Geometry sensing by dendritic cells dictates spatial organization and PGE2-induced dissolution of podosomes

Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E2 (PGE2). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE2 causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE2-mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D and 3-D geometric cues control the spatial organization of podosomes. More importantly, our studies demonstrate the importance of substrate dimensionality in regulating podosome dissolution and suggest that substrate dimensionality plays an important role in controlling DC activation, a key process in initiating immune responses

Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells

Non-Linear Elasticity of Extracellular Matrices Enables Contractile Cells to Communicate Local Position and Orientation

Most tissue cells grown in sparse cultures on linearly elastic substrates typically display a small, round phenotype on soft substrates and become increasingly spread as the modulus of the substrate increases until their spread area reaches a maximum value. As cell density increases, individual cells retain the same stiffness-dependent differences unless they are very close or in molecular contact. On nonlinear strain-stiffening fibrin gels, the same cell types become maximally spread even when the low strain elastic modulus would predict a round morphology, and cells are influenced by the presence of neighbors hundreds of microns away. Time lapse microscopy reveals that fibroblasts and human mesenchymal stem cells on fibrin deform the substrate by several microns up to five cell lengths away from their plasma membrane through a force limited mechanism. Atomic force microscopy and rheology confirm that these strains locally and globally stiffen the gel, depending on cell density, and this effect leads to long distance cell-cell communication and alignment. Thus cells are acutely responsive to the nonlinear elasticity of their substrates and can manipulate this rheological property to induce patterning

Embryogenic cell suspension cultures of garlic (Allium sativum L.) as method for mass propagation and potential material for genetic improvement

4th International Symposium on Edible Alliaceae Location: Beijing, China, 2004/04/21-26International audienceEmbryogenic calluses were induced from young leaf explants of garlic (Allium sativum L). Four cultivars, ‘Rouge de la Réunion’, ‘Messidrome’, ‘Morasol’ and ‘Printanor’ have been successfully tested. These calluses expressed up to 90% of embryogenic calluses differentiating globular somatic embryos after 2 months on N6 modified medium supplemented with 2,4-D (0.1 mg l-1) and kinetin (0.5 mg l-1). Embryogenic calluses were used to establish cell suspension cultures of the above-mentioned cultivars. Friable calluses were induced from compact ones, and could give rise to the production of cell suspension cultures composed of small aggregates of embryogenic cells. These suspension cultures were maintained in liquid medium based on N6 modified macro-nutrients and supplemented with 2,4-D/benzyladenine (0.3 mg l-1/0.1 mg l-1). The packed cell volume (PCV) of the suspension cultures increased 2-fold in a 2-week period. These cell suspension cultures led to successful regeneration of mature embryos and their conversion into plantlets. Optimal embryo regeneration efficiency was obtained after plating on semi-solid medium base on N6 macro-nutrients and a balance in 2,4-D/Kinetin (0.1 mg l-1/0.5 mg l-1). A large number of somatic embryos (potentially 8 x 109 to 1011) could be produced per year for each cultivar. The conversion into plantlet was approximately 50%. Plants were successfully acclimatised in greenhouse. Histological analyses were performed along the suspension cultures and regeneration process, and helped for establishing the sequence of culture media. The somatic embryogenic nature was confirmed by single cell origin and polar development of the regenerants. This protocol was used in a goal of mass propagation of garlic plants true to the original type. It would be a key tool for biotechnologies in genetic improvement of garlic

Establishment of embryogenic cell suspension cultures of garlic (Allium sativum L.), plant regeneration and biochemical analyses

International audienceEmbryogenic cell suspension cultures of garlic (Allium sativum L.) were initiated in liquid medium from friable embryogenic tissue. The optimal parameters for culture maintenance were: (1) an initial cell density of 1–4% (v/v); (2) medium renewal every 14 days and subculturing every 28 days; (3) a low 2,4-dichlorophenoxyacetic acid concentration (0.1–0.3 mg/l). Cultures regenerated during a 14-month period. The cell suspension cultures differentiated embryos following transfer to a semi-solid embryo induction medium, with histological studies confirming and characterising the embryogenic nature of the process. Forty percent of these embryos converted into plantlets, which produced micro bulbs in vitro. The composition of the sulphur compounds of the micro bulbs obtained from cell suspension embryo-derived plantlets differed slightly from those produced by in vitro shoot proliferation-derived plantlets, but after two cycles of multiplication in the field these differences had disappeared