Space experiments identify pathways that transmit external information into cells – Overview of gravity-mediated mitochondrial translation regulation (generated by Issei Takahashi)
[RIKEN] An international collaborative research group consisting of Shintaro Iwasaki, Principal Researcher, Taisei Wakikawa, Special Researcher, Yusuke Kimura, Graduate Student Research Associate (at the time of the research), Mari Mito, Technical Staff I, Hiroki Saito, Graduate Student Research Associate (at the time of the research), and Yuichi Nanano, Senior Researcher (at the time of the research, now Visiting Researcher, Professor at the University of Tsukuba’s Faculty of Medicine and Health Sciences), at the Iwasaki RNA Systems Biochemistry Laboratory, RIKEN (Riken Institute of Physical and Chemical Research ), has discovered through experiments in outer space the mechanism by which cells sense gravity and activate mitochondrial translation [1], which synthesizes proteins in mitochondria.
This research is expected to contribute to the development of drugs and methods to suppress not only aging phenomena during long-term space travel, but also general aging.
In this study, an international collaborative research group cultured cells under microgravity conditions in the Japanese Experiment Module “Kibo” of the International Space Station (ISS) and conducted a comprehensive analysis of ” translation [2] ,” the process by which amino acids are combined within cells to synthesize proteins. They discovered that mitochondrial translation was drastically reduced. This led to the identification of a signaling pathway that transmits external information, such as gravity, into the cell. Furthermore, they revealed that this pathway physiologically controls mitochondrial translation in a manner dependent on mechanical stress, such as exercise.
This research was published online in the scientific journal * Nature Communications * (June 30th: June 30th Japan time).
Schematic diagram of gravity-mediated mitochondrial translation regulation
Overview of gravity-mediated mitochondrial translation regulation (generated by Issei Takahashi)
background
Throughout life’s adaptation under Earth’s gravity, cells have always been exposed to gravity and evolved based on this premise. However, how gravity affects fundamental cellular functions, particularly “translation,” the process of protein synthesis, has not been well understood. Now that humanity is attempting to expand its living space beyond Earth, understanding cellular responses in the near-absent environment of space is crucial for both basic science and space medicine.
Therefore, with the cooperation of the Japan Aerospace Exploration Agency (JAXA), the international joint research group used the Kibo Experiment Module of the International Space Station (ISS) to culture cells under microgravity conditions and attempted a comprehensive translation analysis ( Note) . The cell culture experiment on the ISS was conducted by astronaut Soichi Noguchi.
Note 1)JAXA Human Space Technology Directorate website, “Comprehensive Analysis of Translation Control under Microgravity Opens in a new tab.“
Research methods and results
For a comprehensive analysis of the translation, we used ribosome profiling [3] . This method generates and collects short RNA fragments called ribosome footprints, and by sequencing them with a next-generation sequencer, we can obtain comprehensive information about “where” and “how much” ribosomes [5] are located on messenger RNA (mRNA) [4] .
Human cultured cells were cultured in a microgravity environment using cell culture facilities within the ISS “Kibo” module. For comparison, cells were also cultured in an environment with a gravitational acceleration of 1 g (Figure 1A). After sampling under each condition, the cells were returned to Earth, and ribosome profiling experiments were performed (Figure 1B). Ribosome profiling is a technique that can capture changes in translation comprehensively and quantitatively without bias. As a result of the analysis, it was found that translation of some cytoplasmic mRNAs decreased in the microgravity environment, and furthermore, mitochondrial translation decreased significantly (Figure 1C).
Mitochondria are the primary source of adenosine triphosphate (ATP), which mediates energy exchange in intracellular metabolism; they are essentially the cell’s energy factories. Evolutionarily, mitochondria originate from bacteria and therefore possess their own unique genomic DNA (mitochondrial DNA), which contains the sequence information for 13 proteins necessary for ATP production. Consequently, translation within these energy factories is a crucial reaction essential for energy production, and abnormalities in this process can lead to diseases such as mitochondrial diseases. Mitochondrial translation occurs when mRNA transcribed from mitochondrial DNA is read by specialized ribosomes (mitochondrial ribosomes). Therefore, this translation mechanism is completely different from cytoplasmic translation. Ribosome profiling allows for the simultaneous analysis of both cytoplasmic and mitochondrial translation (Figure 1B).
[English translation via Grok]
Figure 1. Analysis of the effects of microgravity on translation using ribosome profiling.
A. Cells were cultured and samples were prepared on the International Space Station (ISS) under conditions of microgravity and a gravitational acceleration of 1 × g⁻¹ created by a centrifuge.
B. Ribosome profiling allows for the simultaneous analysis of cytoplasmic and mitochondrial translation.
C. Comprehensive quantitative analysis of the effects of microgravity on translation on the ISS. The horizontal axis represents the average and standardized number of reads obtained from all sample data used in the analysis. The vertical axis represents the logarithmic ratio of the amount of translation change that occurred in the microgravity environment compared to the 1× g environment.
To understand whether these results are reproducible in individuals and whether this phenomenon occurs across species, we performed ribosome profiling again using Caenorhabditis elegans nematodes cultured under microgravity conditions on the ISS. Again, a decrease in mitochondrial translation was observed under these conditions.
To gain a more detailed understanding of the mechanism of mitochondrial translation abnormalities caused by microgravity, we conducted replication experiments on Earth. The international collaborative research group used a 3D clinostat [6] . This device rotates the culture vessel in three dimensions, allowing the average acceleration acting on the sample to approach zero (Figure 2A). Ribosome profiling performed under these simulated microgravity conditions revealed a decrease in mitochondrial translation (Figure 2B).
To understand the molecular mechanism of the decrease in mitochondrial translation due to microgravity, an international collaborative research group focused on cell adhesion [7] . Based on the finding from past space experiments that the strength of cell adhesion weakens in cells placed in microgravity, they hypothesized that this leads to a decrease in mitochondrial translation for some reason. First, they used the mito-FUNCAT method [8] to verify whether mitochondrial translation is dependent on cell adhesion, and it became clear that mitochondrial translation increases with an increase in laminin, a basement membrane molecule of the extracellular matrix (Figure 2C). When they actually analyzed translation in pseudo-microgravity under conditions of increased laminin using ribosome profiling, they found that it counteracted the effects of pseudo-microgravity and mitigated the decrease in mitochondrial translation (Figure 2D). In other words, it became clear that the decrease in mitochondrial translation due to microgravity is due to weakened cell adhesion.
[English translation via Grok]
Figure 2. Analysis of the effect of pseudo-microgravity on translation using ribosome profiling.
A. Cell culture and sample preparation in a simulated microgravity environment using a 3D clinostat in a ground-based laboratory.
B. Comprehensive quantitative analysis of the effects of simulated microgravity on translation in ground-based laboratories. The horizontal axis represents the average and standardized number of reads obtained from all sample data used in the analysis. The vertical axis represents the logarithmic ratio of the translation change that occurred in the simulated microgravity environment compared to the 1× g environment.
C. Laminin treatment strengthens cell adhesion, leading to increased mitochondrial translation (detected by the mito-FUNCAT method). The vertical axis represents the relative amount when the mean value under the control experimental conditions is set to 1.
D. Comprehensive quantitative analysis of the effects of simulated microgravity on translation (detection by ribosome profiling). Laminin treatment counteracts the effects on mitochondrial translation, reducing translational repression.
To understand the signaling pathway from cell adhesion to mitochondrial translation, we first focused on the laminin- integrin pathway [9]. Integrins are cell membrane proteins that bind to laminin and, on the cytoplasm, bind to a phosphorylation enzyme called FAK. Knockdown experiments of the FAK gene revealed that FAK plays a crucial role in laminin-dependent mitochondrial translation activation. FAK transmits information through the phosphorylation of various factors. To clarify which downstream factors and pathways transmit information to mitochondrial translation, we conducted a small-scale chemical screening (inhibitor experiments). As a result, we found that PAK1, PAK1, BAD, and Bcl2 family proteins downstream of FAK are carriers of information transmission from FAK to mitochondrial translation (Figure 3A).
The Bcl2 family proteins mentioned above are located on the outer mitochondrial membrane. On the other hand, intramitochondrial translation occurs in the matrix (the part surrounded by the inner membrane), which is the inside of the mitochondria, so we investigated what mechanisms are at work inside the mitochondria. As a result, we found that the fatty acid synthesis pathway [10] plays an important role in the mechanisms inside the mitochondria. Fatty acid synthesis inside the mitochondria uses malonyl-CoA (malonyl coenzyme A) [10] as a substrate, but malonyl-CoA accumulates when fatty acid synthesis weakens. In such cases, it is known that malonyl-CoA, which is highly concentrated, undergoes malonylation, binding to lysine residues of proteins in an enzyme-independent manner.
An international collaborative research group revealed that this fatty acid synthesis is promoted by cell adhesion. On the other hand, they showed that when cell adhesion is weakened, malonylation of the mitochondrial translation machinery occurs, and that this further suppresses mitochondrial translation (Figure 3B).
[English translation via Grok]
Figure 3. Information transduction from cell adhesion to the outer mitochondrial membrane and regulatory mechanisms within mitochondria.
A. Chemical screening (inhibitor experiments) revealed that the signal transduction pathway from cell adhesion to mitochondrial translation followed the order of FAK downstream PAK1 → PAK1 downstream RAC1 → RAC1 downstream BAD → Bcl2 family proteins downstream BAD. p: phosphorus, GTP: guanosine triphosphate.
B. When cell adhesion is strong, fatty acid synthesis proceeds using malonyl-CoA (malonyl-CoA), which is a derivative of acetyl-CoA, as a substrate. On the other hand, when cell adhesion weakens, malonylation occurs in the mitochondrial translation system, and mitochondrial translation is suppressed.
Normally, cells are rarely exposed to microgravity, and the international research group considered that the above response likely has a different significance than being an inherent biological response mechanism to microgravity. In particular, since the laminin-integrin cell adhesion pathway is known to be activated by mechanical stress such as exercise, they hypothesized that cells possess a system that enhances mitochondrial translation in this pathway in response to exercise. When ribosome profiling was performed using a mouse skeletal muscle model in which mechanical stress was minimized, a decrease in mitochondrial translation was observed (Figure 4).
[English translation via Grok]
Figure 4. Analysis of the effect of minimizing mechanical stress on translation using ribosome profiling.
A. Mice were raised under conditions that minimized mechanical stress. Cell extracts were prepared from the skeletal muscle of these mice and from control mice.
B. Comprehensive quantitative analysis of the effects of minimizing mechanical stress on translation from skeletal muscle cell extracts. Mitochondrial mRNA (intramitangial translation) decreased. The horizontal axis shows the average and standardized number of reads obtained from all samples used in the analysis. The vertical axis represents the logarithmic ratio of the change in translation that occurred under the mechanical stress minimization condition compared with the control experimental condition.
Expectations for the future
This research elucidated the molecular mechanisms by which physical stimuli, such as gravity and mechanical stress, are transmitted from the extracellular environment to mitochondrial translational regulation. This finding provides a certain answer to the fundamental question of “what exactly happens to the human body in a weightless environment?” It also presents us with the important proposition that overcoming mitochondrial damage, which stems from mitochondrial translation abnormalities, is necessary to enable long-term space travel. Through these findings, it is expected that our understanding of mitochondrial energy metabolism in gravity-deficient environments will advance in astrobiology and space medicine. Furthermore, the results of this research will directly contribute to elucidating the pathogenesis of diseases related to aging, muscle atrophy, and mechanical stress in everyday life, not just in the space environment. It can also be said that these results will lead to drug discovery research targeting mitochondrial translation.
supplementary explanation
- Intramitochondrial Translation: The process of synthesizing proteins from messenger RNA (mRNA) (see [4]) copied from mitochondrial DNA. This process takes place within mitochondria by specialized ribosomes (see [5]).
- This is the process of converting the base sequence written in translation mRNA into an amino acid sequence, and then synthesizing proteins by combining amino acids in ribosomes.
- Ribosome profiling is a technique that analyzes which genes are being translated and to what extent by extracting ribosomes, which are the translation machinery, and identifying the mRNA sequences bound to the ribosomes. Since ribosomes are large complexes that bind to cover certain mRNA regions, when these ribosome-mRNA complexes are treated with RNA-degrading enzymes, only the mRNA fragments protected by the ribosomes are recovered without being degraded.
- Messenger RNA (mRNA) is RNA that carries the amino acid sequence information (codons) of a protein. Protein synthesis occurs when codons are read by ribosomes.
- A ribosomal complex is a large structure composed of ribosomal RNA (rRNA) and ribosomal proteins.
- A 3D clinostat is a device that simulates microgravity (an environment close to weightlessness) on Earth by averaging the direction of gravity through continuous three-dimensional rotation of a sample. It is mainly used to study the gravitational responses of plants and cells.
- Cell adhesion is a phenomenon in which cells bind to each other or to the extracellular matrix via specific adhesion molecules. This regulates tissue structure maintenance, cell migration, and signal transduction.
- The mito-FUNCAT method is a technique that labels novel synthetic proteins with homopropagyl-glycine, a methionine derivative, and detects them using a click reaction and a fluorescent dye. By suppressing cytoplasmic translation with an inhibitor, only mitochondrial translation can be isolated and detected.
- Integrins are receptor proteins present in the cell membrane that play a role in connecting cells to the extracellular matrix. In addition to cell adhesion, they regulate cell morphology, movement, and proliferation by transmitting external signals into the cell.
- Fatty acid synthesis, specifically malonyl-CoA (malonyl coenzyme A) fatty acid synthesis, is a metabolic pathway that starts with acetyl-CoA (acetyl coenzyme A) and extends the carbon chain to produce fatty acids, primarily occurring in the cytoplasm. Malonyl-CoA is a two-carbon donor used in this process, playing a role in extending the fatty acid chain by two carbons with each reaction.
International collaborative research group
Iwasaki RNA Systems Biochemistry Laboratory, RIKEN Institute for Advanced Research
Principal Investigator: Shintaro Iwasaki
Special Researcher: Taisei Wakigawa
Graduate Student Research Associate (at the time of research): Yusuke Kimura
Technical Staff I: Mari Mito
Graduate Student Research Associate (at the time of research): Hironori Saito
Senior Researcher (at the time of research): Yuichi Shichino
(Currently Visiting Researcher, Currently Professor, Faculty of Medicine, University of Tsukuba)
University of Tokyo
Department of Chemical and Biological Engineering, Faculty of Engineering / Graduate School of Engineering
Associate Professor Yusuke Hirabayashi;
Postdoctoral Researcher (at the time of research) Koki Nakamura;
Graduate School of Medicine / Faculty of Medicine
Associate Professor Taku Saito;
JSPS Research Fellow (at the time of research) Toshiya Tsubaki;
Graduate School of Frontier Sciences
Associate Professor Nono Tomita;
Project Assistant Professor Moo-Hoon Lee
Tohoku University, Graduate School of Life Sciences
Professor: Atsushi Higashitani
Tatsuhisa Tsuboi, Associate Professor, Shenzhen International Graduate School, Tsinghua University (China); Abdul Haseeb Khan, Researcher.
Japan Space Forum, General Incorporated Foundation, Space Utilization Division
Deputy Director (at the time of research): Toru Yamamori
Japan Aerospace Exploration Agency (JAXA)
Principal Research and Development Officer: Tomokazu Yamazaki;
Technical Area Manager: Akira Higashibata
Research support
This research is part of the RIKEN Pioneering Projects “Biology of Intracellular Environments (Research Collaborators: Shintaro Iwasaki, Yuichi Nanano)” and RIKEN TRIP Initiative (AGIS* Cell Response Model Development) (Participating Researcher: Shintaro Iwasaki), Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (Exploratory) “Comprehensive Analysis of Gravity-Based Translational Regulation (Principal Investigator: Shintaro Iwasaki)”, Grants-in-Aid for Scientific Research (B) “Comprehensive Analysis of Organelle Translation by Mitochondrial-Specific Ribosome Profiling Method (Principal Investigator: Shintaro Iwasaki)”, Grants-in-Aid for Scientific Research (A) “Comprehensive Analysis of Interactions Between Heteropolymers Supporting Intracellular Structures (Co-Investigator: Shintaro Iwasaki)”, Grants-in-Aid for Scientific Research (B) “Novel Disome-Seq Method: Comprehensive Exploration of Parametric Ribosome Congestion (Principal Investigator: Shintaro Iwasaki)”, Grants-in-Aid for Scientific Research (A) “Time-Protein Science: Large-Scale Parallel Comprehensive Analysis of Translation Rate (Principal Investigator: Shintaro Iwasaki)”, “APEX-Ribo-Seq: Atypical Local Translation by Neighborhood Labeling” This research was supported by grants from the following organizations: “Comprehensive analysis of (Principal Investigator: Yuichi Nanano)”, “Establishment of the APEX-Ribo-Seq method for comprehensively elucidating atypical local translation (Principal Investigator: Yuichi Nanano)”, the Special Research Fellowship for Young Scientists “Elucidation of the molecular mechanism of mitochondrial translation regulation by cell adhesion (Principal Investigator: Taisei Wakikawa)”, “Development of a single-cell ribosome profiling method and its application to animal tissues (Principal Investigator: Yusuke Kimura)”, the Japan Science and Technology Agency (JST) Strategic Creative Research Promotion Program CREST “Creation of innovative technologies for local translation and RNA dynamics (Principal Investigator: Shintaro Iwasaki)”, and the University of Tokyo Graduate School of Frontier Sciences Challenging New Frontier Doctoral Research Grant Program “Elucidation of the mechanism of mitochondrial translation activation by cell adhesion (Principal Investigator: Taisei Wakikawa)” and “Elucidation of the quality control mechanism that resolves mitochondrial ribosome collisions (Principal Investigator: Taisei Wakikawa)”.
- AGIS: Advanced General Intelligence for Science Program (Science Research Infrastructure Model Development Program)
Original paper information
Taisei Wakigawa, Yusuke Kimura, Mari Mito, Toshiya Tsubaki, Muhoon Lee, Koki Nakamura, Abdul Haseeb Khan, Hironori Saito, Tohru Yamamori, Tomokazu Yamazaki, Akira Higashibata, Tatsuhisa Tsuboi, Yusuke Hirabayashi, Nono Takeuchi-Tomita, Taku Saito, Atsushi Higashitani, Yuichi Shichino, and Shintaro Iwasaki, “Gravitational and mechanical forces shape mitochondrial translation”, Nature Communications , 10.1038/s41467-026-74493-z
Astrobiology, microgravity, genomics,
