Cellular processes are influenced in many ways by changes in gravitational force. In previous studies, we were able to demonstrate, in various cellular systems and research platforms that reactions and adaptation processes occur very rapidly after the onset of altered gravity. In this study we systematically compared differentially expressed gene transcript clusters (TCs) in human Jurkat T cells in microgravity provided by a suborbital ballistic rocket with vector-averaged gravity (vag) provided by a 2D clinostat. Additionally, we included 9× g centrifuge experiments and rigorous controls for excluding other factors of influence than gravity. We found that 11 TCs were significantly altered in 5 min of flight-induced and vector-averaged gravity. Among the annotated clusters were G3BP1, KPNB1, NUDT3, SFT2D2, and POMK. Our results revealed that less than 1% of all examined TCs show the same response in vag and flight-induced microgravity, while 38% of differentially regulated TCs identified during the hypergravity phase of the suborbital ballistic rocket flight could be verified with a 9× g ground centrifuge. In the 2D clinostat system, doing one full rotation per second, vector effects of the gravitational force are only nullified if the sensing mechanism requires 1 s or longer. Due to the fact that vag with an integration period of 1 s was not able to reproduce the results obtained in flight-induced microgravity, we conclude that the initial trigger of gene expression response to microgravity requires less than 1 s reaction time. Additionally, we discovered extensive gene expression differences caused by simple handling of the cell suspension in control experiments, which underlines the need for rigorous standardization regarding mechanical forces during cell culture experiments in general.

The mechanisms underlying gravity perception in mammalian cells are unknown. We have recently discovered that the transcriptome of cells in the immune system, which is the most affected system during a spaceflight, responds rapidly and broadly to altered gravity. To pinpoint potential underlying mechanisms, we compared gene expression and three-dimensional (3D) chromosomal conformational changes in human Jurkat T cells during the short-term gravitational changes in parabolic flight and suborbital ballistic rocket flight experiments. We found that differential gene expression in gravity-responsive chromosomal regions, but not differentially regulated single genes, are highly conserved between different real altered gravity comparisons. These coupled gene expression effects in chromosomal regions could be explained by underlying chromatin structures. Based on a high-throughput chromatin conformation capture (Hi-C) analysis in altered gravity, we found that small chromosomes (chr16–22, with the exception of chr18) showed increased intra- and interchromosomal interactions in altered gravity, whereby large chromosomes showed decreased interactions. Finally, we detected a nonrandom overlap between Hi-C-identified chromosomal interacting regions and gravity-responsive chromosomal regions (GRCRs). We therefore demonstrate the first evidence that gravitational force-induced 3D chromosomal conformational changes are associated with rapid transcriptional response in human T cells. We propose a general model of cellular sensitivity to gravitational forces, where gravitational forces acting on the cellular membrane are rapidly and mechanically transduced through the cytoskeleton into the nucleus, moving chromosome territories to new conformation states and their genes into more expressive or repressive environments, finally resulting in region-specific differential gene expression.

Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed.

Microgravity acts on cellular systems on several levels. Cells of the immune system especially react rapidly to changes in gravity. In this study, we performed a correlative metabolomics analysis on short-term and long-term microgravity effects on primary human macrophages. We could detect an increased amino acid concentration after five minutes of altered gravity, that was inverted after 11 days of microgravity. The amino acids that reacted the most to changes in gravity were tightly clustered. The observed effects indicated protein degradation processes in microgravity. Further, glucogenic and ketogenic amino acids were further degraded to Glucose and Ketoleucine. The latter is robustly accumulated in short-term and long-term microgravity but not in hypergravity. We detected highly dynamic and also robust adaptative metabolic changes in altered gravity. Metabolomic studies could contribute significantly to the understanding of gravity-induced integrative effects in human cells.

Cellular responses to micro- and hypergravity are rapid and complex and appear within the first few seconds of exposure. Transcriptomic analyses are a valuable tool to analyze these genome-wide cellular alterations. For a better understanding of the cellular dynamics upon altered gravity exposure, it is important to compare different time points. However, since most of the experiments are designed as endpoint measurements, the combination of cross-experiment meta-studies is inevitable. Microarray and RNA-Seq analyses are two of the main methods to study transcriptomics. In the field of altered gravity research, both methods are frequently used. However, the generation of these data sets is difficult and time-consuming and therefore the number of available data sets in this research field is limited. In this study, we investigated the comparability of microarray and RNA-Seq data and applied the results to a comparison of the transcriptomics dynamics between the hypergravity conditions during two real flight platforms and a centrifuge experiment to identify temporal adaptation processes. We performed a comparative study on an Affymetrix HTA2.0 microarray and a paired-end RNA-Seq data set originating from the same Jurkat T cell RNA samples from a short-term hypergravity experiment. The overall agreeability was high, with better sensitivity of the RNA-Seq analysis. The microarray data set showed weaknesses on the level of single upregulated genes, likely due to its normalization approach. On an aggregated level of biotypes, chromosomal distribution, and gene sets, both technologies performed equally well. The microarray showed better performance on the detection of altered gravity-related splicing events. We found that all initially altered transcripts fully adapted after 15 min to hypergravity and concluded that the altered gene expression response to hypergravity is transient and fully reversible. Based on the combined multiple-platform meta-analysis, we could demonstrate rapid transcriptional adaptation to hypergravity, the differential expression of the ATPase subunits ATP6V1A and ATP6V1D, and the cluster of differentiation (CD) molecules CD1E, CD2AP, CD46, CD47, CD53, CD69, CD96, CD164, and CD226 in hypergravity. We could experimentally demonstrate that it is possible to develop methodological evidence for the meta-analysis of individual data.

The transcriptome of human immune cells rapidly reacts to altered gravity in a highly dynamic way. We could show in previous experiments that transcriptional patterns show profound adaption after seconds to minutes of altered gravity. To gain further insight into these transcriptional alteration and adaption dynamics, we conducted a highly standardized RNA-Seq experiment with human Jurkat T cells exposed to 9xg hypergravity for 3 and 15 min, respectively. We investigated the frequency with which individual exons were used during transcription and discovered that differential exon usage broadly appeared after 3 min and became less pronounced after 15 min. Additionally, we observed a shift in the transcript pool from coding towards non-coding transcripts. Thus, adaption of gravity-sensitive differentially expressed genes followed a dynamic transcriptional rebound effect. The general dynamics were compatible with previous studies on the transcriptional effects of short hypergravity on human immune cells and suggest that initial up-regulatory changes mostly result from increased elongation rates. The shift correlated with a general downregulation of the affected genes. All chromosome bands carried homogenous numbers of gravity-sensitive genes but showed a specific tendency towards up- or downregulation. Altered gravity affected transcriptional regulation throughout the entire genome, whereby the direction of differential expression was strongly dependent on the structural location in the genome. A correlation analysis with potential mediators of the early transcriptional response identified a link between initially upregulated genes with certain transcription factors. Based on these findings, we have been able to further develop our model of the transcriptional response to altered gravity.