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Projects. Life and Earth Sciences

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

Lead Researcher:
Miguel Vicente Manzanares

Research Centre: 
Universidad Autónoma de Madrid. 

Synopsis: 

Miguel Vicente ManzanaresWithin 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.

Scientific production
3 articles published in Journals
1 paper at international conferences

 

Researcher's web address:
 http://web.uam.es/personal_pdi/medicina/mvicente/


Miguel Vicente Manzanares

Miguel Vicente Manzanares received a Degree in Chemistry from the Complutense University, Madrid, in 1996. He undertook his postgraduate studies in the Autonomous University, Madrid, under the direction of Prof. Francisco Sánchez-Madrid. He was awarded the Extraordinary Prize for his Doctoral thesis (2002), which revealed the details of the inflammatory signalling that attracts T lymphocytes to inflammation sites and their effects on the migratory morphology of the same. In 2004, he moved to the United States, where he lived until March, 2011, working with Prof. Alan "Rick" Hurwitz. His pioneering work in the field of cellular migration has deciphered some of the key mechanisms in the morphological changes in cells during the processes of migration and differentiation. All these works have been published in journals of the very highest renown in the field of cell biology, such as Nature Cell Biology, Nature Reviews Molecular Cell Biology, Journal of Cell Biology, etc. After returning to Spain in March, 2011, he set up a dynamic and original line of research in the "Princesa" University Hospital in Madrid, with the aim of deciphering the complicated mechanical and physical relationships between cells and their micro-environment, especially during the migration and differentiation of stem cells. The details of the said mechanisms are essential for the development of regenerative therapies based on the reprogramming of these cells.


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