Advanced Plant EXperiments-02-2 (APEX-02-2) - 08.05.14
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The Advanced Plant EXperiments-02-2 (APEX-02-2) utilizes the NanoRacks Plate Reader facility on the International Space Station. The investigation aims to learn how cells adapt to the unique aspects of the space environment, using the model eukaryotic organism, Saccharomyces cerevisiae (yeast). By identifying specific mechanisms which are regulated within the regions of genes that respond to growth in microgravity, the science team identifies factors associated with how genetic information transfers and the associated signaling pathways involved in microbial growth and physiological responses in the space environment.
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Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
Human Exploration and Operations Mission Directorate (HEOMD)
ISS Expedition Duration
March 2014 - September 2014
Previous ISS Missions
- Mechanical forces, and more specifically, changes in mechanical forces as seen in microgravity, are environmental stresses to all cells. A hallmark of environmental stresses is that they induce physiological responses, primarily in gene expression. Earth-based studies have mapped how cells respond to environmental stresses. Previous experiments have discovered that microgravity activates a unique set of genes whose roles are to coordinate and regulate both rapid and long-term cell adaptations to new environments. The specific molecular mechanisms by which microgravity modulates gene expression remains incompletely defined.
- The goal of the APEX-02-2 research is to identify specific functions responsible for regulating the regions of genes that respond to growth in microgravity.
- The hope is that knowledge gained from this investigation will build upon previous data to better understand how cells adapt to new environments. The expectation is that what is observed in yeast is likely to have a comparable effect in the more complex mammalian cells, leading to vast opportunities for new discoveries.
The APEX-02-2 research investigates microbial growth and physiological responses to the multiple stimuli encountered in space flight environments, using Saccharomyces cerevisiae as a model system. Yeast are an ideal model because their complete gene sequence is known; there are extensive and sophisticated clone libraries available for study; and they can easily be flown in space, held at +4°C in a quiescent state, and activated by simple warming. Regulatory mechanisms in yeast are highly similar to mammalian cells. The science team has shown that microgravity activates a unique set of genes in yeast, but the specific molecular mechanisms remain incompletely defined.
The hypothesis being tested is that, by identifying specific regulatory motifs within the promoter regions of microgravity-responsive genes, it will be possible to determine transcription factors that bind the motifs, and thus the signaling pathways involved in microbial growth and physiological responses.
The APEX-02-2 experiments are timely because (a) recent improvements in promoter analysis have vastly increased the accuracy of promoter elements predictions; (b) novel yeast clones allow direct measurement of gene expression by fluorescence using the Nanorack’s Plate Reader that is already in place on the International Space Station (ISS); (c) yeast on agar plates can be assayed repetitively, thereby eliminating sample to sample variability to increasing the statistical power of the data; (d) new reagents allow design of experiments with minimal up mass; and (e) all data can be returned by telemetry, eliminating the need for down mass.
To test the APEX-02-2 hypothesis, the science team will:
1) Identify promoter motifs associated with genes responding to Rotating Wall Vessel (RWV)-emulated microgravity compared to static cultures. The RWV will be used to emulate microgravity as the science team has already demonstrated that it induces changes in yeast gene expression that are similar to those of yeast flown in space. The Yeast Green Fluorescent Protein clone collection will permit identification, by changes in fluorescence, of genes whose expression changes in response to real and simulated microgravity. After extensive normalization and analytic procedures, microgravity-responsive genes will be grouped into clusters on the basis of similar kinetics, under the assumption that these are likely to be co-regulated. We will then search the upstream sequence of co-expressed genes and identify candidate binding motifs for transcription factors.
2) Confirm the role of regulatory sequences in directly activating transcription of a set of genes changed by RWV-simulated microgravity and to identify transcription factors that interact with these regulatory motifs. This specific aim makes novel use of the yeast deletion collection, a systematic gene-by-gene set of complete gene deletions, the only such resource that exists for any group of organisms. Transcription factors will be identified by robotically introducing a reporter plasmid that codes for 1) a suicide-inducing enzyme which is activated in all clones except the one(s) with a deletion in the gene for the transcription activating factor that bind to the promoter motif or 2) an endogenously activated essential gene to identify repressors.
3) Confirm and extend the results obtained in the science team’s ground-based studies on the selective growth pressure in the true microgravity environment. Utilizing the genome wide yeast GFP clone collections spotted on agar plates, a parallel flight study is proposed. The science team’s questions can ultimately only be definitively answered in the true microgravity of space, because on the ground gravity must always be onset with an equal and opposite force.
APEX-02-2 measures a specific set of genes that have been tagged with a fluorescent marker, monitoring how their expression changes in response to microgravity. Researchers use state-of-the-art technology to analyze genetic expression, or cells’ use of genetic information, in space. The investigation also examines the role of gravity in development of yeast cells’ internal and external structures. This will provide insight for genetic engineering efforts aimed at designing plants that can grow better in microgravity. This could enable large plant habitats on the International Space Station and for future space missions.
Environmental stresses, such as changing gravitational conditions, cause physical changes to cells. These especially include differences in how genes are expressed. Yeast is a model organism, meaning studies of yeast can be used to interpret the molecular responses of more complicated plant and animal cells. Understanding how environmental changes affect genetic expression could benefit human health in a variety of ways.
- Time between launch and installation into Plate Reader: up to 6 weeks if kept at +4°C
- Experiment run duration: 11 days
- Each plate is read automatically in the plate reader every 10 minutes for 18 hours. Plate change out is scheduled according to astronaut scheduling convenience. Downlink of Excel data file required
The first petri plate will be removed from Cold Stowage and transferred to the Nanorack’s Plate Reader. Activation of the yeast requires a temperature of +30°C for yeast incubation. GFP and Cherry red fluorescent protein light images (108 fluorescent reads/18 hours) will be recorded and downlinked. According to astronaut scheduling convenience after more than 18 hours,, the plate will be removed from the GFP Imager, place back into cold stowage or stowed at ambient and a new plate will be installed. This operation will be repeated nine times, for a total of ten petri plates. The APEX-02-2 relies on telescience data (Excel data files) downlinked to the ground and does not require any harvests or other crew manipulations. The petri plates will return to the ground at ambient and +4 C.