The impact of microgravity-based proteomics research

Expert Review of Proteomics

ISSN: 1478-9450 (Print) 1744-8387 (Online) Journal homepage: http://www.tandfonline.com/loi/ieru20

The impact of microgravity-based proteomics research Daniela Grimm, Jessica Pietsch, Markus Wehland, Peter Richter, Sebastian M Strauch, Michael Lebert, Nils Erik Magnusson, Petra Wise & Johann Bauer To cite this article: Daniela Grimm, Jessica Pietsch, Markus Wehland, Peter Richter, Sebastian M Strauch, Michael Lebert, Nils Erik Magnusson, Petra Wise & Johann Bauer (2014) The impact of microgravity-based proteomics research, Expert Review of Proteomics, 11:4, 465-476 To link to this article: http://dx.doi.org/10.1586/14789450.2014.926221

Published online: 24 Jun 2014.

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The impact of microgravity-based proteomics research Downloaded by [Statsbiblioteket Tidsskriftafdeling] at 00:13 24 October 2015

Expert Rev. Proteomics 11(4), 465–476 (2014)

Daniela Grimm*1, Jessica Pietsch2, Markus Wehland2, Peter Richter3, Sebastian M Strauch3, Michael Lebert3, Nils Erik Magnusson4, Petra Wise5 and Johann Bauer6 1 Institute of Biomedicine, Pharmacology, Aarhus University, 8000 Aarhus C, Denmark 2 Clinic for Plastic, Aesthetic and Hand Surgery, Otto-von-Guericke-University Magdeburg, 39120 Magdeburg, Germany 3 Department of Biology, Cell Biology, Friedrich-Alexander University Erlangen-Nuremberg, 91058 Erlangen, Germany 4 Medical Research Laboratories, Department of Clinical Medicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark 5 Hematology/Oncology, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA 90027, USA 6 Max-Planck Institute for Biochemistry, 82152 Martinsried, Germany *Author for correspondence: Tel.: +45 8716 7693 Fax: +45 8612 8804 [email protected]

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Proteomics is performed in microgravity research in order to determine protein alterations occurring qualitatively and quantitatively, when single cells or whole organisms are exposed to real or simulated microgravity. To this purpose, antibody-dependent (Western blotting, flow cytometry, Luminex technology) and antibody-independent (mass spectrometry, gene array) techniques are applied. The anticipated findings will help to understand microgravity-specific behavior, which has been observed in bacteria, as well as in plant, animal and human cells. To date, the analyses revealed that cell cultures are more sensitive to microgravity than cells embedded in organisms and that proteins changing under microgravity are highly interactive. Furthermore, one has to distinguish between primary gravity-induced and subsequent interaction-dependent changes of proteins, as well as between direct microgravity-related effects and indirect stress responses. Progress in this field will impact on tissue engineering and medicine and will uncover possibilities of counteracting alterations of protein expression at lowered gravity. KEYWORDS: 3D cell growth • bed rest • clinostat • random positioning machine • spaceflight • tail suspension

In recent years, it has become clear that many microgravity-related changes of normal biological functions in unicellular organisms, plants, animals and humans are due to alteration in the expression of various genes and the subsequent changes in the composition of proteins [1–5]. Shifts have been observed in the production of cell wall components, of extracellular and intracellular proteins, of growth, apoptotic and nuclear factors as well as of receptors, kinases and other components of signaling pathways [6–12]. This understanding is currently stimulating a series of analyses and comparisons of proteins, which are present either in cells living under gravity or in cells exposed to microgravity. To expose cells and tissues to reduced gravity, scientists use platforms in real microgravity [13–18]. These include orbital platforms such as satellites or space stations (microgravity time of weeks to years) [13,14], sounding rockets (microgravity time 3–13 min) [15], parabolic flight campaigns (microgravity time about 20–30 s) [16–18] and drop towers (microgravity time about 5 s) [19]. Due to the high costs of a stay in real microgravity, ground-based

10.1586/14789450.2014.926221

platforms simulating microgravity can be additionally used [20,21]. The most commonly used ground-based platforms for simulating microgravity are the random positioning machine (RPM) [22], the 2D clinostat [21] and the rotating wall vessel [23]. Although gravitational unloading is not achieved by these devices, they inhibit sedimentation of cell or tissue samples by rotation of the samples around one or two axes [5]. In this way, the gravity vector is randomized, but fluid convection and shear stress is not completely avoided. Hence, results obtained from cells incubated in real microgravity are frequently similar, but not completely identical [21,24,25]. Recently, diamagnetic levitation was introduced. It enables microgravity and low gravity-like effects in a stable environment [26]. In this review, the authors want to summarize alterations of cellular protein contents induced by culturing cells, tissues or whole organisms under the various conditions of annulling gravity. Although many results show platform dependence, the study may point to effects that microgravity exerts on the protein content of cells. The knowledge about

2014 Informa UK Ltd

ISSN 1478-9450

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Grimm, Pietsch, Wehland et al.

microgravity-related proteomics gathered so far is gradually having impact on biotechnology, medicine and space travel.

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Figure 1. Detection of lymphocte cytosolic protein 1. Western blot analyses of (A) LCP-1 and (B) phosphorylated LCP-1. Western blot analysis of FTC-133 cells cultured up to 3 days under 1 g or random positioning machine conditions (mg) are shown. Cells grew adherently or in form of multicellular tumor spheroids. Photographs of western blots and their densitometric evaluation are given. The black bars indicate protein concentrations found in controls (1 g) and grey bars show the results for the random positioning machine grown adherently cells and the white bars the values for the multicellular tumor spheroids samples. Values are given as mean ± standard deviation. *p < 0.05. LCP1: Lymphocyte cytosolic protein 1 (plastin 2).

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Proteins may be quantified and qualified by western blotting, flow cytometry, Luminex technology or mass spectrometry (MS) or may be measured by gene array analyses. Western blotting and flow cytometry are well-established methods [27,28]. Using western blot analysis, it is possible to quantify the amount of distinct cellular proteins (FIGURE 1). Applying flow cytometry, it is possible to count the cells of a population that bear a protein of interest. Luminex technology is based on a multiplex bead assay and may be used to determine proteins secreted by cells into culture supernatants or body fluids. To this purpose, antibodies against a protein of interest are being conjugated to unique fluorescent synthetic microbeads, incubated with a relevant supernatant and processed [29]. Finally, a Luminex sorter returns mean fluorescence intensity values of sorted beads and correlates each mean fluorescence intensity to the appropriate concentration of each analyte using a standard curve of each analyte [30]. Antibodies are essential for the application of all these methods. Choice of antibodies may be based on observations of biological behavior such as apoptosis or cell shape alteration. However, in most cases, antibodies must be selected by using pathway analyses or by presumption. This makes a comprehensive determination of various types of proteins very time consuming and expensive. Therefore, MS has been utilized for comprehensive determination of thousands of proteins. This method enables the detection of unforeseen types of proteins. Especially if a cell lysate was preseparated, rare proteins become visible in addition to the abundant ones [31–33]. However, for a long time it was difficult to quantify the amount of a protein present in a cell [34,35]. Only a rough estimation of change in protein contents induced by alteration of gravity conditions during culture was achieved, when the proteomes were determined of cells Expert Rev. Proteomics 11(4), (2014)


The impact of microgravity-based proteomics research

incubated in parallel, either under normal or lowered gravity [33,36] and considered qualitative indicators of protein abundance [34,37]. The information gained by MS could be enriched if the cellular subpopulations or intracellular organelles are purified before the proteome analysis [38] or isoelectric point of protein samples is determined before MS is applied [39]. Nowadays, liquid chromatography coupled with highresolution tandem MS (MS/MS) is used to identify and quantify peptides on a large scale. The peptide intensity information returned by the mass spectrometer is employed to identify and quantify proteins [40,41]. In this way, even quantitative determination of a cell’s number of copies of a distinct protein is possible [42]. The new technology is now ready for routine analysis. Its application in space research will surely provide a deeper insight into protein compositions. Gravity-dependent protein changes in microorganisms & plants Bacteria

Mainly two observations drive protein determination in microorganisms and plants. First, a number of studies have revealed that space conditions increase virulence and reduce susceptibility of pathogenic microorganisms against antimicrobial agents [43]. One reason for this phenomenon may be an impaired function of the immune system of infected humans or animals [44]. But a change of the protein production capability of microorganisms must also be considered as a cause of the enhanced infection rate, because incubation of Escherichia coli under simulated microgravity showed that randomization of the gravity vector triggers these bacteria to secrete higher amounts of heat-labile enterotoxin [45]. Furthermore, Pseudomonas aeruginosa, an opportunistic pathogen that is present in the space habitat, responded to spaceflight conditions through differential regulation of 28 proteins [46] and Rhodospirillum rubrum showed altered intracellular amounts of stress and ribosomal proteins after exposure to microgravity [47]. Plants

Second, the gravity vector is important for the orientation of swimming unicellular organisms, such as fresh water flagellate Euglena gracilis as well as for the orientation of the growth of roots and stems in cells [48]. In both cases, orientation is impaired or prevented when the gravity vector is randomized or annulled [12,49]. In addition, changes in the expression of selected types of mRNA were detected when protists or plants were exposed to microgravity and simultaneously to ground-based platforms. In a recent Space experiment (Sino-German Simbox/ Shenzhou-8 Space mission), it was found that several investigated genes in E. gracilis were differentially expressed in microgravity compared with an identical 1 g reference sample [49]. Quantitative RT-PCR revealed that genes involved in signal transduction, oxidative stress, defense, cell cycle regulation and heat shock response were upregulated. Correll et al. [50] investigated in 2013 the transcriptome of Arabidopsis thaliana seedlings in space (TROPI-2 experiment on the International Space Station). It informahealthcare.com

Review

was found that the transcription of 278 genes (twofold differentially expressed) was different from simultaneous ground controls and about 27 from in-flight 1 g controls. Next to many undefined genes, a large number of genes involved in salt stress, oxidative stress, detoxification, biotic-induced stress, water stress and defense response (and other stress responses) were among the differentially expressed ones. Also Zupanska et al. found significant stress responses (effects on heat shock proteins [HSP] and chaperons) of Arabidopsis tissue cultures in space [51]. In the course of parabolic flights, Paul et al. studied in 2011 the effect of subsequent short-term microgravity phases (each about 20 s) achieved by means of parabolic flight maneuvers (parabolas) and gene expression in A. thaliana was investigated after 20 or 40 parabolas, respectively [52]. The authors detected significant changes in the expression of various genes. Similar to other reports, strong changes were found in the following Gene Ontology categories: signal transduction, stress response, response to biotic and abiotic stimuli. The expression profiles were changing with the number of parabolas. During a spaceflight experiment (BRIC-16 on STS-131, 2010), the same authors compared the transcriptome of A. thaliana seedlings or A. thaliana cell cultures [53]. Callus cells were found to be more impaired by microgravity compared with seedlings. Among others in seedlings genes involved in pathogen response and wounding, stress response (cold and drought stress), auxin-mediated root development and calcium signaling (downregulation) were differentially expressed compared with ground controls. The pattern of differentially expressed genes was different between seedlings and cell cultures. The transcription profiles of Ceratopteris richardii spores in mg and 1 g were analyzed and compared at different subsequent time points during the NASA shuttle mission STS-93 [54]. Various genes were altered, for example, transferases and hydrolases, as well as HSP. Interestingly, almost no sequence, which was shown to be up- or downregulated at any time point showed the same expression at all (three) time points. These results indicate that gene expression changes in microgravity are not stable. It seems that after an initial time of stimulation, gene expression goes back to previous level or to a new equilibrium. During a space shuttle flight and subsequent installation on the International Space Station, Stutte et al. determined the effect of microgravity on dwarf wheat grown in real microgravity (located in a Biomass Production System device) [55]. They found only marginal changes in morphology compared with a 1 g ground control and no significant changes in the transcription level. The experiments pointed to a number of proteins that may change in plants or flagellates when gravity is omitted. In this way, they challenged determinations of gravity-dependent protein composition changes. Barjaktarovic et al. investigated A. thaliana callus cells after incubation on the RPM by the means of 2D-gel electrophoresis and subsequent ESI-MS/MS. Mainly proteins involved in radical scavenging and detoxification of reactive oxygen species, primary metabolism and signaling were found to be different from controls [56]. Furthermore, significant changes in phosphorylation of stress proteins became obvious [57]. Wang et al. investigated the effect of vertical clinorotation on the 467


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proteome of Arabidopsis calli [58]. After incubation, proteins were analyzed by 2D-gel electrophoresis. It was found that about 80 protein spots of clinorotated and control cells were different in abundance and/or position on the gel. When the proteins of the spots were further investigated by liquid chromatography-ion trap-MS, those of 18 spots could be identified. Among others, stress response proteins, enzymes of carbohydrate metabolism, signaling proteins, ribosomal proteins and transcription factors were altered. In another study, when the effects of a clinostat on the proteome of root cells were investigated, the authors came to a similar result [59]. Proteins involved in general stress response, proteins of carbohydrate metabolism (e.g., glyceraldehyde3-phosphate dehydrogenase, triose-phosphate isomerase) and signaling proteins were changed upon vertical clinorotation [59]. HSP-70 and HSP-90 protein levels in Pea seedlings remained higher during germination under horizontal clinorotation as compared with controls [60]. A change of proteins involved in abiotic stress and secondary metabolism was also observed when in vitro Arabidopsis callus cultures were incubated on various Earth-based microgravity simulation devices [61]. In Arabidopsis callus cells, the proteins involved in primary metabolism and detoxification of reactive oxygen species appear to respond very quickly to a gravity change, because these proteins were already modified during parabolic flights [62]. Furthermore, seeds of rice were flown on the Chinese recoverable satellite, JB-1, for 15 days. Afterward, their proteins were investigated by 2D-gel electrophoresis and MS. Four proteins (hydrogen peroxide oxidase, ascorbate peroxidase, a manganese-stabilizing protein and a ribosomal protein) could be identified as differently expressed [63]. Stout et al. found the activity of antidiuretic hormone to be increased several-fold in microgravity compared with the ground, after they had incubated roots of Brassica rapa on the Mir station for 13 days. They considered hypoxia as a main reason for this change [64]. Recent experiments of Scherer and Quader, who investigated endocytosis of tobacco pollen tubes in real microgravity in the course of a sounding rocket flight, indicate impacts of gravity or microgravity on the cytoskeleton [15]. Conclusion from studies on microorganisms & plants

Surprisingly, the proteome analyses hitherto performed on microorganisms and plants revealed mainly proteins of cell structure maintenance, metabolism and stress response being changed under microgravity, while our current knowledge about gravity perception as well as the connected signal transduction chains remains very limited. Further determination of proteins changed in the various cells of plants and microorganisms is urgently required. In addition, it is very important to distinguish stress- and microgravity-related effects. Studies on human cells cultured in vitro

Proteome analysis on human cells is driven by two observations: first, an impairment of the immune response of astronauts is evident already after a few days in space and after long-term spaceflights [65]; second, culturing cells under 468

microgravity revealed an onset of 3D growth of human tissue cells, which grow exclusively in a 2D monolayer when cultured in a normal laboratory incubator [66–71]. Hence, there are biotechnological and medical reasons for searching proteins affected by microgravity. Transition from 2D to 3D growth of tissue cells occurs under real microgravity as well as under simulated microgravity [14,66]. This process is always accompanied by a profound change of gene expression patterns. During different spaceflights, primary human renal cell cultures [3], human fibroblasts [72], human lymphocytes [73], osteoblasts [74] and human cervical 48A9 CaSki cancer cells [75] up- or downregulated the expression of a number of genes. Even after incubation on groundbased platforms, simulating microgravity, tremendous alterations of gene expression patterns were found in cancer cells and benign human cells [76]. Frequently, early alterations can be observed within 22 s of exposure to microgravity [17,18]. In a recent paper, we found several genes such as IL-8 and von Willebrand factor the most prominently affected by simulated microgravity using a RPM. Another 20 proteins that are important in cell structure maintenance and angiogenesis extended their network of interaction. Thus, this study reveals numerous genes, which mutually influence each other during initiation of 3D growth of endothelial cells between 5 and 7 days [76]. However, the influence of microgravity on gene expression only suggests a subsequent shift of cellular protein composition, because the translation from mRNAs to functional proteins is intricately regulated [77]. However, such information helps to perform protein analysis more efficiently. Thyroid cancer cells

For over two decades, we have studied the proteins of thyroid cancer cells. Initially, a lot of background knowledge was accumulated through thoroughly studying this type of cancer cell under normal laboratory conditions. On ground, the 2D growth behavior of thyroid cancer cells deriving from differentiated tumors was compared with their 3D growth in liquid overlay systems and in spinner flasks [78]. In addition, the thyroid cancer cells’ interaction with endothelial cells was investigated [79] as well as their response to treatment with radiation [80,81] and sunitinib [82]. Furthermore, the thyroid cancer cell line ML-1 was established [83]. Later on, the thyroid cancer cells were exposed to the RPM. The subsequent analyses of cells revealed changes in differentiation and migration, an increase in apoptosis, an elevation of extracellular matrix proteins and cell adhesion molecules as well as a transition from a 2D to the 3D growth behavior [11,66]. These behavioral and physiological alterations of the thyroid cancer cells were accompanied by shifts in the cellular protein compositions. Western blotting and flow cytometry analyses unfolded changes of intracellular concentrations of apoptosisrelated proteins, extracellular matrix components and growth factors in ML-1 [66], HTU-5 [84] and other cells [85]. To further explore the thyroid cell proteome, we modified a protocol of Grun et al. [86], and incubated human thyroid cancer cells either on ground or on the RPM, lysed them, Expert Rev. Proteomics 11(4), (2014)



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Figure 2. Immunofluorescence of b-tubulin of follicular thyroid cancer cells. The cells are also stained with F-actin (rhodamine-phalloidin stain) and the nuclei are counterstained with Hoechst 33342 stain. (A) Normal static 1 g control cells grown on a slide flask for 4 h; (B) follicular thyroid cancer 133 cells cultured on a 2D clinostat for 4 h and (C) follicular thyroid cancer 133 cells grown on a random positioning machine for 4 h.

pre-separated their proteins by free flow electrophoresis and applied selected fractions of proteins to MS [33,35]. The MS analysis returned exponentially modified protein abundance index values [34] that hinted at a number of potential gravisensitive proteins [33]. Some of them were proved to be gravisensitive by subsequent western blots [33,35]. A further evaluation of the MS data indicated that the expression of surface proteins binding fibronectin may accelerate spheroid formation and strengthen 3D cell cohesion [39]. Also, microgravity diminished enhanced glycolytic rates, which cancer cells turn on in vivo in order to survive in a low oxygen environment [87] and downregulated lymphocyte cytosolic protein 1 (plastin 2) (FIGURE 1), which had been earlier identified as an ovarian cancer tumor biomarker [88]. Despite the accumulation of knowledge about microgravity-dependent protein alterations, it is still difficult to decide whether a protein shift is either a cause or a byproduct of a microgravity-dependent alteration in the biology of a cell, since many of the proteins found are members of networks of proteins whose expression is regulated mutually [89]. Moreover, we could show that follicular thyroid cancer 133 thyroid cancer cells when cultured under simulated microgravity conditions on a 2D clinostat and on an RPM show differences in the microtubule network (FIGURE 2). In addition, we saw that stress factors, such as vibration and transient hypergravity normally accompanying a spaceflight, significantly affect the gene expression patterns of thyroid cells, although these factors alone do not trigger 3D growth [90]. However, in combination with microgravity, they might contribute favorably to 3D growth, as the thyroid cancer cells formed extraordinary large spheroids during a spaceflight [14]. Neuroblastoma cancer cells

In another approach, Zhang et al. investigated up- and downregulated proteins in SH-SY5Y neuroblastoma cells exposed to a clinostat: simulating microgravity. No apoptosis was informahealthcare.com

detectable in these cells, but the cell microfilament network was disrupted under simulated microgravity [91]. Applying the comparative proteomic method based on the 18O-labeling technique, the authors detected 22 differentially abundant proteins, for example, proteins interacting with the cytoskeleton, HSP and others. Interestingly, several 14-3-3 proteins, 78 kDa glucose-regulated protein and heat shock cognate 71 kDa proteins had also attracted our attention in our studies on thyroid proteomes [33,35]. Endothelial cells

In contrast to liquid overlay and Spinner flask technology, exposure to microgravity also triggers benign human cells to switch from 2D to 3D growth. For example, endothelial cells form 3D aggregates when cultured on a RPM for 5–7 days [67,92–95]. Interestingly, these aggregates are oblong and sometimes even resemble tubular structures. The cells begin to change very soon after the application of microgravity conditions. During parabolic flights, 22 s of weightlessness are enough to cause early structural alterations of the cytoskeleton [17]. When studied after hours or days of incubation on a RPM, the cells showed alterations of the actin cytoskeleton coupled with increased nitric oxide synthesis [96] as well as microfilament thinning and intracellular redistribution [97]. In addition, extracellular matrix proteins were upregulated [30], the von Willebrand factor was downregulated [92] and the secretion of various growth factors was up- or downregulated [98]. Interestingly, the concentrations of matrix metalloproteinases (e.g., MMP-2) remain stable during incubation under simulated microgravity, while the tissue inhibitor of metalloproteinases is upregulated [67,99]. Recently, MS was applied in combination with free flow electrophoresis to compare the proteins of two types of endothelial cells, the EA.hy926 and the human microvascular endothelial cells (HMVEC). These cells transit between the fifth and the seventh day of the RPM from 2D to 3D growth, but 469

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the HMVEC cells proceeded faster than the EA.hy926 cells. A total of 1175 types of proteins were found in EA.hy926 cells and 846 in HMVEC. Of these, 584 proteins were present in both types of cells. These included a number of metabolic enzymes, structure-related proteins and stress proteins. Their evaluation suggested a downregulation of 26S proteasomes and a transient upregulation of ribosomal proteins during tube formation [36]. It still remains to be elucidated which mechanism is the gravisensitive one, because stress and mutual regulation can also influence the expression of a cellular protein, as we have learned from thyroid cells [90]. Chondrocytes

Chondrocytes represent another type of benign human cells that adjust to microgravity by a transition from 2D to 3D growth. In a scaffold-free manner, they start forming 3D cell assemblies within 5 days on the RPM, which subsequently develop into small fragments of cartilage [71]. The beginning of this process is marked by an alteration of gene and protein expression after 4 and 24 h of culture on the RPM, respectively [71,100]. During the subsequent 4 days until forming the 3D aggregates, chondrocytes exposed to simulated microgravity change their extracellular matrix production behavior and rearrange their cytoskeleton. The proteins most strongly affected are collagens type I, II and X, laminin, chondroitin sulfate and aggrecan as well as b-tubulin, vimentin, TGF-b1 and osteopontin. Immune system

It is well-documented that the immune response of astronauts is reduced during their stay in space [65]. A possible explanation for this phenomenon was provided when isolated lymphocytes were investigated during the first Spacelab mission. In this experiment, a strong impairment of the response of lymphocytes to mitogenic stimulation was observed, which was clearly due to the lack of gravity [101,102]. When gravitational forces were absent, the structure of the cytoskeleton was rearranged. Subsequently, the signal transduction processes required for T-cell activation were disturbed [103,104] and apoptosis-associated Fas/APO-1 proteins were enhanced [7]. In addition, 5-lipoxygenase activity was induced and cytochrome c release into the cytosol augmented, which promotes apoptosis [105]. Furthermore, PKC translocation required for transducing an activation signal was reduced [106], as well as the abundance of the phosphorylated cAMP-response element binding protein [107]. From these results, it was concluded that the protein kinase A signaling could be affected by removing gravity [107]. Conclusions from human cell culture experiments

Taking together, all proteome studies performed in vitro on isolated cells, one may conclude that a striking number of proteins are obviously changed when cells are exposed to microgravity. Looking at all these proteins described, one wonders how an animal or human can survive an extended stay in the 470

orbit, unless these effects are either prevented or counterregulated in a whole organism. As survival without sequential damage is possible at least for months, it appears necessary to also look at whole organisms and investigate which microgravityinduced changes can be found there. Studies on organisms

So far, approximately 500 people have been in space for a time frame of up to 1 year. Although humans seem to adapt well to weightlessness, prolonged exposure to microgravity causes a variety of adaptive changes in the human organism [108]. These changes include muscle loss, bone loss, changes in kidney function as well as potential changes in the regulatory responses of the immune system as seen in impaired wound healing [109], altered monocyte and granulocyte function [110–112] and altered cytokine production patterns [113]. Presently, there is an interest to study and understand the impact of long-term space travel on whole organisms [114]. There is a specific need to uncover the gravity-sensitive molecular signaling being altered, which may explain the observed physiological changes. Mapping key regulators of cell signaling in different cell systems [11,107,113] will surely help to better dissect and understand the biological effects that weightlessness causes in the organism as a whole. Samples of tissue and fluids of organisms have been prepared for analyzing their protein content. Moriggi et al. applied muscle biopsies to investigate the effects of microgravity on myosin heavy and light chain isoform distribution in the muscles of humans [115]. Muscle samples were taken on the second day of bed rest and close to the end of the bed rest period of 55 days. The proteins were extracted and their proteins were analyzed with the help of 2D electrophoresis and MS. Proteins from the Z-disk region and from costameres (sub-sarcolemmal protein assemblies) were differently dysregulated during bed rest [115]. In a further human study, the effect of weightlessness on urinary proteome composition of cosmonauts was studied before and after spaceflight from a molecular perspective. A total of 15 proteins originating from the renal tissues were identified as differentially expressed and 10 of these were present in the urine [116]. The presence of neutral endopeptidase, afamin, aquaporin-2, aminopeptidase A and dipeptidyl peptidase 4 in the urine was dependent on spaceflight exposure. Further studies were done on tissue samples of animals. Changes in peptide and protein abundance expression caused by microgravity in mouse cardiac tissue were measured with MS. This approach was shown to efficiently identify proteins related to basic heart functions, cardiac stress and energy supply [117]. Ding et al. examined the morphological damage and molecular changes in rat kidneys following 2 months of tail suspension [118]. The authors identified necrosis of renal tubular epithelial cells and glomerular atrophy as well as mitochondrial swelling and increase in apoptosis. Protein analyses revealed that HSP-70 and GRP78 were possible candidates of microgravity-regulated proteins. Lung function is also affected by weightlessness. In a study using the tail suspension model with male Sprague-Dawley rats, the lung proteome was Expert Rev. Proteomics 11(4), (2014)


Review

Table 1. Recent publications (2011–2013) describing protein alterations under microgravity. Kind of exposure to mg

Major results

Spaceflight

Changes in 28 proteins

[46]

Arabidopsis thaliana

Clinorotation

Annexin-2 change

[59]

Pisum sativum seedling

Clinorotation

Alteration of HSP-70, HSP-90 regulation

[60]

Arabidopsis callus

Diamagnetic levitation

Alteration of proteomic signature

[61]

Tobacco pollen tube

Sounding rocket

Increase in endocytosis, mg-effects on cytoskeleton (relaxation) with effects on certain membrane channels

[15]

A. thaliana callus cells

RPM

25 proteins altered on RPM

[57]

A. thaliana cell cultures

Parabolic flight

Rapid changes in protein phosphorylation pattern

[62]

Rat pulmonary tissue

Tail suspended

13 proteins upregulated, 4 proteins downregulated

[119]

Human urine

6-month space

Presence of proteins of kidneys and urinary tract

[116]

Mice liver

13 days in space

Alteration of proteome signature

[120]

Rat hippocampus

Hind limb suspension

4 proteins upregulated, 6 proteins downregulated

[121]

Human thyroid cells

RPM

Downregulation of glycolytic enzymes

[87]

Human thyroid cells

RPM

NF-kB production, IL-6 secretion

[11]

Human neuroblastoma cells

Clinorotation

22 differentially expressed proteins, microfilament disruption

[91]

Human chondrocytes

RPM

TGF-b1 enhancement

[100]

Human monocytes

Blood of astronauts

Reduction of cytokine production

[111]

Xenopus cells

RPM

Alterations of cytoskeletal proteins

[122]

Samples

Ref.

Microbiology Pseudomonas aeruginosa


Plant physiology

Physiology

Cellular physiology

HSP: Heat shock protein; RPM: Random positioning machine.

analyzed using 2D-gel electrophoresis [119]. Differentially expressed proteins, selected by 2D-gel electrophoresis were identified by MS and 13 and 4 proteins were up- and downregulated, respectively. These proteins were related to cellular energy metabolism, stress and inflammatory response, cell injury, repair and intracellular signaling. After a NASA flight, formalin-fixed paraffin-embedded tissue sections of mice livers were analyzed with MS [120]. Downregulation of aldolase B, regucalcin, ribonuclease UK114, a-enolase, glycine N-methyltransferase and S-adenosylmethionine synthetase isoform type-1 was observed, while the 60 kDa HSP was elevated. In addition, after 14 days of hindlimb suspension, four and six proteins of the hippocampus of 3-week-old male Wistar Hannover rats were up- and downregulated, respectively, as compared with controls [121]. A proteomic approach was applied to compare the protein profiles of Xenopus laevis embryos developed in simulated microgravity and in normal conditions. The results strongly suggest that some of the major components of the cytoskeleton are differently expressed in X. laevis embryos exposed to weightlessness as compared with those living under 1 g [122]. informahealthcare.com

Conclusions from studies on whole organisms

Taken together, proteomic studies on organisms exposed to microgravity clearly indicated that cellular protein contents are altered when whole organisms are exposed to microgravity. Further studies are required to establish correlations between in vitro and in vivo protein expression. The efforts appear worthwhile, because they will show which of the many protein content alterations found in in vitro cultured cells are counterregulated in whole organisms and which are not. Expert commentary

The data concerning alterations of cellular contents of proteins induced by altered gravity conditions in cells of bacteria, plants, animals and humans are slowly increasing (TABLE 1). However, the distinction between primary gravity-induced and subsequent interaction-dependent changes of proteins as well as between direct microgravity-related effects and indirect stress responses is not clear. Understanding the signaling pathways turned on or off by the basic gravisensitive elements is necessary, because it enables the development of effective countermeasures to spaceflight-induced health concerns (e.g., osteoporosis and 471

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muscle atrophy), and helps to set up life support systems in long-term flights. In addition, this work fosters exploitation of weightlessness for earth-based research and space research on diseases such as cancer and for advancing the development of scaffold-free tissue engineering [123]. Therefore, it is important to use the information gathered so far to design improved protein determination experiments considering whole organisms after bed rest or spaceflight in comparison with cells exposed to the various platforms of simulated and real microgravity.


Five-year view

In the next 5 years, several spaceflights will be launched in the USA, PR China, Russia and other countries. In addition, rockets will be launched into the near-Earth orbit and parabolic flights will be performed. In most of these flights, cells of various sources will be taken along and will be used afterward to analyze their proteins or even their whole proteome. In addition, more and more devices aiming to create microgravity on the Earth, such as the RPM, the 2D clinostat or the rotating wall vessel will become available, which allow easy, cheap and frequent exposure of cells to conditions of simulated microgravity. Studies to compare the effects of real microgravity in space with simulated microgravity are just published or will be performed in the future [25]. Hence, extended possibilities will be provided in the near future to explore gravity-dependent proteome alterations in several cell models of humans, animals, plants and microorganisms and to study shifts of proteins in whole organisms exposed to microgravity. In addition, MS technology is developing very quickly [40–42]. In the future, it will be possible to determine up to 5000 different types of proteins using samples that are so small that they can easily be provided from cell cultures or biopsies. Therefore, we expect a rather comprehensive knowledge of many, even rare proteins present or modified in cells of interest. From today’s perspective, all opportunities to perform microgravity-related experiments should be used to ascertain and to broaden the proteome data known so far; to identify these proteins of human, animal, plant and microbial cells, which sense the change of gravity immediately; to unveil the cellular signaling pathways as well as the protein–protein and protein–gene interaction mechanisms activated or inhibited by loss of gravity; to investigate the potential of growth factors, hormones or other whole body messengers such as cytokines (animals) or auxins (plants) in counterregulating microgravity induced dysfunction of single cells; to exploit the knowledge gained so far from proteome research in space medicine, in tissue engineering and in developing life support systems for space transporters or space stations with reduced gravity. It could be an advantage, if each research team uses as many as possible space research opportunities in a comparative manner, as each kind of microgravity exposure has its inherent side effects. For example, during space flights, vibration of the transporter as well as a transient exposure to hypergravity cannot be avoided. Both may influence the physiology of single cells as well as of whole organisms. Examinations of short-term 472

effects can best be achieved in parabolic flight experiments. Bed rest studies can also be performed. However, a bed rest proband does not experience as much radiation and stress as an astronaut during spaceflight. Research on whole organisms may show the outcome, but does not reveal the underlying mechanisms. For this purpose, the RPM is a cheap and easily accessible technique, but shear forces cannot be completely excluded in cell cultures and animals may not be investigated at all with the help of such a machine. If research is done on several tracks and corresponding results of each type of experiment are compared with each other, it will be easier to differentiate between microgravity effects and accompanying vibration, hypergravity or radiation effects [90,124]. Furthermore, a physiologic (whole organism) effect will become discriminable from a cellular effect. Research on several tracks with the help of high-throughput techniques such as MS or gene array analysis will provide a huge amount of data. These data have to be evaluated for each technique. In addition, the information gained by each method separately has to be reconciled. This procedure may be facilitated, if the various results are collected in databases, which allow communication among each other. These authors are convinced that the ongoing work in space research will give a good return even in the near future. The accumulation of knowledge will have impact on improvement of security, health and cushiness of space travelers as well as on life on the Earth. The enhanced pathogenicity of microorganism during spaceflight will become opposable, if the change of bacterial protein production is known together with protein changes in the human immune system. The establishment of bioregenerative life support systems such as biological air and water revitalization will be facilitated, if the behavior of plants under microgravity is better understood. The latter will be an enormous advantage concerning the weight, cost and psychological issues of future long-term space missions. As soon as we know how annulling gravity is changing gene and protein expression, which triggers the transition from the 2D to the 3D growth, we may understand why some cells leave a tissue, while others aggregate to 3D structures [123]. This knowledge may enable us to develop strategies against cancer development and metastasis. Acknowledgements

D Grimm gratefully acknowledges support from the German Space Agency DLR (grants 50WB0524; 50WB0824; 50WB1124) and M Lebert acknowledges support from the German Space Agency DLR on behalf of the BMBF (50WB1228). Financial & competing interests disclosure

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. The writing assistance (English editing) of Expert Reviews of Proteomics was utilized in the production of this manuscript. Expert Rev. Proteomics 11(4), (2014)


Review

Key issues • Proteome analysis is performed in microgravity research in order to understand the effects of microgravity on biological systems. • Antibody-dependent techniques of proteomics such as western blotting, flow cytometry and Luminex technology are useful in quantifying the proteins. • Antibody-independent techniques of proteomics such as mass spectrometry are helpful in gaining a comprehensive overview. • Simulated and real microgravity should be applied to identify and quantify microgravity-dependent protein alterations. • It is important to distinguish between primary gravity-induced and subsequent interaction-dependent changes of proteins as well as between direct microgravity-related effects and indirect stress responses. • Analyses of more general patterns of observed response to microgravity (e.g., stress response, effects on carbohydrate metabolism) and more individual species/cell type-specific responses are important to identify a possible common underlying mechanism how complex organisms like humans, animals, plants and microorganisms react to changes in gravity forces.


• Proteomics data gained in space research is of interest to improve journeys to microgravity and to promote medical and biotechnological issues on the Earth. The knowledge from microgravity-induced transition from 2D to 3D growth may be applied in cancer research and tissue engineering.

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