Changes in the effective gravitational field strength affect the state of phosphorylation of stress-related proteins in callus cultures of Arabidopsis thaliana.
ABSTRACT: In a recent study it was shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by changes in protein expression. Using ESI-MS/MS for proteins with differential abundance after separation by 2D-PAGE, 28 spots which changed reproducibly and significantly in amount (P <0.05) after 2 h of hypergravity (18 up-regulated, 10 down-regulated) could be identified. The corresponding proteins were largely involved in stress responses, including the detoxification of reactive oxygen species (ROS). In the present study, these investigations are extended to phosphorylated proteins. For this purpose, callus cell cultures of Arabidopsis thaliana were exposed to hypergravity (8 g) and simulated weightlessness (random positioning; RP) for up to 30 min, a period of time which yielded the most reliable data. The first changes, however, were visible as early as 10 min after the start of treatment. In comparison to 1 g controls, exposure to hypergravity resulted in 18 protein spots, and random positioning in 25, respectively, with increased/decreased signal intensity by at least 2-fold (P <0.05). Only one spot (alanine aminotransferase) responded the same way under both treatments. After 30 min of RP, four spots appeared, which could not be detected in control samples. Among the protein spots altered in phosphorylation, it was possible to identify 24 from those responding to random positioning and 12 which responded to 8 g. These 12 proteins (8 g) are partly (5 out of 12) the same as those changed in expression after exposure to 2 h of hypergravity. The respective proteins are involved in scavenging and detoxification of ROS (32%), primary metabolism (20.5%), general signalling (14.7%), protein translation and proteolysis (14.7%), and ion homeostasis (8.8%). Together with our recent data on protein expression, it is assumed that changes in gravitational fields induce the production of ROS. Our data further indicate that responses toward RP are more by post-translational protein modulation (most changes in the degree of phosphorylation occur under RP-treatment) than by protein expression (hypergravity).
Project description:BACKGROUND:Root gravitropsim has been proposed to require the coordinated, redistribution of the plant signaling molecule auxin within the root meristem, but the underlying molecular mechanisms are still unknown. PIN proteins are membrane transporters that mediate the efflux of auxin from cells. The PIN2 is important for the basipetal transport of auxin in roots and plays a critical role in the transmission of gravity signals perceived in the root cap to the root elongation zone. The loss of function pin2 mutant exhibits a gravity-insensitive root growth phenotype. By comparing the proteomes of wild type and the pin2 mutant root tips under different gravitational conditions, we hope to identify proteins involved in the gravity-related signal transduction. RESULTS:To identify novel proteins involved in the gravity signal transduction pathway we have carried out a comparative proteomic analysis of Arabidopsis pin2 mutant and wild type (WT) roots subjected to different gravitational conditions. These conditions included horizontal (H) and vertical (V) clinorotation, hypergravity (G) and the stationary control (S). Analysis of silver-stained two-dimensional SDS-PAGE gels revealed 28 protein spots that showed significant expression changes in altered gravity (H or G) compared to control roots (V and S). Whereas the majority of these proteins exhibited similar expression patterns in WT and pin2 roots, a significant number displayed different patterns of response between WT and pin2 roots. The latter group included 11 protein spots in the H samples and two protein spots in the G samples that exhibited an altered expression exclusively in WT but not in pin2 roots. One of these proteins was identified as annexin2, which was induced in the root cap columella cells under altered gravitational conditions. CONCLUSIONS:The most interesting observation in this study is that distinctly different patterns of protein expression were found in WT and pin2 mutant roots subjected to altered gravity conditions. The data also demonstrate that PIN2 mutation not only affects the basipetal transport of auxin to the elongation zone, but also results in an altered expression of proteins in the root columella.
Project description:Using diamagnetic levitation, we have exposed A. thaliana in vitro callus cultures to five environments with different levels of effective gravity (from levitation i.e. simulated mg* to 2g*) and magnetic fields (10.1 to 16.5 Tesla) and we have compared the results with those of similar experiments done in a Random Position Machine (simulated micro g) and a Large Diameter Centrifuge (2g) free of high magnetic fields. Microarray analysis indicates that there are changes in overall gene expression of the cultured cells exposed to these unusual environments but also that gravitational and magnetic field produce synergic variations in the steady state of the transcriptional profile of A. thaliana. Significant changes in the expression of structural, abiotic stress and secondary metabolism genes were observed into the magnet field. These results confirm that the strong magnetic field, both at micro g* or 2g*, has a significant effect on the expression of these genes but subtle gravitational effects are still observable. These subtle responses to microgravity environments are opposite to the ones observed in a hypergravity one. Overall design: seven-condition experiment, MM2D Arabidopsis culture callus control vs. Treatment (altered gravity simulation, GBF). Three GBF were used (LDC (2g) + control, RPM (mg) + control and Magnet (mg*, 0.1g*, 1g*, 1.9g*, 2g*) + control). Biological replicates: 3 replicates in all conditions and controls except 1.9g* (2 replicates)
Project description:Biological systems respond to changes in both the Earth's magnetic and gravitational fields, but as experiments in space are expensive and infrequent, Earth-based simulation techniques are required. A high gradient magnetic field can be used to levitate biological material, thereby simulating microgravity and can also create environments with a reduced or an enhanced level of gravity (g), although special attention should be paid to the possible effects of the magnetic field (B) itself.Using diamagnetic levitation, we exposed Arabidopsis thaliana in vitro callus cultures to five environments with different levels of effective gravity and magnetic field strengths. The environments included levitation, i.e. simulated ?g* (close to 0 g* at B = 10.1 T), intermediate g* (0.1 g* at B = 14.7 T) and enhanced gravity levels (1.9 g* at B = 14.7 T and 2 g* at B = 10.1 T) plus an internal 1 g* control (B = 16.5 T). The asterisk denotes the presence of the background magnetic field, as opposed to the effective gravity environments in the absence of an applied magnetic field, created using a Random Position Machine (simulated ?g) and a Large Diameter Centrifuge (2 g).Microarray analysis indicates that changes in the overall gene expression of cultured cells exposed to these unusual environments barely reach significance using an FDR algorithm. However, it was found that gravitational and magnetic fields produce synergistic variations in the steady state of the transcriptional profile of plants. Transcriptomic results confirm that high gradient magnetic fields (i.e. to create ?g* and 2 g* conditions) have a significant effect, mainly on structural, abiotic stress genes and secondary metabolism genes, but these subtle gravitational effects are only observable using clustering methodologies.A detailed microarray dataset analysis, based on clustering of similarly expressed genes (GEDI software), can detect underlying global-scale responses, which cannot be detected by means of individual gene expression techniques using raw or corrected p values (FDR). A subtle, but consistent, genome-scale response to hypogravity environments was found, which was opposite to the response in a hypergravity environment.
Project description:During spaceflight, organisms are subjected to mechanical force changes (gravity (G) changes) that affect the immune system. However, gravitational effects on lymphopoiesis have rarely been studied. Consequently, we investigated whether the TCR? repertoire, created by V(D)J recombination during T lymphopoiesis, is affected by hypergravity exposure during murine development. To address this question, C57BL/6j mice were mated in a centrifuge so that embryonic development, birth and TCR? rearrangements occurred at 2G. Pups were sacrificed at birth, and their thymus used to quantify transcripts coding for factors required for V(D)J recombination and T lymphopoiesis. We also created cDNA mini-libraries of TCR? transcripts to study the impact of hypergravity on TCR? diversity. Our data show that hypergravity exposure increases the transcription of TCR? chains, and of genes whose products are involved in TCR signaling, and affects the V(D)J recombination process. We also observed that ~85% of the TCR? repertoire is different between hypergravity and control pups. These data indicate that changing a mechanical force (the gravity) during ontogeny will likely affect host immunity because properties of loops constituting TCR antigen-binding sites are modified in hypergravity newborns. The spectrum of peptides recognized by TCR will therefore likely be different.
Project description:The peripheral vestibular organs are sensors for linear acceleration (gravity and head tilt) and rotation. Further, they regulate various body functions, including body stability, ocular movement, autonomic nerve activity, arterial pressure, body temperature, and muscle and bone metabolism. The gravitational environment influences these functions given the highly plastic responsiveness of the vestibular system. This review demonstrates that hypergravity or microgravity induces changes in vestibular-related physiological functions, including arterial pressure, muscle and bone metabolism, feeding behavior, and body temperature. Hopefully, this review contributes to understanding how human beings can adapt to a new gravitational environment, including the moon and Mars, in future.
Project description:Using diamagnetic levitation, we have exposed A. thaliana in vitro callus cultures to five environments with different levels of effective gravity (from levitation i.e. simulated mg* to 2g*) and magnetic fields (10.1 to 16.5 Tesla) and we have compared the results with those of similar experiments done in a Random Position Machine (simulated micro g) and a Large Diameter Centrifuge (2g) free of high magnetic fields. Microarray analysis indicates that there are changes in overall gene expression of the cultured cells exposed to these unusual environments but also that gravitational and magnetic field produce synergic variations in the steady state of the transcriptional profile of A. thaliana. Significant changes in the expression of structural, abiotic stress and secondary metabolism genes were observed into the magnet field. These results confirm that the strong magnetic field, both at micro g* or 2g*, has a significant effect on the expression of these genes but subtle gravitational effects are still observable. These subtle responses to microgravity environments are opposite to the ones observed in a hypergravity one. seven-condition experiment, MM2D Arabidopsis culture callus control vs. Treatment (altered gravity simulation, GBF). Three GBF were used (LDC (2g) + control, RPM (mg) + control and Magnet (mg*, 0.1g*, 1g*, 1.9g*, 2g*) + control). Biological replicates: 3 replicates in all conditions and controls except 1.9g* (2 replicates)
Project description:BACKGROUND:we aimed at investigating the influence of weightlessness and hypergravity by means of parabolic flight on the levels of the heart failure biomarkers H-FABP, sST2, IL-33, GDF-15, suPAR and Fetuin-A. METHODS:14 healthy volunteers (males: eight; mean age: 28.9) undergoing 31 short-term phases of weightlessness and hypergravity were included. At different time points (baseline, 1 h/24 h after parabolic flight), venous blood was drawn and analyzed by the use of ELISA. RESULTS:sST2 evidenced a significant decrease 24 h after parabolic flight (baseline vs. 24, p = 0.009; 1 h vs. 24 h, p = 0.004). A similar finding was observed for GDF-15 (baseline vs. 24 h, p = 0.002; 1 h vs. 24 h, p = 0.025). The suPAR showed a significant decrease 24 h after parabolic flight (baseline vs. 24 h, p = 0.1726; 1 h vs. 24 h, p = 0.009). Fetuin-A showed a significant increase at 1 h and 24 h after parabolic flight (baseline vs. 24 h, p = 0.007; 1 h vs. 24 h, p = 0.04). H-FABP and IL-33 showed no significant differences at all time points. CONCLUSION:Our results suggest a reduction in cardiac stress induced by exposure to gravitational changes. Moreover, our findings indicate an influence of gravitational changes on proliferative processes and calcium homeostasis.
Project description:The gravitational force has been constant throughout Earth's evolutionary history. Since the cell nucleus is subjected to permanent forces induced by Earth's gravity, we addressed the question, if gene expression homeostasis is constantly shaped by the gravitational force on Earth. We therefore investigated the transcriptome in force-free conditions of microgravity, determined the time frame of initial gravitational force-transduction to the transcriptome and assessed the role of cation channels. We combined a parabolic flight experiment campaign with a suborbital ballistic rocket experiment employing the human myelomonocytic cell line U937 and analyzed the whole gene transcription by microarray, using rigorous controls for exclusion of effects not related to gravitational force and cross-validation through two fully independent research campaigns. Experiments with the wide range ion channel inhibitor SKF-96365 in combination with whole transcriptome analysis were conducted to study the functional role of ion channels in the transduction of gravitational forces at an integrative level. We detected profound alterations in the transcriptome already after 20 s of microgravity or hypergravity. In microgravity, 99.43% of all initially altered transcripts adapted after 5 min. In hypergravity, 98.93% of all initially altered transcripts adapted after 75 s. Only 2.4% of all microgravity-regulated transcripts were sensitive to the cation channel inhibitor SKF-96365. Inter-platform comparison of differentially regulated transcripts revealed 57 annotated gravity-sensitive transcripts. We assume that gravitational forces are rapidly and constantly transduced into the nucleus as omnipresent condition for nuclear and chromatin structure as well as homeostasis of gene expression.
Project description:Euglena gracilis is a unicellular freshwater flagellate, which uses the gravitational vector for orientation in the water column in the dark. This allows the cell to reach areas in the water column for reproduction and growth. How exactly the gravitational vector is perceived, and which intracellular pathways are involved in the signaling is not very well understood so far. In the past, parabolic flight campaigns were used to study the swimming behavior of Euglena gracilis under altered gravitational accelerations. It was shown that cells adapt their swimming direction very fast: in the dark under 1xg cell show negative gravitaxis, i.e. they move upwards in the water column of the experiment hardware and against the gravitational vector. With onset of the first hypergravity period of 1.8xg the precision of upward swimming increases slightly. This first hyper gravity lasts for only 20 seconds and is followed by 22 sec of microgravity. During this period no gravitational vector is perceived by the cells, therefore they lack a cue for orientation and move randomly in all direction. In the subsequent hyper gravity period of 1.8xg, which also lasts for 20 sec, cells direct their movement again and swim upwards. Over different parabolic flight campaigns and other experiments it was shown that this gravitactic behavior is linked to changes in membrane potential, calcium and cAMP concentration. However, due to the lack of genomic and transcriptomic data, it was so far not possible to link the differential movement to the abundance of distinct mRNA transcripts. In contrast, other model organisms, such as Arabidopsis thaliana, have been analyzed by means of gene expression with respect to the effects of altered gravitational accelerations. Also various human cell lines have shown to adapt their gene expression in dependence of the prevailing acceleration. With the recently published Euglena gracilis transcriptome, we now aimed at analyzing effects of altered acceleration on the gene expression in the flagellate. Therefore, Euglena gracilis samples were taken in the course of the 29th DLR parabolic flight campaign during parabola 1 and 31 (time difference of 2 hours and 30 additional parabolas). During both parabolas samples were fixed with TRIzol at 1xg just before onset of the first hyper gravity period, 20 sec into hyper gravity (1.8xg), 20 sec into microgravity (µg) and 20 sec into the last hypergravity period.
Project description:The objective of this study was to determine the response of the lumbar spinal motor control in different gravitational conditions. This was accomplished by measuring indicators of lumbar motor control, specifically lumbar spinal stiffness, activity of lumbar extensor and flexor muscles and lumbar curvature, in hypergravity and microgravity during parabolic flights. Three female and five male subjects participated in this study. The mean age was 35.5 years (standard deviation: 8.5 years). Spinal stiffness of the L3 vertebra was measured using impulse response; activity of the erector spinae, multifidi, transversus abdominis, and psoas muscles was recorded using surface electromyography; and lumbar curvature was measured using distance sensors mounted on the back-plate of a full-body harness. An effect of gravity condition on spinal stiffness, activity of all muscles assessed and lumbar curvature (p's < 0.007) was observed (Friedman tests). Post hoc analysis showed a significant reduction in stiffness during hypergravity (p < 0.001) and an increase in stiffness during microgravity (p < 0.001). Activity in all muscles significantly increased during hypergravity (p's < 0.001). During microgravity, the multifidi (p < 0.002) and transversus abdominis (p < 0.001) increased significantly in muscle activity while no significant difference was found for the psoas (p = 0.850) and erector spinae muscles (p = 0.813). Lumbar curvature flattened in hypergravity as well as microgravity, albeit in different ways: during hypergravity, the distance to the skin decreased for the upper (p = 0.016) and the lower sensor (p = 0.036). During microgravity, the upper sensor showed a significant increase (p = 0.016), and the lower showed a decrease (p = 0.005) in distance. This study emphasizes the role of spinal motor control adaptations in changing gravity conditions. Both hypergravity and microgravity lead to changes in spinal motor control. The decrease in spinal stiffness during hypergravity is interpreted as a shift of the axial load from the spine to the pelvis and thoracic cage. In microgravity, activity of the multifidi and of the psoas muscles seems to ensure the integrity of the spine. Swiss (BASEC-NR: 2018-00051)/French "EST-III" (Nr-ID-RCB: 2018-A011294-51/Nr-CPP: 18.06.09).