Experiment

A: It is thought that, like human beings, cells detect the stiffness of an object (such as surrounding cells and substrates) by pulling it.

B: Ca²⁺ response is low on a soft substrate (above) and high on a rigid substrate (below).

Figure 1: Active-touch sensing and Ca²⁺ response at a focal adhesion

Figure 2: Molecular model of gravity sensing. A stress fiber in a cell generates tensile force depending on substrate rigidity. We hypothesize that additional tension caused by mitochondria, which has a high specific gravity, is involved in gravity sensing. Variation in the tension can cause variation in membrane tension, which may lead to the regulation of the activities of Ca²⁺-permeable mechanosensitive channels and mechanosensitive membrane enzymes, and resultant muscle hypertrophy or muscle atrophy.

In the space experiment to be performed, cellular reactions to changes in substrate rigidity will be investigated. If differences in cellular reactions to the same scaffold are observed between the ground experiment and the space experiment, it will indicate that the traction force generated by the cell pulling on the scaffold changes, and that cells sense gravity via fluctuations in cell membrane tension. We will investigate the mechanism by detailed observations of sites where the cytoskeleton adheres to the substrate and mitochondrial behavior (Figure 3).

Muscles are known to atrophy in microgravity conditions because of lack of exercise, and it has been suggested that special enzymes regulate this muscle atrophy. The Cell Mechanosensing experiment is being carried out with Professor Takeshi Nikawa of the University of Tokushima, who has studied the mechanism of muscle atrophy in rat cells in a space shuttle experiment, and in the Myo Lab experiment performed in the International Space Station (ISS) in 2010. Medicines selected on the basis of Professor Nikawa’s results from these past experiments will be used in the Cell Mechanosensing experiment to verify their effect on muscle cells in space.

The problem of muscle atrophy in space may be resolved by elucidating the mechanisms by which cells sense the presence or absence of gravity, by identifying which enzymes act in cells in response, and by developing medicines that can successfully control the mechanisms. This research may also contribute to treatment of muscle atrophy on Earth.

Figure 3: We will investigate the mechanism by detailed observation of sites where the cytoskeleton adheres to the substrate and mitochondrial behavior. We hypothesize that mitochondria, which sink in the presence of gravity, will float upwards and fragment in the absence of gravity, producing an adverse effect on cells.

Outline of the Experiment

First experiment (Gene expression experiment)

From February 2014 onward, equipment and materials for this experiment will be delivered to and collected from the ISS by the Commercial Resupply Services-3 (CRS-3) mission of the Dragon cargo spacecraft, developed by the Space Exploration Technologies Corporation (SpaceX).

Rat L6 myotube cells (L6 cells) will be cultured in the Cell Biology Experiment Facility (CBEF) onboard Kibo Agents that inhibit a substance leading to muscle atrophy will be added to the culture medium, and the L6 cells will continue to be cultured to confirm the effect of the agents. Length, thickness, and changes in gene expression of muscle fibers will be investigated in detail.

The culture surfaces of cell culture vessels will be coated with silicone-based agents of varying rigidity, and mouse mesenchymal stem cells (MSCs) will be cultured in the vessels. Based on our hypothesis described on the Background and Objectives page, we will investigate the mechanism of gravity sensing by examining the reactions of cells on the culture surfaces with different rigidities.

The cultured cells will be treated with RNAlater, a tissue storage reagent that can preserve genes and be frozen for return.

After returning to Earth, the effect of the inhibition agents will be assessed by gene expression profiling.

Second and Third experiment (Fluorescent microscopic observation experiment)

From April 2014 onward, equipment and materials for the second experiment will be delivered to and collected from the ISS by the SpaceX CRS-4 mission. From June 2015 onward, the third experiment will be delivered by the SpaceX CRS-7 mission. In the second experiment, MSCs and A6 cells derived from the kidneys of Xenopus laevis will be used. In the third experiment, L6 cells and MSCs will be used.

Using cells into which fluorescent proteins have been incorporated, focal adhesions and mitochondria will be observed with a microscope onboard Kibo (Figure 4). We expect that under microgravity conditions, the tension of stress fibers and cell membranes will change, and as a result, mitochondria will move away from the fibers, will detect that the situation is abnormal, and will fragment into small pieces. We are looking forward to observing what will actually occur.

In addition, cells will be cultured in the culture vessels coated with silicone-based agents of varying rigidity, and cell migration will be observed. Although cell migration has not been observed in space, cellular behavior is strongly influenced by behaviors of focal adhesions and stress fibers. Therefore, we expect that the behavior of the cells will change in space. Observing and analyzing the behavior of the cells is very important to understanding the influence of microgravity on human body functions because the behavior of the cells is directly linked to tissue metabolism and wound repair. By observing the behavior of the cells on scaffolds of differing rigidity, the relationship between the behavior of the cells and their gravity-sensing mechanism, as studied in the first experiment, will be also examined.

The cultured cells will be chemically fixed with paraformaldehyde and returned to Earth under refrigeration. On the basis of the images obtained in orbit and from the chemically fixed cells on returning to Earth, the influence of gravity on the adherence, organelles, and motility of the cells will be assessed.

This is the Point!

How cells sense mechanical stress, including gravity, and connect the perception with their growth (including differentiation and proliferation) is unknown. Our primary objective is to reveal the gravity-sensing mechanism of cells by culturing muscle cells and mesenchymal stem cells in microgravity conditions. At the same time, we aim to elucidate the signaling mechanisms leading to muscle atrophy, changes in cell motility, and mitochondrial behavior under microgravity conditions. Analysis of multiple responses from cells of different species will allow us to assess gravity- or mechanical stress-sensing mechanisms that not only a certain cell of a certain species may have but also those that various cells will universally have.

Development of measures against the muscle atrophy caused by microgravity is indispensable for human beings to make long-term visits to space. Because this research also involves experiments on the expression mechanisms of genes related to muscle atrophy and inhibition of muscle atrophy by medicines under microgravity conditions, the results will also be helpful in developing a treatment for muscle atrophy.

The average age of Japanese citizens continues to increase, and the Cabinet Office estimates that one in every four people will be over the age of 75 in 2055. As a result, the bedridden population is also progressively increasing, and these factors have become a large social problem. A preventive medicine for muscle atrophy, which may be developed based on this research, is highly anticipated for the clinical setting. It is expected to be helpful for the prevention of conditions leading to bedridden patients, as well as the treatment of bedridden people.

Detailed explanation for those who want to learn more

The human body consists of about 60 trillion cells, and in fact, each cell is affected by gravity and can sense its presence or absence. However, the mechanism by which cells sense gravity remains unclear.

Until recently, it was thought that membrane proteins in cells simply float in the cell membranes, which act as a barrier. However, a group led by Professor Sokabe discovered in 2012 that membrane lipids and proteins are very strongly linked through some of their amino acid residues near the surface of the cell membrane (Sawada Y, Murase M, *Sokabe M (2012) Channels, 6(4): 317-331). This discovery has suggested that fluctuations of the cell membrane exert a large influence on the three-dimensional structure and activity of membrane proteins via the linkage, and inspired researchers to hypothesize that cells may sense mechanical stress via fluctuations in the cell membrane and the cytoskeleton, which is linked to the membranes.

Previous studies of mechanisms of sensing mechanical stress had focused on finding stress sensors. In addition, structural analysis of proteins in a high-pressure environment and other studies have revealed that microgravity does not have a direct influence on protein structure. On the other hand, when cells are cultured on expandable silicone sheets and put under tensile stress, calcium (Ca²⁺) channels and proteases in the cell membranes are activated. Furthermore, it has been reported that membrane lipids of cells cultured under microgravity conditions for a long period of time show changes in lipid components. Professor Sokabe has also proposed a hypothesis based on results (unpublished) obtained from a ground experiment under simulated microgravity conditions (using a clinostat) that fluctuations of the cell membrane and actin cytoskeleton plays an important role in how cells sense microgravity stress. This experiment aimed to reveal, for the first time, that sensing of microgravity stress is achieved by mechanosensor molecules that are connected to cell membranes and actin cytoskeletons, and can sense the changes in tension fluctuations in the membrane/actin cytoskeleton caused by microgravity. Microgravity stress, as described here, means stress changes in cells caused by loss of gravity.

Identifying mechanosensitive channels and mechanosensitive membrane proteases in skeletal muscle cells－the activity of which varies with microgravity-inducing changes in the tension of the cell membrane and cytoskeleton－and downstream signaling molecules will lead to the elucidation of microgravity sensors in muscle cells and their signaling systems. Furthermore, the use of inhibitors and activators of the molecular components identified will allow us to explore potential drugs for treating muscle atrophy.

What do you do when you don’t know how rigid an object is? You will probably check by pushing and pulling it with your fingers. When you push a rigid object, the object will push back and won’t budge an inch; when you pull it, it will pull back and won’t stretch. On the other hand, when you push or pull a soft object, the object will change its shape, and you will know that it is soft. It is thought that cells also sense the rigidity of substrates by doing likewise. Cells adhere to substrates by using foot-like “focal adhesions.” The focal adhesions are part of their cell membranes, and actomyosin fibers called cytoskeletons (stress fibers) extend from the focal adhesions in the cells. The actonyosin fibers in cytoskeletons can produce force and can contract like muscles, and exert force on the substrate through the focal adhesions and connecting fibers. The tension in the actin cytoskeletons and the connected cell membrane changes in accordance with the rigidity of the substrate. It appears that, like human beings, cells test the rigidity of substrates by pulling. If the substrate reacts strongly when pulled, the cell will “know” that the substrate is rigid.

Our cells contain organelles, such as mitochondria and nuclei, which are heavy and have a high specific gravity. These organelles are connected by the actin cytoskeleton previously described. Imagine that a weight, such as a nucleus or mitochondrion, is attached to the cytoskeleton. Also imagine that the actin cytoskeleton is pulling on its substrates with tension generated by contractile force. When cells are on Earth, the tension increases compared to the tension generated by the cytoskeleton only because additional force is applied to the cytoskeleton due to the gravitational force applied to weights such as nuclei and mitochondria, which have a high specific gravity and are connected to the cytoskeleton. When cells are under microgravity conditions, the additional force generated by these weights disappears. The cells may feel light or may feel changes in the force applied to the cytoskeleton when the weights disappear. When the influence of gravity on the cells is changed by turning the cells upside down, the cells reconstruct their cytoskeletons, and the structure of the mitochondria also changes. This may be because the cells sense changes in force applied to the skeletons. When the cells pull the substrate with their actin cytoskeleton in space, the cells may sense that the substrate is soft because the tension on the cytoskeleton is less than it is on Earth, and may thus sense the lack of gravity.

If cells use similar systems to sense the rigidity of substrates and to sense microgravity conditions, how cells sense rigidity may change in space. Every cell has its own preferred level of rigidity. Cells preferring harder substrates move to harder ones. Surprisingly, it was recently discovered that differentiation of multipotent mesenchymal stem cells is regulated by substrate rigidity. Multipotent mesenchymal stem cells differentiate into nerve cells on soft substrates, into cells regulating bone formation on very rigid substrates, and into muscle cells on medium-rigid substrates. Accordingly, the differentiation of stem cells may change in space. This possibility will also be examined in this space experiment.

In the human body, breakdown and repair of tissues is in continuous progress in various organs, including the skin. This repair and regeneration function is indispensable for living things and is extremely complicated. For example, in the case of repair of the epidermis, healthy cells remaining in the damaged area transform (dedifferentiate), migrate, proliferate, and finally redifferentiate and stop proliferating. For the cell migration step, a mechanism for sensing substrate rigidity is important. In addition, once cells fully cover the damaged area and become crowded, the cells sense the mechanical stress generated by the crowded situation and stop proliferating. Because cancer cells have a defect in this function, the cells continue to proliferate and form a tumor. Therefore, studies of how cells sense mechanical stress reveal not only fundamental information, including the mechanism of sensing gravity, but connect directly with the solution of essential medical challenges, including muscle atrophy, osteoporosis, cancer, and regenerative medicine.