sensitive ionic channels.     

An Explanation for the Limited Contractility of Stentor amethystinus Leidy, 1880

SYNOPSIS. The contractile ability of Stentor amethystinus was compared to that of other highly contractile stentors. S. amethystinus contracted very little. A structural examination was made in an attempt to explain this difference in behavior. The results suggested that the endoplasm might act in this species as a hydrostatic skeleton contained within a continuous sheath of contractile material. It is also suggested that S. amethystinus might in fact be only slightly extensible as opposed to showing limited contraction.     

Plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity

We investigated the assembly of the TOM complex within skeletal muscle under conditions of chronic contractile activity-induced mitochondrial biogenesis. Tom40 import into mitochondria was increased by chronic contractile activity, as was its time-dependent assembly into the TOM complex. These changes coincided with contractile activity-induced augmentations in the expression of key protein import machinery components Tim17, Tim23, and Tom22, as well as the cytosolic chaperone Hsp90. These data indicate the adaptability of the TOM protein import complex and suggest a regulatory role for the assembly of this complex in exercise-induced mitochondrial biogenesis.     

Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers

O-N-acetylglucosaminylation is a reversible post-translational modification which presents a dynamic and highly regulated interplay with phosphorylation. New insights suggest that O-GlcNAcylation might be involved in striated muscle physiology, in particular in contractile properties such as the calcium activation parameters. By the inhibition of O-GlcNAcase, we investigated the effect of the increase of soleus O-GlcNAcylation level on the contractile properties by establishing T/pCa relationships. We increased the O-GlcNAcylation level on soleus biopsies performing an organ culture of soleus treated or not with PUGNAc or Thiamet-G, two O-GlcNAcase inhibitors. The enhancement of O-GlcNAcylation pattern was associated with an increase of calcium affinity on slow soleus skinned fibers. Analysis of the glycoproteins pattern showed that this effect is solely due to O-GlcNAcylation of proteins extracted from skinned biopsies. We also characterized the O-GlcNAcylated contractile proteins using a proteomic approach, and identified among others troponin T and I as being O-GlcNAc modified. We quantified the variation of O-GlcNAc level on all these identified proteins, and showed that several regulatory contractile proteins, predominantly fast isoforms, presented a drastic increase in their O-GlcNAc level. Since the only slow isoform of contractile protein presenting an increase of O-GlcNAc level was MLC2, the effect of enhanced O-GlcNAcylation pattern on calcium activation parameters could involve the O-GlcNAcylation of sMLC2, without excluding that an unidentified O-GlcNAc proteins, such as TnC, could be potentially involved in this mechanism. All these data strongly linked O-GlcNAcylation to the modulation of contractile activity of skeletal muscle.     

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.     

Cyclic nucleotides in the cellular action of neurohypophyseal hormones.

Cyclic 3',5'-nucleotides play an important role in the action of neurohypophyseal hormones on peripheral tissues. All available evidence indicates that cyclic AMP serves as an intracellular mediator in the regulatory action of neurohypophyseal hormones on transport of fluids and solutes across both mammalian and nonmammalian epithelial membranes. There is a close association among binding of neurohypophyseal hormones on membrane, stimulation of cyclic AMP generation, and the functional response. On the other hand, neurohypophyseal hormones have no similar effect on cyclic AMP metabolism in contractile tissues such as smooth muscle. It appears likely that neurohypophyseal hormones stimulate primarily generation of cyclic GMP in contractile tissues, and the increase in cyclic GMP levels may be associated with the contractile response. While the role of cyclic AMP in neurohypophyseal hormone effects in epithelia is firmly established, the possible role of cyclic GMP in contractile responses is largely hypothetical at the present time.     

Contractile properties of smooth muscles of bronchial tubes at formation of hyperreactance of airways

Contractile properties of smooth muscles of bronchial tubes of porpoises in formation of hyperreactance airways, were studied. Sensitization by ovalbumin results in formation of hyperresponsiveness of the airways as shown in increase in amplitude of contractile responses to histaminergic influences and oppression of adrenergic relaxations of segments. Respiratory epithelium oppresses contractile responses of airways in intact animals to histaminergic influences. Influence of epithelium on adrenergic responses depends on precontractile factor: they increase in prereduction by histamine. In formation of hyperresponse, the epithelium loses ability to modulate responses of smooth muscles of the airways. Incubation segments of smooth muscles of the airways in interleukin 5 increases contractile responses to histaminergic influences. Receptor of interleukin 5 (the soluble fraction) neutralizes the effect of exogenous interleukin 5, and incubation of receptor of sensitized animals oppresses histaminergic contractile responses.     

Contractility of rat testicular seminiferous tubules in vitro: prostaglandin f 1 alpha and indomethacin-1,2.

The frequency of spontaneous in vitro contractions of seminiferous tubules of the rat appeared to be increased in a dose-dependent manner by prostaglandin F1alpha. PGF1alpha treatment increased the tonus of the smooth muscle cells in the wall of the tubules as indicated by a reduction in the diameter of the tubules. When the tubules were rinsed successively with fresh Tyrode's solution, the contractile frequency was diminished. Returning the original bathing medium to the tubules restored their contractile frequency, as did treatment of the rinsed tubules with PGF1alpha (10(-7) M). Preinjecting ther rats with indomethacin tended to reduce the contractile frequency of the extirpated tubules. Treating the tubules with a solution of indomethacin for 90 min. in vitro was more effective than pretreatment in vivo in reducing contractile frequency, but a combination of these two procedures produced the greatest inhibition. PGF1alpha restored the contractile frequency of the indomethacin-treated tubules. Our results indicate that PGs modulate the in vitro contractility of the tubules.     

α2—肾上腺素体介导大鼠主动脉收缩的特性

为阐明功能性α2－肾上腺素受体（α2－AR）的特性及其与α1－AR的相互关系,以离体大鼠主动脉为模型,进行收缩功能实验,发现在大鼠主动脉中,α1－AR和α2－AR产可介导其收缩效应并以α1－AR折作用为主,α1－AR可增强α2－AR介导的收缩效应,而α2－AR则对α1－AR介导的收缩效应无影响,在不可逆阻断α1－AR而保留α2－AR的条件下,α2－AR介导的缩血管效应消失,仅在阈值浓度的KCl存在下才能显示,且收缩幅度较对照组显著降低,结果表明,在离体大鼠主体动脉中存在功能性α2－AR,α2－AR的缩血管作用依赖于α1－AR的激动,其最大收缩反应远远小于α1－AR。     

Beneficial and harmful effects of oscillatory mechanical strain on airway smooth muscle

Airway smooth muscle (ASM) cells are constantly under mechanical strain as the lung cyclically expands and deflates, and this stretch is now known to modulate the contractile function of ASM. However, depending on the experimental conditions, stretch is either beneficial or harmful limiting or enhancing contractile force generation, respectively. Stretch caused by a deep inspiration is known to be beneficial in limiting or reversing airway constriction in healthy individuals, and oscillatory stretch lowers contractile force and stiffness or lengthens muscle in excised airway tissue strips. Stretch in ASM culture has generally been reported to cause increased contractile function through increases in proliferation, contractile protein content, and organization of the cell cytoskeleton. Recent evidence indicates the type of stretch is critically important. Growing cells on flexible membranes where stretch is non-uniform and anisotropic leads to pro-contractile changes, whereas uniform biaxial stretch causes the opposite effects. Furthermore, the role of contractile tone might be important in modulating the response to mechanical stretch in cultured cells. This report will review the contrasting evidence for modulation of contractile function of ASM, both in vivo and in vitro, and summarize the recent evidence that mechanical stress applied either acutely within 2 h or chronically over 11 d is a potent stimulus for cytoskeletal remodelling and stiffening. We will also point to new data suggesting that perhaps some of the difference in response to stretch might lie with one of the fundamental differences in the ASM environment in asthma and in culture--the presence of elevated contractile tone.     

Characterization of contractile function and expression of muscarinic receptors,G proteins and adenylate cyclase in cultured tracheal smooth muscle of Swine

Smooth muscle cells lose their contractile function and phenotype very rapidly when placed in culture. During organ culture of smooth muscle strips, phenotype is lost more slowly. In the present studies, we established an organ culture model to study contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase in different serum concentrations in tracheal smooth muscle from swine. The results show that contractile function and the amounts of M(3) receptors, G proteins and adenylyl cyclase were maintained for up to 5 days in culture. The expression of M(2) receptors was significantly decreased in culture when compared to freshly isolated muscles. Maximal isometric tension was significantly increased in cultured muscles compared with freshly isolated muscles. Different serum concentrations did not significantly affect contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase. In conclusion, our studies suggest that cultured smooth muscle might be used as a model to study the regulation of contractile function of smooth muscle by various signal transduction pathways.     

The response of contractile and non-contractile vacuoles of Paramecium calkinsi to widely varying salinities.

Paramecium calkinsi from tidal marshes survive a wide salinity range. Fluid output of contractile vacuoles of these cells decreased as salinity of the medium to which they were acclimated increased, and both pulse rate and vacuole volume were used to regulate output. When cells were first exposed to more dilute medium, contractile vacuoles greatly increased volume so that fluid output increased even though pulse rate decreased. In cells shifted to a more concentrated medium, contractile vacuole output decreased by decreasing pulse rate. The contractile vacuole is surrounded by a set of collecting structures which change form as the salinity changes. Distensible ampullae are found in media of low salinity and collecting canals are found in media of high salinity. When cells are shifted from high salinity to low, the number of ampullae increases and the number of canals decreases. When cells are shifted from low salinity to high, the number of ampullae decreases and the number of canals decreases. Other non-contracting vacuoles also appear in response to a hypoosmotic shock. These include vacuoles within the cell as well as "blisters" on the surface. The number and frequency of blisters increases with the size of the hypoosmotic shock. They detach from cells without resulting in any visible loss of cytoplasm. Non-contractile vacuoles may play a role in sequestering and removing excess water that the contractile vacuoles cannot handle.     

Contractile activity of duodenum, jejunum, and ileum at psychogenic stress in rabbits before and after muscarinic or nicotinic cholinoceptor blockade

In chronic experiments on rabbits, myoelectric activity (contractile activity index) in distal part of the duodenum, proximal and distal parts of the jejunum and proximal part of the ileum was studied under psychogenic stress caused by rigid fastening rabbit to a table in supine position. In duodenum, the stressor impact rendered stimulating, and in an ileum--inhibitory influence on their motility. In a jejunum the inhibition with the subsequent stimulation was observed, the latter being more expressed in a proximal part of the intestine. The proximo-distal gradient of exitatory and inhibitory influences of the psychogenic stress on contractile activity of the small bowel was revealed: in distal direction, inhibitory influences strengthen and stimulatory ones weaken. The muscarinic receptor blockade abolished increase of the duodenal and jejunal contractile activity obsereved in the control. The nicotinic cholinoceptor blockade abolished increase of the duodenal contractile activity in the 1-st phase of the stressor response and did not exclude an increase of the duodenal contractile activity in the 2-nd phase of the response. Muscarinic or nicotinic blockade did not influence the manifestation of the inhibitory reaction of proximal part of the ileum. In the period after release of the animal, the duodenal and jejunal contractile activity exeeded its initial level. This exeeding did not preserve after muscarinic cholinoceptor blockade but did preserve after nicotinic one in duodenum and proximal jejunum. The received data allow to conclude, that produced by the stress increase of the contractile activity of the distal part of duodenum, proximal and distal parts of jejunum produced by the stress, as well as exceeding the initial contractile activity level in the period after release of an animal, are mediated by cholinergic effector neurones of the enteric nervous system.