Effects of altered gravity on the actin and microtubule cytoskeleton of human SH-SY5Y neuroblastoma cells

Summary. Human SH-SY5Y neuroblastoma cells were used to study the effects of altered gravity on the actin and microtubule cytoskeleton dynamics. A cholinergic stimulation of the cells during a 6 min period of changing gravity (3 parabolas) resulted in an enhanced actin-driven protrusion of evoked lamellipodia. Likewise, the spontaneous protrusive activity of nonactivated cells was promoted during exposure to changing gravity (6 up to 31 parabolas). Ground-based experiments revealed a similar enhancement of the spontaneous and evoked lamellar protrusive activity when the cells were kept at 2 g hypergravity for at least 6 min. This gravity response was independent of the direction of the acceleration vector in respect to the cells. Exposure of the cells to “simulated weightlessness” (clinorotation) had no obvious influence on this type of lamellar actin cytoskeleton dynamics. A 20 min exposure of the cells to simulated weightlessness or to changing gravity (6 to 31 parabolas) – but not to 2 g (hypergravity, centrifugation) – resulted in an altered arrangement of microtubules indicated by bending, turning, and loop formation. A similar altered arrangement was shown by microtubules which had polymerized into lamellipodia after release from a taxol block at simulated weightlessness (clinorotation) or during changing gravity (5 parabolas). Our data suggest that in human SH-SY5Y neuroblastoma cells, microgravity affects the dynamics and spatial arrangement of microtubules but has no influence on the Rac-controlled lamellar actin cytoskeleton dynamics and cell spreading. The latter, however, seems to be promoted at hypergravity. Correspondence and reprints: Cell and Developmental Neurobiology, Institute of Zoology, University of Hohenheim, Garbenstrasse 30, 70593 Stuttgart, Federal Republic of Germany.     

Global and temporal regulation of gene expression in Xenopus kidney cells in response to presumed microgravity generated by 3D clinostats.

We documented changes in morphology and gene expression of the renal epithelial cell line A6 derived from Xenopus leavis adult kidney induced by long-term culturing with three dimensional clinostats. An oligo microarray analysis on A6 cells showed that mRNA levels of 52 out of 8091 genes were significantly altered in response to clinorotation. Upregulation or downregulation of gene expression became evident on day 8 and day 10 while there was no significant change on day 5. However, on day 15, expression of 18 out of 52 genes resumed to the levels similar to its original levels while remaining 33 genes maintained altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in mRNA levels of selected genes were found only under clinorotation but not under hypergravity (7 G) and ground control (1 G). Morphological changes including loss of dome-like structures, disassembly of E-cadherin adherence junctions and disassembly of cortical actin were also observed over 10 days of culturing with clinorotation. The results revealed genes which expression was altered specifically in A6 cells cultured under clinorotation.     

Cell proliferation of Paramecium tetraurelia under clinorotation.

It has been reported that Paramecium proliferates faster under microgravity in space, and slower under hypergravity (Kato et al., 2003). Effects of gravity on cell proliferation could be discussed in terms of energetics of swimming. Because of the characteristics of 'gravikinesis' as well as 'gravitaxis', Paramecium would decrease the energy expenditure under microgravity and increase it under hypergravity. The larger stock of energy would enhance the proliferation under microgravity. In order to simulate the effect of microgravity, we investigated the proliferation under clinorotation. When cells were rotated at 2.5 rpm, the proliferation rate decreased. Similar but less pronounced decrease was also found under low speed clinorotation (0.2 rpm).     

Time-course of changes in amounts of specific proteins upon exposure to hyper-g, 2-D clinorotation, and 3-D random positioning of Arabidopsis cell cultures

In previous studies it has been shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by altered gene expression. In this study an investigation was carried out into how different g conditions affect the proteome of such cells. For this purpose, callus cells were exposed to 8 g (centrifugation) and simulated microgravity (2-D clinorotation: fast rotating clinostat, yielding 0.0016 g at maximum; and 3-D random positioning) for up to 16 h. Extracts containing total soluble protein were subjected to 2-D SDS-PAGE. Image analysis of Sypro Ruby-stained gels showed that approximately 28 spots reproducibly and significantly (P <0.05) changed in amount after 2 h of hypergravity (18 up- and 10 down-regulated). These spots were analysed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the case of 2-D clinorotation, 19 proteins changed in a manner similar to hypergravity, while random positioning affected only eight spots. Identified proteins were mainly stress related, and are involved in detoxification of reactive oxygen species, signalling, and calcium binding. Surprisingly, centrifugation and clinorotation showed homologies which were not detected for random positioning. The data indicate that simulation of weightlessness is different between clinorotation and random positioning.     

Enhanced expression of the LDH-A gene after gravity-changing stress in human RSa cells.

A major issue in radiation and space biology is whether gene expression levels are altered in cells exposed to gravity-changing stress. In the present study, genes up- or down-regulated in radiation-sensitive human RSa cells cultured under gravity-changing conditions, were identified using a PCR-based mRNA differential display method. Exposure of cells to gravity-changing stress was performed by free-fall with a drop-shaft facility or by an airplane-conducted parabolic flight. Among the candidates for gravity-changing stress-responsive genes obtained by the differential display analysis, the lactate dehydrogenase A gene (LDH-A) was confirmed by Northern blotting analysis to exhibit increased expression levels. The gravity-changing stress consisted of a combination of microgravity and hypergravity. However, exposure of the cells to hypergravity produced by centrifuge only slightly affected the LDH-A mRNA expression. Thus, LDH-A was found to be a candidate for the genes which play a role in the cellular response to gravity-changing stress, and mainly to microgravity.     

Caveolae and caveolae constituents in mechanosensing

Studies in modeled microgravity or during orbital space flights have clearly demonstrated that endothelial cell physiology is strongly affected by the reduction of gravity. Nevertheless, the molecular mechanisms by which endothelial cells may sense gravity force remain unclear. We previously hypothesized that endothelial cell caveolae could be a mechanosensing system involved in hypergravity adaptation of human endothelial cells. In this study, we analyzed the effect on the physiology of human umbilical vein endothelial cell monolayers of short exposure to modeled microgravity (24–48h) obtained by clinorotation. For this purpose, we evaluated the levels of compounds, such as nitric oxide and prostacyclin, involved in vascular tone regulation and synthesized starting from caveolae-related enzymes. Furthermore, we examined posttranslational modifications of Caveolin (Cav)-1 induced by simulated microgravity. The results we collected clearly indicated that short microgravity exposure strongly affected endothelial nitrix oxide synthase activity associated with Cav-1 (Tyr 14) phosphorylation, without modifying the angiogenic response of human umbilical vein endothelial cells. We propose here that one of the early molecular mechanisms responsible for gravity sensing of endothelium involves endothelial cell caveolae and Cav-1 phosphorylation.     

The oxidative burst reaction in mammalian cells depends on gravity

Gravity has been a constant force throughout the Earth’s evolutionary history. Thus, one of the fundamental biological questions is if and how complex cellular and molecular functions of life on Earth require gravity. In this study, we investigated the influence of gravity on the oxidative burst reaction in macrophages, one of the key elements in innate immune response and cellular signaling. An important step is the production of superoxide by the NADPH oxidase, which is rapidly converted to H₂O₂ by spontaneous and enzymatic dismutation. The phagozytosis-mediated oxidative burst under altered gravity conditions was studied in NR8383 rat alveolar macrophages by means of a luminol assay. Ground-based experiments in “functional weightlessness” were performed using a 2 D clinostat combined with a photomultiplier (PMT clinostat). The same technical set-up was used during the 13th DLR and 51st ESA parabolic flight campaign. Furthermore, hypergravity conditions were provided by using the Multi-Sample Incubation Centrifuge (MuSIC) and the Short Arm Human Centrifuge (SAHC). The results demonstrate that release of reactive oxygen species (ROS) during the oxidative burst reaction depends greatly on gravity conditions. ROS release is 1.) reduced in microgravity, 2.) enhanced in hypergravity and 3.) responds rapidly and reversible to altered gravity within seconds. We substantiated the effect of altered gravity on oxidative burst reaction in two independent experimental systems, parabolic flights and 2D clinostat / centrifuge experiments. Furthermore, the results obtained in simulated microgravity (2D clinorotation experiments) were proven by experiments in real microgravity as in both cases a pronounced reduction in ROS was observed. Our experiments indicate that gravity-sensitive steps are located both in the initial activation pathways and in the final oxidative burst reaction itself, which could be explained by the role of cytoskeletal dynamics in the assembly and function of the NADPH oxidase complex.     

T cell activation responses are differentially regulated during clinorotation and in spaceflight.

Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground-based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti-CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL-2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL-1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR-mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead-Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead-Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell proliferative response in microgravity culture is a result of alterations in signaling events within the first few hours of activation, which are required for the expression of important regulatory molecules.     

The effect of clinorotation on vestibular compensation in upside-down swimming catfish.

Upside-down swimming catfish Synodontis nigriventris can keep upside-down swimming posture stably under pseudo-microgravity generated by clinostat. When the vestibular organ is unilaterally ablated, the operated S. nigriventris shows disturbed swimming postures under the clinorotation condition. However, about 1 month after the operation, unilateral vestibular organ-ablated S. nigriventris shows stable upside-down swimming posture under the condition (vestibular compensation). In contrast, a closely related upside-up swimming catfish Synodontis multipunctatus belonging to same Synodontis family can not keep stable swimming postures under the clinorotation conditions. In this study, we examined the effect of continuous clinorotation on vestibular compensation in intact and unilateral vestibular organ-ablated Synodontis nigriventris and Synodontis multipunctatus. After the exposure to continuous clinorotation, the postures of the catfish were observed under microgravity provided by parabolic flights of an aircraft. Unilateral vestibular organ-ablated S. nigriventris which had been exposed to continuous clinorotation showed stable swimming postures and did not show dorsal light reaction (DLR) under microgravity. This postural control pattern of the operated catfish was similar to that of intact catfish. Intact and unilateral vestibular organ-ablated S. multipunctatus showed DLR during microgravity. Our results confirmed that S. nigriventris has a novel balance sensation which is not affected by microgravity. DLR seems not to play an important role in postural control. It remains unclear that the continuous clinorotation effects on vestibular compensation because we could not keep used unilateral vestibular organ-ablated fish alive under continuous clinorotation for uninterrupted 25 days. This study suggests that space flight experiments are required to explore whether gravity information is essential for vestibular compensation.     

Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells

Many space missions have shown that prolonged space flights may increase the risk of cardiovascular problems. Using a three-dimensional clinostat, we investigated human endothelial EA.hy926 cells up to 10 days under conditions of simulated microgravity (microg) to distinguish transient from long-term effects of microg and 1g. Maximum expression of all selected genes occurred after 10 min of clinorotation. Gene expression (osteopontin, Fas, TGF-beta(1)) declined to slightly upregulated levels or rose again (caspase-3) after the fourth day of clinorotation. Caspase-3, Bax, and Bcl-2 protein content was enhanced for 10 days of microgravity. In addition, long-term accumulation of collagen type I and III and alterations of the cytoskeletal alpha- and beta-tubulins and F-actin were detectable. A significantly reduced release of soluble factors in simulated microgravity was measured for brain-derived neurotrophic factor, tissue factor, vascular endothelial growth factor (VEGF), and interestingly for endothelin-1, which is important in keeping cardiovascular balances. The gene expression of endothelin-1 was suppressed under microg conditions at days 7 and 10. Alterations of the vascular endothelium together with a decreased release of endothelin-1 may entail post-flight health hazards for astronauts.     

Effect of hypergravity stimulus on XTH gene expression in Arabidopsis thaliana.

There are several reports indicating that hypergravity and microgravity influence the mechanical properties of cell walls in shoots, resulting in changes in the growth rate. The mechanical properties of cell walls in dicots are mainly determined by the physicochemical properties of xyloglucan, a matrix polysaccharide. An increase in the molecular mass of xyloglucan correlated with a decrease in cell wall extensibility. Hypergravity is known to increase the molecular mass of xyloglucan. The cell wall enzyme, xyloglucan endotransglucosylase/hydrolase (XTH) is involved in xyloglucan metabolism. Using Arabidopsis, it was examined whether or not the expression of XTH genes in the floral stem and rosette leaf is influenced by hypergravity. RT-PCR analysis revealed that the expression of XTH genes changes in response to hypergravity of 300 g.     

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

ABSTRACT: In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.     

Effect of spaceflight on isoflavonoid accumulation in etiolated soybean seedlings

In order to explore the potential impact of microgravity on flavonoid biosynthesis, we examined isoflavonoid levels in soybean (Glycine max) tissues generated under both spaceflight and clinorotation conditions. A 6-day Space Shuttle-based microgravity exposure resulted in enhanced accumulation of isoflavone glycosides (daidzin, 6"-O-malonyl-7-O-glucosyl daidzein, genistin, 6"-O-malonyl-7-O-glucosyl genistein) in hypocotyl and root tissues, but reduced levels in cotyledons (relative to 1g controls on Earth). Soybean seedlings grown on a horizontally rotating clinostat for 3, 4 and 5 days exhibited (relative to a vertical clinorotation control) an isoflavonoid accumulation pattern similar to the space-grown tissues. Elevated isoflavonoid levels attributable to the clinorotation treatment were transient, with the greatest increase observed in the three-day-treated tissues and smaller increases in the four- and five-day-treated tissues. Differences between stresses presented by spaceflight and clinorotation and the resulting biochemical adaptations are discussed, as is whether the increase in isoflavonoid concentrations were due to differential rates of development under the "gravity" treatments employed. Results suggest that spaceflight exposure does not impair isoflavonoid accumulation in developing soybean tissues and that isoflavonoids respond positively to microgravity as a biochemical strategy of adaptation.     

The development of vestibular connections in rat embryos in microgravity

Existing experimental embryological data suggests that the vestibular system initially develops in a very rigid and genetically controlled manner. Nevertheless, gravity appears to be a critical factor in the normal development of the vestibular system that monitors position with respect to gravity (saccule and utricle). In fact several studies have shown that prenatal exposure to microgravity causes temporary deficits in gravity-dependent righting behaviors, and prolonged exposure to hypergravity from conception to weaning causes permanent deficits in gravity-dependent righting behaviors. Data on hypergravity and microgravity exposure suggest some changes in the otolith formation during development, in particular the size although these changes may actually vary with the species involved. In adults exposed to microgravity there is a change in the synaptic density in the optic sensory epithelia suggesting that some adaptation may occur there. However, effects have also been reported in the brainstem. Several studies have shown synaptic changes in the lateral vestibular nucleus and in the nodulus of the cerebellum after neonatal exposure to hypergravity. We report here that synaptogenesis in the medial vestibular nucleus is retarded in developing rat embryos that were exposed to microgravity from gestation days 9 to 19.     

Dome formation and tubule morphogenesis by Xenopus kidney A6 cell cultures exposed to microgravity simulated with a 3D-clinostat and to hypergravity

Confluent high-density cell cultures of A6 cells derived from adult male Xenopus kidney exhibit spontaneous dome-formation at 1 g. To determine whether this morphogenetic property is altered by gravity, we used a three-dimensional (3D) clinostat to subject the cells to simulated microgravity, and a centrifuge to subject them to hypergravity. We used the generation orbit control method as the new rotation control system of the 3D-clinostat, not the random method. The growth of A6 cells was significantly enhanced by hypergravity, but significantly reduced by simulated microgravity. Dome formation by A6 cells at high confluence was inhibited under simulated microgravity conditions, whereas hypergravity promoted dome formation and induced tubule morphogenesis, compared to the control at 1 g. These results indicated that changes in gravity influence the morphogenetic properties of A6 cells, such as dome formation and tubule morphogenesis. When dome formation by A6 cells at high confluence was induced spontaneously in the control 1 g culture, the gene expression of the HGF family of pleiotropic factors, such as HGF-like protein (HLP) and growth factor-Livertine (GF-l.ivertine), an epithelial serine protease of channel activating protease 1 (CAP1), and Na+, K+-adenosine triphosphatase (ATPase), increased. Simulated microgravity increased the gene expression of activin A and reduced the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. Hypergravity, on the other hand, decreased the gene expression of activin A and increased the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. These results suggest that the effects of gravitational changes on expression of the HGF family member gene, CAP1, and Na+, K+-ATPase gene may be important for the cell growth, tubule morphogenesis, and dome formation of A6 cells in altered     

Changes in gravitational forces induce modifications of gene expression in<Emphasis Type="Italic"> A. thaliana</Emphasis> seedlings

By comparing the expression patterns of selected genes from Arabidopsis thaliana (L.) Heynh. grown either at 1 g or on a clinostat (horizontally or vertically inverted, 1 rpm), and either used directly or after hypergravity stimulation, we have shown that the pattern of expression did not proceed in a stereotypical manner. Rather, the selected genes fell into different classes. These classes include (i) those insensitive to the gravitational conditions, (ii) those that are regulated in an opposite manner by hypergravity and clinostat conditions, (iii) those that are desensitised to hypergravity by long-term culture on a clinostat, and (iv) those enhanced by such a treatment. Our data suggest that rapid reorientation of gene expression is likely to occur in response to changes in the gravitational conditions.     

Analysis of the expression levels of genes that encode cytoskeletal proteins in Drosophila melanogaster larvae during micro- and hypergravity effect simulations of different durations

The goal of this study was to find genes that encode cytoskeletal proteins that are potential candidates for the role of triggers in cell mechanosensitivity in the fruit fly. Centrifugation was used to simulate the hypergravity effects (2g group); the constantly changing orientation of the larvae in the gravity field was performed in order to simulate the effects of microgravity (0g group) for 1.5, 6, 12 and 24 h. mRNA levels of different genes that encode the components of both tubulin and actin cytoskeleton were assessed by qRT-PCR. In the 0g group the mRNA levels of beta-tubulin and Msps were reduced after 1.5 h of the exposure and remained unchanged until 12 h, while they exceeded the control level after 24 h. The mRNA level of chaperonin containing T-complex 1 polypeptide subunits recovered earlier: after 6 and 12 h of the microgravity exposure. At the same time, the hypergravity effect led to more significant changes in the mRNA level of TCP1 complex components compared with those of tubulin and Msps. The mRNA level of beta-actin isoforms under micro- and hypergravity was decreased up to 12 h of the exposure, however, it remained reduced under microgravity conditions, while it recovered (Act87E) and even exceeded (Act57B) the reference level under hypergravity conditions. The mRNA level of supervillin was almost unchanged. Under microgravity conditions the mRNA level of fimbrin was decreased (it recovered by the 24 h time point), while the mRNA level of alpha-actinin was significantly increased by the 12 h time point of the exposure and after 24 h it was reduced to the control level. In contrast, under hypergravity conditions the mRNA level of fimbrin initially increased, and after 24 h it dropped below the control, while the mRNA level of alpha-actinin was significantly reduced, and after 24 h it was higher than the reference level. Similar results were obtained earlier in the experiments in rodents, but similar dynamics were observed for alpha-actinin isoforms 1 and 4, although no changes were observed for fimbrin. Since Drosophila melanogaster has no alpha-actinin isoform 4, it is hypothesized that its role in the cell is played by fimbrin.