RhoGTPases as Key players in mammalian cell adaptation to microgravity.

A growing number of studies are revealing that cells reorganize their cytoskeleton when exposed to conditions of microgravity. Most, if not all, of the structural changes observed on flown cells can be explained by modulation of RhoGTPases, which are mechanosensitive switches responsible for cytoskeletal dynamics control. This review identifies general principles defining cell sensitivity to gravitational stresses. We discuss what is known about changes in cell shape, nucleus, and focal adhesions and try to establish the relationship with specific RhoGTPase activities. We conclude by considering the potential relevance of live imaging of RhoGTPase activity or cytoskeletal structures in order to enhance our understanding of cell adaptation to microgravity-related conditions

Cyclic strain promotes shuttling of PYK2/Hic-5 complex from focal contacts in osteoblast-like cells.

International audienceWe showed that cyclic strain (CS) of osteoblastic cells induced tyrosine phosphorylation of two homologous tyrosine kinases FAK and PYK2, and of two homologous adaptor proteins paxillin and Hic5, with similar kinetics. Immunostaining showed that all four proteins were localized to focal contacts in controls. In contrast, the dynamics of their subcellular localization observed after CS differed. While FAK and paxillin remained at the focal contact, Hic-5 and PYK2 translocated outside ventral focal contacts as early as 30 min after CS and were sequestered by the cytoskeleton. Co-immunoprecipitation showed that the association of PYK2/Hic-5 and PYK2/FAK increased with time after strain while that of paxillin and Hic-5 decreased. Altogether these results suggested that CS regulates focal contact activity in osteoblasts by modulating PYK2-containing complexes in particular by shuttling out of the focal contact the adaptor Hic-5 and favoring the anchorage of FAK within contacts

Traction Force Measurements of Human Aortic Smooth Muscle Cells Reveal a Motor-Clutch Behavior

International audienceThe contractile behavior of smooth muscle cells (SMCs) in the aorta is an important determinant of growth, remodeling, and homeostasis. However, quantitative values of SMC basal tone have never been characterized precisely on individual SMCs. Therefore, to address this lack, we developed an in vitro technique based on Traction Force Microscopy (TFM). Aortic SMCs from a human lineage at low passages (4-7) were cultured 2 days in conditions promoting the development of their contractile apparatus and seeded on hydrogels of varying elastic modulus (1, 4, 12 and 25 kPa) with embedded fluorescent microspheres. After complete adhesion, SMCs were artificially detached from the gel by trypsin treatment. The microbeads movement was tracked and the deformation fields were processed with a mechanical model, assuming linear elasticity, isotropic material, plane strain, to extract the traction forces formerly applied by individual SMCs on the gel. Two major interesting and original observations about SMC traction forces were deduced from the obtained results: 1. they are variable but driven by cell dynamics and show an exponential distribution, with 40% to 80% of traction forces in the range 0-10 µN. 2. They depend on the substrate stiffness: the fraction of adhesion forces below 10 µN tend to decrease when the substrate stiffness increases, whereas the fraction of higher adhesion forces increases. As these two aspects of cell adhesion (variability and stiffness dependence) and the distribution of their traction forces can be predicted by the probabilistic motor-clutch model, we conclude that this model could be applied to SMCs. Further studies will consider stimulated contractility and primary culture of cells extracted from aneurysmal human aortic tissue

Le strontium comme inhibiteur de l'adipogenèse et modulateur du statut redox des cellules souches mésenchymateuses

L ostéoporose liée à l âge se caractérise par une perte osseuse et une augmentation de l adiposité médullaire tout en s accompagnant d un stress oxydant général. L ostéoblaste et l adipocyte ont un précurseur commun, la cellule souche mésenchymateuse (CSM), dont la capacité à se renouveler et à se différencier est influencée par le statut redox cellulaire. Le Strontium (Sr) est un élément possédant un effet antifracturaire significatif in vivo cependant, il n affecte que peu les marqueurs d activités des cellules osseuses différenciées. Partant de ce constat, nous avons émis l hypothèse que les CSMs pouvaient être une cible cellulaire du Sr, et notamment que l inhibition de leur différenciation adipocytaire pouvait diminuer la lipotoxicité médullaire néfaste à la survie des ostéoblastes au cours du vieillissement. Nous montrons chez des souris traitées 3 semaines au Sr une diminution de l adiposité médullaire et une augmentation du volume osseux trabéculaire par rapport aux animaux témoins. Nos résultats démontrent que le Sr inhibe rapidement l adipogenèse des cellules multipotentes mésenchymateuses (CMMs) C3H10T1/2 en réprimant PPARg2 et l accumulation des gouttelettes lipidiques de façon partiellement dépendante de la voie ERK. Ce mécanisme serait dépendant de son effet proliférateur puisque que nous observons qu en présence de Sr plus la Cycline D1 est exprimée, plus PPARg2 est réprimé. De plus, le Sr prévient la mise en place de processus impliqués dans le statut redox cellulaire et nécessaires à la maturation d un adipocyte comme la biogenèse mitochondriale, l accumulation de Rac1 (une sous unité régulatrice de l activité de la Nadph oxydase) et l augmentation de l expression des enzymes antioxydantes. Nous montrons aussi que le Sr diminue la production d espèces réactives de l oxygène (ERO) de façon précoce ce qui pourrait expliquer son action anti-adipogénique. En effet, les ERO sont indispensables à l engagement des CSMs vers l adipogenèse et elles oxydent des lipides qui sont alors activateurs de PPARg. L ensemble de ces données nous montre que le Sr, en modifiant la production d ERO intracellulaire, maintiendrait un statut redox favorable à la prolifération des CMMs et défavorable à leur différenciation adipocytaire. Ainsi la capacité antioxydante et antiadipogénique de futures molécules pourraient définir de nouvelles approches dans le traitement de l ostéoporoseAge-related osteoporosis is associated with both an increased marrow adiposity while bone mass decreased and an increased oxidative stress. Mesenchymal stem cells (MSCs) differentiate into osteoblasts or adipocytes and their capacity of self-renewal and differentiation is influenced by cell redox status. Strontium (Sr) have an anti-fracture effect in vivo however, it doesn t clearly modulate markers of mature bone cell activities. Starting from this observation, we hypothesized that MSCs could be a cellular target of Sr, and particularly the inhibition of their adipocyte differentiation could reduce the marrow lipotoxicity which is deleterious for the osteoblast survival during aging. Our study showed that Sr-treated mice presented a lower medullary adiposity and a higher trabecular bone volume as compared to control animals. It was demonstrated that Sr rapidly inhibited adipogenesis of multipotent mesenchymal cells (MMCs) C3H10T1/2 by repressing PPARg2 and droplet lipid formation in a partially ERK-dependant pathway. This mechanism was linked to its proliferative effect since in presence of Sr the higher Cyclin D1 gene expression; the lower was that of PPARg2. Moreover, Sr prevented the establishment of processes involved in the cell redox status and necessary for the adipocyte maturation such as mitochondrial biogenesis, Rac1 protein accumulation (a NADPH oxidase regulatory subunit) and increase of the antioxidant enzyme expression. Sr also induced intracellular reactive oxygen species (ROS) decrease that could explain its anti-adipogenic action. Indeed, ROS are essential for the CSM commitment toward adipogenesis and they oxidize lipids which could in turn activate PPAR. Taken together, these data showed that Sr by modulating the intracellular ROS production maintained a redox status supporting the MMCs proliferation and preventing adipocyte differentiation. Thus, the antioxidant and anti-adipogenic capacities of future molecules could define new therapeutic approaches for osteoporosis treatmentST ETIENNE-Bib. électronique (422189901) / SudocSudocFranceF

Osteocytes and Weightlessness

International audienceAbstract Purpose of Review Osteocytes are considered to be the cells responsible for mastering the remodeling process that follows the exposure to unloading conditions. Given the invasiveness of bone biopsies in humans, both rodents and in vitro culture systems are largely adopted as models for studies in space missions or in simulated microgravity conditions models on Earth. Recent Findings After a brief recall of the main changes in bone mass and osteoclastic and osteoblastic activities in space-related models, this review focuses on the potential role of osteocytes in directing these changes. The role of the best-known signalling molecules is questioned, in particular in relation to osteocyte apoptosis. Summary The mechanotransduction actors identified in spatial conditions and the problems related to fluid flow and shear stress changes, probably enhanced by the alteration in fluid flow and lack of convection during spaceflight, are recalled and discussed

Why do State-of-the-art Super-Resolution Methods not work well for Bone Microstructure CT Imaging?

International audience3D Computerized Tomography (CT) is a gold standard technique to assess bone microstructure in the context of bone diseases such as osteoporosis. However, when acquired invivo, bone images may suffer from a low spatial resolution and the presence of noise due to the limited tolerable radiation exposure. One way to overcome this issue consists in applying Super-Resolution (SR) techniques that aim at recovering high resolution images. Significant progress has been recently made thanks to deep learning SR methods trained on natural image datasets. To measure the reconstruction quality, Peak Signal to Noise Ratio (PSNR) and Structural Similarity (SSIM) are commonly used in the SR literature. In this paper, we give evidence of the limitation of these two criteria. Through extensive experiments performed from a dataset of mice tibias specifically collected and imaged for this study, we show that state of the art deep learning-based SR methods miss important details about the bone microstructure which is not reflected by the PSNR and SSIM values. This study opens the door to future promising lines of research including new SR methods regularized with respect to morphometric and topological parameters of bone microstructures

Why do State-of-the-art Super-Resolution Methods not work well for Bone Microstructure CT Imaging?

International audience3D Computerized Tomography (CT) is a gold standard technique to assess bone microstructure in the context of bone diseases such as osteoporosis. However, when acquired invivo, bone images may suffer from a low spatial resolution and the presence of noise due to the limited tolerable radiation exposure. One way to overcome this issue consists in applying Super-Resolution (SR) techniques that aim at recovering high resolution images. Significant progress has been recently made thanks to deep learning SR methods trained on natural image datasets. To measure the reconstruction quality, Peak Signal to Noise Ratio (PSNR) and Structural Similarity (SSIM) are commonly used in the SR literature. In this paper, we give evidence of the limitation of these two criteria. Through extensive experiments performed from a dataset of mice tibias specifically collected and imaged for this study, we show that state of the art deep learning-based SR methods miss important details about the bone microstructure which is not reflected by the PSNR and SSIM values. This study opens the door to future promising lines of research including new SR methods regularized with respect to morphometric and topological parameters of bone microstructures