Gravity-sensing Cells Research Articles

Simulated Microgravity-Induced Changes to Drug Response in Cancer Cells Quantified Using Fluorescence Morphometry.

Unlike plants that have special gravity-sensing cells, such special cells in animals are yet to be discovered. However, microgravity, the condition of apparent weightlessness, causes bone, muscular and immune system dysfunctions in astronauts following spaceflights. Decades of investigations show correlations between these organ and system-level dysfunctions with changes induced at the cellular level both by simulated microgravity as well as microgravity conditions in outer space. Changes in single bone, muscle and immune cells include morphological abnormalities, altered gene expression, protein expression, metabolic pathways and signaling pathways. These suggest that human cells mount some response to microgravity. However, the implications of such adjustments on many cellular functions and responses are not clear. Here, we addressed the question whether microgravity induces alterations to drug response in cancer cells. We used both adherent cancer cells (T98G) and cancer cells in suspension (K562) to confirm the known effects of simulated microgravity and then treated the K562 cells with common cancer drugs (hydroxyurea and paclitaxel) following 48 h of exposure to simulated microgravity via a NASA-developed rotary cell culture system. Through fluorescence-guided morphometry, we found that microgravity abolished a significant reduction (p < 0.01) in the nuclear-to-cytoplasm ratio of cancer cells treated with hydroxyurea. Our results call for more studies on the impact of microgravity on cellular drug response, in light of the growing need for space medicine, as space exploration grows.

Understanding the role of starch sheath layer in graviception of Alternanthera philoxeroides: a biophysical and microscopical study.

Plants' ability to sense and respond to gravity is a unique and fundamental process. When a plant organ is tilted, it adjusts its growth orientation relative to gravity direction, which is achieved by a curvature of the organ. In higher, multicellular plants, it is thought that the relative directional change of gravity is detected by starch-filled organelles that occur inside specialized cells called statocytes, and this is followed by signal conversion from physical information to physiological information within the statocytes. The classic starch statolith hypothesis, i.e., the starch accumulating amyloplasts movement along the gravity vector within gravity-sensing cells (statocytes) is the probable trigger of subsequent intracellular signaling, is widely accepted. Acharya Jagadish Chandra Bose through his pioneering research had investigated whether the fundamental reaction of geocurvature is contractile or expansive and whether the geo-sensing cells are diffusedly distributed in the organ or are present in the form of a definite layer. In this backdrop, a microscopy based experimental study was undertaken to understand the distribution pattern of the gravisensing layer, along the length (node-node) of the model plant Alternanthera philoxeroides and to study the microrheological property of the mobile starch-filled statocytes following inclination-induced graviception in the stem of the model plant. The study indicated a prominent difference in the pattern of distribution of the gravisensing layer along the length of the model plant. The study also indicated that upon changing the orientation of the plant from vertical position to horizontal position there was a characteristic change in orientation of the mobile starch granules within the statocytes. In the present study for the analysis of the microscopic images of the stem tissue cross sections, a specialized and modified microscopic illumination setup was developed in the laboratory in order to enhance the resolution and contrast of the starch granules.

Gravity sensing and responses in the coordination of the shoot gravitropic setpoint angle.

Gravity is one of the fundamental environmental cues that affect plant development. Indeed, the plant architecture in the shoots and roots is modulated by gravity. Stems grow vertically upward, whereas lateral organs, such as the lateral branches in shoots, tend to grow at a specific angle according to a gravity vector known as the gravitropic setpoint angle (GSA). During this process, gravity is sensed in specialised gravity-sensing cells named statocytes, which convert gravity information into biochemical signals, leading to asymmetric auxin distribution and driving asymmetric cell division/expansion in the organs to achieve gravitropism. As a hypothetical offset mechanism against gravitropism to determine the GSA, the anti-gravitropic offset (AGO) has been proposed. According to this concept, the GSA is a balance of two antagonistic growth components, that is gravitropism and the AGO. Although the nature of the AGO has not been clarified, studies have suggested that gravitropism and the AGO share a common gravity-sensing mechanism in statocytes. This review discusses the molecular mechanisms underlying gravitropism as well as the hypothetical AGO in the control of the GSA.

Autophagy promotes organelle clearance and organized cell separation of living root cap cells in Arabidopsis thaliana.

ABSTRACTThe root cap is a multilayered tissue covering the tip of a plant root that directs root growth through its unique functions, such as gravity sensing and rhizosphere interaction. To maintain the structure and function of the root cap, its constituent cells are constantly turned over through balanced cell division and cell detachment in the inner and outer cell layers, respectively. Upon displacement toward the outermost layer, columella cells at the central root cap domain functionally transition from gravity-sensing cells to secretory cells, but the mechanisms underlying this drastic cell fate transition are largely unknown. Here, using live-cell tracking microscopy, we show that organelles in the outermost cell layer undergo dramatic rearrangements. This rearrangement depends, at least partially, on spatiotemporally regulated activation of autophagy. Notably, this root cap autophagy does not lead to immediate cell death, but is instead necessary for organized separation of living root cap cells, highlighting a previously undescribed role of developmentally regulated autophagy in plants. This article has an associated ‘The people behind the papers’ interview.

LAZY2 controls rice tiller angle through regulating starch biosynthesis in gravity-sensing cells.

Rice (Oryza sativa) tiller angle is a key component for achieving ideal plant architecture and higher grain yield. However, the molecular mechanism underlying rice tiller angle remains elusive. We characterized a novel rice tiller angle mutant lazy2 (la2) and isolated the causative gene LA2 through map-based cloning. Biochemical, molecular and genetic studies were conducted to elucidate the LA2-involved tiller angle regulatory mechanism. The la2 mutant shows large tiller angle with impaired shoot gravitropism and defective asymmetric distribution of auxin. We found that starch granules in amyloplasts are completely lost in the gravity-sensing leaf sheath base cells of la2, whereas the seed development is not affected. LA2 encodes a novel chloroplastic protein that can interact with the starch biosynthetic enzyme Oryza sativa plastidic phosphoglucomutase (OspPGM) to regulate starch biosynthesis in rice shoot gravity-sensing cells. Genetic analysis showed that LA2 regulates shoot gravitropism and tiller angle by acting upstream of LA1 to mediate lateral auxin transport. Our studies revealed that LA2 acts as a novel regulator of rice tiller angle by specifically regulating starch biosynthesis in gravity-sensing cells, and established the framework of the starch-statolith-dependent rice tiller angle regulatory pathway, providing new insights into the rice tiller angle regulatory network.

A single-cell analysis of the Arabidopsis vegetative shoot apex.

The shoot apical meristem allows for reiterative formation of new aerial structures throughout the life cycle of a plant. We use single-cell RNA sequencing to define the cellular taxonomy of the Arabidopsis vegetative shoot apex at the transcriptome level. We find that the shoot apex is composed of highly heterogeneous cells, which can be partitioned into 7 broad populations with 23 transcriptionally distinct cell clusters. We delineate cell-cycle continuums and developmental trajectories of epidermal cells, vascular tissue, and leaf mesophyll cells and infer transcription factors and gene expression signatures associated with cell fate decisions. Integrative analysis of shoot and root apical cell populations further reveals common and distinct features of epidermal and vascular tissues. Our results, thus, offer a valuable resource for investigating the basic principles underlying cell division and differentiation in plants at single-cell resolution.

Gravity-Sensing Tissues for Gravitropism Are Required for "Anti-Gravitropic" Phenotypes of Lzy Multiple Mutants in Arabidopsis.

Plant posture is controlled by various environmental cues, such as light, temperature, and gravity. The overall architecture is determined by the growth angles of lateral organs, such as roots and branches. The branch growth angle affected by gravity is known as the gravitropic setpoint angle (GSA), and it has been proposed that the GSA is determined by balancing two opposing growth components: gravitropism and anti-gravitropic offset (AGO). The molecular mechanisms underlying gravitropism have been studied extensively, but little is known about the nature of the AGO. Recent studies reported the importance of LAZY1-LIKE (LZY) family genes in the signaling process for gravitropism, such that loss-of-function mutants of LZY family genes resulted in reversed gravitropism, which we term it here as the “anti-gravitropic” phenotype. We assume that this peculiar phenotype manifests as the AGO due to the loss of gravitropism, we characterized the “anti-gravitropic” phenotype of Arabidopsis lzy multiple mutant genetically and physiologically. Our genetic interaction analyses strongly suggested that gravity-sensing cells are required for the “anti-gravitropic” phenotype in roots and lateral branches. We also show that starch-filled amyloplasts play a significant role in the “anti-gravitropic” phenotype, especially in the root of the lzy multiple mutant.

Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control

In many plant species, roots maintain specific growth angles relative to the direction of gravity, known as gravitropic set point angles (GSAs). These contribute to the efficient acquisition of water and nutrients. AtLAZY1/LAZY1-LIKE (LZY) genes are involved in GSA control by regulating auxin flow toward the direction of gravity in Arabidopsis. Here, we demonstrate that RCC1-like domain (RLD) proteins, identified as LZY interactors, are essential regulators of polar auxin transport. We show that interaction of the CCL domain of LZY with the BRX domain of RLD is important for the recruitment of RLD from the cytoplasm to the plasma membrane by LZY. A structural analysis reveals the mode of the interaction as an intermolecular β-sheet in addition to the structure of the BRX domain. Our results offer a molecular framework in which gravity signal first emerges as polarized LZY3 localization in gravity-sensing cells, followed by polar RLD1 localization and PIN3 relocalization to modulate auxin flow.

Gravity sensing and signal conversion in plant gravitropism.

Plant organs control their growth orientation in response to gravity. Within gravity-sensing cells, the input (gravity sensing) and signal conversion (gravity signalling) progress sequentially. The cells contain a number of high-density, starch-accumulating amyloplasts, which sense gravity when they reposition themselves by sedimentation to the bottom of the cell when the plant organ is re-orientated. This triggers the next step of gravity signalling, when the physical signal generated by the sedimentation of the amyloplasts is converted into a biochemical signal, which redirects auxin transport towards the lower flank of the plant organ. This review focuses on recent advances in our knowledge of the regulatory mechanisms that underlie amyloplast sedimentation and the system by which this is perceived, and on recent progress in characterising the factors that play significant roles in gravity signalling by which the sedimentation is linked to the regulation of directional auxin transport. Finally, we discuss the contribution of gravity signalling factors to the mechanisms that control the gravitropic set-point angle.

Auxin methylation is required for differential growth in Arabidopsis

Asymmetric auxin distribution is instrumental for the differential growth that causes organ bending on tropic stimuli and curvatures during plant development. Local differences in auxin concentrations are achieved mainly by polarized cellular distribution of PIN auxin transporters, but whether other mechanisms involving auxin homeostasis are also relevant for the formation of auxin gradients is not clear. Here we show that auxin methylation is required for asymmetric auxin distribution across the hypocotyl, particularly during its response to gravity. We found that loss-of-function mutants in Arabidopsis IAA CARBOXYL METHYLTRANSFERASE1 (IAMT1) prematurely unfold the apical hook, and that their hypocotyls are impaired in gravitropic reorientation. This defect is linked to an auxin-dependent increase in PIN gene expression, leading to an increased polar auxin transport and lack of asymmetric distribution of PIN3 in the iamt1 mutant. Gravitropic reorientation in the iamt1 mutant could be restored with either endodermis-specific expression of IAMT1 or partial inhibition of polar auxin transport, which also results in normal PIN gene expression levels. We propose that IAA methylation is necessary in gravity-sensing cells to restrict polar auxin transport within the range of auxin levels that allow for differential responses.

Plants work out which way is up

Plant Science Gravity-sensing cells in plants contain tiny grains of starch called statoliths. The orientation of the statoliths changes with the plant's orientation. The gravity-sensing cells respond to even the slightest tilt off of the established plane. Plant statoliths seem to evade the rules of physics that govern other granular materials. In live-cell imaging of young wheat shoots, Berut et al. observed that statolith piles behave more like slowly creeping liquids than like granular accumulations. The reason is that the individual statoliths are always jiggling around, perhaps because of interactions with the plant cytoskeleton. Proc. Natl. Acad. Sci. U.S.A. 10.1073/pnas.1801895115 (2018).

The role of Arabidopsis Actin-Related Protein 3 in amyloplast sedimentation and polar auxin transport in root gravitropism.

Gravitropism is vital for shaping directional plant growth in response to the forces of gravity. Signals perceived in the gravity-sensing cells can be converted into biochemical signals and transmitted. Sedimentation of amyloplasts in the columella cells triggers asymmetric auxin redistribution in root tips, leading to downward root growth. The actin cytoskeleton is thought to play an important role in root gravitropism, although the molecular mechanism has not been resolved. DISTORTED1 (DIS1) encodes the ARP3 subunit of the Arabidopsis Actin-Related Protein 2/3 (ARP2/3) complex, and the ARP3/DIS1 mutant dis1-1 showed delayed root curvature after gravity stimulation. Microrheological analysis revealed that the high apparent viscosity within dis1-1 central columella cells is closely associated with abnormal movement trajectories of amyloplasts. Analysis using a sensitive auxin input reporter DII-VENUS showed that asymmetric auxin redistribution was reduced in the root tips of dis1-1, and the actin-disrupting drug Latrunculin B increased the asymmetric auxin redistribution. An uptake assay using the membrane-selective dye FM4-64 indicated that endocytosis was decelerated in dis1-1 root epidermal cells. Treatment and wash-out with Brefeldin A, which inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus, showed that cycling of the auxin-transporter PIN-FORMED (PIN) proteins to the plasma membrane was also suppressed in dis1-1 roots. The results reveal that ARP3/DIS1 acts in root gravitropism by affecting amyloplast sedimentation and PIN-mediated polar auxin transport through regulation of PIN protein trafficking.

Transcriptional regulation of PIN genes by FOUR LIPS and MYB88 during Arabidopsis root gravitropism.

PIN proteins are auxin export carriers that direct intercellular auxin flow and in turn regulate many aspects of plant growth and development including responses to environmental changes. The Arabidopsis R2R3-MYB transcription factor FOUR LIPS (FLP) and its paralogue MYB88 regulate terminal divisions during stomatal development, as well as female reproductive development and stress responses. Here we show that FLP and MYB88 act redundantly but differentially in regulating the transcription of PIN3 and PIN7 in gravity-sensing cells of primary and lateral roots. On the one hand, FLP is involved in responses to gravity stimulation in primary roots, whereas on the other, FLP and MYB88 function complementarily in establishing the gravitropic set-point angles of lateral roots. Our results support a model in which FLP and MYB88 expression specifically determines the temporal-spatial patterns of PIN3 and PIN7 transcription that are closely associated with their preferential functions during root responses to gravity.

Dynamical and Microrheological Analysis of Amyloplasts in the Plant Root Gravity-Sensing Cells

Gravitropism in plants is one of the most controversial issues. In the most wildly accepted starch-statolith hypothesis the sedimentation movement of amyloplasts in the gravisensing columella cells primarily triggers the asymmetric distribution of auxin which leads to the differential growth of the plant root. It has been gradually recognized that the inhomogeneous structures in statocytes arising from intracellular components such as cytoskeletons significantly affect the complex movements of amyloplasts and the final gravimorphogenesis. In this letter, we implement a diffusive dynamics measurement and inplanta microrheological analysis of amyloplasts in the wild-type plants and actin cytoskeleton mutants for the first time. We found that the intracellular environment of columella cells exhibits the spatial heterogeneity and the cage-confinement on amyloplasts which is the typically characteristics in colloidal suspensions. By comparing the distinct diffusive dynamics of amyloplasts in different types of plants with the behaviors of colloidal systems in different states, we quantitatively characterized the influence of the actin organization dominated intracellular envoronments on the amyloplast movements. Furthermore, the cage-confinement strength was measured by calculating the spatial fluctuation of local apparent viscosity within the columella cells. Finally, a linear association between the initial mechanical stimulation in the columella cells the subsequent intercellular signal transduction and the final gravity response was observed and a possible gravity sensing mechanism was suggested. It suggests the existence of a potential gravity-sensing mechanism that dictates a linear frustration effect of the actin cytoskeleton on the conversion of the mechanical stimulation of amyloplasts into gravitropic signals.

A Unique HEAT Repeat-Containing Protein SHOOT GRAVITROPISM6 is Involved in Vacuolar Membrane Dynamics in Gravity-Sensing Cells of Arabidopsis Inflorescence Stem

Plant vacuoles play critical roles in development, growth and stress responses. In mature cells, vacuolar membranes (VMs) display several types of structures, which are formed by invagination and folding of VMs into the lumenal side and can gradually move and change shape. Although such VM structures are observed in a broad range of tissue types and plant species, the molecular mechanism underlying their formation and maintenance remains unclear. Here, we report that a novel HEAT-repeat protein, SHOOT GRAVITROPISM6 (SGR6), of Arabidopsis is involved in the control of morphological changes and dynamics of VM structures in endodermal cells, which are the gravity-sensing cells in shoots. SGR6 is a membrane-associated protein that is mainly localized to the VM in stem endodermal cells. The sgr6 mutant stem exhibits a reduced gravitropic response. Higher plants utilize amyloplast sedimentation as a means to sense gravity direction. Amyloplasts are surrounded by VMs in Arabidopsis endodermal cells, and the flexible and dynamic structure of VMs is important for amyloplast sedimentation. We demonstrated that such dynamic features of VMs are gradually lost in sgr6 endodermal cells during a 30 min observation period. Histological analysis revealed that amyloplast sedimentation was impaired in sgr6. Detailed live-cell imaging analyses revealed that the VM structures in sgr6 had severe defects in morphological changes and dynamics. Our results suggest that SGR6 is a novel protein involved in the formation and/or maintenance of invaginated VM structures in gravity-sensing cells.

Auxin Controls Gravitropic Setpoint Angle in Higher Plant Lateral Branches

Lateral branches in higher plants are often maintained at specific angles with respect to gravity, a quantity known as the gravitropic setpoint angle (GSA) [1]. Despite the importance of GSA control as a fundamental determinant of plant form, the mechanisms underlying gravity-dependent angled growth are not known. Here we address the central questions of how stable isotropic growth of a branch at a nonvertical angle is maintained and of how the value of that angle is set. We show that nonvertical lateral root and shoot branches are distinguished from the primary axis by the existence of an auxin-dependent antigravitropic offset mechanism that operates in tension with gravitropic response to generate angled isotropic growth. Further, we show that the GSA of lateral roots and shoots is dependent upon the magnitude of the antigravitropic offset component. Finally, we show that auxin specifies GSA values dynamically throughout development by regulating the magnitude of the antigravitropic offset component via TIR1/AFB-Aux/IAA-ARF-dependent auxin signaling within the gravity-sensing cells of the root and shoot. The involvement of auxin in controlling GSA is yet another example of auxin's remarkable capacity to self-organize in development [2] and provides a conceptual framework for understanding the specification of GSA throughout nature.

Directional Gravity Sensing in Gravitropism

Plants can reorient their growth direction by sensing organ tilt relative to the direction of gravity. With respect to gravity sensing in gravitropism, the classic starch statolith hypothesis, i.e., that starch-accumulating amyloplast movement along the gravity vector within gravity-sensing cells (statocytes) is the probable trigger of subsequent intracellular signaling, is widely accepted. Several lines of experimental evidence have demonstrated that starch is important but not essential for gravity sensing and have suggested that it is reasonable to regard plastids (containers of starch) as statoliths. Although the word statolith means sedimented stone, actual amyloplasts are not static but instead possess dynamic movement. Recent studies combining genetic and cell biological approaches, using Arabidopsis thaliana, have demonstrated that amyloplast movement is an intricate process involving vacuolar membrane structures and the actin cytoskeleton. This review covers current knowledge regarding gravity sensing, particularly gravity susception, and the factors modulating the function of amyloplasts for sensing the directional change of gravity. Specific emphasis is made on the remarkable differences in the cytological properties, developmental origins, tissue locations, and response of statocytes between root and shoot systems. Such an approach reveals a common theme in directional gravity-sensing mechanisms in these two disparate organs.

Gravity sensing and signaling

Gravitropism has attracted much attention from plant biologists. Recent studies have provided molecular evidence supporting two long-surviving hypotheses about the mechanism of gravitropism: the starch-statolith hypothesis and the Cholodney-Went hypothesis. Amyloplast movement along the gravity vector within gravity-sensing cells in the root and shoot is the most likely trigger of subsequent intracellular signaling. Several possible events leading from this signaling to differential auxin distribution within the sensing cells have been suggested recently.

Involvement of the Vacuoles of the Endodermis in the Early Process of Shoot Gravitropism in Arabidopsis

The endodermal cells of the shoot are thought to be the gravity-sensing cells in Arabidopsis. The amyloplasts in the endodermis that sediment in the direction of gravity may act as statoliths. Endodermis-specific expression of SGR2 and ZIG using the SCR promoter could complement the abnormal shoot gravitropism of the sgr2 and zig mutants, respectively. The abnormalities in amyloplast sedimentation observed in both mutants recovered simultaneously. These results indicate that both genes in the endodermal cell layer are crucial for shoot gravitropism. ZIG encodes AtVTI11, which is a SNARE involved in vesicle transport to the vacuole. The fusion protein of SGR2 and green fluorescent protein localized to the vacuole and small organelles. These observations indicate that ZIG and SGR2 are involved in the formation and function of the vacuole, a notion supported by the results of subcellular analysis of the sgr2 and zig mutants with electron microscopy. These results strongly suggest that the vacuole participates in the early events of gravitropism and that SGR2 and ZIG functions are involved.

Gravitropism in tip-growing cells.

Unicellular tip-growing cells are excellent experimental systems in which to study gravitropism because cell extension, gravity sensing and the gravity response are all confined to the apical dome. Thus various approaches can be used to determine the distinct steps of the short gravitropic signal-transduction chain, which lacks a signal-transmission phase between the gravity-sensing cells and the competent responding target cells. Single-cell systems readily allow in-vivo observation of cellular processes during gravistimulation at 1 g, centrifugation, clinostatting and in microgravity, as well as permitting fluorescence labeling. Such diverse studies have revealed fascinating information on the mechanism of gravitropic tip growth, especially on the important role of the cytoskeleton in the positioning of the statoliths and in organizing and adjusting the Spitzenkorper. A hypothesis explaining the negative and positive gravitropism of Chara rhizoids and Chara protonemata has been put forward, which emphasizes the role of the actin cytoskeleton in the process of gravitropic tip-growth. Differences in the gravitropic responses of single-cell systems, however, reflect a diversity of gravitropic mechanisms, and represent an example of parallel evolution.