Elucidating the âGravomeâ: Quantitative Proteomic Profiling of the

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Elucidating the ‘Gravome’: Quantitative Proteomic Profiling of the Response to Chronic Hypergravity in Drosophila Ravikumar Hosamani, Ryan Leib, Shilpa R. Bhardwaj, Christopher M. Adams, and Sharmila Bhattacharya J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00030 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 22, 2016

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Journal of Proteome Research

Elucidating the ‘Gravome’: Quantitative Proteomic Profiling of the Response to Chronic Hypergravity in Drosophila

Ravikumar Hosamani1, Ryan Leib2, Shilpa R. Bhardwaj1, Christopher M. Adams2 and *Sharmila Bhattacharya1 1

Space Biosciences Division, NASA Ames Research Center, Moffett field, CA-94035, USA

2

Stanford University Mass Spectrometry (SUMS), Palo Alto, CA-94305, USA

Running title: Hypergravity-induced proteomic changes in Drosophila melanogaster

*Corresponding Author Sharmila Bhattacharya, Ph.D. Principal Investigator Space Bioscience Division NASA Ames Research Center Moffett field, CA-94035 Email: [email protected] Phone # +1 (650) 604-1531 Fax # +1 (650) 604-3159

1 ACS Paragon Plus Environment


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ABSTRACT

Altered gravity conditions, such as experienced by organisms during spaceflight, is known to cause transcriptomic and proteomic changes.

We describe the proteomic changes in whole adult

Drosophila melanogaster (fruit fly), but focus specifically on the localized changes in the adult head in response to chronic hypergravity (3g) treatment. Canton S adult female flies (2-3 days old) were exposed to chronic hypergravity for 9 days and compared with 1g controls. After hypergravity treatment, either whole flies (body + head) or fly head only samples were isolated and evaluated for quantitative comparison of the two gravity conditions using an isobaric tagging liquid chromatography-tandem mass spectrometry approach. A total of 1948 proteins from whole flies and 1480 proteins from fly heads, were differentially present in hypergravity-treated flies. Gene Ontology analysis of head specific proteomics revealed host immune response and humoral stress proteins were significantly upregulated. Proteins related to calcium regulation, ion transport and ATPase were decreased. Increased expression of cuticular proteins may suggest an alteration in chitin metabolism and in chitin-based cuticle development. We therefore present a comprehensive quantitative survey of proteomic changes in response to chronic hypergravity in Drosophila, which will help elucidate the underlying molecular mechanism/s associated with altered gravity environments.

Keywords: Drosophila melanogaster, hypergravity, proteomics, immune response, stress response, cuticle development, transport proteins.


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INTRODUCTION

Evolutionarily, all known organisms are inherently adapted to Earth’s gravitational force of 1g. It is therefore fascinating to examine the response of an organism, at a molecular level, as it attempts to adapt to changes in the perceived gravity vector. Transcriptional and proteomic responses are crucial for a cell and/or organism to acclimate to extreme environmental conditions such as altered gravity. To aid in the study of these responses, researchers have developed several experimental methods to understand altered gravity effects while remaining in a laboratory on Earth. Hypergravity can be readily modeled using a standard centrifugation approach, which has been used extensively to study and predict microgravity effects on Earth.1 NASA is particularly interested in both microgravity and hypergravity research because transit into space for astronauts results in chronic exposure to microgravity over the course of a mission, and acute hypergravity during launch and reentry. Launch loads on astronauts for the space shuttle were approximately 3g (or 3 times Earth’s gravitational force) 2 and upon reentry the g-loads were around 1.4 to 3g for the space shuttle and approximately 4g for Soyuz. Thus, there is a need to study these physiological changes in model organisms to help better understand the adverse physiological effects of space flight in humans. Previous spaceflight experiments from our lab and others demonstrate the usefulness of Drosophila melanogaster as a model organism for understanding the effects of altered gravity.3 This organism is ideal for such studies due to its small size, rapid reproduction, genetic homology to mammals, and extensive understanding of its behavior and physiology under normal gravity conditions. Studies with hypergravity (2g) and simulated microgravity (0g) using magnetic levitation have shown extensive changes in global gene expression in adult flies.4 Several



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physiological functions, including development, behavior, immune and stress response, were negatively affected under these simulated microgravity conditions.4 The Drosophila ‘GENE’ experiment carried out on the ISS during the ‘Cervantes’ 11 days Soyuz mission revealed dramatic changes in whole-genome transcriptional profile in pupae suggesting an adverse impact on fly metamorphosis and development.5 Later, these experiments were mimicked on the ground under simulated microgravity and hypergravity conditions, and a similar transcriptional response was observed.5 Microarray data from our previous flight experiment (STS-121- FIT) and others demonstrate significant compromise of the fly immune response and impairment of various other metabolic processes. 3, 6, 7 Similarly, acute and chronic hypergravity (3g) studies in our laboratory indicate elevated oxidative stress across the life stages, and impaired locomotor behavior in adults (unpublished data). The Le Bourg laboratory has extensively investigated the effect of hypergravity on Drosophila lifespan, behavior, and aging.8-10 They demonstrated that short-term exposure to hypergravity results in a positive influence on the physiological and behavioral traits in male flies; whereas life-long exposure causes negative effects.10 A recent study by Taylor et al. suggests that hypergravity increases the survival of flies after pathogenic fungus infection.6 Combination of cold and mild hypergravity stress has a negative impact on the phenotypic outcome in flies, including aging and stress resistance.11 Notably, this combination has a worse effect in females compared to males, indicating the possibility of a sexually dimorphic response to hypergravity.11 Similar findings from our own laboratory suggest that females are more responsive to both acute and chronic hypergravity in terms of induction of oxidative stress related genes in the head and associated locomotor behavior changes of the fly (Unpublished data). Despite numerous studies detailing the profound influence of gravity on diverse biological processes at the transcriptomics level, an understanding of the underlying molecular responses in


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terms of the changes in the levels of functional proteins, is currently lacking. As expressed proteins are the primary effectors of a host of physiological processes, a proteomic response to altered gravity is critical to the understanding of observed phenotypic and functional changes. In the present study we used liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) to determine the global proteomic alterations to chronic hypergravity in whole flies and also specifically in fly heads. The Tandem Mass Tag (TMT) isobaric labeling technique was used to quantify the relative abundance of proteins across pooled biological replicates of 1g and hypergravity (3g) exposed flies. This effort serves as a first description of the quantitative proteomic changes resulting from chronic exposure to hypergravity, and thus serves as a direct extension of previous transcriptomics and behavioral studies.

MATERIALS AND METHODS

Drosophila culture and maintenance

Adult Canton S wild-type flies were reared at 250 C and at 50% relative humidity (RH) on standard laboratory food (25.6g torula yeast, 103.2g dextrose, 48.8g cornmeal, 7.44g agar per liter of fly food). To generate adult flies for the experiment, parent flies (males and females) were allowed to mate and lay eggs in bottles for 48hrs. The parent flies were then discarded, and the eggs were allowed to develop until they eclosed into adult flies. When the adult flies from the F1 generation were 2-3 days old, the flies were sorted and the adult female flies were subjected either to hypergravity exposure or kept at 1g as a control. We specifically used female flies in this study because our preliminary data with both acute and chronic hypergravity indicated that females



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showed a significant response in terms of hypergravity-induced oxidative stress. Future studies can utilize the same methodology to query the response in male flies.

Hypergravity exposure and sample isolation

In our study, hypergravity is defined as 3 times Earth’s gravitational force, or 3g. Using standard centrifugation methods in a BECKMAN GS-6R CENTRIFUGE (modified to maintain low gravitational accelerations of 1.5g and higher; the rotor chamber is 18 inches in diameter with a 1 foot diameter rotor) where 97rpm resulted in a force equivalent to 3g, adult (2-3 days old) female flies were subjected to chronic hypergravity for 9 days. On day 5 the centrifuge was stopped briefly (10 minutes) to place fresh food in the vials. For statistical significance three independent biological replicates were centrifuged under each condition where each fly vial housed 20 female flies. 1g control flies were maintained in the dark under similar environmental conditions such as temperature, relative humidity and in identical vials to match the 3g exposed flies. While centrifugation has a rotational component that does not exactly mimic the g-loads imposed on a system during the launch of a spacecraft, it does however present a useful ground-based tool to study the effects of altered gravity.1,12 Samples can be subjected to well-defined gravitational loads for specific durations of time and monitored in real time or immediately after gravity treatment, complex experiment protocols can also be followed without the mass and volume constraints imposed by spaceflight. Therefore the method of centrifugation has been used widely to create a hypergravity environment for a variety of biological systems for studies relating to altered gravity.


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Sample preparation for Mass Spectrometry

After hypergravity exposure, 4 whole flies were used for each replicate (body + head samples) and 20 adult fly heads per replicate (head only samples) were then lysed in 8M-urea solution. There were 3 replicates each for the whole fly samples and 3 for the head only samples. The supernatant was diluted down to 4M by adding 50mM ammonium bicarbonate. Protein concentration was determined using the Bradford method, and approximately 40µg of protein per sample was used for proteomic analyses. Lysate amounts containing 40µg of protein was reduced with 500mM DTT (Dithiothreitol) and alkylated using 1M acrylamide. 50mM Ammonium bicarbonate was added to the sample in order to dilute down the urea concentration to 1M. Tryptic digestion was carried out overnight using 2µg Trypsin/Lys-C (Promega) per sample, and then samples were acidified with 50% formic acid. Tryptic peptides were labeled using TMT isobaric labels (Thermo Scientific Pierce, TMTsixplex Isobaric Label Reagent Set), as per the manufacturer’s instructions. The resulting labeled peptides were pooled and fractionated into 8 fractions using strong cation exchange (SCX) chromatography. These fractions were then concentrated for injection to LC-MS/MS (Figure 1).

LC-MS/MS Analysis

As is typical for an LC-MS/MS experiment, analytical samples were loaded on a nanoacquity LC (Waters) using a 25 cm analytical C18 reversed-phase column. The column was packed using a 3 µM particle size packing material, and optimum volume was used for each injection. LC-MS/MS acquisition gradients of 2 to 3 hours were used for each sample to facilitate separation on a



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column. Mass spectra were collected using an LTQ (Linear Quadrapole Ion Trap) Orbitrap Velos (Thermo Scientific), set to acquire in DDA (Data Dependent Acquisition) fashion where the top 12 most intense precursor ions were selected for fragmentation using HCD (Higher Energy Collisional Dissociation). Proteins were identified from the RAW output data using a Byonic proteomics node (Protein Metrics) in Proteome Discoverer (Thermo Scientific). All protein identifications were obtained by comparison with canonical proteomic sequences for Drosophila melanogaster (Uniprot), and qualified using a 1% false discovery rate against reverse sequence decoys. TMT label quantification was completed using the Proteome Discoverer Reporter Ion Quantifier node. Quantification was further qualified using statistical analysis scripts prepared in either MatLab (Mathworks) or R (R Project), as described in detail below. The entire experimental process, from sample preparation to LC-MS/MS ready, is summarized in Figure 1.

Statistical analysis

After isobaric tag ratios were identified for each assigned spectrum using Proteome Discover, additional analyses were completed to verify potential proteome expression differences as a function of exposure to enhanced gravity. To a first approximation, the ratio of isobaric tag reporter ions for any given peptide should match those of all other peptides for the same protein. However, a number of factors beyond experimental measurement error can result in an observed distribution of reporter ion ratios, including co-isolation of interferent species which also bear tags, multiple gene products from the same canonical sequence, and/or improper assignment of peptides to a given protein due to algorithm parsimony rules for conserved peptide sequences. As such, more than a simple algebraic ratio across conditions is required to identify proteins that


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differ significantly and to exclude those proteins that are influenced heavily by interference of their isobaric tags. To estimate the effect of isobaric tag interference across all identified proteins, non-parametric ‘bootstrap’ fits were obtained for each protein with at least five peptides. Additionally, these five peptides must each contain observable product ions corresponding to the isobaric tag in all six sample sets i.e., the three control and three hypergravity samples, to be included. These bootstrapped data are then used to obtain estimated population mean and standard deviation values for each protein under both gravity conditions, and obtain p-values. Using this approach, tags that vary due to interference effects are readily identified as multimodal distributions in the bootstrapped data, and expected variance can be estimated. These sample-wide estimated values provide bounds for proteins that have less than five peptides, even when the bootstrap and p-value approach used for more extensively observed proteins is not available.

RESULTS AND DISCUSSION

Gene Ontology (GO) analysis of the proteomic data obtained from whole flies and fly heads provides insight into the diverse biological processes affected by chronic exposure to hypergravity (Figure 5B). Significant differences from the Earth gravity vector controls are observed in a number of critical pathways, including the immune pathway, the domeless pathway, calcium signaling, cuticle formation, iron metabolism, redox homeostasis and proteins related to electrolyte transport/ATPase, ribosome, odorant binding and ATP synthase (Figure 5A & 5B). Figure 2 represents the total number of hypergravity induced proteins with whole adult fly data represented in red, and isolated Drosophila head tissue in blue (Figure 2). This diagram



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contains proteins with at least one peptide observed to result in measureable reporter ion signal for three biological replicates, and removes those proteins associated with common exogenous contaminants. We observe 24% fewer expressed proteins in the head region (1480) compared to proteins from the whole Drosophila (1948). There is significant overlap for whole Drosophila exposed to hypergravity vs. the head alone (47% overlap). The observed protein differences in the whole fly largely correspond to locomotor proteins present in the whole drosophila and are not present in the head, as determined by gene ontology. This is consistent with the increased requirement for locomotor protein to aid in movement in this increased gravity environment (Figure 2). Controls at normal Earth gravity vector were also collected for both the whole Drosophila and the head only samples, and differences between the hypergravity and normal Earth gravity vector are discussed in detail below.

Overview of the impact of chronic hypergravity treatment on whole fly v/s head extracts

To better understand the broad proteomic changes observed following hypergravity exposure, isobarically tagged peptides were used to determine expression quantitatively. The volcano plot of these changes is based on the proteins containing at least 5 unique peptides in which all 6reporter ions can be measured (Figure 3). Since the whole body proteome did not yield many significant changes in protein levels, we studied the head-specific proteome (Figure 4) in order to understand the tissue-specific response to hypergravity. It is interesting to note that, among the 1948 proteins identified in the whole body, only two proteins Jon99 peptidase family (P-17205; Fold change 1.456; p-value 0,039), and Peroxisomal Multifunctional enzyme type 2 (Q9VXJ0; fold change 1.296; p-value 0.008) showed significant differences at p-value of 0.05 (Figure 4).


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However, head specific proteomics yielded many more proteins (104) that were significantly altered in response to chronic hypergravity treatment. When comparing the relatively homogeneous tissue-specific ‘head’ sample to the more disparate whole Drosophila lysate comprised of many tissue types, we are able to observe a greater apparent change for many proteins. This is largely explained by the vast number of proteins that are shared between different tissues, but differ dramatically in their relative expression on a tissue-dependent basis. Thus, depending on the particular expression changes within that tissue in response to stimulus (and the relative amount of that tissue within the drosophila as a whole), lysing the whole drosophila will result in a masking of many of the effects observable within a single tissue. We thus use the ‘head’ region as an example of the tissue-specific dynamic range improvements one might anticipate for other organs (brain, gut, testis, etc.) in the absence of confounding gene expression from other tissues representing the aggregate of all proteoforms amongst all tissue types. In addition, we specifically focused on the fly head for our subsequent proteomic analyses because our previous data indicated that the fly brain plays an important role in responding to the hypergravity environment. We have seen significant changes in oxidative stress in terms of ROS generation and antioxidant defense in the fly brain in response to hypergravity, and site-specific gene expression changes in the head that correlate with altered behavior in increased gravity environments (Bhattacharya, unpublished data). The head specific data was subjected to protein interactome analysis (Figure 5A). This will help us understand the intracellular interactions between the proteins and their functional links, which are not typically derived from the protein expression analysis. By studying the protein interactome, we can compile specific biological pathways, and determine the functional role of previously uncharacterized proteins and



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biological processes. The detailed discussion of the changes seen in expressed proteins after hypergravity treatment in this paper will therefore focus on the fly head proteomic data.

Chronic hypergravity alters host immune response

Drosomycin (1.8150 fold; p-value 0.001), Immune-induced molecule 10 (1.398 fold; p-value 0.0158), and CG13551 (1.4292 fold; p-value 0.0199) are the major immune-related proteins that are significantly upregulated following hypergravity exposure compared to the 1g control (Figure 5A, Table S-1). These proteins are involved in innate and humoral immune response, and are a part of the Toll-signaling pathway.13 Typically these proteins are known to be strongly upregulated in response to a microbial infection via a proteolytic cascade.6 However, the upregulation observed here in the hypergravity condition is in the absence of an induced microbial infection, suggesting that hypergravity could cause an increased host immune response in flies. Considering the hormetic effects of hypergravity exposure reported by [14]– this data is suggestive of a constitutive increase in the regulation of the Toll pathway. Studies carried out by the Le bourge lab suggest that fruit flies exposed to hypergravity (3g & 5g) conditions at a young age will increase the longevity of male flies, the resistance to heat stress in both sexes, and substantially delay behavioral aging.15 More recently, Taylor et al., provided evidence for the positive effects of hypergravity on immune function by demonstrating increased survival in hypergravity post-infection in wild type and immune mutants (Imd and Thor).6 In agreement with this, microarray data from our own previous spaceflight experiment (STS-121), which was carried out aboard the space shuttle Discovery, indicated that the Toll signaling pathway is negatively affected by microgravity as evidenced by a substantial decrease in mRNA levels of


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Drs and Mtk genes in space returned flies. 3 Given the fact that microgravity is associated with compromised immune function in both humans and in Drosophila.

3, 6, 16

it is plausible that the

proteomic impact of hypergravity might be the inverse of what is observed in microgravity, and as a result boosts certain aspects of host immune function. This contradictory effect of microgravity and hypergravity is demonstrated for the immune pathway in other organisms, for example on platelet function in mice.17 A study by Herranz et al. revealed a significant change in global transcriptomics in response to microgravity that included respiratory, developmental and stress related functions. 18 Under hypergravity condition, many of the same genes were affected but in some cases a subset of them were changed in the opposite direction compared to spaceflight.18 Alterations of genes/proteins of the innate immune pathway, that are linked to neurodegenerative conditions, have been shown previously.19 For instance recent study shown that dominant role of Toll and autophagy pathways in expanded RNA repeat pathogenesis and in Huntington’s disease in Drosophila.20 Hence expression of these proteins specifically in the head region may suggest a correlation between neuronal function and immune response under hypergravity treatment.

Chronic hypergravity induces humoral stress response and heat shock proteins

A significant upregulation of humoral stress factor proteins such as TotA (2.128 fold; p-value 0.001) and TotC (2.540 fold; p-value 0.021) were observed following chronic exposure to the hypergravity condition (Figure 5A, Table S-1). These proteins are known to respond to a wide spectrum of stressors including heat stress, mechanical stimulus, UV irradiation, oxidative agents, and bacterial challenge.21 These proteins have a distinct functional response to stress



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compared to heat shock proteins (HSPs). Heat shock proteins are effectively induced in response to high temperature, UV- irradiation, osmosis and heavy metal exposure, but are thought to be a transient and rapid response to such stressors. Heat shock proteins confer cellular protection by stabilizing, aggregating and degrading specific proteins at the site of cellular damage.22 In contrast, Turandot proteins are secreted into the hemolymph and act systemically and are induced with different kinetics than heat shock proteins. Turandot proteins are induced more slowly but more persistently than heat shock proteins and are often induced under more extreme environmental stress conditions than are heat shock proteins.21 Given that Turandot proteins are induced in response to non-transient stressors,22 chronic hypergravity is possibly exerting a relatively intense and persistent stress on the organism resulting in the induction of Turandot A and Turandot C expression. These proteins also play a significant role in immune response via the domeless JNK cascade; for instance, Turandot A is strongly induced in response to an immune challenge. Unlike Toll and Imd, only limited information is currently known about the domeless JNK pathway, but it is known to be critical for processes relating to phagocytosis. Interestingly, heat shock proteins such as Hsp60 (1.2412 fold; p-value 0.0434), and Hsp70 (1.3229 fold; p-value 0.0208) were also significantly upregulated in the head-specific proteomic data set (Figure 5A, Table S-1). Increased expression of these molecular chaperones is vital in protecting cells from heat and other environmental stressors. It is therefore not surprising that chronic hypergravity-induced stress can induce change in expression of heat shock proteins. This data complements studies in Xenopus laevis embryos, where embryos exposed to 3g showed a significant upregulation of Hsp60 and 70 proteins.23 Similarly, Minois et al., found that induction of Hsp70 may be responsible for the increased thermotolerance of hypergravity-treated flies.24


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These data strongly support that altered gravity conditions induce physiological and heat stress in biological systems.

Chronic hypergravity affects calcium-signaling pathway

Many calcium ion regulatory proteins were significantly down regulated upon hypergravity exposure (Figure 5A, Table S-2). These proteins, including endoplasmic reticulum-specific calcium-transporting ATPase (calcium ATPase at 60A) (0.6205 fold; p-value 0.0343), plasma membrane calcium ATPase, isoform K (0.6885 fold; p-value 0.0366), isoform 4 of calcium/calmodulin-dependent protein kinase type II alpha chain (0.6663 fold; p-value 0.0493) and Bruchpilot (0.8193 fold; p-value 0.0208) are all proteins that are integral to the calcium signaling pathway. Calcium has evolved as a major signaling molecule in eukaryotes, and plays a critical role in diverse cellular processes such as neurotransmission, neural plasticity, and muscle contraction.25 Under ideal conditions, calcium-dependent calmodulin facilitates both plasma membrane and ER specific Ca2+ATPase to eliminate excess Ca2+ ions from within the cell to the extracellular space and to the lumen of the ER respectively. These regulatory proteins work in concert to maintain optimal reserve pools of Ca2+ ions inside the cell for efficient neurotransmission, neural plasticity and muscle contraction.26 Given that the abundance of these proteins is significantly decreased in response to hypergravity in our study, it is possible that these proteins are unable to remove the excess Ca2+ ions efficiently from the cell, thus leading to potential Ca2+ build up inside the cell. From the literature we know that accumulated Ca2+ ions can trigger apoptotic pathways and ultimately lead to cell death.27 Changes in calcium metabolism and bone health under altered gravity are well established in mammalian models.27



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Space flight studies in mice suggest that microgravity-induced decrease in muscle fiber, its contractile property, and myosin heavy chain expression pattern are largely dependent on calcium homeostasis.28 In accordance, our proteomic data also shows significant changes in muscle related proteins such as Myosin regulatory light chain 2, Muscle-specific protein 20 and Myosin-2 essential light chain and will be discussed in a later section.

Effects of chronic hypergravity on specific transport proteins/ ATPase

We find 5 transport proteins and ATPases whose relative abundance is significantly decreased in response to hypergravity (Figure 5A, Table S-2). Although these proteins play a vital role in many physiological processes, they are mainly involved in the movement of ions and small molecules across cell membranes. These proteins are therefore critical to a number of signaling pathways, including cell volume regulation, and electrolyte transport as an energy source for different physiological functions.29 GO analysis indicates these proteins are ATP and metal ion binding in nature, and involved in various biological processes such as response to mechanical stimulus, locomotor behavior, neuromuscular processes, synaptic transmission, phosphate transport, dopamine transport, and circadian sleep wake cycles. Bent, isoform F, a protein involved in ATP binding, myosin light chain kinase activity, and protein serine threonine kinase activity was markedly decreased in its expression (0.6836 fold; p-value 0.0127) in response to hypergravity. This suggests that hypergravity could have specific effects on processes such as protein phosphorylation, and sarcomere organization. Similarly, another protein (CG9090) with a phosphate ion transmembrane transport activity was also significantly decreased (0.5789 fold; p-value 0.0055) in its expression in hypergravity condition, suggesting direct impact on the


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intercellular phosphate ion transport. Interestingly, this data correlates with previous findings in microbes that describe the regulatory role of phosphate ion in Salmonella virulence in spaceflight. 30 The kazachoc (Kcc, CG5594) isoform c protein is significantly down regulated (0.6494 fold; pvalue 0.0484) in response to hypergravity (Figure 5A, Table S-2). Kazachoc encodes for potassium chloride co-transporter, and GO analysis indicates kazachoc is involved in K+/Clsymporter activity and amino acid transporter activity. Studies suggest that Kcc is an essential gene, and loss-of-function leads to lethality in flies.31 Reduced Kcc level may reflect increased neurological excitability defects and greater vulnerability to epileptic-like seizures.31 Although we have not seen any epileptic-like seizures among flies exposed to chronic hypergravity, altered behavior in hypergravity that we have observed (data not shown) could be partially attributed to change in neuronal excitability. In parallel with this, Le Bourg’s group showed altered behavior upon hypergravity exposure in adult flies.8,11

Effects on muscle proteins

Studies in mammalian models have reported the ability of muscular and neuromuscular fibers to adapt differentially to environmental stimuli including changes in gravity.32 Space flight data from different organisms highlight how challenging it is to manage muscle density and prevent muscle loss in space. In the present study, we identified flight muscle, contractile muscle and non-muscle proteins such as Mlc2 (1.3633 fold; p-value 0.0272), Mhc (1.1757 fold; p-value 0.0442), Mf (1.3602 fold; p-value 0.0222), Upheld (1.4497 fold; p-value 0.0123) and Tm1 (1.4077 fold; p-value 0.0134) to be significantly upregulated in response to chronic hypergravity



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exposure (Figure 5A, Table S-3). GO analysis revealed that Mlc2, Mhcc and Cam belong to the evolutionarily related troponin superfamily. These are protein/calcium ion binding proteins, and are involved in myofibril assembly, muscle system processes and muscle contraction. Actn (Alpha-actinin) (0.7243 fold; p-value 0.0291) and Shot (0.7709 fold; p-value 0.0181) proteins were also significantly down-regulated in response to hypergravity exposure. All of these muscle related proteins are directly or indirectly associated with the larger troponin and tropomyosin complexes and are known to be involved in muscle contraction through a series of calcium dependent changes. To further support this, in the present study calcium regulating proteins such as isoform A of calcium-transporting ATPase sarcoplasmic/endoplasmic reticulum (ER), plasma membrane calcium ATPase, isoform K, isoform 4 of calcium/calmodulin-dependent protein kinase type II alpha chain were significantly down regulated indicating close association between muscle contraction and calcium homeostasis in the muscle cell. In spaceflight or simulated microgravity conditions muscle tissue are known to atrophy differentially based on the function and muscle type.

32,33

Space flight conditions significantly reduced the muscle contractile force

by altering the myosin heavy chains and light chains in relation to the calcium changes.33 Rats exposed to 2g from conception, to birth, and reared continuously in hypergravity until the age of 100 days, showed a significant decrease of muscle mass in soleus and plantaris muscles specifically. There was also a decrease in the diameter of isolated skinned fibers from the soleus muscle from mice that were conceived, born and raised (CBR) in hypergravity and these fibers showed an increased affinity for Ca2+.

34

All these studies correlate well with our own findings

that muscle related proteins adapt and function differently under altered gravity conditions.


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Effects on cuticle development and chitin metabolism

In this study cuticular proteins are consistently upregulated, and Figure 5A, Table S-4 lists the 13 cuticular proteins that show a significant response to hypergravity GO analysis reveals cuticular proteins (Cpr’s) such as Cpr72Ec, Cpr49Ae, CG42367, Cpr65Au, Cpr62Bc, CG34461, CG8192, Cpr67B, Cpr47Ea, Cpr49Ab, Cpr64Ac, Ccp84Ag and crystallin are structural constituents of cuticle. However, among the l3 mentioned above, crystallin protein is additionally involved in compound eye corneal lens development and visual perception. These results corroborate the findings by Herranz R at al., (2013), where a number of genes that showed significant alterations in all three conditions tested (microgravity, hypergravity and simulated microgravity) was related to cuticular development. These proteins impact the downstream chitin-based cuticle development in hypergravity. Note that although the exoskeletal system of invertebrates and the skeletomuscular system of mammals evolved through different lineages, they perform similar structural functions. We know from the literature that mechanical loading or unloading has a profound impact on the skeletal-muscular system of mammalian models.

35

Several lines of

evidence support the fact that mechanical unloading of tissue leads to various degenerative conditions.35 For instance, exposure to microgravity causes significant bone and muscle loss in mammalian models.36,37 However, hypergravity-induced mechanical loading supports the tissue regenerative health in bone and muscle tissue.38, 39 In this context, it is interesting to note the similarities between the invertebrates’ cuticular system and the mammalian skeletal system in their response to gravity. The increased expression of cuticle and chitin metabolism protein could imply the thickening of exoskeleton in response to mechanical loading caused by chronic hypergravity. Developmental and histochemical studies should be conducted in the future to



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check whether the cuticle thickness is differentially impacted in flies that are subjected to different gravity loads.

Effects on proteolytic and ribosomal proteins

Proteasomal proteins play a critical role in the catabolic pathway to provide demand driven amino acids for protein synthesis. In this context, we found that a set of proteins with cysteine type-endopeptidase activity, and corresponding inhibitory proteins, were affected by hypergravity. CG4847 (1.1890 fold; p-value 0.0447) and Cp1 (1.2293 fold; p-value 0.0482) were significantly upregulated (Figure 5A, Table S-5). These proteins have cysteine-type endopeptidase activity, and are involved in proteolysis. Essentially, these proteins participate in hydrolysis of internal, alpha-peptide bonds in the polypeptide chain by a catalytic reaction, which consist of a cysteine at the active center acting as a nucleophile.40 Typically proteolytic processing of polypeptide chain happens in the organism by the digestive enzymes to provide the required amino acids to organism in case of starvation/stress. However, these proteins are not only located in digestive tract but also have a role in stress response and protein degradation pathway specific to the head region.

Hence, possibly these proteins were upregulated to

compensate for the demand driven synthesis of active proteins in the head. Interestingly, expression of corresponding inhibitory proteins such as Cys (1.3193 fold; p-value 0.0195) and Tep4 (1.2026 fold; p-value 0.0294) were also significantly increased in response to hypergravity exposure suggesting the role of feedback mechanism in regulating the proteolytic pathways. Figure 5A, Table S-6 indicates significant upregulation of ribosomal proteins (RPs) in response to chronic hypergravity. Among the 79 cytoplasmic RPs in the present study, 7 RPs


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showed significant upregulation in the head. GO categorization suggests that these ribosomal proteins are cytoplasm specific, and are an integral part of the ribosomal structure. These proteins are interesting for several reasons. Firstly, as mentioned earlier, they play a critical role in translation. Secondly, apart from RPs being involved in translation, some RPs perform other physiological and developmental functions, such as RpS8’s involvement in neurogenesis; RpS30 and RpL31 are involved in mitotic spindle elongation and organization; and RpS21 has a role in cell proliferation and lymph gland development. These specific proteins, RpS8, RpS21, RpS30, RpL31 are significantly upregulated in the present study. Thirdly, there is a very high degree of similarity (69%) between human and drosophila RPs in terms of amino acids identity.41 The altered expression of some of the human cytoplasmic RPs are implicated in human diseases including Diamond-Blackfan anemia, Turner syndrome, hearing loss and cancer.42 More recently, Drosophila RPs have been exploited as a tool to understand the growth and developmental aspects of organisms.43 In this study, the upregulation of RPs in response to hypergravity could be explained by the possible increase in demand for protein synthesis and could also be implicated in extra-ribosomal functions.

Effects on ATP synthase and iron metabolism

Mitochondrial ATPsynβ (1.355 fold; p-value 0.0307), ATPsynδ (1.4433 fold; p-value 0.0082), and ATPsynCF6 (1.4362 fold; p-value 0.0078) were significantly upregulated in hypergravity (Figure 5A, Table S-6). These proteins are an integral subunit of the F1 complex, and catalyze the synthesis of ATP in the inner mitochondrial membrane by effectively utilizing an electrochemical gradient generated during oxidative phosphorylation. Expression of these ATP



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synthases at the translation level are well justified in the present study given that the flies will be experiencing mechanical loading that demands more energy for normal physiological and metabolic processes. Our findings are consistent with a previous study showing hypergravity (2g and 3g) stimulates release of ATP in bovine endothelial cells.44 In Drosophila, Fer1HCH (CG2216) and Fer2LCH (CG1469) are two subunits of ferritin. Ferritin is present in the vacuolar system, and iron loaded ferritin molecules transfer through golgi complex and are secreted into the hemolymph.45 Maintaining tissue specific, systemic cellular iron homeostasis is critical for various physiological functions such as locomotor rhythm, hemolymph

coagulation, cell proliferation, postembryonic development and

detoxification of iron ion. In the present study, both Fer1HCH (1.2641 fold; p-value 0.0159) and Fer2LCH (1.2720 fold; p-value 0.0380) production were significantly increased in response to chronic hypergravity condition (Figure 5A, Table S-1). The increased expression of ferritin related proteins might be an effort to compensate for a change in iron metabolism induced by chronic hypergravity.

General odorant-binding, mRNA and protein binding proteins

Odorant binding proteins (OBP) are necessary to recognize chemical stimuli in insects. These proteins are generally expressed in olfactory sensilla including antenna, maxillary palp and proboscis. OBPs are thought to serve as passive odorant shuttles, however recently it has been demonstrated that OBPs can also play an active role in recognizing the odorant itself.46 In this context, upregulation of specific OBP’s such as Obp56d (1.3625 fold; p-value 0.0104), Obp19d (1.2242 fold; p-value 0.0500) in response to chronic hypergravity might suggest their role in


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gravity perception (Figure 5A, Table S-1). Previously, Johnston’s organ (JO) containing the primary sensory neurons in the second segment of the antenna has been shown to have a role in gravity sensing.47,

48, 49

The olfactory sensillum that hosts odorant-binding proteins is located

close to the distal antennal segment. In Drosophila, several members of the odorant binding protein family are known to express exclusively in the antenna.50 Hence, in the present study we hypothesize that based on the cellular localization of these proteins in the antenna, perhaps these proteins also play a direct or indirect role in perceiving altered gravity. A group of mRNA binding proteins, such as Vig, B52, Yps, Glo and RNA-binding protein 2 were significantly upregulated in response to hypergravity (Figure 5A, Table S-7). These proteins are generally cytoplasmic and nuclear bound proteins, and play a vital role in gene expression. They participate in splicing, localizing, and stabilizing mRNA and thereby play a critical role in post-transcriptional regulation of gene expression.51 Proteins such as Yps and Glo are reported to play an important role in developmental oogenesis. Interestingly spaceflight has been shown to affect oogenesis.52 Protein binding proteins such as CG8759, CG11267, dSmt3, Lam were also upregulated in response to hypergravity. These proteins play diverse roles from oogenesis to de novo protein folding, regulation of response to stimulus and sensory organ development and locomotor behavior.

Proteins related to glycolysis, electron transport chain and redox homeostasis

Energy production is a result of the closely choreographed processes of citric acid cycle (or the tricarboxylic acid, TCA, cycle) and electron transport chain (ETC) activity in the cell. Succinate and NADH generated by the TCA cycle go through the series of enzyme complexes in ETC to



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produce ATP. In this study, the down regulation of CG6439 (0.8248 fold; p-value 0.0286)) which converts isocitrate into alpha ketoglutarate releasing NADH, and upregulation of CG7430 (1.2931 fold; p-value 0.0271) which has dihydrolipoyl dehydrogenase activity suggests that hypergravity has effects on the TCA cycle. Similarly, protein expression of complex I, NADH dehydrogenase (ubiquinone) 51 kDa subunit) (0.7276 fold; p-value 0.0062) was significantly down regulated, whereas cytochrome c oxidase subunit 5A (complex III) (1.3127 fold; p-value 0.0289) was upregulated in response to hypergravity exposure. Not having optimal function of these two complex enzymes in ETC can potentially cause electron leakage, which in turn can react with oxygen molecules leading to oxidative stress. It is also possible that hypergravity has differential effects on each of the specific complex enzymes in the ETC cycle. In either case, being essential components of the mitochondrial respiratory complex, these proteins likely have direct impact on ATP synthesis. Maintaining optimum redox homeostasis is critical for a cell to protect against oxidative stress. In the present study, increased expression of redox proteins (Pdh, CG3902, ERp60, Pdi and CG12171) in response to chronic hypergravity indicates an imbalance in redox homeostasis (Figure 5A, Table S-8). Redox regulation becomes especially important when organism/cells are exposed to oxidative insults. Redox status and molecular chaperones are closely associated in order to combat oxidative stress. An abrupt imbalance in redox homeostasis leads to the induction of hsps/molecular chaperones. Interestingly in the present study Hsp60 and Hsp70 proteins were significantly upregulated. We know that, flies exposed to acute hypergravity show signs of oxidative stress as evidenced by increased ROS levels and altered oxidative stress and antioxidant defense gene expression in fly head extracts (Bhattacharya, unpublished data). Therefore the observed changes in protein expression of these redox and stress related genes are likely a reflection of the oxidative stress experienced by the fly in


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hypergravity. Similarly, expression of the phosphoglucose isomerase (0.8031 fold; p-value 0.0284) protein was downregulated in hypergravity condition (Figure 5A, Table S-8). This protein has dual function unlike many other proteins involved in glycolysis. In the cytoplasm, this protein is involved in gluconeogenesis, whereas outside the cell, it can work as a neurotrophic factor for sensory neurons.53 Given that we observed this change in the head specific proteome, it could indicate a fruit fly sensory neuronal response to gravitational change in addition to its role in carbohydrate metabolism. Hausmann et al. showed that plant cells exposed to parabolic flights showed an immediate increase in Ca+2 ion and H2O2 levels with the onset of microgravity. Gene expression analysis showed that several of the transcripts that were altered consisted of several Ca2+ and ROS-related gene products. Subsequent phosphopeptide analyses of the same plant cell extracts demonstrated that a large number of the proteins with altered phosphorylation states after microgravity or hypergravity exposure consisted of proteins involved in primary metabolism such as glycolysis, gluconeogenesis and the TCA cycle. The authors propose a mechanism whereby gravity is sensed by the plasma membrane and results in increased Ca+2 ion in the cell. The Ca+2 is then thought to activate a Ca+2 –dependent protein kinase which phosphorylates NADPH oxidase and facilitates the synthesis of H2O2. On the one hand, this initiates subsequent gene expression changes to supply ROS detoxifying enzymes in the cell. In parallel the Ca+2 influx can also result in protein modulation via Ca+2 binding proteins and protein kinases and recruit enzymes involved in primary metabolism leading to immediate changes in processes such as those involved with carbohydrate metabolism within the cell.

54

These findings with Arabidopsis cells

are in keeping with our own results from fruit flies that after exposure to an altered gravity



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environment, a signaling cascade could be triggered by Ca+2, that is then followed by changes such as metabolic adaptation and the control of ROS within the organism. Among the proteins that were significantly affected by hypergravity, nearly 20% of them had either no clear molecular function by GO analysis or their involvement in a specific biological process are currently unknown (Figure 5A, Table S-9).

CONCLUSION

To summarize, this study provides the first comprehensive picture of the global proteomic changes induced by chronic hypergravity in Drosophila heads. The data confirms that chronic hypergravity does affect many different metabolic and physiological processes in Drosophila (Figure 5A & 5B). Since most earth-based life is well adapted to earth’s gravitational force, it is understandable that when an organism is exposed to a novel environment such as hypergravity, it responds by manipulating the levels of its expressed proteins to counteract the effects of the new condition. By studying the details of the molecular pathways that are changed to react to such environments, we can then start to understand the underlying biological effects of exposing an organism to altered gravity situations.


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ASSOCIATED CONTENT Supporting information SUPPORTING TABLES: Head specific (and whole body) proteomic changes in hypergravity Table S-1. Chronic hypergravity (3g) affects immune related proteins, humoral and heat stress proteins, iron metabolism proteins and odorant binding proteins in fly heads (p-value of ≤ 0.05). (Quantitative 3g:1g ratio for the whole fly extract were represented in parenthesis below the fly head data (p-value of ≥ 0.05). Table S-2. Chronic hypergravity affects calcium-signaling pathway and specific transport proteins/ ATPase in fly heads (p-value of ≤ 0.05). (Quantitative 3g:1g ratio for the whole fly extract were represented in parenthesis below the fly head data (p-value of ≥ 0.05). Table S-3. Muscle proteins are significantly altered in response to chronic hypergravity in fly heads (p-value of ≤ 0.05). (Quantitative 3g:1g ratio for the whole fly extract were represented in parenthesis below the fly head data (p-value of ≥ 0.05). Table S-4. Effects of hypergravity on cuticle development and chitin metabolism in fly heads (pvalue of ≤ 0.05). (Quantitative 3g:1g ratio for the whole fly extract were represented in parenthesis below the fly head data (p-value of ≥ 0.05). Table S-5. Chronic hypergravity affects proteolytic proteins, GTP and histone related proteins in fly heads (p-value of ≤ 0.05). (Quantitative 3g:1g ratio for the whole fly extract were represented in parenthesis below the fly head data (p-value of ≥ 0.05).



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Table S-6. Effects on ribosomal proteins and ATP synthase in fly heads (p-value of ≤ 0.05). (Quantitative 3g:1g ratio for the whole fly extract were represented in parenthesis below the fly head data (p-value of ≥ 0.05). Table S-7. Effect of hypergravity on mRNA and protein binding proteins in fly heads (p-value of ≤ 0.05). (Quantitative 3g:1g ratio for the whole fly extract were represented in parenthesis below the fly head data (p-value of ≥ 0.05). Table S-8. Effect of hypergravity on proteins related to glycolysis, electron transport chain and redox homeostasis in fly heads (p-value of ≤ 0.05). (Quantitative 3g:1g ratio for the whole fly extract were represented in parenthesis below the fly head data (p-value of ≥ 0.05). Table S-9. Unknown proteins affected by hypergravity in fly heads (p-value of ≤ 0.05). (Quantitative 3g:1g ratio for the whole fly extract were represented in parenthesis below the fly head data (p-value of ≥ 0.05).


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AUTHOR INFORMATION Corresponding author *(S.B) Email: [email protected]; Phone # +1 (650) 604-1531 Author’s contribution (R.H, S.R.B, S.B)-Contributed to experimental conception and design; (R.H, S.R.B) – Carried out the experiments; (R.H, R.L, C.A, S.B) - Contributed to data analysis, manuscript drafting and editing Notes The authors declare no competing financial interest



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ACKNOWLEDGMENTS We thank Allis Chien and Anna Okumu of Stanford University Mass Spectrometry (SUMS) for the facility and the Stanford Dean of Research for support of the Fusion Mass Spectrometer. FUNDING SUPPORT This work was funded by NASA grants to SB (NNX15AB42G and NNX13AN38G). RH was supported by a NASA Post-Doctoral Program (NPP) Fellowship. ABBREVIATIONS Cpr’s - Cuticular proteins; 3g-hypergravity; Hsp’s- Heat Shock Proteins; Mlc2-Myosin light chain; Mhc-Myosin heavy chain; Mf-Myofilin; Tm1-Tropomyosin; Pdh- Photoreceptor dehydrogenase


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(43) Lin, J.I,; Mitchell, N.C,; Kalcina, M,; Tchoubrieva, E,; Stewart, M.J,; Marygold, S.J,; et al. Drosophila Ribosomal Protein Mutants Control Tissue Growth Non-Autonomously via Effects on the Prothoracic Gland and Ecdysone. PLoS Genet. 2011, 7(12), e1002408. Doi: 10.1371/journal.pgen.1002408 (44) Koyama, T.; Kimura, C.; Hayashi, M.; Watanabe, M.; Karashima, Y.; et al., (2009) Hypergravity induces ATP release and actin reorganization via tyrosine phosphorylation and RhoA activation in bovine endothelial cells. Eur. J. Physiol. 2009, 457, 711-719. (45) Anuja, M.; Abhyuday, D.; Fanis, M. Genetic screening for novel Drosophila mutants with discrepancies in iron metabolism. Biochem. Soc. Transac. 2008, 36, 1313–1316. (46) Swarup, S.; Williams, T. I.; Anholt, R. R. Functional dissection of odorant binding protein genes in Drosophila melanogaster. Gen. Brain Behav. 2011, 10, 648-657. (47) Beckingham, K.; M.; Texada, M. J.; Baker, D. A.; Munjaal, R.; Armstrong, J. D. Genetics of graviperception in animals. Adv. Genet. 2005, 55, 105–145. (48) Baker, D. A.; Beckingham, K. M.; Armstrong, J. D. Functional dissection of the neural substrates for gravitaxic maze behavior in Drosophila melanogaster. J. Comp. Neurol. 2007, 501, 756–764. (49) McKenna M. P.; Hekmat-Scafe D. S.; Gaines P.; Carlson J. R. Putative pheromone-binding proteins expressed in a subregion of the olfactory system. J. Biol. Chem. 1994, 269, 1–8. (50) Hekmat-Scafe D.; Steinbrecht R.; Carlson J. R. Coexpression of two odorant binding homologs in Drosophila: implications for olfactory coding. J. Neurosci. 1997, 17, 1616–1624. (51) Gamberi, C.; Johnstone, O.; Lasko, P. Drosophila RNA binding proteins. Int. Rev. Cytol. 2006, 248: 43-139.


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(52) Vernos, I.; Gonzalez-Jurado, J.; Calleja, M., Marco, R. Microgravity effects on the oogenesis and development of embryos of Drosophila melanogaster laid in the Space shuttle during the Biorack experiment (ESA). Int. J. Dev. Biol. 1989, 33(2), 213-26. (53) Volkenhoff A, Weiler A, Letzel M, Stehling M, Klambt C, Schirmeier S. Glial glycolysis is essential for neuronal survival in Drosophila. Cell Metabol. 2015, 22(3), 437-47. (54) Hausmann, N.; Fengler, S.; Hennig, A.; Franz-Wachtel, M.; Hampp, R.; Neef, M. Cytosolic calcium, hydrogen peroxide and related gene expression and protein modulation in Arabidopsis thaliana cell cultures respond immediately to altered gravitation: parabolic flight data. Plant Biol 2014, Suppl 1:120-8. doi: 10.1111/plb.12051.



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FIGURE LEGENDS

Figure1. Experimental design

Adult Drosophila exposed to either hypergravity (blue) or normal gravity (red) were pooled and lysed to obtain sufficient protein quantities for this study. These pooled samples were then digested, isobarically tagged, and pooled again into a single sample for LC-MS/MS analysis. Two independent sets of proteomics studies were carried out using this methodology one with the whole fly (4 flies includes body +head) and the other one with only fly heads isolated from 20 adults. Four adult flies and 20 heads were used in each replicate, and three such independent biological replicates were used for statistical analysis.

Figure 2. Relative changes in protein profile in response to hypergravity

Describes the number of proteins that were observed to have changed in response to chronic hypergravity for whole Drosophila (red), and isolated heads (blue).

Figure 3. Tissue specificity of protein expression

The log2 fold change of proteins observed in both the whole fly (x-axis) and head-specific (yaxis) samples, shows that greater tissue specificity corresponds with greater observed changes between gravity conditions for the same set of proteins, consistent with the fact that localized tissue effects on the proteome can be masked by a global ‘whole fly’ analysis.


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Figure 4. Volcano plot of log2 fold change in protein expression

Volcano plots of the –log2 probability as a function of the log2 fold change for A) the whole fly and B) the isolated head region. These values are derived using the bootstrapping method (see text). Proteins that are up-regulated will appear on the right, with down-regulated proteins on the left of the central axis. A dashed line is provided in both graphs corresponding the p-value of 0.05 to give an indication of significance.

Figure 5A. Interactome of functional proteins from the fly head, in response to chronic hypergravity exposure.

Each functional group of proteins is classified with the same color of arrows and bubbles. Up and down arrows indicate upregulation and down-regulation of protein expression respectively, in comparison with the 1g controls.

Figure 5B. Overview of the diverse functional protein groups in the fly head affected by hypergravity

Different functional groups of proteins are altered in Drosophila heads due to chronic hypergravity exposure. The chart indicates the number of proteins significantly altered in each category of a physiological function that is affected by chronic hypergravity.



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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5A


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Figure 5B



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For TOC only


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