Architecture and functions of stomatal cell walls in eudicots and grasses.

BACKGROUND: Like all plant cells, the guard cells of stomatal complexes are encased in cell walls that are composed of diverse, interacting networks of polysaccharide polymers. The properties of these cell walls underpin the dynamic deformations that occur in guard cells as they expand and contract to drive the opening and closing of the stomatal pore, the regulation of which is critical for photosynthesis and water transport in plants. SCOPE: Our understanding of how cell wall mechanics are influenced by the nanoscale assembly of cell wall polymers in guard cell walls, how this architecture changes over stomatal development, maturation, and aging, and how the cell walls of stomatal guard cells might be tuned to optimize stomatal responses to dynamic environmental stimuli is still in its infancy. CONCLUSION: In this review, we discuss advances in our ability to experimentally probe and quantitatively model the structure and dynamics of guard cell walls across a range of plant species, highlighting new ideas and exciting opportunities for further research into these actively moving plant cells.

Young guard cells function dynamically despite low mechanical anisotropy but gain efficiency during stomatal maturation in Arabidopsis thaliana.

Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized in Arabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D-nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild-type and cellulose-deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation.

Stomatal opening efficiency is controlled by cell wall organization in Arabidopsis thaliana.

Stomatal function in plants is regulated by the nanoscale architecture of the cell wall and turgor pressure, which together control stomatal pore size to facilitate gas exchange and photosynthesis. The mechanical properties of the cell wall and cell geometry are critical determinants of stomatal dynamics. However, the specific biomechanical functions of wall constituents, for example, cellulose and pectins, and their impact on the work required to open or close the stomatal pore are unclear. Here, we use nanoindentation in normal and lateral directions, computational modeling, and microscopic imaging of cells from the model plant Arabidopsis thaliana to investigate the precise influences of wall architecture and turgor pressure on stomatal biomechanics. This approach allows us to quantify and compare the unique anisotropic properties of guard cells with normal composition, lower cellulose content, or alterations in pectin molecular weight. Using these data to calculate the work required to open the stomata reveals that the wild type, with a circumferential-to-longitudinal modulus ratio of 3:1, is the most energy-efficient of those studied. In addition, the tested genotypes displayed similar changes in their pore size despite large differences in wall thickness and biomechanical properties. These findings imply that homeostasis in stomatal function is maintained in the face of varying wall compositions and biomechanics by tuning wall thickness.

Automated 3D segmentation of guard cells enables volumetric analysis of stomatal biomechanics.

Automating the three-dimensional (3D) segmentation of stomatal guard cells and other confocal microscopy data is extremely challenging due to hardware limitations, hard-to-localize regions, and limited optical resolution. We present a memory-efficient, attention-based, one-stage segmentation neural network for 3D images of stomatal guard cells. Our model is trained end to end and achieved expert-level accuracy while leveraging only eight human-labeled volume images. As a proof of concept, we applied our model to 3D confocal data from a cell ablation experiment that tests the "polar stiffening" model of stomatal biomechanics. The resulting data allow us to refine this polar stiffening model. This work presents a comprehensive, automated, computer-based volumetric analysis of fluorescent guard cell images. We anticipate that our model will allow biologists to rapidly test cell mechanics and dynamics and help them identify plants that more efficiently use water, a major limiting factor in global agricultural production and an area of critical concern during climate change.

StarD13 negatively regulates invadopodia formation and invasion in high-grade serous (HGS) ovarian adenocarcinoma cells by inhibiting Cdc42.

Metastasis remains the main challenge to overcome for treating ovarian cancers. In this study, we investigate the potential role of the Cdc42 GAP StarD13 in the modulation of cell motility, invasion in ovarian cancer cells. StarD13 depletion does not affect the 2D motility of ovarian cancer cells. More importantly, StarD13 inhibits matrix degradation, invadopodia formation and cell invasion through the inhibition of Cdc42. StarD13 does not localize to mature TKS4-labeled invadopodia that possess matrix degradation ability, while a Cdc42 FRET biosensor, detects Cdc42 activation in these invadopodia. In fact, StarD13 localization and Cdc42 activation appear mutually exclusive in invadopodial structures. Finally, for the first time we uncover a potential role of Cdc42 in the direct recruitment of TKS4 to invadopodia. This study emphasizes the specific role of StarD13 as a narrow spatial regulator of Cdc42, inhibiting invasion, suggesting the suitability of StarD13 for targeted therapy.

StarD13 differentially regulates migration and invasion in prostate cancer cells.

Prostate cancer is the second most commonly diagnosed cancer in men and one of the main leading causes of cancer deaths among men worldwide. Rapid uncontrolled growth and the ability to metastasize to other sites are key hallmarks in cancer development and progression. The Rho family of GTPases and its activators the GTPase-activating proteins (GAPs) are required for regulating cancer cell proliferation and migration. StarD13 is a GAP for Rho GTPases, specifically for RhoA and Cdc42. We have previously shown that StarD13 acts as a tumor suppressor in astrocytoma as well as breast and colorectal cancer. In this study, we performed a functional comparative analysis of StarD13 targets/and or interacting molecules to understand the general role that StarD13 plays in cancers. Our data highlight the importance of StarD13 in modulating several hallmarks of cancer. Findings from database mining and immunohistochemistry revealed that StarD13 is underexpressed in prostate cancers, in addition knocking down Stard13 increased cancer cell proliferation, consistent with its role as a tumor suppressor. Stard13 depletion, however, led to an increase in cell adhesion, which inhibited 2D cell migration. Most interestingly, StarD13 depletion increases invasion and matrix degradation, at least in part, through its regulation of Cdc42. Altogether, the data presented suggest that StarD13 acts as a tumor suppressor inhibiting prostate cancer cell invasion.

Differential regulation of rho GTPases during lung adenocarcinoma migration and invasion reveals a novel role of the tumor suppressor StarD13 in invadopodia regulation.

BACKGROUND: Lung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549. METHODS: We knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation. RESULTS: We observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation. CONCLUSION: In conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases. Video abstract.

Rho A and Rac1: Antagonists moving forward.

Cells detect external stimuli through cell-surface receptors. In cases where the stimulus is a cytokine or a growth factor, the cell responds by inducing modifications in the actin cytoskeleton. These changes are mediated through the Rho family of GTPases. Among these GTPases, RhoA, Rac1 and Cdc42 have been extensively studied. The activity of these proteins is closely monitored and tightly regulated through Guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that turn the "switch" on and off respectively. Crosstalk between Rho GTPases has been long studied; yet many questions are raised regarding the spatiotemporal regulation of these GTPases, particularly RhoA and Rac1. This review sheds a light on the antagonistic relationship between both GTPases and puts emphasis on the importance of cycling of RhoA activation at the focal adhesions for optimal cell migration.

The Role of Rho GTPases in VEGF Signaling in Cancer Cells.

Vascular endothelial growth factors (VEGFs) consist of five molecules (VEGFA through D as well as placental growth factor) which are crucial for regulating key cellular and tissue functions. The role of VEGF and its intracellular signaling and downstream molecular pathways have been thoroughly studied. Activation of VEGF signal transduction can be initiated by the molecules' binding to two classes of transmembrane receptors: (1) the VEGF tyrosine kinase receptors (VEGF receptors 1 through 3) and (2) the neuropilins (NRP1 and 2). The involvement of Rho GTPases in modulating VEGFA signaling in both cancer cells and endothelial cells has also been well established. Additionally, different isoforms of Rho GTPases, namely, RhoA, RhoC, and RhoG, have been shown to regulate VEGF expression as well as blood vessel formation. This review article will explore how Rho GTPases modulate VEGF signaling and the consequences of such interaction on cancer progression.

StarD13: a potential star target for tumor therapeutics.

StarD13 is a tumor suppressor and a GTPase activating protein (GAP) for Rho GTPases. Thus, StarD13 regulates cell survival pathways and induces apoptosis in a p53-dependent and independent manners. In tumors, StarD13 is either downregulated or completely inhibited, depending on the tumor type. As such, and through the dysregulation of Rho GTPases, this affects adhesion dynamics, actin dynamics, and leads to an increase or a decrease in tumor metastasis depending on the tumor grade and type. Being a key regulatory protein, StarD13 is a potential promising candidate for therapeutic approaches. This paper reviews the key characteristics of this protein and its role in tumor malignancies.

The Role of Rho GTPases in Motility and Invasion of Glioblastoma Cells.

Astrocytomas are primary malignant brain tumors that originate from astrocytes. Grade IV astrocytoma or glioblastoma is a highly invasive tumor that occur within the brain parenchyma. The Rho family of small GTPases, which includes Rac1, Cdc42, and RhoA, is an important family whose members are key regulators of the invasion and migration of glioblastoma cells. In this review, we describe the role played by the Rho family of GTPases in the regulation of the invasion and migration of glioblastoma cells. Specifically, we focus on the role played by RhoA, Rac1, RhoG, and Cdc42 in cell migration through rearrangement of actin cytoskeleton, cell adhesion, and invasion. Finally, we highlight the importance of potentially targeting Rho GTPases in the treatment of glioblastoma.

Effect of surface pre-treatments on biocompatibility of magnesium.

This study reports the influence of Mg surface passivation on the survival rate of human HeLa cells and mouse fibroblasts in cell culture experiments. Polished samples of commercially pure Mg show high reactivity in the cell culture medium, leading to a pH shift in the alkaline direction, and therefore cell adhesion and survival is strongly impaired. Passivation of the Mg surface in 1M NaOH can strongly enhance cell survival. The best initial cell adhesion is observed for Mg samples incubated in simulated body fluid (M-SBF), which leads to the formation of a biomimetic, amorphous Ca/Mg-phosphate layer with high surface roughness. This surface layer, however, passivates and seals the Mg surface only partially. Subsequent Mg dissolution leads to a significantly stronger pH increase compared to NaOH-passivated samples, which prevents long-term cell survival. These results demonstrate that surface passivation with NaOH and M-SBF together with the associated changes of surface reactivity, chemistry and roughness provide a viable strategy to facilitate cell survival on otherwise non-biocompatible Mg surfaces.