Ruth Hemmersbach

Ground-based facilities for simulation of microgravity: organism-specific recommendations for their use, and recommended terminology.

Research in microgravity is indispensable to disclose the impact of gravity on biological processes and organisms. However, research in the near-Earth orbit is severely constrained by the limited number of flight opportunities. Ground-based simulators of microgravity are valuable tools for preparing spaceflight experiments, but they also facilitate stand-alone studies and thus provide additional and cost-efficient platforms for gravitational research. The various microgravity simulators that are frequently used by gravitational biologists are based on different physical principles. This comparative study gives an overview of the most frequently used microgravity simulators and demonstrates their individual capacities and limitations. The range of applicability of the various ground-based microgravity simulators for biological specimens was carefully evaluated by using organisms that have been studied extensively under the conditions of real microgravity in space. In addition, current heterogeneous terminology is discussed critically, and recommendations are given for appropriate selection of adequate simulators and consistent use of nomenclature.

Short-term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells

This study focused on the effects of short‐term microgravity (22 s) on the gene expression and morphology of endothelial cells (ECs) and evaluated gravisensitive signaling elements. ECs were investigated during four German Space Agency (Deutsches Zentrum für Luft‐ und Raumfahrt) parabolic flight campaigns. Hoechst 33342 and acridine orange/ethidium bromide staining showed no signs of cell death in ECs after 31 parabolas (P31). Gene array analysis revealed 320 significantly regulated genes after the first parabola (P1) and P31. COL4A5, COL8A1, ITGA6, ITGA10, and ITGB3 mRNAs were down‐regulated after P1. EDN1 and TNFRSF12A mRNAs were up‐regulated. ADAM19, CARD8, CD40, GSN, PRKCA (all down‐regulated after P1), and PRKAA1 (AMPKα1) mRNAs (up‐regulated) provide a very early protective mechanism of cell survival induced by 22 s microgravity. The ABL2 gene was significantly up‐regulated after P1 and P31, TUBB was slightly induced, but ACTA2 and VIM mRNAs were not changed. β‐Tubulin immunofluorescence revealed a cytoplasmic rearrangement. Vibration had no effect. Hypergravity reduced CARD8, NOS3, VASH1, SERPINH1 (all P1), CAV2, ADAM19, TNFRSF12A, CD40, and ITGA6 (P31) mRNAs. These data suggest that microgravity alters the gene expression patterns and the cytoskeleton of ECs very early. Several gravisensitive signaling elements, such as AMPKα1 and integrins, are involved in the reaction of ECs to altered gravity.—Grosse, J., Wehland, M., Pietsch, J., Ma, X., Ulbrich, C., Schulz, H., Saar, K., Hübner, N., Hauslage, J., Hemmersbach, R., Braun, M., van Loon, J., Vagt, N., Infanger, M., Eilles, C., Egli, M., Richter, P., Baltz, T., Einspanier, R., Sharbati, S., Grimm, D. Short‐term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells. FASEB J. 26, 639–655 (2012). www.fasebj.org

Graviorientation in protists and plants.

Gravitaxis, gravikinesis, and gravitropism are different graviresponses found in protists and plants. The phenomena have been intensively studied under variable stimulations ranging from microgravity to hypergravity. A huge amount of information is now available, e.g. about the time course of these events, their adaptation capacity, thresholds, and interaction between gravity and other environmental stimuli. There is growing evidence that a pure physical mechanism can be excluded for orientation of protists in the gravity field. Similarly, a physiological signal transduction chain has been postulated in plants. Current investigations focus on the question whether gravity is perceived by intracellular gravireceptors (e.g. the Muller organelle of the ciliate Loxodes, barium sulfate vacuoles in Chara rhizoids or starch statoliths in higher plants) or whether the whole cell acts as a sedimenting body exerting pressure on the lower membrane. Behavioral studies in density adjusted media, effects of inhibitors of mechano-sensitive ion channels or manipulations of the proposed gravireceptor structures revealed that both mechanisms have been developed in protists and plants. The threshold values for graviresponses indicate that even 10% of the normal gravitational field can be detected, which demands a focusing and amplifying system such as the cytoskeleton and second messengers.

Gravitaxis in the Flagellate Euglena gracilis is Controlled by an Active Gravireceptor

Gravitactic orientation was investigated in the unicellular photosynthetic flagellate, Euglena gracilis, under different accelerations between 0 and 1.5 x g during a recent space flight on board the American shuttle Columbia. The threshold for gravitaxis was found at < or = 0.16 x g. Above the threshold the precision of orientation increased with acceleration in a sigmoidal fashion and reached saturation at about 0.32 x g, a behavior typical for physiological receptors. At accelerations above the saturation point the cells were closely aligned with the gravity vector (negative gravitaxis) and deviated more and more as the acceleration decreased. Obviously the gravireceptor responds to an error signal that elicits a course correction, again indicating the involvement of an active physiological gravireceptor. No adaptation of the cells to the conditions of weightlessness could be observed over the duration of the space mission (12 days). After landing, the cells showed a normal gravitactic behavior at 1 x g.

Differential gene expression profile and altered cytokine secretion of thyroid cancer cells in space

This study focuses on the effects of short‐term [22 s, parabolic flight campaign (PFC)] and long‐term (10 d, Shenzhou 8 space mission) real microgravity on changes in cytokine secretion and gene expression patterns in poorly differentiated thyroid cancer cells. FTC‐133 cells were cultured in space and on a random positioning machine (RPM) for 10 d, to evaluate differences between real and simulated microgravity. Multianalyte profiling was used to evaluate 128 secreted cytokines. Microarray analysis revealed 63 significantly regulated transcripts after 22 s of microgravity during a PFC and 2881 after 10 d on the RPM or in space. Genes in several biological processes, including apoptosis (n=182), cytoskeleton (n=80), adhesion/extracellular matrix (n=98), proliferation (n=184), stress response (n=268), migration (n=63), angiogenesis (n=39), and signal transduction (n=429), were differentially expressed. Genes and proteins involved in the regulation of cancer cell proliferation and metastasis, such as IL6, IL8, IL15, OPN, VEGFA, VEGFD, FGF17, MMP2, MMP3, TIMP1, PRKAA, and PRKACA, were similarly regulated under RPM and spaceflight conditions. The resulting effect was mostly antiproliferative. Gene expression during the PFC was often regulated in the opposite direction. In summary, microgravity is an invaluable tool for exploring new targets in anticancer therapy and can be simulated in some aspects in ground‐based facilities.—Ma, X., Pietsch, J., Wehland, M., Schulz, H., Saar, K., Hübner, N., Bauer, J., Braun, M., Schwarzwälder, A., Segerer, J., Birlem, M., Horn, A., Hemmersbach, R., Waβer, K., Grosse, J., Infanger, M., Grimm, D. Differential gene expression profile and altered cytokine secretion of thyroid cancer cells in space. FASEB J. 28, 813–835 (2014). www.fasebj.org

Differential gene regulation under altered gravity conditions in follicular thyroid cancer cells: relationship between the extracellular matrix and the cytoskeleton

Extracellular matrix proteins, adhesion molecules, and cytoskeletal proteins form a dynamic network interacting with signalling molecules as an adaptive response to altered gravity. An important issue is the exact differentiation between real microgravity responses of the cells or cellular reactions to hypergravity and/or vibrations. To determine the effects of real microgravity on human cells, we used four DLR parabolic flight campaigns and focused on the effects of short-term microgravity (22 s), hypergravity (1.8 g), and vibrations on ML-1 thyroid cancer cells. No signs of apoptosis or necrosis were detectable. Gene array analysis revealed 2430 significantly changed transcripts. After 22 s microgravity, the F-actin and cytokeratin cytoskeleton was altered, and ACTB and KRT80 mRNAs were significantly upregulated after the first and thirty-first parabolas. The COL4A5 mRNA was downregulated under microgravity, whereas OPN and FN were significantly upregulated. Hypergravity and vibrations did not change ACTB, KRT-80 or COL4A5 mRNA. MTSS1 and LIMA1 mRNAs were downregulated/slightly upregulated under microgravity, upregulated in hypergravity and unchanged by vibrations. These data indicate that the graviresponse of ML-1 cells occurred very early, within the first few seconds. Downregulated MTSS1 and upregulated LIMA1 may be an adaptive mechanism of human cells for stabilizing the cytoskeleton under microgravity conditions.

Gravity-sensitive signaling drives 3-dimensional formation of multicellular thyroid cancer spheroids

This study focused on the effects induced by a random positioning machine (RPM) on FTC‐133 thyroid cancer cells and evaluated signaling elements involved in 3‐dimensional multicellular tumor spheroid (MCTS) formation. The cells were cultured on the RPM, a device developed to simulate microgravity, and under static 1‐g conditions. After 24 h on the RPM, MCTSs swimming in culture supernatants were found, in addition to growth of adherent (AD) cells. Cells grown on the RPM showed higher levels of NF‐κB p65 protein and apoptosis than 1‐g controls, a result also found earlier in endothelial cells. Employing microarray analysis, we found 487 significantly regulated transcripts belonging not only to the apoptosis pathway but also to other biological processes. Selected transcripts were analyzed with quantitative real‐time PCR using the same samples. Compared with 1‐g IL‐6, IL‐8, CD44, and OPN were significantly up‐regulated in AD cells but not in MCTSs, while ERK1/2, CAV2, TLN1, and CTGF were significantly down‐regulated in AD cells. Simultaneously, the expression of ERK2, IL‐6, CAV2, TLN1, and CTGF was reduced in MCTSs. IL‐6 protein expression and secretion mirrored its gene expression. Thus, we concluded that the signaling elements IL‐6, IL‐8, OPN, TLN1, and CTGF are involved with NF‐κB p65 in RPM‐dependent thyroid carcinoma cell spheroid formation.—Grosse, J., Wehland, M., Pietsch, J., Schulz, H., Saar, K., Hübner, N., Eilles, C., Bauer, J., Abou‐El‐Ardat, K., Baatout, S., Ma, X., Infanger, M., Hemmersbach, R., Grimm, D. Gravity‐sensitive signaling drives 3‐dimensional formation of multicellular thyroid cancer spheroids. FASEB J. 26, 5124–5140 (2012). www.fasebj.org

Salt-dependent chemotaxis of macrophages

Besides their role in immune system host defense, there is growing evidence that macrophages may also be important regulators of salt homeostasis and blood pressure by a TonEBP-VEGF-C dependent buffering mechanism. As macrophages are known to accumulate in the skin of rats fed under high salt diet conditions and are pivotal for removal of high salt storage, the question arose how macrophages sense sites of high sodium storage. Interestingly, we observed that macrophage-like RAW264.7 cells, murine bone marrow-derived macrophages and peritoneal macrophages recognize NaCl hypertonicity as a chemotactic stimulus and migrate in the direction of excess salt concentration by using an in vitro transwell migration assay. While RAW264.7 cells migrated toward NaCl in a dose-dependent fashion, no migratory response toward isotonic or hypotonic media controls, or other osmo-active agents, e.g. urea or mannitol, could be detected. Interestingly, we could not establish a specific role of the osmoprotective transcription factor TonEBP in regulating salt-dependent chemotaxis, since the specific migration of bone marrow-derived macrophages following RNAi of TonEBP toward NaCl was not altered. Although the underlying mechanism remains unidentified, these data point to a thus far unappreciated role for NaCl-dependent chemotaxis of macrophages in the clearance of excess salt, and suggest the existence of novel NaCl sensor/effector circuits, which are independent of the TonEBP system.

Graviperception and graviorientation in flagellates

Abstract. In the flagellate Euglena gracilis Klebs,?gravitaxis is mediated by an active physiological receptor and is not the result of passive alignment of the cells in the water column. The threshold of this response was found at 0.08?< threshold <0.16?g during a recent space flight on the American shuttle Columbia, where the cells were subjected to different accelerations between 0 and 1.5?g; the response saturated at 0.32?<?saturation???0.64?g. Over the whole duration of the mission no adaptation of the response to microgravity was observed. The whole body of the cell, rather than intracellular organelles, seems to act as statolith since suspending the cells in a density-adjusted medium (Ficoll) resulted in an inhibition of gravitaxis and even reversal of orientation at higher densities. Thus, the cytoplasm seems to exert a pressure on the respective lower membrane where it is hypothesized to activate stretch-sensitive specific ion channels, as indicated by inhibitor studies with gadolinium. One of the early steps in the sensory transduction chain seems to be a modulation of the membrane potential since ion-channel blockers, ionophores and ATPase inhibitors strongly inhibit gravitaxis in this flagellate without seriously affecting motility and phototaxis.

Influence of accelerations on the spatial orientation of Loxodes and Paramecium

The gravitactic ciliates Paramecium and Loxodes were cultivated for 15 days in space during the IML-2 spacelab mission. At dedicated times their behavioral responses to different accelerations between 10(-3) x g and 1.5 x g were investigated by using a slow rotating centrifuge microscope (NIZEMI). The threshold for gravitaxis of Paramecium was found to be at > 0.16 x g and < or = 0.3 x g. No adaptation of Paramecium to the conditions of weightlessness was observed over the duration of 15 days. Loxodes showed no graviresponses to increasing accelerations, though it demonstrated gravitaxis after return to earth.