A microfluidic method generating monodispersed microparticles with controllable sizes and mechanical properties

International audienceSeeking to produce microparticles that mimic red blood cells (RBCs), we present a microfluidic method of generating monodispersed hydrogel microparticles of Na-/Ca-alginate with controllable sizes (micrometer range) and mechanical properties. No surfactant is used. Transformation from Na-alginate to Ca-alginate microparticles is realized via ex situ gelation, which proves essential to obtaining desired microparticle properties, such as insolubility in water and RBC-like mechanical properties. For both Na-alginate and Ca-alginate microparticles, a smooth surface and a porous inner structure are observed under a scanning electron microscope. A platform of microgrippers is successfully developed to manipulate the microparticles. The Young’s modulus measured using an atomic force microscope on the surface of Ca-alginate microparticles is of the same order as that of RBCs

Régulation de la mécanique cellulaire par la dynamique du cytosquelette : microscopie à force atomique et optique combinée

Dans le processus complexe d'invasion du cancer et de formation de métastases, les cellules cancéreuses se détachent de la tumeur primaire et migrent, entrant et sortant des cavités exiguës entre d'autres cellules qui forment les tissus environnants. Dans ce cheminement si ardu, les cellules cancéreuses doivent se déformer sous des micro-constrictions et résister et survivre à de fortes contraintes mécaniques. Ceci suggère que la formation de métastases n'est pas seulement le résultat d'une différenciation cellulaire, de mutations génétiques, de signalation, mais aussi d'un équilibre entre forces et contraintes mécaniques. En effet, il a été démontré que les cellules cancéreuses malignes sont plus molles que les cellules bénignes, une caractéristique qui peut être importante pour comprendre les métastases. Les signaux mécaniques sont générés par le cytosquelette, un réseau complexe de filaments protéiques, qui peut maintenir une tension s’il est ancré au substrat, maintenir la rigidité cellulaire et ainsi former l’échafaudage structurel de la cellule. Le continue remodelage du cytosquelette et sa structure influencent la déformabilité des cellules. Ainsi, la compréhension de la déformabilité et de la mécanique des cellules cancéreuses et de la dynamique du cytosquelette est importante pour mieux comprendre le cancer. Cependant, cette interaction entre la structure et la mécanique du cytosquelette est mal comprise, principalement en raison du manque d’outils disponibles combinant la mécanique et les informations moléculaires structurelles. L’objectif principal de cette thèse est de corréler la réponse mécanique avec la structure du cytosquelette des cellules cancéreuses.In the complex process of cancer invasion and metastasis formation, cancer cells detach from the primary tumor and migrate, entering and exiting the cramped cavities between other cells that form the surrounding tissues. In this so arduous path cancer cells need to deform under micro-constrictions and to resist and survive strong mechanical stress. This suggests that the metastasis formation is not only the result of cell differentiation, genetic mutations, signaling, but also a balance between forces and mechanical constraints. Indeed, it has been shown that malignant cancer cells are softer than benign cells, a feature that may be important to understand metastasis. Cells can produce and sense forces. These mechanical signals are involved in many relevant cellular processes and functions. Mechanical signals are generated by the cytoskeleton, a complex network of protein filaments, that can sustain tension if anchored to the substrate or to junctions with other cells, maintain cell rigidity, and thus form the structural scaffold of the cell. Cytoskeleton is in continuous transformation and its remodelling and structure influences cell deformability. Thus, the comprehension of cancer cell deformability and mechanics and the cytoskeleton dynamics is important to better understand the complex mechanisms of cancer. However, this interplay between cytoskeleton’s structure and mechanics is poorly understood, mainly due to the lack of available tools combining mechanics and structural molecular information. The main goal of this thesis project is to correlate the mechanical response with the structure of the cytoskeleton of cancer cells

αvβ3 integrin expression increases elasticity in human melanoma cells

International audienceLiving cells interact with the extracellular matrix (ECM) transducing biochemical signals into mechanical cues and vice versa. Thanks to this mechano-transduction process, cells modify their internal organization and upregulate their physiological functions differently. In this complex mechanism integrins play a fundamental role, connecting the extracellular matrix with the cytoskeleton. Cytoskeletal rearrangements, such as the increase of the overall contractility, impact cell mechanical properties, the entire cell stiffness, and cell deformability. How cell mechanics is influenced via different integrins and their interaction with ECM in health and disease is still unclear. Here, we investigated the influence of αvβ3 integrin expression on the mechanics of human melanoma M21 cells using atomic force microscopy and micro-constriction. Evidence is provided that (i) αvβ3 integrin expression in human melanoma cells increases cell stiffness in both adherent and non-adherent conditions; (ii) replacing αvβ3 with αIIbβ3 integrin in melanoma cells, cell stiffness is increased under adherent, while decreased under non-adherent conditions; (iii) αvβ3 integrin cell stiffening is also maintained when cells adhere to fibronectin, but this phenomenon does not strongly depend on the fibronectin concentration. In all, this study sheds light on the role of αvβ3 in regulating cellular mechanics

Interplay between HGAL and Grb2 proteins regulates B-cell receptor signaling

International audienceKey Points• HGAL and Gb2 proteins directly interact upon BCR stimulation.• HGAL and Gb2 interaction plays a role in BCR clustering in sig-nalosomes and regulates BCR-induced biochemical signaling.Human germinal center (GC)-associated lymphoma (HGAL) is an adaptor protein expressed in GC B cells. HGAL regulates cell motility and B-cell receptor (BCR) signaling, processes that are central for the successful completion of the GC reaction. Herein, we demonstrate phosphorylation of HGAL by Syk and Lyn kinases at tyrosines Y80, Y86, Y106Y107, Y128, and Y148. The HGAL YEN motif (amino acids 107-109) is similar to the phosphopeptide motif pYXN used as a binding site to the growth factor receptor-bound protein 2 (Grb2). We demonstrate by biochemical and molecular methodologies that HGAL directly interacts with Grb2. Concordantly, microscopy studies demonstrate HGAL-Grb2 colocalization in the membrane central supramolecular activation clusters (cSMAC) following BCR activation. Mutation of the HGAL putative binding site to Grb2 abrogates the interaction between these proteins. Further, this HGAL mutant localizes exclusively in the peripheral SMAC and decreases the rate and intensity of BCR accumulation in the cSMAC. Furthermore, we demonstrate that Grb2, HGAL, and Syk interact in the same complex, but Grb2 does not modulate the effects of HGAL on Syk kinase activity. Overall, the interplay between the HGAL and Grb2 regulates the magnitude of BCR signaling and synapse formation