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Gravity sensing in plant and animal cells

Gravity sensing in plant and animal cells www.nature.com/npjmgrav REVIEW ARTICLE OPEN 1✉ 2 3 4 5 6 Ken Takahashi , Hideyuki Takahashi , Takuya Furuichi , Masatsugu Toyota , Makoto Furutani-Seiki , Takeshi Kobayashi , 7 8 9 10 10 Haruko Watanabe-Takano , Masahiro Shinohara , Takuro Numaga-Tomita , Asako Sakaue-Sawano , Atsushi Miyawaki and Keiji Naruse Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space. npj Microgravity (2021) 7:2 ; https://doi.org/10.1038/s41526-020-00130-8 INTRODUCTION obtain light (negative gravitropism), whereas roots grow down- ward to acquire water and minerals (positive gravitropism). When Gravity defines the morphology of life on Earth. It affects the plants in a vertical position are reoriented horizontally, the pattern growth and development of plants and animals by regulating the of gravitropic response differs among plant species and organs. proliferation of their constituent cells . Gravity also plays crucial Other aspects of plant growth and development are also roles in cellular function. For example, plants grow leaves and regulated by gravity. Accordingly, the terms “gravimorphism” or roots in the correct direction by sensing gravity . Animals regulate “gravimorphogenesis” can be used for gravity-regulated phenom- the densities of bones and muscles in response to gravitational 3,4 ena in plants. load . A response to gravity is an active activity inherent to the The plant hormone auxin plays an important role in plant physiology of plants and animals. gravimorphogenesis. A major endogenous auxin is indole-3-acetic Historically, elucidation of gravity sensing mechanisms in life acid (IAA) that regulates various aspects of plant growth and originates from the study of plants. Abundant research in plant development. In auxin signaling pathway, Aux/IAA proteins biology has laid the foundation for studying gravity sensing mechanisms in animals. In the first half of this article, we review inactivate the auxin response factor (ARF) when auxin level is the mechanisms of gravity sensing in plants from the viewpoint of low . A high level of auxin results in forming a complex of the cellular and molecular biology. Subsequently, the mechanisms of transcriptional repressor, Aux/IAA, and the AUXIN SIGNALING F- gravity sensing in animal cells will be discussed. Since both lunar BOX PROTEIN co-repressor/auxin receptor, TIR/AFBs, which allows base construction and manned Mars exploration plans are being the degradation of Aux/IAA and the release of ARF repression 5,6 7 discussed at present , discussions regarding bone loss and for modulating the expressions of auxin-related genes . Auxin muscle atrophy in microgravity environments are inevitable. This concentration in the tissues is determined by regulating its paper also summarizes the recent findings in this field. Through biosynthesis, inactivation, and transport. A unique feature of auxin these discussions, we outline the common mechanisms of gravity is the polar auxin transport that contributes to most of the sensing in plants and animals. directional auxin transport and local auxin concentration. This This paper expands on our understanding of gravity sensing in polar auxin transport is regulated by auxin efflux carriers PIN- plant and animal cells and discusses the future direction of FORMED (PIN) family and auxin influx carriers AUX/LAX family gravitational biology, with the ultimate purpose of contributing to proteins. It is considered that auxin is directionally transported the development of living in space. across the plasma membrane where PINs localize with a 8,9 polarity . ATP-binding cassette ABC transporters of the B class 10,11 family (ABCB) also play a role in polar auxin transport . GRAVITY SENSING IN PLANTS TWISTED DWARF1 (TWD1) has been shown to interact with ABCB Auxin regulation of gravimorphogenesis in plants protein for auxin transport. ABCB could export auxin indepen- dently of PIN proteins, but the functional interaction of ABCB-PIN The survival of sessile organisms, such as plants, depends upon their ability to avoid or mitigate various environmental stresses to pairs for auxin transport is also considered. Abbreviations are which they are subjected. As one of such strategies, plants possess listed in Table 1. an ability to control directional growth by gravitropism (Fig. 1). Gravisensing apparatuses reside in the endodermal cells of Typically, coleoptiles and stems (shoots) grow upward in order to shoots and columella cells of roots, where amyloplast statoliths 1 2 Department of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Graduate School of Life 3 4 Sciences, Tohoku University, Sendai, Japan. Faculty of Human Life Sciences, Hagoromo University of International Studies, Sakai, Japan. Department of Biochemistry and Molecular Biology, Saitama University, Saitama, Japan. Department of Systems Biochemistry in Regeneration and Pathology, Graduate School of Medicine, Yamaguchi University, 6 7 Yamaguchi, Japan. Department of Integrative Physiology, Graduate School of Medicine, Nagoya University, Nagoya, Japan. Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan. Department of Rehabilitation for the Movement Functions, Research Institute, National Rehabilitation Center for 9 10 Persons with Disabilities, Tokorozawa, Japan. Department of Molecular Pharmacology, Shinshu University School of Medicine, Matsumoto, Japan. Lab for Cell Function and Dynamics, CBS, RIKEN, Wakō, Saitama, Japan. email: takah-k2@okayama-u.ac.jp Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; K. Takahashi et al. gravistimulation gravistimulation (lay down) (lay down) a b asymmetric gravitropism auxin flow a a b b gravistimulation phosphorylation/dephosphorylation (upside down) of PINs LAZY –associated Directional change Relocation of amyloplast RLD polarization of auxin flow PINs sedimentation iii ii 2+ Ca 2+ Ca gravismulaon amyloplast sedimentaon iv 2+ Ca 2+ Ca Fig. 1 Mechanism for directional growth in response to gravity in plants. a Model of gravitropism. b Asymmetric auxin flow in horizontally reoriented plants, c cellular responses in gravisensing (endodermal) cells. At first, gravity causes sedimentation of amyloplasts. RLD proteins associated with LAZY proteins get polarized to the new bottom side. The LAZY proteins regulate the localization of PIN proteins, which are efflux carriers of plant hormone auxin. Finally, the change in direction of auxin flow causes asymmetric growth of plants. d Activation of mechanosensitive ion channels in plasma- and endomembrane upon amyloplast sedimentation (i, ii), deformation, compression and shear stress (iii), displacement of amyloplast (iv). 12 10,11 sediment upon plant reorientation . However, the mechanisms of during gravitropic response . In roots, for example, PIN3 and graviperception remain unsolved. The Cholodny–Went theory PIN7 localize to the plasma membrane of gravisensing columella explains differential growth in tropisms through the redistribution cells in the root cap. Within 10 min of gravistimulation by of the plant hormone auxin in elongating organs . In gravi- reorienting plant seedlings, PIN3 and PIN7 change their location stimulated plants, more auxin accumulates in the lower side than to the new bottom of the plasma membrane, allowing auxin to the upper side of shoots and roots in a horizontal position, causing move to the bottom side of the root cap. Thereafter, PIN2 the upward bending of shoots and downward bending of roots. localizing to the proximal side of the plasma membrane in the Auxin redistribution following gravistimulation has been verified. lateral root cap and the epidermis, plays a role in the basipetal Indeed, molecular genetics with many gravitropic mutants has transportation of auxin from the bottom side of the root cap to revealed the importance of the roles of auxin transport, the elongation zone. In gravistimulated roots, auxin redistribution redistribution, and response in gravitropism . In particular, the is thus established, and transcriptional regulation depending on identification of an auxin efflux carrier PIN-FORMED (PIN) was a the auxin level on the upper and lower sides leads to downward significant breakthrough in our understanding of the mechanisms bending. Spaceflight experiments with cucumber seedlings in the for asymmetric auxin transport and distribution in gravistimulated Space Shuttle and the International Space Station (ISS) showed 15,16 shoots and roots . However, unlike some PINs, the pattern of that the endodermal cells relocalize an auxin efflux carrier, CsPIN1, ABCB expression and the phenotypes of ABCB mutants indicate because of gravistimualtion in space and laterally transport auxin 17–19 that it is not directly involved in asymmetric redistribution of auxin from the upper to lower flank . npj Microgravity (2021) 2 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; K. Takahashi et al. gravitropic response. Thus, amyloplast position itself may play an Table. 1. List of abbreviations. important role in gravity sensing/signaling as discussed in next section “Gravity sensor in plants.” However, the mechanism ARHGAP Rho GTPase Activating Protein underling the LAZY polarization upon amyloplast sedimentation BRX Brevis radix in gravisensing cells still remains unknown. CaN Calcineurin Auxin biosynthesis and distribution in microgravity were also CBF/DREB1 C-repeat binding factor / dehydration-responsive examined by spaceflight experiments in some plant species. Most element-binding 1 of those results showed no differences between space- and ground-grown seedlings. Recently, transformed Arabidopsis lines CCL Conserved C terminus in the LAZY1 family 2+ 2+ with GFP reporter gene, pDR5r::GFP, pTAA1::TAA1-GFP, pSCR:: CICR Ca -induced Ca -release SCR–GFP and pARR5::GFP, were used for spaceflight experiments D6PK Serine/threonine-protein kinase on ISS . The expressions of the auxin artificial AuxRE promoter DCC Disposable cell culture construct (pDR5r::GFP), Tryptophan Aminotransferase of Arabi- DEK1 Defective Kernel 1 dopsis fusion (pTAA1::TAA1-GFP) and Scarecrow fusion (pSCR:: SCR–GFP) were used to monitor auxin level, auxin production and GNOM ARF guanine-nucleotide exchange factor auxin-related signals, respectively. There were no differences in HEK Human Embryonic Kidney the expression patterns and levels of those genes in the primary ISS International Space Station root tips of seedlings grown under microgravity and 1 G ground LAZY1 Plant-specific genes with unknown molecular functions conditions. These results implied that auxin gradient in plants is that are involved in gravitropism established independently of gravity. On the other hand, space- MCA Mid1 (Yeast mechanosensitive channel) complementing flight experiments with pea and maize seedlings showed altered activity protein polar auxin transport in microgravity; polar auxin transport in MSC Mesenchymal stem cells microgravity was decreased in pea epicotyls and accelerated in MSL MscS (bacterial mechanosensitive channel of small maize coleoptiles and mesocotyls compared with the 1 G 28,29 conductance) like protein controls . Recent spaceflight experiments immunohistochemi- cally compared PsPIN1 localization in etiolated pea epicotyls NFAT Nuclear factor of activated T cells grown under microgravity and 1 G conditions in space . PsPIN1 NOX NADPH oxidases proteins were detected in the lower side of the plasma membrane 2+ OSCA reduced hyperosmolality- induced [Ca ] increase 1 of 80–90% endodermal cells under artificial 1 G conditions, PDZ Domains bind to a short region of the C-terminus of other whereas number of those endodermal cells showing polarized specific proteins PsPIN1 localization significantly decreased in microgravity. The PLC Phospholipase C authors consider the change in PsPIN1 localization pattern as a RANKL Receptor activator of nuclear factor-B ligand possible cause for the reduction of polar auxin transport in pea epicotyls under microgravity conditions. In maize seedlings, RCC Regulator of chromosome condensation interestingly, the enhanced accumulation of ZmPIN1and the RCN Reticulocalbin alteration of ZmPIN1a localization in parenchymatous cells of RLD Regulator of chromosome condensation-like domains the coleoptiles were likely responsible for the enhanced polar ROS Reactive oxygen species auxin transport in microgravity . However, species differences of TRPC Canonical transient receptor potential channel polar auxin transport in microgravity are mysterious. Thus, the PIN-mediated auxin transport and distribution are essential parts of plant gravimorphogenesis. It should be Some factors that could be involved in PIN polarization and emphasized that some PIN proteins were verified to be gravity thereby asymmetric auxin flow have been reported. That is, responsive in their relocalization on the plasma membrane of the proteins such as RCN1, PINOID, and D6PK regulate PIN phosphor- gravisensing cells by spaceflight experiments. Polarization and 2,20,21 ylation/dephosphorylation . The phosphorylation status of function of LAZY1/LAZY1-like proteins appear to play a key role PIN proteins, together with GNOM-dependent PIN recycling in the gravity-induced PIN relocalization and thereby asymmetric processes, is hypothesized to participate in polar localization of auxin flow. To understand the entire regulatory mechanism of PIN proteins on the plasma membrane. Dynamics of microfila- plant gravimorphogenesis, it is important to clarify the grav- ments and microtubules (MTs) is an important factor involved in iperception mechanism that leads to the regulation of LAZY1/ the regulation of trafficking of auxin transporters. It is reported LAZY1-like proteins polarization and PIN relocalization in that Sorting Nexin 1 (SNX1) plays a role in PIN2 recycling via gravisensing cells. interaction with MTs-associated protein CLASP . Recently, it was found that LAZY1 regulates PIN relocalization in Gravity sensor in plants gravisensing cells and determines negative gravitropism in shoots 23,24 As discussed above, plants have a mechanism for gravity sensing and positive gravitropism in roots . Interestingly, the alteration using the sedimentation of organelles in order to establish the of two amino acids in LAZY1 was found to successfully switch asymmetric transport of hormones. The most widely accepted negative gravitropism to positive gravitropism in Arabidopsis model for plant gravity sensing is the starch-statolith hypothesis, shoots . Furthermore, it was revealed that upon amyloplast in which intracellular sedimentation of the starch-filled organelle sedimentation, LAZY1/LAZY1-like proteins get polarized to the (amyloplast) plays a crucial role in the events triggering the initial plasma membrane of the bottom side of gravisensing cells . The 32–34 phases of gravity sensing in plants . Recent live-cell imaging conserved C terminus in the LAZY1 family (CCL) domains interact technology has revealed, however, that the movement of the with the Brevis radix (BRX) domains of the regulator of amyloplast is not static but saltatory because its dynamics are chromosome condensation (RCC)-like domains (RLD) proteins, dependent on both the gravity vector and intracellular environ- thereby polarly recruiting RLD from the cytoplasm to the plasma ments such as those of the cytoskeleton and vacuole . In the membrane . It was demonstrated that RLD1–4 localize in the root cap and modulate auxin transport through regulation of PIN shoot statocytes (endodermal cells) of Arabidopsis thaliana, localization, possibly via a GNOM-like function in PIN trafficking . the amyloplasts are tightly surrounded by the vacuolar membrane This process is required for controlling polarized auxin flow and and are supposed to interact with actin filaments . The abnormal Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 2 K. Takahashi et al. behavior of the vacuolar membrane, however, pushes the segment of MscS (mechanosensitive channel of small conduc- amyloplasts to the periphery of the cell in the agravitropic tance) in E. coli . Among the 10 members of the MSL protein in mutant, shoot gravitropism (sgr) 2, which restricts the movement of Arabidopsis, MSL1 localizes to the inner mitochondrial membrane, the amyloplasts and renders them nonsedimentable . Therefore, whereas MSL2 and 3 are found at the inner plastid membrane, inflorescence stems in sgr2 mutants do not sense gravity and do and MSL8, 9, and 10 localize to the plasma membrane . MSL2 and not show a gravitropic response because of nonsedimentable 3 regulate the size and shape of the plastid, and MSL8 is required amyloplasts . distorted1 (dis1)/actin-related protein 3 (arp3) for the rehydration of the pollen grain, indicating that a major role mutants possess irregular thick actin bundles surrounding of MSL proteins is osmotic regulation. Yeast two-hybrid assays amyloplasts in their root statocytes (columella cells), and demonstrated that MSL2 and 3 interact with each other, consequently, the amyloplasts do not sediment fully from the suggesting that some MSLs can form heteromeric channels . actin filaments, resulting in a reduced gravitropic response in MCA proteins have been identified as complements of the mid1 2+ 50 roots . sgr9 mutants also have nonsedimentable, clustered mutant of the yeast that is defective in Ca influx . MCA1 and amyloplasts entangled with actin filaments in the endodermal MCA2 localize in the plasma membrane and mediate the cold- 2+ cells because of an excess of interaction between the amyloplasts induced Ca response that leads to cold tolerance according to and the actin filaments and exhibit a weak gravitropic response . the CBF/DREB1-independent pathway . MCA1, but not MCA2, is These abnormal phenotypes in both dis1 and sgr9 mutants were required for the penetration of roots into harder agar and results compensated by disrupting actin filaments, such as through the in the retardation of the leafing and bolting of the mutant. The 36,38 use of the actin filament-depolarizing drug latrunculin B , expression levels of MCA1 and MCA2 were increased under suggesting that the actin filament is not an essential component hypergravity conditions in the absence of light, and the hypocotyl for either gravity sensing or gravitropic responses, but rather acts elongation under these conditions was attenuated in the over- as an intracellular component affecting amyloplast dynamics. expressing seedlings. Therefore, MCA proteins might be respon- Taken together, the dynamics of the vacuolar membrane and sible for resistance to gravity . OSCA proteins, homologous to the actin filaments could diffuse amyloplasts from the bottom of the TMEM63 family of proteins known throughout eukaryotes, have cell, leading to the nonstatic, saltatory behavior of the plant been identified from Arabidopsis mutants exhibiting a low 2+ 53 statolith and amyloplast. hyperosmolality-induced Ca response . An ortholog of animal A long-lasting question regarding gravity sensing in plants is Piezo protein, the mechanosensitive cation channel for touch how the physical process of amyloplast sedimentation is sensation and vascular development, is commonly conserved in converted into intracellular signals. The most reasonable models, monocots and suppresses the systemic movement of viruses . such as the inner ear (hair cell) system of vertebrates, suggest that Mechanosensitive cation channel activity in MCAs and OSCAs amyloplast sedimentation activates mechanosensitive ion chan- has been recorded using patch-clamp techniques with hetero- 39–41 55,56 nels via actin, resulting in intracellular ionic signaling . logous expression in Xenopus oocytes and HEK cells . Electro- Changes in the gravity vector (inclining the specimens) elevate physiological studies using Arabidopsis mutants and Xenopus 42,43 cytosolic calcium concentrations in Arabidopsis seedlings . This oocytes have revealed that MSL proteins show a preference for 2+ Ca response is attenuated with latrunculin B and mechan- anions over cations, leading to depolarization of the plasma 3+ 42 2+ osensitive ion channel blockers such as Gd , supporting a membrane and the following Ca response through the model involving actin filaments that function as a tether to activation of voltage-dependent cation channels . Most recently, 2+ activate mechanosensitive channels . Actin filaments may have a lack of rapidly activated mechanosensitive Ca -permeable both a positive role in the activation of mechanosensitive channel activity (RMA) was reported in Arabidopsis DEK1 channels upon gravity stimulation and a negative role in the mutants . Although there is no sequence homology, AtDEK1 sedimentary dynamics of amyloplasts as discussed above. To has a high number of transmembrane helices as with mammalian 2+ demonstrate this, direct observations of Ca responses in both Piezo proteins, and RMA shows low conductance and rapid the shoot endodermal and root columella cells are needed. An inactivation. These electrophysiological studies have demon- alternative model is the position-sensing hypothesis in which the strated that the thresholds of membrane stretch for the activation, spatial distribution of amyloplasts upon gravity stimulation is conductance, and inactivation time constant of plant mechan- detected as a signal for gravity sensing . In this hypothesis, a osensitive channels are varied, even within the same family. putative machinery, rather than mechanosensitive channels Although series of proteins and their physiological roles have sensing the gravitational force exerted on the amyloplasts, detects been characterized in numerous aspects, the mechanosensitive the position (state) of the amyloplasts in gravity sensing cells, channels responsible for gravitropism have not yet been consistent with data indicating that gravitropic responses in wheat identified. Overlapping tissue expression patterns suggest that coleoptile are dependent on the angle of inclination of the mechanosensitive channels in the same tissue share physiological specimens but not on the amplitude of the gravitational force . functions. Thus, most mutants, even those that lacking five MSLs These data suggest a variety of gravity sensing mechanisms in 47 (MSL4, 5, 6, 9, and 10), do not show a significant phenotype . diverse plant species such as monocots or dicots which are, Interestingly, most mechanosensitive channels are expressed in however, quite different from those of animals. 2+ the vasculature, where gravity-induced Ca response is observed . In root statocytes of Brassica grown in ISS, ten- Mechanosensitive channels in plants minute onset (µg to 1 g) or removal (1 g to µg) of a gravity- 2+ induced Ca response in absence of a significant statolith As discussed above, gravity is the force that generates several displacement . Changes in gravity vector and magnitude effects such as the weight leading to the sedimentation of 2+ 46 promote a Ca response with similar kinetics . It suggests that amyloplast statoliths, deformation and compression of the cells, and fluid shift in vasculature, all of which generate mechanical multiple mechanosensitive channels in plasma- and endomem- stresses in plasma and endomembranes. One of the earliest branes could be differentially activated by gravity, and promotes a 2+ 2+ response to changes in gravity vector and magnitude is the Ca small Ca response that is amplified by common intracellular response, which has been reported in many plant species . Thus, machineries. Thus, pharmacological studies suggest that gravity- 2+ 2+ 2+ it is plausible that mechanosensitive channels are the primary induced Ca response is greatly amplified by Ca -induced Ca - 2+ sensors of plant graviperception evoking the Ca response. release (CICR) from organelles through signaling cascades, MSL proteins share the C-terminal transmembrane (TM) including PLC activation . Subcellular and tissue-specific distribu- 2+ segment corresponding to the pore-forming transmembrane tions of gravity-induced Ca response and the underlying npj Microgravity (2021) 2 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA K. Takahashi et al. Fig. 2 YAP-mediated 3D organ/tissue formation withstanding gravity. a Mechanical negative feedback maintaining YAP activity in a cell. b YAP-mediated 3D organ/tissue formation withstanding gravity. In (a), YAP/TAZ acts as a mechanotransducer and mechanoeffector. As a mechanotransducer, it provides physical inputs, including gravity activation of YAP/TAZ that leads to an expansion of organ size. As a mechanoeffector, it activates YAP, which, in turn, controls F-actin turnover, leading to the suppression of YAP as part of a negative feedback mechanism. F-actin turnover controls the cell/tissue tension that mediates 3D organogenesis. b YAP is essential for the formation of complex 3D organs by coordinating 3D tissue shape (left) and tissue alignment (right). In response to external forces, including gravity, YAP activates (1) ARHGAP18 expression, which mediates (2) contractile actomyosin formation controlling (3) tissue tension. Tissue tension is required for both (4) cell stacking to form a 3D tissue shape and (5) fibronectin assembly required for adjacent tissue alignment, e.g., the alignment of the lens and eye-cup. molecular mechanisms should be investigated more deeply in the lens and eye cup (Fig. 2b). It is hypothesized that a YAP- order to understand graviperception mechanisms in plants. mediated response to gravity is involved in the maintenance of bones and skeletal muscles, since YAP is known to control the organ size through Hippo signaling and is expressed in the stem GRAVITY SENSING IN ANIMALS cells of many organs, including skeletal muscles. YAP-mediated gravity response and 3D organ growth and YAP orchestrates the response to gravity by controlling maintenance actomyosin contractility by negatively regulating F-actin polymer- ization through its target gene ARHGAP18. Since actomyosin both Although plants and animals share common mechanisms for generates mechanical forces and acts as a mechanical sensor , gravity sensing, such as the homologous mechanosensitive ion actomyosin is a putative gravity sensor. Gravity could promote channels discussed above, the transcriptional coactivator Yes- F-actin polymerization, activating YAP in order to maintain a 3D associated protein (YAP) is a mechanosensitive machinery specific organ size. This is consistent with reports that simulated to animals. The relationship between gravity and YAP was first microgravity inhibits the osteogenic differentiation of mesench- revealed by the analysis of the medaka fish YAP mutant. The body ymal stem cells via the de-polymerization of F-actin that inhibits of this mutant was flattened because of its inability to withstand TAZ nuclear translocation . Further detailed studies are necessary gravity . This demonstration that YAP is required for withstanding to elucidate the mechanisms by which the YAP-mediated gravity gravity in generating a 3D body/organ shape first suggested that response is linked with organ growth and maintenance. These YAP not only transduces gravity responses as a mechano- studies will be useful in alleviating compromises to health, such as transducer , but more strikingly acts as a mechano-effector for the loss of bones and skeletal muscles that arises from periods of withstanding gravity, forming a mechanical negative-feedback . “life in space.” Since YAP is the key regulator orchestrating organ growth , this review will focus on the role of YAP in linking gravity response with organ growth and maintenance. Gravity sensing in bones YAP and its paralog TAZ (transcriptional coactivator with PDZ- Bone loss is one of the major health problems facing organisms binding motif) act as transcriptional co-activators, mainly in the that experience life in space. The structure of bones is shown in 63,64 nucleus . YAP nuclear localization is controlled mainly by the Fig. 3. Here we discuss the sensing mechanisms of gravitational Hippo pathway and F-actin-mediated signaling responses to loads in the trabecular bone and cortical bone. diverse signals, e.g., growth factors and mechanical stimuli . YAP is able to expand the organ size when constitutively Mechanical sensing by osteocytes, a commander for activated and is involved in such diseases as cancer and osteoclasts and osteoblasts fibrosis . Bone homeostasis is maintained as a result of the balanced action The discovery that YAP could act as a mechanoeffector of osteoblasts for bone formation and osteoclasts for bone uncovered a negative feedback control of YAP activity: F-actin resorption . In bone destructive diseases, such as osteoporosis, polymerization activates YAP and its target gene ARHGAP18 and bone resorption is favored over bone formation, leading to bone then negatively regulates F-actin polymerization, suppressing YAP activity (Fig. 2a). This is a mechanical negative feedback since loss. In particular, bone loss in unloading conditions, such as the negative regulation of F-actin polymerization by YAP microgravity in space, is caused by enhanced bone resorption by optimizes F-actin turnover and maximizes actomyosin contrac- osteoclasts and suppressed bone formation by osteoblasts. tility, i.e., cell/tissue tension. Cell/tissue tension then controls 3D Osteocytes, another type of bone cell, are differentiated from tissue formation and tissue alignment necessary for generating a osteoblasts and embedded in the bone matrix have many 3D organ consisting of multiple tissues, e.g., an eye consisting of dendrites formed through osteocytogenesis that communicate Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 2 K. Takahashi et al. Periosteum Fibrous layer Long bone Cortical bone Fibroblast Periosteum SSC Osteoblast Blood vessel Osteocyte Lacuna Canaliculi Fig. 3 The structure of the long bone, cortical bone, and periosteum. Osteocytes are embedded in the lacuna of the bone matrix and are connected with each other through dendrites surrounded by canaliculi. At the periphery of the bone, SSCs, and fibroblasts form the periosteum together with osteoblasts. Osteocytes and the periosteum are mechanical sensors in the bone tissue. either with each other or with the osteoblasts and osteoclasts at bones except for joints, generates the cortical bone in physiolo- the bone surface . Osteocytes also have a critical role in bone gical and pathological situations through the provision of homeostasis, functioning as a commander for osteoclasts and osteoblasts . The periosteum contains two layers: an outer layer osteoblasts by regulating the expression of genes involved in the of fibroblasts and an inner layer composed of bone-forming receptor activator of nuclear factor-B ligand (RANKL), an osteoclast osteoblasts. Although the periosteum is not highly sensitive to differentiation factor, and as a negative regulator of osteoblast mechanical loading compared to the endocortical surface, it differentiation by sclerostin (Sost) . Importantly, expressions of nonetheless responds to loading and gives rise to bones in a these genes vary in response to mechanical loading or unloading variety of animal models. The unloading model of the hind limb to osteocytes in the bone. Osteocyte cell body and dendrites reduces bone formation in the cortical bone as well as in the reside in the lacunae and canaliculi, respectively, and mechanical trabecular bone . Conversely, periosteal bone formation is loading induces minute changes in the structure of the bone that stimulated by enhanced loading using in vivo models of axial 79,80 generate interstitial fluid flow in the lacuno-canalicular system. loading and three-point bending . The alteration of gene This flow acts as a mechanical loading, similar as pressure and expression patterns and cell morphologies within the periosteum shear stress, and affects osteocytes directly . after loading provides evidence that periosteal cells sense loading 80,81 Such mechanical loadings to osteocytes as described above can stimuli . activate mechanotransduction mediators such as ion channels, connexins, integrins, and cytoskeleton-related molecules .In Mechanical loading is possibly translated into bone formation addition, the cytosolic signaling adapter protein p130Cas, a through the periosteal skeletal stem cell cellular mechanosensing molecule , is involved in the regulation Among the cells responsible for sensing physical loading is of bone homeostasis in response to mechanical loading in skeletal stem cell (SSC) because loading-induced bone formation osteocytes . Interestingly, p130Cas translocates into the nucleus requires activation of the periosteal SSC to give rise to osteoblasts. and negatively regulates NF-κB activity to suppress bone The periosteal SSC displays a unique gene expression pattern and resorption by downregulating the expression of RANKL. These exhibits high regenerative capacity in response to bone injury findings suggest that the p130Cas- NF-κB axis in osteocytes is a 82 when compared to bone marrow skeletal stem cells (BMSC) .A potential target for treatment against disuse osteoporosis. recent study has revealed that Cathepsin K (CTSK)-lineage Although the critical significance of mechanical loading to the populations within the periosteum contain postnatal self- bone has been clearly elucidated, a large portion of the molecular renewing and multipotent stem cells . The deletion of Osterix, mechanisms underlying the mechanical regulation of bone encoded by the Sp7 gene, in CTSK-lineage cells results in impaired homeostasis is not understood. Efforts to clarify these mechanisms bone formation and fracture healing . Furthermore, Prx1-lineage will be a promising strategy to prevent bone loss during future mesenchymal cells that contain SSC sense loading stimuli through space missions. their primary cilia indicate loading-dependent bone forma- 78,82 + + tion . More recently, Nestin and Leptin cells have been Periosteum might sense physical loading shown to generate osteoblasts for periosteal bone formation .On The deterioration of the bone microarchitecture during spaceflight the other hand, some studies have shown that loading alters gene occurs not only in the trabecular bone but also in the cortical expression patterns, including extracellular molecules in osteo- 76 85 bone . It is thought to be triggered by enhanced osteoclast- blasts . Thus, the osteoblast may function as a mechanotransdu- mediated bone resorption at the endocortical surface and cer that induces osteogenic differentiation of SSCs in the suppressed bone-forming activity in the periosteum. The perios- periosteum. Accordingly, direct or indirect loading can activate teum, the highly vascularized outer membrane that covers all several types of SSC to induce cortical bone formation. npj Microgravity (2021) 2 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA K. Takahashi et al. Fig. 4 Stress fiber remodeling in MSCs exposed to simulated microgravity as analyzed by confocal fluorescence microscopy. MSCs expressing Lifeact-TagGFP2 contained thick stress fibers under 1 G conditions (a), whereas an exposure to simulated microgravity for 6 h led to the appearance of thinner stress fibers (b). The images in (a) and (b) showing the same field of view, were recorded, processed and presented in an identical condition (Kobayashi, unpublished). Scale bar: 20 μm. Gravity sensing in muscles them from proteasomal degradation . ROS production mediated by TRPC3 and NOX2 coupling causes cardiac muscle atrophy in Muscle atrophy is another major health problem in life in space 94,95 stressed hearts, in which the hemodynamic load is reduced . and involves the decrease of muscle mass in response to the Therefore, TRPC channels might have dual roles in unloading- reduction of hemodynamic loads. It is known to be caused by induced muscle atrophy: the first is the regulation of myoblast microgravity, long term bed rest, and cancer cachexia . proliferation via CaN activation, and the second is the production Unloading-induced muscle wasting is mediated by a decrease of of ROS, which induces catabolic remodeling of muscle tissue. protein synthesis in the homeostasis of muscle cells and an increase of catabolism. Consequently, there must be a molecule that senses and transduces the signals originating from mechan- Gravity sensing in mesenchymal stem cells ical loading. One of the candidates for such a load transducer is a Mesenchymal stem cells (MSCs) are crucial in the field of nonselective cation channel, the canonical transient receptor regenerative medicine by virtue of their self-renewal and multi- potential channel (TRPC). Members of the TRPC channels, namely, differentiation potentials . MSC self-renewal and differentiation TRPC1, TRPC3, and TRPC6, are reportedly activated downstream of are known to be controlled by a diverse set of soluble factors, mechanical signals in addition to phospholipase C-coupled cell including growth factors or cytokines. In addition, the fate of MSCs surface receptor activation . TRPC channels play important roles has been shown to be influenced by mechanical stresses or in the activation of protein phosphatase calcineurin (CaN). CaN surrounding physical microenvironments, such as substrate 2+ regulates the Ca -dependent transcription factor, the nuclear stiffness , or changes in gravity. Many space experiments and factor of activated T cells (NFAT), and the peroxisome proliferator- ground-based studies have demonstrated that MSCs are very activated receptor γ . Both proteins are important for myogen- sensitive to the modulation of gravitational stimuli and exhibit esis. Exposure of C2C12 skeletal myoblasts to microgravity induces various responses against such effects . The exposure of MSCs to the reduction of TRPC1 expression, which arrests the cell cycle at microgravity or simulated microgravity induces characteristic the G2/M phase, thereby inhibiting myoblast proliferation . The physiological responses, including remodeling of the cytoskeleton 99,100 importance of TRPC1 has also been demonstrated in muscle and the disruption of the stress fiber , reduced activity in regrowth after unloading-induced atrophy. Hind limb unloading transcriptional coactivator YAP/TAZ , suppression of osteoblastic 101,102 induces the reduction of TRPC1 expression, which persists even differentiation, and the promotion of adipogenesis , some of after reloading . The expression of the TRPC3 channel is also which were also observed in other nonspecialized animal cells. suppressed at complete atrophy and in the early recovered How can MSCs sense a microgravity environment? Ordinary phase . These changes in the expression of the TRPC1 and TRPC3 mechanical forces, including stretch or shear stress, can be sensed channels are consistent with the muscle mass, suggesting by animal cells through cell mechanosensors that convert that these channels play important roles in load-dependent mechanical stimuli into electrical or chemical signals. To date, muscle growth. mechanosensitive channels, focal adhesion proteins (p130Cas and It is widely accepted that oxidative stresses caused by the Talin), and actin fibers have been established to function as aberrant production of reactive oxygen species (ROS) or reactive mechanosensors for various types of cells. It has been postulated nitrogen species are key regulators for catabolic muscle wasting . that MSCs also utilize these common sensors to detect changes in ROS are produced as a byproduct of the mitochondrial respiratory gravity, since MSCs have no specific gravity sensors, as is the case chain or are produced enzymatically by NADPH oxidases (NOX) for organs such as the animal gravity sensor statocyst. Recent within the cell. In cardiac muscles, ROS production by the NOX2 studies have proposed that the cytoskeleton may function as an 2+ protein is physiologically important for Ca homeostasis and is initial sensor for microgravity . In the early phase (30 min to 6 h) activated mechanically during diastole . However, in pathological of exposure to microgravity, environmental changes experienced conditions, NOX2-mediated ROS production causes cardiac by the cytoskeleton have been observed, including a reduced remodeling in response to various stresses. It has also been noted amount or thinning of stress fibers (unpublished data in Fig. 4) that pathological situations in muscle tissue can engender and the redistribution of microtubules. In addition, genetic 2+ 2+ abnormal Ca signaling. Since some NOX isoforms require Ca restoration of the arrangement of actin fibers or the pharmaco- for activation, it is plausible that there exists a crosstalk between logical stabilization of actin cytoskeleton could maintain the 2+ pathological NOX activation and abnormal Ca signaling. TRPC3 osteogenic differentiation of MSCs under modeled micrograv- 68,99 and NOX2 proteins exist at this crossroads of signaling pathways. ity . This indicates that changes in the actin cytoskeleton in the Additionally, it has been demonstrated that the TRPC3 channels cells transferred under microgravity conditions could have a play an important role in NOX2 protein stabilization by protecting crucial role in cellular responses against changed gravity. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 2 K. Takahashi et al. However, it remains unclear if the cytoskeleton acts as an initial understanding cellular responses to gravity will form the and primary mechanosensor for gravity sensing . It has been foundations of living in space. proposed that the loss of gravitational forces acting on heavy organelles, including the nucleus and mitochondria, could affect DATA AVAILABILITY the cytoskeleton. Further studies will provide deeper insight All data are available in the main text. regarding gravity sensing and transduction. Received: 5 February 2020; Accepted: 14 December 2020; Gravity sensitivity of the cell cycle Regulation of the cell cycle is crucial for the maintenance of organs, such as bones and muscles. 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J. & van Loon, J. J. The role article’s Creative Commons license and your intended use is not permitted by statutory of the cytoskeleton in sensing changes in gravity by nonspecialized cells. FASEB regulation or exceeds the permitted use, you will need to obtain permission directly J. 28, 536–547 (2014). from the copyright holder. To view a copy of this license, visit http://creativecommons. 104. Miyawaki, A. & Niino, Y. Molecular spies for bioimaging–fluorescent protein- org/licenses/by/4.0/. based probes. Mol. Cell 58, 632–643 (2015). 105. Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008). © The Author(s) 2021 npj Microgravity (2021) 2 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals

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www.nature.com/npjmgrav REVIEW ARTICLE OPEN 1✉ 2 3 4 5 6 Ken Takahashi , Hideyuki Takahashi , Takuya Furuichi , Masatsugu Toyota , Makoto Furutani-Seiki , Takeshi Kobayashi , 7 8 9 10 10 Haruko Watanabe-Takano , Masahiro Shinohara , Takuro Numaga-Tomita , Asako Sakaue-Sawano , Atsushi Miyawaki and Keiji Naruse Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space. npj Microgravity (2021) 7:2 ; https://doi.org/10.1038/s41526-020-00130-8 INTRODUCTION obtain light (negative gravitropism), whereas roots grow down- ward to acquire water and minerals (positive gravitropism). When Gravity defines the morphology of life on Earth. It affects the plants in a vertical position are reoriented horizontally, the pattern growth and development of plants and animals by regulating the of gravitropic response differs among plant species and organs. proliferation of their constituent cells . Gravity also plays crucial Other aspects of plant growth and development are also roles in cellular function. For example, plants grow leaves and regulated by gravity. Accordingly, the terms “gravimorphism” or roots in the correct direction by sensing gravity . Animals regulate “gravimorphogenesis” can be used for gravity-regulated phenom- the densities of bones and muscles in response to gravitational 3,4 ena in plants. load . A response to gravity is an active activity inherent to the The plant hormone auxin plays an important role in plant physiology of plants and animals. gravimorphogenesis. A major endogenous auxin is indole-3-acetic Historically, elucidation of gravity sensing mechanisms in life acid (IAA) that regulates various aspects of plant growth and originates from the study of plants. Abundant research in plant development. In auxin signaling pathway, Aux/IAA proteins biology has laid the foundation for studying gravity sensing mechanisms in animals. In the first half of this article, we review inactivate the auxin response factor (ARF) when auxin level is the mechanisms of gravity sensing in plants from the viewpoint of low . A high level of auxin results in forming a complex of the cellular and molecular biology. Subsequently, the mechanisms of transcriptional repressor, Aux/IAA, and the AUXIN SIGNALING F- gravity sensing in animal cells will be discussed. Since both lunar BOX PROTEIN co-repressor/auxin receptor, TIR/AFBs, which allows base construction and manned Mars exploration plans are being the degradation of Aux/IAA and the release of ARF repression 5,6 7 discussed at present , discussions regarding bone loss and for modulating the expressions of auxin-related genes . Auxin muscle atrophy in microgravity environments are inevitable. This concentration in the tissues is determined by regulating its paper also summarizes the recent findings in this field. Through biosynthesis, inactivation, and transport. A unique feature of auxin these discussions, we outline the common mechanisms of gravity is the polar auxin transport that contributes to most of the sensing in plants and animals. directional auxin transport and local auxin concentration. This This paper expands on our understanding of gravity sensing in polar auxin transport is regulated by auxin efflux carriers PIN- plant and animal cells and discusses the future direction of FORMED (PIN) family and auxin influx carriers AUX/LAX family gravitational biology, with the ultimate purpose of contributing to proteins. It is considered that auxin is directionally transported the development of living in space. across the plasma membrane where PINs localize with a 8,9 polarity . ATP-binding cassette ABC transporters of the B class 10,11 family (ABCB) also play a role in polar auxin transport . GRAVITY SENSING IN PLANTS TWISTED DWARF1 (TWD1) has been shown to interact with ABCB Auxin regulation of gravimorphogenesis in plants protein for auxin transport. ABCB could export auxin indepen- dently of PIN proteins, but the functional interaction of ABCB-PIN The survival of sessile organisms, such as plants, depends upon their ability to avoid or mitigate various environmental stresses to pairs for auxin transport is also considered. Abbreviations are which they are subjected. As one of such strategies, plants possess listed in Table 1. an ability to control directional growth by gravitropism (Fig. 1). Gravisensing apparatuses reside in the endodermal cells of Typically, coleoptiles and stems (shoots) grow upward in order to shoots and columella cells of roots, where amyloplast statoliths 1 2 Department of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Graduate School of Life 3 4 Sciences, Tohoku University, Sendai, Japan. Faculty of Human Life Sciences, Hagoromo University of International Studies, Sakai, Japan. Department of Biochemistry and Molecular Biology, Saitama University, Saitama, Japan. Department of Systems Biochemistry in Regeneration and Pathology, Graduate School of Medicine, Yamaguchi University, 6 7 Yamaguchi, Japan. Department of Integrative Physiology, Graduate School of Medicine, Nagoya University, Nagoya, Japan. Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan. Department of Rehabilitation for the Movement Functions, Research Institute, National Rehabilitation Center for 9 10 Persons with Disabilities, Tokorozawa, Japan. Department of Molecular Pharmacology, Shinshu University School of Medicine, Matsumoto, Japan. Lab for Cell Function and Dynamics, CBS, RIKEN, Wakō, Saitama, Japan. email: takah-k2@okayama-u.ac.jp Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; K. Takahashi et al. gravistimulation gravistimulation (lay down) (lay down) a b asymmetric gravitropism auxin flow a a b b gravistimulation phosphorylation/dephosphorylation (upside down) of PINs LAZY –associated Directional change Relocation of amyloplast RLD polarization of auxin flow PINs sedimentation iii ii 2+ Ca 2+ Ca gravismulaon amyloplast sedimentaon iv 2+ Ca 2+ Ca Fig. 1 Mechanism for directional growth in response to gravity in plants. a Model of gravitropism. b Asymmetric auxin flow in horizontally reoriented plants, c cellular responses in gravisensing (endodermal) cells. At first, gravity causes sedimentation of amyloplasts. RLD proteins associated with LAZY proteins get polarized to the new bottom side. The LAZY proteins regulate the localization of PIN proteins, which are efflux carriers of plant hormone auxin. Finally, the change in direction of auxin flow causes asymmetric growth of plants. d Activation of mechanosensitive ion channels in plasma- and endomembrane upon amyloplast sedimentation (i, ii), deformation, compression and shear stress (iii), displacement of amyloplast (iv). 12 10,11 sediment upon plant reorientation . However, the mechanisms of during gravitropic response . In roots, for example, PIN3 and graviperception remain unsolved. The Cholodny–Went theory PIN7 localize to the plasma membrane of gravisensing columella explains differential growth in tropisms through the redistribution cells in the root cap. Within 10 min of gravistimulation by of the plant hormone auxin in elongating organs . In gravi- reorienting plant seedlings, PIN3 and PIN7 change their location stimulated plants, more auxin accumulates in the lower side than to the new bottom of the plasma membrane, allowing auxin to the upper side of shoots and roots in a horizontal position, causing move to the bottom side of the root cap. Thereafter, PIN2 the upward bending of shoots and downward bending of roots. localizing to the proximal side of the plasma membrane in the Auxin redistribution following gravistimulation has been verified. lateral root cap and the epidermis, plays a role in the basipetal Indeed, molecular genetics with many gravitropic mutants has transportation of auxin from the bottom side of the root cap to revealed the importance of the roles of auxin transport, the elongation zone. In gravistimulated roots, auxin redistribution redistribution, and response in gravitropism . In particular, the is thus established, and transcriptional regulation depending on identification of an auxin efflux carrier PIN-FORMED (PIN) was a the auxin level on the upper and lower sides leads to downward significant breakthrough in our understanding of the mechanisms bending. Spaceflight experiments with cucumber seedlings in the for asymmetric auxin transport and distribution in gravistimulated Space Shuttle and the International Space Station (ISS) showed 15,16 shoots and roots . However, unlike some PINs, the pattern of that the endodermal cells relocalize an auxin efflux carrier, CsPIN1, ABCB expression and the phenotypes of ABCB mutants indicate because of gravistimualtion in space and laterally transport auxin 17–19 that it is not directly involved in asymmetric redistribution of auxin from the upper to lower flank . npj Microgravity (2021) 2 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; K. Takahashi et al. gravitropic response. Thus, amyloplast position itself may play an Table. 1. List of abbreviations. important role in gravity sensing/signaling as discussed in next section “Gravity sensor in plants.” However, the mechanism ARHGAP Rho GTPase Activating Protein underling the LAZY polarization upon amyloplast sedimentation BRX Brevis radix in gravisensing cells still remains unknown. CaN Calcineurin Auxin biosynthesis and distribution in microgravity were also CBF/DREB1 C-repeat binding factor / dehydration-responsive examined by spaceflight experiments in some plant species. Most element-binding 1 of those results showed no differences between space- and ground-grown seedlings. Recently, transformed Arabidopsis lines CCL Conserved C terminus in the LAZY1 family 2+ 2+ with GFP reporter gene, pDR5r::GFP, pTAA1::TAA1-GFP, pSCR:: CICR Ca -induced Ca -release SCR–GFP and pARR5::GFP, were used for spaceflight experiments D6PK Serine/threonine-protein kinase on ISS . The expressions of the auxin artificial AuxRE promoter DCC Disposable cell culture construct (pDR5r::GFP), Tryptophan Aminotransferase of Arabi- DEK1 Defective Kernel 1 dopsis fusion (pTAA1::TAA1-GFP) and Scarecrow fusion (pSCR:: SCR–GFP) were used to monitor auxin level, auxin production and GNOM ARF guanine-nucleotide exchange factor auxin-related signals, respectively. There were no differences in HEK Human Embryonic Kidney the expression patterns and levels of those genes in the primary ISS International Space Station root tips of seedlings grown under microgravity and 1 G ground LAZY1 Plant-specific genes with unknown molecular functions conditions. These results implied that auxin gradient in plants is that are involved in gravitropism established independently of gravity. On the other hand, space- MCA Mid1 (Yeast mechanosensitive channel) complementing flight experiments with pea and maize seedlings showed altered activity protein polar auxin transport in microgravity; polar auxin transport in MSC Mesenchymal stem cells microgravity was decreased in pea epicotyls and accelerated in MSL MscS (bacterial mechanosensitive channel of small maize coleoptiles and mesocotyls compared with the 1 G 28,29 conductance) like protein controls . Recent spaceflight experiments immunohistochemi- cally compared PsPIN1 localization in etiolated pea epicotyls NFAT Nuclear factor of activated T cells grown under microgravity and 1 G conditions in space . PsPIN1 NOX NADPH oxidases proteins were detected in the lower side of the plasma membrane 2+ OSCA reduced hyperosmolality- induced [Ca ] increase 1 of 80–90% endodermal cells under artificial 1 G conditions, PDZ Domains bind to a short region of the C-terminus of other whereas number of those endodermal cells showing polarized specific proteins PsPIN1 localization significantly decreased in microgravity. The PLC Phospholipase C authors consider the change in PsPIN1 localization pattern as a RANKL Receptor activator of nuclear factor-B ligand possible cause for the reduction of polar auxin transport in pea epicotyls under microgravity conditions. In maize seedlings, RCC Regulator of chromosome condensation interestingly, the enhanced accumulation of ZmPIN1and the RCN Reticulocalbin alteration of ZmPIN1a localization in parenchymatous cells of RLD Regulator of chromosome condensation-like domains the coleoptiles were likely responsible for the enhanced polar ROS Reactive oxygen species auxin transport in microgravity . However, species differences of TRPC Canonical transient receptor potential channel polar auxin transport in microgravity are mysterious. Thus, the PIN-mediated auxin transport and distribution are essential parts of plant gravimorphogenesis. It should be Some factors that could be involved in PIN polarization and emphasized that some PIN proteins were verified to be gravity thereby asymmetric auxin flow have been reported. That is, responsive in their relocalization on the plasma membrane of the proteins such as RCN1, PINOID, and D6PK regulate PIN phosphor- gravisensing cells by spaceflight experiments. Polarization and 2,20,21 ylation/dephosphorylation . The phosphorylation status of function of LAZY1/LAZY1-like proteins appear to play a key role PIN proteins, together with GNOM-dependent PIN recycling in the gravity-induced PIN relocalization and thereby asymmetric processes, is hypothesized to participate in polar localization of auxin flow. To understand the entire regulatory mechanism of PIN proteins on the plasma membrane. Dynamics of microfila- plant gravimorphogenesis, it is important to clarify the grav- ments and microtubules (MTs) is an important factor involved in iperception mechanism that leads to the regulation of LAZY1/ the regulation of trafficking of auxin transporters. It is reported LAZY1-like proteins polarization and PIN relocalization in that Sorting Nexin 1 (SNX1) plays a role in PIN2 recycling via gravisensing cells. interaction with MTs-associated protein CLASP . Recently, it was found that LAZY1 regulates PIN relocalization in Gravity sensor in plants gravisensing cells and determines negative gravitropism in shoots 23,24 As discussed above, plants have a mechanism for gravity sensing and positive gravitropism in roots . Interestingly, the alteration using the sedimentation of organelles in order to establish the of two amino acids in LAZY1 was found to successfully switch asymmetric transport of hormones. The most widely accepted negative gravitropism to positive gravitropism in Arabidopsis model for plant gravity sensing is the starch-statolith hypothesis, shoots . Furthermore, it was revealed that upon amyloplast in which intracellular sedimentation of the starch-filled organelle sedimentation, LAZY1/LAZY1-like proteins get polarized to the (amyloplast) plays a crucial role in the events triggering the initial plasma membrane of the bottom side of gravisensing cells . The 32–34 phases of gravity sensing in plants . Recent live-cell imaging conserved C terminus in the LAZY1 family (CCL) domains interact technology has revealed, however, that the movement of the with the Brevis radix (BRX) domains of the regulator of amyloplast is not static but saltatory because its dynamics are chromosome condensation (RCC)-like domains (RLD) proteins, dependent on both the gravity vector and intracellular environ- thereby polarly recruiting RLD from the cytoplasm to the plasma ments such as those of the cytoskeleton and vacuole . In the membrane . It was demonstrated that RLD1–4 localize in the root cap and modulate auxin transport through regulation of PIN shoot statocytes (endodermal cells) of Arabidopsis thaliana, localization, possibly via a GNOM-like function in PIN trafficking . the amyloplasts are tightly surrounded by the vacuolar membrane This process is required for controlling polarized auxin flow and and are supposed to interact with actin filaments . The abnormal Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 2 K. Takahashi et al. behavior of the vacuolar membrane, however, pushes the segment of MscS (mechanosensitive channel of small conduc- amyloplasts to the periphery of the cell in the agravitropic tance) in E. coli . Among the 10 members of the MSL protein in mutant, shoot gravitropism (sgr) 2, which restricts the movement of Arabidopsis, MSL1 localizes to the inner mitochondrial membrane, the amyloplasts and renders them nonsedimentable . Therefore, whereas MSL2 and 3 are found at the inner plastid membrane, inflorescence stems in sgr2 mutants do not sense gravity and do and MSL8, 9, and 10 localize to the plasma membrane . MSL2 and not show a gravitropic response because of nonsedimentable 3 regulate the size and shape of the plastid, and MSL8 is required amyloplasts . distorted1 (dis1)/actin-related protein 3 (arp3) for the rehydration of the pollen grain, indicating that a major role mutants possess irregular thick actin bundles surrounding of MSL proteins is osmotic regulation. Yeast two-hybrid assays amyloplasts in their root statocytes (columella cells), and demonstrated that MSL2 and 3 interact with each other, consequently, the amyloplasts do not sediment fully from the suggesting that some MSLs can form heteromeric channels . actin filaments, resulting in a reduced gravitropic response in MCA proteins have been identified as complements of the mid1 2+ 50 roots . sgr9 mutants also have nonsedimentable, clustered mutant of the yeast that is defective in Ca influx . MCA1 and amyloplasts entangled with actin filaments in the endodermal MCA2 localize in the plasma membrane and mediate the cold- 2+ cells because of an excess of interaction between the amyloplasts induced Ca response that leads to cold tolerance according to and the actin filaments and exhibit a weak gravitropic response . the CBF/DREB1-independent pathway . MCA1, but not MCA2, is These abnormal phenotypes in both dis1 and sgr9 mutants were required for the penetration of roots into harder agar and results compensated by disrupting actin filaments, such as through the in the retardation of the leafing and bolting of the mutant. The 36,38 use of the actin filament-depolarizing drug latrunculin B , expression levels of MCA1 and MCA2 were increased under suggesting that the actin filament is not an essential component hypergravity conditions in the absence of light, and the hypocotyl for either gravity sensing or gravitropic responses, but rather acts elongation under these conditions was attenuated in the over- as an intracellular component affecting amyloplast dynamics. expressing seedlings. Therefore, MCA proteins might be respon- Taken together, the dynamics of the vacuolar membrane and sible for resistance to gravity . OSCA proteins, homologous to the actin filaments could diffuse amyloplasts from the bottom of the TMEM63 family of proteins known throughout eukaryotes, have cell, leading to the nonstatic, saltatory behavior of the plant been identified from Arabidopsis mutants exhibiting a low 2+ 53 statolith and amyloplast. hyperosmolality-induced Ca response . An ortholog of animal A long-lasting question regarding gravity sensing in plants is Piezo protein, the mechanosensitive cation channel for touch how the physical process of amyloplast sedimentation is sensation and vascular development, is commonly conserved in converted into intracellular signals. The most reasonable models, monocots and suppresses the systemic movement of viruses . such as the inner ear (hair cell) system of vertebrates, suggest that Mechanosensitive cation channel activity in MCAs and OSCAs amyloplast sedimentation activates mechanosensitive ion chan- has been recorded using patch-clamp techniques with hetero- 39–41 55,56 nels via actin, resulting in intracellular ionic signaling . logous expression in Xenopus oocytes and HEK cells . Electro- Changes in the gravity vector (inclining the specimens) elevate physiological studies using Arabidopsis mutants and Xenopus 42,43 cytosolic calcium concentrations in Arabidopsis seedlings . This oocytes have revealed that MSL proteins show a preference for 2+ Ca response is attenuated with latrunculin B and mechan- anions over cations, leading to depolarization of the plasma 3+ 42 2+ osensitive ion channel blockers such as Gd , supporting a membrane and the following Ca response through the model involving actin filaments that function as a tether to activation of voltage-dependent cation channels . Most recently, 2+ activate mechanosensitive channels . Actin filaments may have a lack of rapidly activated mechanosensitive Ca -permeable both a positive role in the activation of mechanosensitive channel activity (RMA) was reported in Arabidopsis DEK1 channels upon gravity stimulation and a negative role in the mutants . Although there is no sequence homology, AtDEK1 sedimentary dynamics of amyloplasts as discussed above. To has a high number of transmembrane helices as with mammalian 2+ demonstrate this, direct observations of Ca responses in both Piezo proteins, and RMA shows low conductance and rapid the shoot endodermal and root columella cells are needed. An inactivation. These electrophysiological studies have demon- alternative model is the position-sensing hypothesis in which the strated that the thresholds of membrane stretch for the activation, spatial distribution of amyloplasts upon gravity stimulation is conductance, and inactivation time constant of plant mechan- detected as a signal for gravity sensing . In this hypothesis, a osensitive channels are varied, even within the same family. putative machinery, rather than mechanosensitive channels Although series of proteins and their physiological roles have sensing the gravitational force exerted on the amyloplasts, detects been characterized in numerous aspects, the mechanosensitive the position (state) of the amyloplasts in gravity sensing cells, channels responsible for gravitropism have not yet been consistent with data indicating that gravitropic responses in wheat identified. Overlapping tissue expression patterns suggest that coleoptile are dependent on the angle of inclination of the mechanosensitive channels in the same tissue share physiological specimens but not on the amplitude of the gravitational force . functions. Thus, most mutants, even those that lacking five MSLs These data suggest a variety of gravity sensing mechanisms in 47 (MSL4, 5, 6, 9, and 10), do not show a significant phenotype . diverse plant species such as monocots or dicots which are, Interestingly, most mechanosensitive channels are expressed in however, quite different from those of animals. 2+ the vasculature, where gravity-induced Ca response is observed . In root statocytes of Brassica grown in ISS, ten- Mechanosensitive channels in plants minute onset (µg to 1 g) or removal (1 g to µg) of a gravity- 2+ induced Ca response in absence of a significant statolith As discussed above, gravity is the force that generates several displacement . Changes in gravity vector and magnitude effects such as the weight leading to the sedimentation of 2+ 46 promote a Ca response with similar kinetics . It suggests that amyloplast statoliths, deformation and compression of the cells, and fluid shift in vasculature, all of which generate mechanical multiple mechanosensitive channels in plasma- and endomem- stresses in plasma and endomembranes. One of the earliest branes could be differentially activated by gravity, and promotes a 2+ 2+ response to changes in gravity vector and magnitude is the Ca small Ca response that is amplified by common intracellular response, which has been reported in many plant species . Thus, machineries. Thus, pharmacological studies suggest that gravity- 2+ 2+ 2+ it is plausible that mechanosensitive channels are the primary induced Ca response is greatly amplified by Ca -induced Ca - 2+ sensors of plant graviperception evoking the Ca response. release (CICR) from organelles through signaling cascades, MSL proteins share the C-terminal transmembrane (TM) including PLC activation . Subcellular and tissue-specific distribu- 2+ segment corresponding to the pore-forming transmembrane tions of gravity-induced Ca response and the underlying npj Microgravity (2021) 2 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA K. Takahashi et al. Fig. 2 YAP-mediated 3D organ/tissue formation withstanding gravity. a Mechanical negative feedback maintaining YAP activity in a cell. b YAP-mediated 3D organ/tissue formation withstanding gravity. In (a), YAP/TAZ acts as a mechanotransducer and mechanoeffector. As a mechanotransducer, it provides physical inputs, including gravity activation of YAP/TAZ that leads to an expansion of organ size. As a mechanoeffector, it activates YAP, which, in turn, controls F-actin turnover, leading to the suppression of YAP as part of a negative feedback mechanism. F-actin turnover controls the cell/tissue tension that mediates 3D organogenesis. b YAP is essential for the formation of complex 3D organs by coordinating 3D tissue shape (left) and tissue alignment (right). In response to external forces, including gravity, YAP activates (1) ARHGAP18 expression, which mediates (2) contractile actomyosin formation controlling (3) tissue tension. Tissue tension is required for both (4) cell stacking to form a 3D tissue shape and (5) fibronectin assembly required for adjacent tissue alignment, e.g., the alignment of the lens and eye-cup. molecular mechanisms should be investigated more deeply in the lens and eye cup (Fig. 2b). It is hypothesized that a YAP- order to understand graviperception mechanisms in plants. mediated response to gravity is involved in the maintenance of bones and skeletal muscles, since YAP is known to control the organ size through Hippo signaling and is expressed in the stem GRAVITY SENSING IN ANIMALS cells of many organs, including skeletal muscles. YAP-mediated gravity response and 3D organ growth and YAP orchestrates the response to gravity by controlling maintenance actomyosin contractility by negatively regulating F-actin polymer- ization through its target gene ARHGAP18. Since actomyosin both Although plants and animals share common mechanisms for generates mechanical forces and acts as a mechanical sensor , gravity sensing, such as the homologous mechanosensitive ion actomyosin is a putative gravity sensor. Gravity could promote channels discussed above, the transcriptional coactivator Yes- F-actin polymerization, activating YAP in order to maintain a 3D associated protein (YAP) is a mechanosensitive machinery specific organ size. This is consistent with reports that simulated to animals. The relationship between gravity and YAP was first microgravity inhibits the osteogenic differentiation of mesench- revealed by the analysis of the medaka fish YAP mutant. The body ymal stem cells via the de-polymerization of F-actin that inhibits of this mutant was flattened because of its inability to withstand TAZ nuclear translocation . Further detailed studies are necessary gravity . This demonstration that YAP is required for withstanding to elucidate the mechanisms by which the YAP-mediated gravity gravity in generating a 3D body/organ shape first suggested that response is linked with organ growth and maintenance. These YAP not only transduces gravity responses as a mechano- studies will be useful in alleviating compromises to health, such as transducer , but more strikingly acts as a mechano-effector for the loss of bones and skeletal muscles that arises from periods of withstanding gravity, forming a mechanical negative-feedback . “life in space.” Since YAP is the key regulator orchestrating organ growth , this review will focus on the role of YAP in linking gravity response with organ growth and maintenance. Gravity sensing in bones YAP and its paralog TAZ (transcriptional coactivator with PDZ- Bone loss is one of the major health problems facing organisms binding motif) act as transcriptional co-activators, mainly in the that experience life in space. The structure of bones is shown in 63,64 nucleus . YAP nuclear localization is controlled mainly by the Fig. 3. Here we discuss the sensing mechanisms of gravitational Hippo pathway and F-actin-mediated signaling responses to loads in the trabecular bone and cortical bone. diverse signals, e.g., growth factors and mechanical stimuli . YAP is able to expand the organ size when constitutively Mechanical sensing by osteocytes, a commander for activated and is involved in such diseases as cancer and osteoclasts and osteoblasts fibrosis . Bone homeostasis is maintained as a result of the balanced action The discovery that YAP could act as a mechanoeffector of osteoblasts for bone formation and osteoclasts for bone uncovered a negative feedback control of YAP activity: F-actin resorption . In bone destructive diseases, such as osteoporosis, polymerization activates YAP and its target gene ARHGAP18 and bone resorption is favored over bone formation, leading to bone then negatively regulates F-actin polymerization, suppressing YAP activity (Fig. 2a). This is a mechanical negative feedback since loss. In particular, bone loss in unloading conditions, such as the negative regulation of F-actin polymerization by YAP microgravity in space, is caused by enhanced bone resorption by optimizes F-actin turnover and maximizes actomyosin contrac- osteoclasts and suppressed bone formation by osteoblasts. tility, i.e., cell/tissue tension. Cell/tissue tension then controls 3D Osteocytes, another type of bone cell, are differentiated from tissue formation and tissue alignment necessary for generating a osteoblasts and embedded in the bone matrix have many 3D organ consisting of multiple tissues, e.g., an eye consisting of dendrites formed through osteocytogenesis that communicate Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 2 K. Takahashi et al. Periosteum Fibrous layer Long bone Cortical bone Fibroblast Periosteum SSC Osteoblast Blood vessel Osteocyte Lacuna Canaliculi Fig. 3 The structure of the long bone, cortical bone, and periosteum. Osteocytes are embedded in the lacuna of the bone matrix and are connected with each other through dendrites surrounded by canaliculi. At the periphery of the bone, SSCs, and fibroblasts form the periosteum together with osteoblasts. Osteocytes and the periosteum are mechanical sensors in the bone tissue. either with each other or with the osteoblasts and osteoclasts at bones except for joints, generates the cortical bone in physiolo- the bone surface . Osteocytes also have a critical role in bone gical and pathological situations through the provision of homeostasis, functioning as a commander for osteoclasts and osteoblasts . The periosteum contains two layers: an outer layer osteoblasts by regulating the expression of genes involved in the of fibroblasts and an inner layer composed of bone-forming receptor activator of nuclear factor-B ligand (RANKL), an osteoclast osteoblasts. Although the periosteum is not highly sensitive to differentiation factor, and as a negative regulator of osteoblast mechanical loading compared to the endocortical surface, it differentiation by sclerostin (Sost) . Importantly, expressions of nonetheless responds to loading and gives rise to bones in a these genes vary in response to mechanical loading or unloading variety of animal models. The unloading model of the hind limb to osteocytes in the bone. Osteocyte cell body and dendrites reduces bone formation in the cortical bone as well as in the reside in the lacunae and canaliculi, respectively, and mechanical trabecular bone . Conversely, periosteal bone formation is loading induces minute changes in the structure of the bone that stimulated by enhanced loading using in vivo models of axial 79,80 generate interstitial fluid flow in the lacuno-canalicular system. loading and three-point bending . The alteration of gene This flow acts as a mechanical loading, similar as pressure and expression patterns and cell morphologies within the periosteum shear stress, and affects osteocytes directly . after loading provides evidence that periosteal cells sense loading 80,81 Such mechanical loadings to osteocytes as described above can stimuli . activate mechanotransduction mediators such as ion channels, connexins, integrins, and cytoskeleton-related molecules .In Mechanical loading is possibly translated into bone formation addition, the cytosolic signaling adapter protein p130Cas, a through the periosteal skeletal stem cell cellular mechanosensing molecule , is involved in the regulation Among the cells responsible for sensing physical loading is of bone homeostasis in response to mechanical loading in skeletal stem cell (SSC) because loading-induced bone formation osteocytes . Interestingly, p130Cas translocates into the nucleus requires activation of the periosteal SSC to give rise to osteoblasts. and negatively regulates NF-κB activity to suppress bone The periosteal SSC displays a unique gene expression pattern and resorption by downregulating the expression of RANKL. These exhibits high regenerative capacity in response to bone injury findings suggest that the p130Cas- NF-κB axis in osteocytes is a 82 when compared to bone marrow skeletal stem cells (BMSC) .A potential target for treatment against disuse osteoporosis. recent study has revealed that Cathepsin K (CTSK)-lineage Although the critical significance of mechanical loading to the populations within the periosteum contain postnatal self- bone has been clearly elucidated, a large portion of the molecular renewing and multipotent stem cells . The deletion of Osterix, mechanisms underlying the mechanical regulation of bone encoded by the Sp7 gene, in CTSK-lineage cells results in impaired homeostasis is not understood. Efforts to clarify these mechanisms bone formation and fracture healing . Furthermore, Prx1-lineage will be a promising strategy to prevent bone loss during future mesenchymal cells that contain SSC sense loading stimuli through space missions. their primary cilia indicate loading-dependent bone forma- 78,82 + + tion . More recently, Nestin and Leptin cells have been Periosteum might sense physical loading shown to generate osteoblasts for periosteal bone formation .On The deterioration of the bone microarchitecture during spaceflight the other hand, some studies have shown that loading alters gene occurs not only in the trabecular bone but also in the cortical expression patterns, including extracellular molecules in osteo- 76 85 bone . It is thought to be triggered by enhanced osteoclast- blasts . Thus, the osteoblast may function as a mechanotransdu- mediated bone resorption at the endocortical surface and cer that induces osteogenic differentiation of SSCs in the suppressed bone-forming activity in the periosteum. The perios- periosteum. Accordingly, direct or indirect loading can activate teum, the highly vascularized outer membrane that covers all several types of SSC to induce cortical bone formation. npj Microgravity (2021) 2 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA K. Takahashi et al. Fig. 4 Stress fiber remodeling in MSCs exposed to simulated microgravity as analyzed by confocal fluorescence microscopy. MSCs expressing Lifeact-TagGFP2 contained thick stress fibers under 1 G conditions (a), whereas an exposure to simulated microgravity for 6 h led to the appearance of thinner stress fibers (b). The images in (a) and (b) showing the same field of view, were recorded, processed and presented in an identical condition (Kobayashi, unpublished). Scale bar: 20 μm. Gravity sensing in muscles them from proteasomal degradation . ROS production mediated by TRPC3 and NOX2 coupling causes cardiac muscle atrophy in Muscle atrophy is another major health problem in life in space 94,95 stressed hearts, in which the hemodynamic load is reduced . and involves the decrease of muscle mass in response to the Therefore, TRPC channels might have dual roles in unloading- reduction of hemodynamic loads. It is known to be caused by induced muscle atrophy: the first is the regulation of myoblast microgravity, long term bed rest, and cancer cachexia . proliferation via CaN activation, and the second is the production Unloading-induced muscle wasting is mediated by a decrease of of ROS, which induces catabolic remodeling of muscle tissue. protein synthesis in the homeostasis of muscle cells and an increase of catabolism. Consequently, there must be a molecule that senses and transduces the signals originating from mechan- Gravity sensing in mesenchymal stem cells ical loading. One of the candidates for such a load transducer is a Mesenchymal stem cells (MSCs) are crucial in the field of nonselective cation channel, the canonical transient receptor regenerative medicine by virtue of their self-renewal and multi- potential channel (TRPC). Members of the TRPC channels, namely, differentiation potentials . MSC self-renewal and differentiation TRPC1, TRPC3, and TRPC6, are reportedly activated downstream of are known to be controlled by a diverse set of soluble factors, mechanical signals in addition to phospholipase C-coupled cell including growth factors or cytokines. In addition, the fate of MSCs surface receptor activation . TRPC channels play important roles has been shown to be influenced by mechanical stresses or in the activation of protein phosphatase calcineurin (CaN). CaN surrounding physical microenvironments, such as substrate 2+ regulates the Ca -dependent transcription factor, the nuclear stiffness , or changes in gravity. Many space experiments and factor of activated T cells (NFAT), and the peroxisome proliferator- ground-based studies have demonstrated that MSCs are very activated receptor γ . Both proteins are important for myogen- sensitive to the modulation of gravitational stimuli and exhibit esis. Exposure of C2C12 skeletal myoblasts to microgravity induces various responses against such effects . The exposure of MSCs to the reduction of TRPC1 expression, which arrests the cell cycle at microgravity or simulated microgravity induces characteristic the G2/M phase, thereby inhibiting myoblast proliferation . The physiological responses, including remodeling of the cytoskeleton 99,100 importance of TRPC1 has also been demonstrated in muscle and the disruption of the stress fiber , reduced activity in regrowth after unloading-induced atrophy. Hind limb unloading transcriptional coactivator YAP/TAZ , suppression of osteoblastic 101,102 induces the reduction of TRPC1 expression, which persists even differentiation, and the promotion of adipogenesis , some of after reloading . The expression of the TRPC3 channel is also which were also observed in other nonspecialized animal cells. suppressed at complete atrophy and in the early recovered How can MSCs sense a microgravity environment? Ordinary phase . These changes in the expression of the TRPC1 and TRPC3 mechanical forces, including stretch or shear stress, can be sensed channels are consistent with the muscle mass, suggesting by animal cells through cell mechanosensors that convert that these channels play important roles in load-dependent mechanical stimuli into electrical or chemical signals. To date, muscle growth. mechanosensitive channels, focal adhesion proteins (p130Cas and It is widely accepted that oxidative stresses caused by the Talin), and actin fibers have been established to function as aberrant production of reactive oxygen species (ROS) or reactive mechanosensors for various types of cells. It has been postulated nitrogen species are key regulators for catabolic muscle wasting . that MSCs also utilize these common sensors to detect changes in ROS are produced as a byproduct of the mitochondrial respiratory gravity, since MSCs have no specific gravity sensors, as is the case chain or are produced enzymatically by NADPH oxidases (NOX) for organs such as the animal gravity sensor statocyst. Recent within the cell. In cardiac muscles, ROS production by the NOX2 studies have proposed that the cytoskeleton may function as an 2+ protein is physiologically important for Ca homeostasis and is initial sensor for microgravity . In the early phase (30 min to 6 h) activated mechanically during diastole . However, in pathological of exposure to microgravity, environmental changes experienced conditions, NOX2-mediated ROS production causes cardiac by the cytoskeleton have been observed, including a reduced remodeling in response to various stresses. It has also been noted amount or thinning of stress fibers (unpublished data in Fig. 4) that pathological situations in muscle tissue can engender and the redistribution of microtubules. In addition, genetic 2+ 2+ abnormal Ca signaling. Since some NOX isoforms require Ca restoration of the arrangement of actin fibers or the pharmaco- for activation, it is plausible that there exists a crosstalk between logical stabilization of actin cytoskeleton could maintain the 2+ pathological NOX activation and abnormal Ca signaling. TRPC3 osteogenic differentiation of MSCs under modeled micrograv- 68,99 and NOX2 proteins exist at this crossroads of signaling pathways. ity . This indicates that changes in the actin cytoskeleton in the Additionally, it has been demonstrated that the TRPC3 channels cells transferred under microgravity conditions could have a play an important role in NOX2 protein stabilization by protecting crucial role in cellular responses against changed gravity. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 2 K. Takahashi et al. However, it remains unclear if the cytoskeleton acts as an initial understanding cellular responses to gravity will form the and primary mechanosensor for gravity sensing . It has been foundations of living in space. proposed that the loss of gravitational forces acting on heavy organelles, including the nucleus and mitochondria, could affect DATA AVAILABILITY the cytoskeleton. Further studies will provide deeper insight All data are available in the main text. regarding gravity sensing and transduction. Received: 5 February 2020; Accepted: 14 December 2020; Gravity sensitivity of the cell cycle Regulation of the cell cycle is crucial for the maintenance of organs, such as bones and muscles. 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J. & van Loon, J. J. The role article’s Creative Commons license and your intended use is not permitted by statutory of the cytoskeleton in sensing changes in gravity by nonspecialized cells. FASEB regulation or exceeds the permitted use, you will need to obtain permission directly J. 28, 536–547 (2014). from the copyright holder. To view a copy of this license, visit http://creativecommons. 104. Miyawaki, A. & Niino, Y. Molecular spies for bioimaging–fluorescent protein- org/licenses/by/4.0/. based probes. Mol. Cell 58, 632–643 (2015). 105. Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008). © The Author(s) 2021 npj Microgravity (2021) 2 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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