CONTENT OF CYTOSKELETON PROTEINS IN OOCYTES OF THIRD GENERATION DROSOPHILA MELANOGASTER FRUIT FLIES OBTAINED AFTER A 44.5-DAY SPACE FLIGHT.

Third-stage larvae of the third generation of Drosophila melanogaster Canton S fruit flies were obtained after a 44.5-day flight of spacecraft a (Russia, 2014). In laboratory, the larvae grew into a third generation of flies. On the second day of emergence ovaries were extracted from female flies (group pF). Similarly, a third generation of flies (group C) was raised in the synchronous experiment. Real-time RT-PCR and Western blotting were used to determine the content of cytoskeletal proteins and mRNA of respective encoding genes. The content of mRNA genes encoding beta (Act57B and Act87E), gamma-actin (Act5C), acting-binding proteins (Arpc3, Tmod, Svil, Fim and Actn), and tubulin (Betatub85D) and tubulin-binding proteins (Msps, T-cpl, Cct5, T-cpleta) was less in group pF but not in the control group. The relative content of actin cytoskeleton components reduced significantly both in membrane and cytoplasmic fractions in group pF as compared to group C; however, reduction of the cytoplasmic fraction was more dramatic. On the contrary, content of beta-tubulin, a component of the tubulin cytoskeleton, reduced more significantly in the membrane fraction in group pF but not in group C. However, the acetylated tubulin content of ovarian membrane or cytoplasmic protein fraction was found unchanged both in the pF and C groups. The data suggest the presence in animal cells, oocytes specifically, of a mechanism regulating segregation of these proteins depending on external mechanical factors.

[Early Development under Microgravity Conditions].

The review is devoted to various aspects of early development under the space flight conditions. The different possible cell mechanosensors are considered. Structural and functional changes in the cells, predominantly, in non-muscle ones, were discussed. The results of the different experiments with the embryos of fish, amphibians, birds and mammals under microgravity conditions are shown discussing possible reasons for the development of morphological changes.

[Biophysical Model of Contractile Activity of Muscle Cells].

Muscle cells have the specific structure, the advanced cytoskeleton, which takes most of cell volume and forms a contractile apparatus. On the basis of the equations of continuum mechanics we proposed a mathematical model of the biomechanical behavior of cells as a whole, and it was modified to describe the contractile activity in muscle cells as an elastic rod. The model takes into account the result of transduction of external influences, namely the development of internal deformation, and allows for estimation of mobility and/or tension developed in muscle cells in the case of change in external influence.

[Structure of the cortical cytoskeleton of the left cardiac ventricle hystiocytes and of soleus muscle fibers in rats following short-time suspension].

Purpose of the investigation was to examine structural rearrangements in the cortical cytoskeleton as a result of changes in external mechanic conditions. Objects of the investigation were Wistar rat's m. soleus fibers and left ventricle hystiocytes. Suspension for 6, 12, 18, 24 and 72 hours was performed according to the Ilyin-Novikov procedure modified by Morey-Holton. Cell stiffness, non-muscle actin and actin-binding proteins in the protein membrane and cytoplasmic fractions, and respective gene expression were evaluated. In addition, corticosterone levels were measured in blood serum. After 6 hours of suspension, actin-binding proteins went down in the membrane fraction, i.e. alpha-actinin-1 decreased in hystiocytes and alpha-actinin-4 in m. soleus fibers. On the contrary, their content in the cytoplasmatic fraction increased. Expression of genes coding beta- and gamma-actin, alpha-actinin-1 and alpha-actinin-4 in m. soleus fibers showed a decrease. However, the apha-actinin-1 gene regained its baseline expression rate in 72 hours. Following 18 to 24 hours of suspension, expression of the beta-actin and alpha-actinin-4 genes in hystiocytes grew in comparison with baseline values, while expression of alpha-actinin-1 gene decreased. After 12 hours of suspension, beta- and gamma-actin levels were reduced in membrane proteins and increased in cytoplasmatic proteins in hystiocytes and m. soleus fibers. Stiffness showed a decline in each type of cell. Further into suspension, membrane proteins of hystiocytes increased levels of non-muscle actin and actin-binding proteins, and stiffness of these cells. Levels of these proteins decreased in m. soleus fibers, as also did cell stiffness. Serum corticosterone hardly altered but was slightly increased after 6 hours of suspension.

[Development of Drosophila melanogaster in space flight].

The review deals with the available literary data on different aspects of Drosophila melanogaster vital functions in the conditions of real and modeled microgravity. The developmental stages, embryogenesis and aging, specifically, and behavioral reactions are discussed. The presented results of morphological as well as molecular genetic analyses are indicative of structural changes in early Drosophila embryos and their compensation during subsequent development, and formation of an adaptive gene-expression pattern in microgravity.

Structure of cortical cytoskeleton in fibers of mouse muscle cells after being exposed to a 30-day space flight on board the BION-M1 biosatellite.

The aim of the work was to analyze changes in the organization of the cortical cytoskeleton in fibers of the mouse soleus muscle, tibialis anterior muscle and left ventricular cardiomyocytes after completion of a 30-day space flight on board the BION-M1 biosatellite (Russia, 2013). The transversal stiffness of the cortical cytoskeleton of the cardiomyocytes and fibers of the skeletal muscles did not differ significantly within the study groups compared with the vivarium control group. The content of beta- and gamma-actin in the membranous fraction of proteins in the left ventricular cardiomyocytes did not differ significantly within all study groups and correlated with the transversal stiffness. A similar situation was revealed in fibers of the soleus muscle and tibialis anterior muscle. At the same time, the content of beta-actin in the cytoplasmic fraction of proteins was found to be decreased in all types of studied tissues compared with the control levels in the postflight group, with lowered beta-actin gene expression rates in the postflight group. After completion of the space flight, the content of alpha-actinin-4 was found to be reduced in the membranous fraction of proteins from the mouse cardiomyocytes, while its content in the cytoplasmic fraction of proteins did not change significantly. Furthermore, gene expression rates of this protein were decreased at the time of dissection (it was started after 13 h after landing). At the same time, the content of alpha-actinin-1 decreased in the membranous fraction and increased in the cytoplasmic fraction of proteins from the soleus muscle fibers.

[Desmin content and transversal stiffness of the left ventricle mouse cardiomyocytes and skeletal muscle fibers after a 30-day space flight on board "BION-M1" biosatellite].

The aim of this study was to determine the transversal stiffness of the cortical cytoskeleton and the cytoskeletal protein desmin content in the left ventricle cardiomyocytes, fibers of the mouse soleus and tibialis anterior muscle after a 30-day space flight on board the "BION-M1" biosatellite (Russia, 2013). The dissection was made after 13-16.5 h after landing. The transversal stiffness was measured in relaxed and calcium activated state by, atomic force microscopy. The desmin content was estimated by western blotting, and the expression level of desmin-coding gene was detected using real-time PCR. The results indicate that, the transversal stiffness of the left ventricle cardiomyocytes and fibers of the soleus muscle in relaxed and activated states did not differ from the control. The transversal stiffness of the tibialis muscle fibers in relaxed and activated state was increased in the mice group after space flight. At the same time, in all types of studied tissues the desmin content and the expression level of desmin-coding gene did not differ from the control level.

[Cytochrome C content and mitochondrial activity in rat's cardiac and skeletal muscle cells in the course of 3-day gravitational unloading].

Purpose of the investigation was to determine the cytochrome c content and associated gene expression in cardiomyocytes and m. soleus fibers obtained from rats following 6, 12, 18, 24 and 72 hours of tail suspension. Cellular respiration was measured using Sax's polarography as a parameter reflecting the mitochondrial activity. In cardiomyocytes, cytochrome c in the protein membrane-mitochondrial fraction grew 34.6% (p < 0.05) after 18 hours of suspension relative the control. Cycs and gapdh expression was not altered, while parameters of the cardiomyocyte cellular respiration were increased. In m. soleus fibers, cytochrome c made a fall following 24-hr suspension. mRNA cycs was found reduced significantly after 6 hours and cellular respiration intensity declined reliably after 72 hours of gravitational unloading.

[Interaction between muscle cell and external mechanical field: a model study].

We have suggested a mathematical model of the muscle cell membrane on the basis of the thin elastic rods theory which takes external mechanic effects into account. We have also performed a quantitative analysis of deformation of rodent shin muscles when exposed to hindlimb suspension. Maximal deformation is shown to be typical for soleus muscle fibers and their magnitude is connected with stiffness of the cortical cytoskeleton.

Cell mechanosensitivity: mechanical properties and interaction with gravitational field.

This paper addressed the possible mechanisms of primary reception of a mechanical stimulus by different cells. Data concerning the stiffness of muscle and nonmuscle cells as measured by atomic force microscopy are provided. The changes in the mechanical properties of cells that occur under changed external mechanical tension are presented, and the initial stages of mechanical signal transduction are considered. The possible mechanism of perception of different external mechanical signals by cells is suggested.

Structure and functional characteristics of rat's left ventricle cardiomyocytes under antiorthostatic suspension of various duration and subsequent reloading.

The goal of the research was to identify the structural and functional characteristics of the rat's left ventricle under antiorthostatic suspension within 1, 3, 7 and 14 days, and subsequent 3 and 7-day reloading after a 14-day suspension. The transversal stiffness of the cardiomyocyte has been determined by the atomic force microscopy, cell respiration--by polarography and proteins content--by Western blotting. Stiffness of the cortical cytoskeleton increases as soon as one day after the suspension and increases up to the 14th day, and starts decreasing during reloading, reaching the control level after 7 days. The stiffness of the contractile apparatus and the intensity of cell respiration also increases. The content of non-muscle isoforms of actin in the cytoplasmic fraction of proteins does not change during the whole experiment, as does not the beta-actin content in the membrane fraction. The content of gamma-actin in the membrane fraction correlates with the change in the transversal stiffness of the cortical cytoskeleton. Increased content of alpha-actinin-1 and alpha-actinin-4 in the membrane fraction of proteins during the suspension is consistent with increased gamma-actin content there. The opposite direction of change of alpha-actinin-1 and alpha-actinin-4 content suggests their involvement into the signal pathways.

[Contractile properties, transversal stiffness and cytoskeletal protein content in Mongolian gerbils soleus fibers under long-term hindlimb suspension].

Structural and functional changes in Mongolian gerbil soleus fibers were analyzed after a 31-day hindlimb suspension. Contractile properties of muscle fibers were studied by means of tensometry; the transversal stiffness of different parts of the contractile apparatus was measured by atomic force microscopy, resting calcium level was estimated by fluorescence microscopy by using Fluo-4-AM; cytoskeletal protein content was determined by western blotting. It was shown that after gravitational unloading the maximal force of contraction and specific tension of fiber were significantly reduced, as well as calcium sensitivity actually lowered. At the same time, the transversal stiffness of Z-disk in the relaxed and activated state was decreased significantly compared to the control group. Desmin content was at the control level, but alpha-actinin-2, main structural protein of Z-disk, became considerably less after a 31-day hindlimb suspension. Besides, resting calcium level remained at control values during the simulated gravitational unloading. The data suggest that Z-disk destruction, as a result of alpha-actinin-2 content reduction, leads to changes in the lattice spacing and decreases contractile properties.

[Parameters of fibers cell respiration and desmin content in rat soleus muscle at early stages of gravitational unloading].

The aim of the work was to study the parameters of fibers cell respiration and desmin content in Wistar rat soleus muscle after 1, 3, 7 and 14 days of gravitational unloading. Gravitational unloading was simulated by antiorthostatic hindlimb suspension. The parameters of cell respiration were determined using the polarography, and desmin content was assessed by means of Western blotting. The results showed that the intensity of cell respiration is reduced after three days of gravitational unloading, reaches a minimum level after seven days and slightly increases by the fourteenth day of hindlimb unloading, as well as the content of desmin, which, however, to the fourteenth day returns to the control level. Taking into account that mitochondrial function depends on the state of cytoskeleton the data allow us to assume that early reduction of the intensity of cell respiration under unloading could be caused by degradation of the protein desmin that determines intracellular localization of mitochondria.

[Desmin content and cell breathing in rats' m. soleus fibers after 3- and 7-day recovery from 14-day suspension].

Cell breathing and relative desmin content were investigated in m. soleus fibers of rats after 3 and 7 days of recovery from 14-day suspension according to the Morey - Holton modification of the Ilyin - Novikov procedure. The cell breathing parameters were determined with the help of polarography; desmin content was measured using the western-blot technique. The results evidence that cell breathing intensity subsided during 14 days of gravitational unloading, reached minimum after 3 days of recovery and regained baseline values after 7 days of recovery. Post suspension desmin content did not differ from control values, made a significant drop in 3 days and returned to baseline values in 7 days of recovery. These data suggest an interdependence between cell breathing and desmin content in m. soleus fibers of rats as during gravitation unloading, so in the period of recovery.

Transversal stiffness and beta-actin and alpha-actinin-4 content of the M. soleus fibers in the conditions of a 3-day reloading after 14-day gravitational unloading.

The aim of the work was to analyze the structural changes in different parts of the sarcolemma and contractile apparatus of muscle fibers by measuring their transversal stiffness by atomic force microscopy in a three-day reloading after a 14-day gravity disuse, which was carried out by hind-limbs suspension. The object of the study was the soleus muscle of the Wistar rat. It was shown that after 14 days of disuse, there was a reduction of transversal stiffness of all points of the sarcolemma and contractile apparatus. Readaptation for 3 days leads to complete recovery of the values of the transversal stiffness of the sarcolemma and to partial value recovery of the contractile apparatus. The changes in transversal stiffness of sarcolemma correlate with beta-actin and alpha-actinin-4 in membrane protein fractions.

[Changes in cell respiration of postural muscle fibers under long-term gravitational unloading after dietary succinate supplementation].

The intensity of cell respiration of the rat m. soleus, m. gastrocnemius c.m. and tibialis anterior fibers during 35-day gravitational unloading, with the addition of succinate in the diet at a dosage rate of 50 mg per 1 kg animal weight has been investigated. The gravitational unloading was modeled by antiorthostatic hindlimb suspension. The intensity of cell respiration was estimated by polarography. It was shown that the rate of oxygen consumption by soleus and gastrocnemius fibers on endogenous and exogenous substrates and with the addition of ADP decreases after the discharge. This may be associated with the transition to the glycolytic energy path due to a decrease in the EMG-activity. At the same time, the respiration rate after the addition of exogenous substrates in soleus fibers did not increase, indicating a disturbance in the function of the NCCR-section of the respiratory chain and more pronounced changes in the structure of muscle fibers. In tibialis anterior fibers, no changes in oxygen consumption velocity were observed. The introduction of succinate to the diet of rats makes it possible to prevent the negative effects of hypokinesia, although it reduces the basal level of intensity of cell respiration.

Effects of long-term microgravitation exposure on cell respiration of the rat musculus soleus fibers.

Cell respiration of the m. soleus fibers was studied in Wistar rats treated with succinic acid and exposed to microgravitation for 35 days. The results indicated that respiration rates during utilization of endogenous and exogenous substrates and the maximum respiration rate decreased in animals subjected to microgravitation without succinate treatment. The respiration rate during utilization of exogenous substrate did not increase in comparison with that on endogenous substrates. Succinic acid prevented the decrease in respiration rate on endogenous substrates and the maximum respiration rate. On the other hand, the respiration rate on exogenous substrates was reduced in vivarium control rats receiving succinate in comparison with intact control group. That could indicate changed efficiency of complex I of the respiratory chain due to reciprocal regulation of the tricarbonic acid cycle.

[Effects of nifedipine on the mechanical properties of sarcolemma and modulation of calcium accumulation dynamics in fibers of the rat soleus muscle under short-term hypogravity conditions].

It has been shown that the modulation of the mechanical properties of sarcolemma by nifedipine may be related to the dynamics of accumulation of calcium ions under short-term rat hindlimb suspension. The basal calcium level was measured by the fluorescence probe Fluo-4AM, the transversal stiffness of different parts of the contractile apparatus and sarcolemma was estimated by atomic force microscopy, and the content of desmin was determined by gel electrophoresis with immunoblotting. It has been found that nifedipine has a protecting effect on muscle fibers under hypogravity by decreasing the degradation of desmin and proteins that determine the transversal stiffness of sarcolemma and the contractile apparatus, and the intensity of the increase in the basal calcium level. It was shown that the selective blocking of L-channels leads to an increase in the basal calcium level in intact soleus fibers. At the same time, the transversal stiffness of sarcolemma and the contractile apparatus increased. The mechanism of this increase is still unclear; however, it is assumed that it mediates the protecting action of nifedipine.