Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

ICAP-1 prevents recruitment of kindlin-2 to β1 integrin to control dynamics of fibrillar adhesion sites, fibronectin deposition, and osteoblast mineralization during bone formation

Mechanotransduction pulls the strings of matrix degradation at invadosome

International audienc

Mechanotransduction pulls the strings of matrix degradation at invadosome

International audienc

Podosomes are dispensable for osteoclast differentiation and migration

International audienc

Effect of RGD functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation

Skeletal muscle tissue engineering holds promise for the replacement of muscle due to an injury and for the treatment of muscle diseases. Although RGD substrates have been widely explored in tissue engineering, there is no study aimed at investigating the combined effects of RGD nanoscale presentation and matrix stiffness on myogenesis. In the present work, we use polyelectrolyte multilayer films made of poly(L-lysine) (PLL) and poly(L-glutamic) acid (PGA) as substrates of tunable stiffness that can be functionalized by a RGD adhesive peptide to investigate important events in myogenesis, including adhesion, migration, proliferation and differentiation. C2C12 myoblasts were used as cellular models. RGD presentation on soft films and increased film stiffness could both induce cell adhesion, but integrins involved in adhesion were different in case of soft and stiff films. Moreover, soft films with RGD peptide appeared to be the most appropriate substrate for myogenic differentiation while the stiff PLL/PGA films significantly induced cell migration, proliferation and inhibited myogenic differentiation. The ROCK kinase was found to be involved in myoblast response to the different films. Indeed, its inhibition was sufficient to rescue the differentiation on stiff films, but no significant changes were observed on stiff films with the RGD peptide. These results suggest that different signaling pathways may be activated depending on mechanical and biochemical properties of the multilayer films. This study emphasizes the superior advantage of the soft PLL/PGA films presenting the RGD peptide in terms of myogenic differentiation. This soft RGD-presenting film may be further used as coating of various polymeric scaffolds for muscle tissue engineering

Micropatterns of BMP-2 and fibronectin regulate myoblast shape and SMAD signaling

International audienc

αII-spectrin regulates invadosome stability and extracellular matrix degradation.

Invadosomes are actin-rich adhesion structures involved in tissue invasion and extracellular matrix (ECM) remodelling. αII-Spectrin, an ubiquitous scaffolding component of the membrane skeleton and a partner of actin regulators (ABI1, VASP and WASL), accumulates highly and specifically in the invadosomes of multiple cell types, such as mouse embryonic fibroblasts (MEFs) expressing SrcY527F, the constitutively active form of Src or activated HMEC-1 endothelial cells. FRAP and live-imaging analysis revealed that αII-spectrin is a highly dynamic component of invadosomes as actin present in the structures core. Knockdown of αII-spectrin expression destabilizes invadosomes and reduces the ability of the remaining invadosomes to digest the ECM and to promote invasion. The ECM degradation defect observed in spectrin-depleted-cells is associated with highly dynamic and unstable invadosome rings. Moreover, FRAP measurement showed the specific involvement of αII-spectrin in the regulation of the mobile/immobile β3-integrin ratio in invadosomes. Our findings suggest that spectrin could regulate invadosome function and maturation by modulating integrin mobility in the membrane, allowing the normal processes of adhesion, invasion and matrix degradation. Altogether, these data highlight a new function for spectrins in the stability of invadosomes and the coupling between actin regulation and ECM degradation

Presentation of bone morphogenetic proteins to cells at their basal side reveals their role in the initiation of cell adhesion

International audienc

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

International audienceIn vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro. Due to their physiological relevance, bone morphogenetic proteins (BMPs) are widely studied for orthopedic clinical applications to enhance the healing of large bone defects 1,2 , as well as for developing new strategies in bone tissue engineering 3-5. BMPs are indeed highly potent growth factors (GFs) that play a crucial role in mor-phogenesis and tissue homeostasis during embryonic development and until adulthood 6,7. In particular, BMP-2 promotes the differentiation of mesenchymal stem cells (MSCs) and osteoblasts toward osteocytes 8,9 , and induces the trans-differentiation of myoblasts into osteoblasts 10. In addition, BMP-2 in solution plays a role in early adhesive events, including adhesion and migration through cytoskeletal reorganization 11,12. Recently, several studies have demonstrated that BMP-2 strongly interacts with extra-cellular matrix (ECM) proteins 13 , especially fibronectin (FN) due to its highly promiscuous GF-binding site (the 12th to 14th type III repeats) 14,15. Moreover, immobilized BMP-2 whether by physical adsorption (i.e. matrix-bound BMP-2 13,16) or by covalent grafting 17 was shown to regulate cell behavior quite distinctly from BMP-2 in solution. This effect is currently poorly known and is likely due to the close proximity and crosstalk of integrin-binding domains of FN and BMP-2 13,17-19. It has indeed been shown that the secretion of FN by cells is necessary for BMP-2-mediated signaling 13. A consequence of this association of BMP-2 with ECM proteins in vivo is that the spatially patterned presentation of BMPs b

Presentation of bone morphogenetic proteins to cells at their basal side reveals their role in the initiation of cell adhesion

International audienc