The FLUMIAS (Fluorescence-Microscopic Analyses System for Life-Cell-Imaging in Space) confocal laser spinning disk fluorescence microscope represents a new imaging capability for live cell imaging experiments on suborbital ballistic rocket missions. During the second pioneer mission of this microscope system on the TEXUS-54 suborbital rocket flight, we developed and performed a live imaging experiment with primary human macrophages. We simultaneously imaged four different cellular structures (nucleus, cytoplasm, lysosomes, actin cytoskeleton) by using four different live cell dyes (Nuclear Violet, Calcein, LysoBrite, SiR-actin) and laser wavelengths (405, 488, 561, and 642 nm), and investigated the cellular morphology in microgravity (10⁻⁴ to 10⁻⁵ g) over a period of about six minutes compared to 1 g controls. For live imaging of the cytoskeleton during spaceflight, we combined confocal laser microscopy with the SiR-actin probe, a fluorogenic silicon-rhodamine (SiR) conjugated jasplakinolide probe that binds to F-actin and displays minimal toxicity. We determined changes in 3D cell volume and surface, nuclear volume and in the actin cytoskeleton, which responded rapidly to the microgravity environment with a significant reduction of SiR-actin fluorescence after 4–19 s microgravity, and adapted subsequently until 126–151 s microgravity. We conclude that microgravity induces geometric cellular changes and rapid response and adaptation of the potential gravity-transducing cytoskeleton in primary human macrophages.

Microgravity is known to affect the organization of the cytoskeleton, cell and nuclear morphology and to elicit differential expression of genes associated with the cytoskeleton, focal adhesions and the extracellular matrix. Although the nucleus is mechanically connected to the cytoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, the role of this group of proteins in these responses to microgravity has yet to be defined. In our study, we used a simulated microgravity device, a 3-D clinostat (Gravite), to investigate whether the LINC complex mediates cellular responses to the simulated microgravity environment. We show that nuclear shape and differential gene expression are both responsive to simulated microgravity in a LINC-dependent manner and that this response changes with the duration of exposure to simulated microgravity. These LINC-dependent genes likely represent elements normally regulated by the mechanical forces imposed by gravity on Earth.

One of the major concerns for long-term exploration missions beyond the Earth’s magnetosphere is consequences from exposures to solar particle event (SPE) protons and galactic cosmic rays (GCR). For long-term crewed Lunar and Mars explorations, the production of fresh food in space will provide both nutritional supplements and psychological benefits to the astronauts. However, the effects of space radiation on plants and plant propagules have not been sufficiently investigated and characterized. In this study, we evaluated the effect of two different compositions of charged particles-simulated GCR, and simulated SPE protons on dry and hydrated seeds of the model plant Arabidopsis thaliana and the crop plant Mizuna mustard [Brassica rapa var. japonica]. Exposures to charged particles, simulated GCRs (up to 80 cGy) or SPEs (up to 200 cGy), were performed either acutely or at a low dose rate using the NASA Space Radiation Laboratory (NSRL) facility at Brookhaven National Lab (BNL). Control and irradiated seeds were planted in a solid phytogel and grown in a controlled environment. Five to seven days after planting, morphological parameters were measured to evaluate radiation-induced damage in the seedlings. After exposure to single types of charged particles, as well as to simulated GCR, the hydrated Arabidopsis seeds showed dose- and quality-dependent responses, with heavier ions causing more severe defects. Seeds exposed to simulated GCR (dry seeds) and SPE (hydrated seeds) had significant, although much less damage than seeds exposed to heavier and higher linear energy transfer (LET) particles. In general, the extent of damage depends on the seed type.

Since opportunities for spaceflight experiments are scarce, ground-based microgravity simulation devices (MSDs) offer accessible and economical alternatives for gravitational biology studies. Among the MSDs, the random positioning machine (RPM) provides simulated microgravity conditions on the ground by randomizing rotating biological samples in two axes to distribute the Earth’s gravity vector in all directions over time. Real-time microscopy and image acquisition during microgravity simulation are of particular interest to enable the study of how basic cell functions, such as division, migration, and proliferation, progress under altered gravity conditions. However, these capabilities have been difficult to implement due to the constantly moving frames of the RPM as well as mechanical noise. Therefore, we developed an image acquisition module that can be mounted on an RPM to capture live images over time while the specimen is in the simulated microgravity (SMG) environment. This module integrates a digital microscope with a magnification range of 20× to 700×, a high-speed data transmission adaptor for the wireless streaming of time-lapse images, and a backlight illuminator to view the sample under brightfield and darkfield modes. With this module, we successfully demonstrated the real-time imaging of human cells cultured on an RPM in brightfield, lasting up to 80 h, and also visualized them in green fluorescent channel. This module was successful in monitoring cell morphology and in quantifying the rate of cell division, cell migration, and wound healing in SMG. It can be easily modified to study the response of other biological specimens to SMG.

Plants have been recognized as key components of bioregenerative life support systems for space exploration, and many experiments have been carried out to evaluate their adaptability to spaceflight. Unfortunately, few of these experiments have involved monocot plants, which constitute most of the crops used on Earth as sources of food, feed, and fiber. To better understand the ability of monocot plants to adapt to spaceflight, we germinated and grew Brachypodium distachyon seedlings of the Bd21, Bd21-3, and Gaz8 accessions in a customized growth unit on the International Space Station, along with 1-g ground controls. At the end of a 4-day growth period, seedling organ’s growth and morphologies were quantified, and root and shoot transcriptomic profiles were investigated using RNA-seq. The roots of all three accessions grew more slowly and displayed longer root hairs under microgravity conditions relative to ground control. On the other hand, the shoots of Bd21-3 and Gaz-8 grew at similar rates between conditions, whereas those of Bd21 grew more slowly under microgravity. The three Brachypodium accessions displayed dramatically different transcriptomic responses to microgravity relative to ground controls, with the largest numbers of differentially expressed genes (DEGs) found in Gaz8 (4527), followed by Bd21 (1353) and Bd21-3 (570). Only 47 and six DEGs were shared between accessions for shoots and roots, respectively, including DEGs encoding wall-associated proteins and photosynthesis-related DEGs. Furthermore, DEGs associated with the “Oxidative Stress Response” GO group were up-regulated in the shoots and down-regulated in the roots of Bd21 and Gaz8, indicating that Brachypodium roots and shoots deploy distinct biological strategies to adapt to the microgravity environment. A comparative analysis of the Brachypodium oxidative-stress response DEGs with the Arabidopsis ROS wheel suggests a connection between retrograde signaling, light response, and decreased expression of photosynthesis-related genes in microgravity-exposed shoots. In Gaz8, DEGs were also found to preferentially associate with the “Plant Hormonal Signaling” and “MAP Kinase Signaling” KEGG pathways. Overall, these data indicate that Brachypodium distachyon seedlings exposed to the microgravity environment of ISS display accession- and organ-specific responses that involve oxidative stress response, wall remodeling, photosynthesis inhibition, expression regulation, ribosome biogenesis, and post-translational modifications. The general characteristics of these responses are similar to those displayed by microgravity-exposed Arabidopsis thaliana seedlings. However, organ- and accession-specific components of the response dramatically differ both within and between species. These results suggest a need to directly evaluate candidate-crop responses to microgravity to better understand their specific adaptability to this novel environment and develop cultivation strategies allowing them to strive during spaceflight.