Exposure to microgravity during spaceflight induces the alterations in endothelial cell function associated with post-flight cardiovascular deconditioning. PIEZO1 is a major mechanosensitive ion channel that regulates endothelial cell function. In this study, we used a two-dimensional clinostat to investigate the expression of PIEZO1 and its regulatory mechanism on human umbilical vein endothelial cells (HUVECs) under simulated microgravity. Utilizing quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis, we observed that PIEZO1 expression was significantly increased in response to simulated microgravity. Moreover, we found microgravity promoted endothelial cells migration by increasing expression of PIEZO1. Proteomics analysis highlighted the importance of C-X-C chemokine receptor type 4(CXCR4) as a main target molecule of PIEZO1 in HUVECs. CXCR4 protein level was increased with simulated microgravity and decreased with PIEZO1 knock down. The mechanistic study showed that PIEZO1 enhances CXCR4 expression via Ca2+ influx. In addition, CXCR4 could promote endothelial cell migration under simulated microgravity. Taken together, these results suggest that the upregulation of PIEZO1 in response to simulated microgravity regulates endothelial cell migration due to enhancing CXCR4 expression via Ca2+ influx.

The necessity to include plants as a component of a Bioregenerative Life Support System leads to investigations to optimize plant growth facilities as well as a better understanding of the plant cell membrane and its numerous activities in the signaling, transport, and sensing of gravity, drought, and other stressors. The cell membrane participates in numerous processes, including endo- and exocytosis and cell division, and is involved in the response to external stimuli. Variable but stabilized microdomains form in membranes that include specific lipids and proteins that became known as (detergent-resistant) membrane microdomains, or lipid rafts with various subclassifications. The composition, especially the sterol-dependent recruitment of specific proteins affects endo- and exo-membrane domains as well as plasmodesmata. The enhanced saturated fatty acid content in lipid rafts after clinorotation suggests increased rigidity and reduced membrane permeability as a primary response to abiotic and mechanical stress. These results can also be obtained with lipid-sensitive stains. The linkage of the CM to the cytoskeleton via rafts is part of the complex interactions between lipid microdomains, mechanosensitive ion channels, and the organization of the cytoskeleton. These intricately linked structures and functions provide multiple future research directions to elucidate the role of lipid rafts in physiological processes.

Gravi-morphoses affect the variability of plants and are the morphogenetic adaptation to different environmental conditions. Gravity-dependent phenotypic plasticity of gametophytes as well as gravi-sensitivity of moss protonemata in microgravity and simulated microgravity conditions are discussed. The moss protonema, a filamentous multicellular system, representing a juvenile stage of moss development, develops as a result of the elongation and division of the apical cell. This apical cell of the protonema is a unique object for research on moss gravi-sensitivity, as graviperception and gravitropic growth occur within the same single cell. Attention is focused on the influence of gravity on bryophyte ontogenesis, including the gravitropic reactivity of moss protonemata, gravi-sensitivity at the stage of leafy shoot development and sporogonium formation, gravity-influenced morphogenesis of apical cell budding, and gravity-dependent spiral growth patterns. The role of gravireceptors in the growth processes of mosses at the cellular level under microgravity conditions are being discussed, as well as the involvement of auxin transport, Ca2+-induced gravitropism and the cytoskeleton in gravitropic reactions.

The plant cell wall is a flexible physical barrier surrounding the cell which functions in growth and differentiation, signaling, and response to environmental stimuli including the Earth gravity force. In the present study, structural and molecular modifications of cell wall components of cultured tobacco (Nicotiana tabacum cv. Burley 21) cells under alternative gravity conditions induced by 7 days exposure to 2-D clinostat have been investigated. In comparison with the control group, clinorotation significantly increased biomass but reduced the total amounts of wall and the contents of cellulose, pectin, uronic acidic, and xyloglucan. Gene expression of H+-ATPase was not changed but of expansin A reduced in clinostat-treated cells. However, the gene expression and activity of xyloglucan endotransglycosylase/hydrolases (XTH; EC 2.4.1.207) and endo-(1,4)-β-D-glucanase (EGase; EC 3.2.1.4), the amount of arabinogalactan proteins (AGP), and the expression of wall-associated kinase (WAK) gene significantly increased by clinorotation. Altered gravity also reduced the activity of polyphenol oxidase and covalently bound peroxidase. The results suggest that altered gravity promoted orchestrated changes of wall-modifying genes and proteins which reduced its stiffness and enhanced cell expansion and division potential.

The present study provides a visual insight into the effects of simulated microgravity (MG) on somatic embryogenesis (SE) in Begonia through the analysis of phytohormone fluctuations and energy metabolism. To investigate this relationship, thin cell layer culture model was first used. The results showed that MG changed the phytohormone content and stimulated starch biosynthesis to convert into sugar to release energy needed for regeneration and proliferation. Moreover, from the results it is likely that MG accelerated the initiation and subsequently maturation and aging of SE via decrease of AUX and increase of ABA. High content of GA, CKs, starch, sugar and low ABA as well as high CKs/ABA ratio were responsible for the increase in the number of embryos under clinorotation which was 1.57-fold higher than control after 90 days. The increase in fresh and dry weight of somatic embryos and chlorophyll content under MG were confirmed as their adaptive responses to gravitational stress. However, long-term exposure to MG (120 days) stimulated biosynthesis of ABA levels 1.85-fold higher than controls, which resulted in a decrease in chlorophyll content, increase in number of mature embryos and stomata length. These results revealed that MG regulated the induction, differentiation and senescence of somatic embryos via a biochemical interaction pathway.

Purpose: The rotary cell culture system (RCCS) is a common clinorotation device for cell culture. It is also used as a low-shear suspension culture bioreactor to form functionalized 3D tissue constructs and to model microgravity. We sought to develop a 3D scaffold composed of type I collagen and hydroxyapatite (collagen-HA) to characterize MLO-Y4 osteocytes following suspension culture or clinorotation.Materials and Methods: MLO-Y4 cells were embedded in collagen-HA. The scaffold was formed into droplets for suspension culture or wall-adhered to the RCCS for clinorotation. AFM, rheometry, immunofluorescence and qRT-PCR were employed to measure the scaffold stiffness, cell viability and gene expression of cells in collagen-HA scaffolds. Dendritic cells were visualized and quantified and gene expression after suspension culture and clinorotation was compared to static controls.Results: The optimized scaffold for the RCCS consisted of collagen with 6 mg/mL HA which had a stiffness of < 1 kPa. MLO-Y4 cell viability was higher in collagen-HA scaffolds, compared to scaffolds without HA. Collagen-HA scaffolds induced higher osteocyte-specific gene expression compared to cells cultured on 2D plastic. Cells in the scaffold downregulated DMP1, E11, IL-6, and RANKL, and had fewer dendritic cells following suspension culture whereas clinorotation downregulated DMP1 and E11 genes, compared to static controls.Conclusions: Suspension culture for 3 days in collagen-HA stimulates growth of osteocytes but may also desensitize them to mechanical cues. Clinorotation for 3 days in collagen-HA does not stimulate proliferation or expression of mechanosensitive genes, indicating that it may be an effective mechanical unloading environment.

Astronauts exposed to a gravity-free environment experience cardiovascular deconditioning that causes post-spaceflight orthostatic intolerance and other pathological conditions. Endothelial dysfunction is an important factor responsible for this alteration. Our previous study showed enhanced autophagy in endothelial cells under simulated microgravity. The present study explored the cytoprotective role of autophagy under microgravity in human umbilical vein endothelial cells (HUVECs). We found that clinorotation for 48 h induced apoptosis and endoplasmic reticulum (ER) stress in HUVECs. ER stress and the unfolded protein response (UPR) partially contributed to apoptosis under clinorotation. Autophagy partially reduced ER stress and restored UPR signaling by autophagic clearance of ubiquitin-protein aggregates, thereby reducing apoptosis. In addition, the ER stress antagonist 4-phenylbutyric acid upregulated autophagy in HUVECs. Taken together, these findings indicate that autophagy plays a protective role against apoptosis under clinorotation by clearing protein aggregates and partially restoring the UPR.

Studies have confirmed that on the ground, the plant cells must expend energy to maintain positional homeostasis against gravity. Under microgravity conditions, such energy may be saved for other process such as biosynthesis of beneficial metabolites for growth. This hypothesis was examined on a cell line of tobacco (Nicotiana tabacum cv. Burley 21). The cells were continuously treated with 2-D clinostat for 1 week. Exposure to clinorotation conditions increased biomass and total protein. Total content of soluble sugar also increased which may provide more precursors for Krebs cycle and adenosine triphosphate (ATP) production. In the case of 2-D clinorotation, the expression and activity of glutamate producing enzyme, glutamate dehydrogenase (GDH) increased, whereas the activity of glutamate decarboxylase (GAD) decreased. Regarding the role of GAD in initiation of gamma amino butyric acid (GABA) shunt, it is plausible that under clinorotation condition, the tobacco cells directed their metabolism toward saving energy for Krebs cycling and more production of ATP rather than shifting to side paths such as GABA shunt. Improvement of radical scavenging enzymes activity and increase of the contents of phenolic compounds and certain peroxide neutralizing amino acids, e.g., His, Pro, Ser, and Asp under clinorotation conditions decreased membrane lipid peroxidation and maintained the growth potential of tobacco cells.

Roots of land plants show gravitropism and hydrotropism in response to gravity and moisture gradients, respectively, for controlling their growth orientation. Gravitropism interferes with hydrotropism, although the mechanistic aspects are poorly understood. Here, we differentiated hydrotropism from gravitropism in cucumber roots by conducting clinorotation and spaceflight experiments. We also compared mechanisms regulating hydrotropism and auxin-regulated gravitropism. Clinorotated or microgravity (μG)-grown cucumber seedling roots hydrotropically bent toward wet substrate in the presence of moisture gradients, but they grew straight in the direction of normal gravitational force at the Earth\'s surface (1G) on the ground or centrifuge-generated 1G in space. The roots appeared to become hydrotropically more sensitive to moisture gradients under μG conditions in space. Auxin transport inhibitors significantly reduced the hydrotropic response of clinorotated seedling roots. The auxin efflux protein CsPIN5 was differentially expressed in roots of both clinorotated and μG-grown seedlings; with higher expression in the high-humidity (concave) side than the low-humidity (convex) side of hydrotropically responding roots. Our results suggest that roots become hydrotropically sensitive in μG, and CsPIN5-mediated auxin transport has an important role in inducing root hydrotropism. Thus, hydrotropic and gravitropic responses in cucumber roots may compete via differential auxin dynamics established in response to moisture gradients and gravity.

Mitochondrial respiration in plants provides energy for biosynthesis, and its balance with photosynthesis determines the rate of plant biomass accumulation. However, there are very limited data on the influence of altered gravity on the functional status of plant mitochondria. In the given paper, we presented the results of our investigations of root respiration, the mitochondrion ultrastructure, and AOX expression of pea 1-, 3- and 5-day old seedlings grown under slow horizontal clinorotation by using an inhibitor analysis, electron microscopy, and quantitative real-time RT-PCR. It was in the first time shown that enhancement of the respiration rate in root apices of pea etiolated seedlings at the 5th day of clinorotation does not connected with increasing of both alternative oxidize capacity and AOX expression. We assumed this phenomenon is provided by more intensive oxidation of respiratory substrates. At the structural level, mitochondria in cells of the distal elongation zone were the most sensitive to clinorotation that confirms the special physiological status of this zone. The performed investigation revealed an enough resistance of plant mitochondria to the influence of altered gravity that, on our opinion, is one of components providing plant adaptation to microgravity in space flight.