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Myosin II integrates the mechanical signals of the cellular micro-environment and controls the migration and differentiation of stem cells

16th national competition for scientific and technical research

Gene and regenerative therapy

Senior Researcher : Miguel Vicente Manzanares

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Research Centre or Institution : Universidad Autónoma de Madrid.

Abstract

Within the context of the aid received, the scientific director has decided, following evaluation of the options existing within the field, to centre the study on human mesenchymal stem cells. The reasoning underlying this decision is the greater scientific rigour and available commercial reagents for characterisation and differentiation. hMSC were obtained from Lonza Inc., together with all of the reagents necessary for their culture and adipogenic and osteogenic differentiation, the two terminal differentiation models to be used. During recent months, work has centred on developing two extremely difficult techniques which will facilitate subsequent work. One is traction force microscopy (TFM), which makes it possible to measure the force exerted by the cells in the substrate. This technique, using polyacrylamide substrates treated with cross-linkers and extracellular matrices, has made it possible to visualise, reliably and repeatably, the forces exerted by cells within the substrate, which are confined to the extremes of cell retraction and protrusion during migration.

It is possible to create gels from 1‑100 kPa, which reproduce the rigidity of most physiological environments. As a proof-of-concept experiment, MIIA was inhibited using specific shRNA. In these cells it was observed that the cells exerted no traction over the substrate, represented using vectorial movement maps. On the contrary, the cells in which MIIB was inhibited present a reduction of the traction force together with depolarisation, and they are not confined to the protrusion and retraction boundaries. The translation of this model to dendritic cells (not in connection with this project, but related to other laboratory lines) and hMSC will permit the detailed analysis of the forces during migration and differentiation of the hMSC.

At molecular level, the contribution of contractile activity is being investigated versus the intercrossing action of the actin in both isoforms using specific mutants with no ATPase activity. The preliminary results indicate that the non-contractile version of MIIA, N93K, induces a small but significant resurge of the traction force, while MIIB R709C (non-contractile) almost completely preserves the inhibition caused by the depletion of endogenous MIIB, indicating that MIIA generates traction through its contractile activity, while MIIB positions the forces generated by itself and MIIA predominantly by means of its intercrossing activity.

The second type of experiment centres on the role of mechanics in cellular signalling. Substrates of differing rigidity are used with different coatings to study the modulation of differentiation signalling. Due to the limitations inherent to the emergent nature of the group, this was limited to signalling by phosphorylation in Tyr. Western blot identified 8+ integrin-dependent and independent bands (range 20‑150 kDa) whose phosphorylation is altered by the rigidity of the substrate and/or by the inhibition of myosin II. In the first months of 2013, these bands will be identified using mass spectrometry and will be studied in the differentiation of hMSC.

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