CELL TRANS

Integrated molecular and cellular mechanotransduction mediated by protein p130Cas

 Partecipanti

Mappa

 Word cloud

Esplora la "nuvola delle parole (Word Cloud) per avere un'idea di massima del progetto.

 Obiettivo del progetto (Objective)

'The sensing of force by cells is determinant for processes like morphogenesis, wound healing, or tumour formation, and can control functions such as proliferation, differentiation, or gene expression. However, the specific mechanisms by which cells sense mechanical forces and respond accordingly remain largely unknown. The proposed project aims to characterize cell mechanotransduction through the study of the Src family kinase substrate p130Cas (Cas, Crk associated substrate), a key protein involved in many cellular processes which is known to be activated (phosphorylated) by force. In the first part of the project, carried out at the outgoing institution, optical and magnetic tweezers and Total Internal Reflection Microscopy (TIRF) will be employed to examine how the phosphorylation of single Cas molecules is regulated by force. Individual Cas molecules will be stretched, and TIRF will be used to determine the force required for phosphorylation, if increased forces lead to increased phosphorylation (providing thus for a mechanism for the sensing of force magnitude), and if discontinuing the application of force leads to dephosphorylation (providing for a mechanism for the sensing of force interruption). In the second part of the project, which will take place mostly at the return institution, magnetic tweezers will be used to study force-dependent Cas phosphorylation in living single cells. The sensing of mechanical forces by cells will be analyzed by studying how these stimuli phosphorylate Cas locally at the site of force application, and globally through cytoskeletal force transmission. The role of the cytoskeleton in integrating local mechanical stimuli will be examined by modifying and measuring its mechanical state through micropatterning technologies. The interest of the project covers disciplines such as biophysics, cell biology nanotechnology, and tissue engineering.'

Introduzione (Teaser)

Cells sense mechanical forces that lead them to create structures playing a role in tumour formation or wound healing. However, little is known about the mechanisms leading to cellular sensing of mechanical forces and their corresponding responses.

Descrizione progetto (Article)

The 'Integrated molecular and cellular mechanotransduction mediated by protein p130Cas' (Cell trans) project had the overall objective of studying the molecular mechanisms of force sensing by cells. The EU-funded project set out to study the mechanotransduction potential of the Src family kinase substrate p130Cas. This is a key protein involved in many cellular processes and is known to be activated (phosphorylated) by force.

Optical and magnetic tweezers and total internal reflection microscopy (TIRF) were used to examine how the phosphorylation of single Cas molecules is regulated by force. TIRF was employed to determine what magnitude of force is required for phosphorylation, whether an increase in force leads to increased phosphorylation and whether interrupting the application of force leads to dephosphorylation.

Experiments revealed that other molecules are involved in mechanotransduction at the pN scale usually present at cell adhesion sites (picoNewtons (pN) are used for the measurement of forces exerted at the molecular scale). This led Cell trans researchers to focus attention on other molecules, integrins, believed to be important in mechanotransduction. Integrins connect cells to the surrounding extracellular matrix. Talin and a-actinin are two key adhesion proteins connecting integrins to the cytoskeleton.

Results of working with talin, proposed as a mechanosensing molecule, offered the first indication that application of force to single molecules can indeed result in mechanotransduction. Further studies on how mechanotransduction is integrated at the cellular level examined the force regime experienced by cell adhesion sites. The functions of maintaining cell adhesive strength and of transducing mechanical signals were found to be differently regulated in the case of talin. This indicates that the adhesion protein is capable of mechanotransduction at the single molecule level.

In studies concentrating on the role of a-actinin in force sensing, it was discovered that a-actinin enables the transmission of force between the cytoskeleton and integrins. In this way, the formation and the dynamics of stable mature adhesion sites between the cell and the extracellular matrix are controlled.

Having characterised how certain molecules function to detect, transmit, resist and respond to forces, Cell trans was able to further understanding of the molecular events dictating cell response to forces. This sets the foundation for future studies to investigate the molecular mechanisms underlying processes and pathologies affected by mechanical stimuli and the cellular mechanical environment, important for research in areas such as cancer or wound healing.