Periodic Report Summary 2 - CELL TRANS (Integrated molecular and cellular mechanotransduction mediated by protein p130Cas)
Mechanical stimuli can determine the creation of structure that occurs in processes like morphogenesis and wound healing, and the disruption of organisation given in tumour formation. At the single cell level, mechanical stresses can also control functions ranging from proliferation to differentiation or gene expression. However, the mechanical mechanisms by which this occurs remain unknown. The overall objective of this project was thus to study the molecular mechanisms of force sensing by cells.
The first molecule that we chose to analyse was Cas. This molecule has been described to phosphorylate upon force application. To this end, single Cas molecules were stretched, showing that even very low forces of a few pN completely stretched Cas molecules and indicating that other molecules are involved in mechanotransduction at the pN scale. We thus expanded our attention to other molecules believed to be important in mechanotransduction: integrins, which connect cells to the surrounding extracellular matrix, and two key adhesion proteins which connect integrins to the cytoskeleton: talin and alpha-actinin.
We first focused on talin, which has been proposed as a mechanosensing molecule. By using the developed magnetic tweezers system and the TIRF detection system, we found that stretching talin molecules unfolded cryptic domains, exposing binding sites to vinculin and resulting in vinculin binding. This provided the first evidence that application of force to single molecules can result in mechanotransduction (del Rio et al., 2009, Science 323:638 - 641).
We next studied how mechanotransduction is integrated at the cellular level by using magnetic tweezers at a higher nN force range, thus probing the force regime experienced by cell adhesion sites. Our study focused on talin and its connection to fibronectin through integrins alpha-5-beta1 and alpha-v-beta3. We found that the functions of maintaining cell adhesive strength to the extracellular matrix and of transducing mechanical signals were differently regulated. That is, while talin and integrin alpha-v-beta3 were necessary for mechanotransduction, it was integrin alpha-5-beta1 that withheld applied forces and maintained adhesion strength (Roca-Cusachs et al., 2009, PNAS 106: 16245 - 16250). Thus, we have observed that talin is capable of mechanotransduction at the single molecule level, and that this ability is required at the cellular level.
To further understand the molecular interplay that takes place during force sensing, we next studied the role of alpha-actinin. This study combined several techniques to analyse the role of alpha-actinin in detecting and responding to forces. We found that talin is responsible to establish initial links between integrins and actin, enabling the formation of nascent adhesions. However, talin then competes with alpha-actinin for binding to integrins, progressively incorporating into adhesions. Then, the force transmitted by the new connection that alpha-actinin establishes between actin and integrins enables adhesion maturation, and the formation of actin stress fibers (Roca-Cusachs et al., submitted).
This project has thus characterised how key molecules in the communication of cells with their environment carry the functions of detecting, transmitting, resisting, and responding to forces. The understanding of the molecular events that dictate cell response to forces has significantly improved. This provides an excellent departure point to begin understanding the molecular mechanisms behind all the different processes and pathologies that are affected by mechanical stimuli and the cellular mechanical environment, such as development, cancer, or wound healing. In the case of cancer, for example, some therapies have already begun to target integrin alpha-v-beta3 due to its prevalent role in certain types of tumours. Our finding that it is required for mechanotransduction points to a key role of force detection in cancer.
The first molecule that we chose to analyse was Cas. This molecule has been described to phosphorylate upon force application. To this end, single Cas molecules were stretched, showing that even very low forces of a few pN completely stretched Cas molecules and indicating that other molecules are involved in mechanotransduction at the pN scale. We thus expanded our attention to other molecules believed to be important in mechanotransduction: integrins, which connect cells to the surrounding extracellular matrix, and two key adhesion proteins which connect integrins to the cytoskeleton: talin and alpha-actinin.
We first focused on talin, which has been proposed as a mechanosensing molecule. By using the developed magnetic tweezers system and the TIRF detection system, we found that stretching talin molecules unfolded cryptic domains, exposing binding sites to vinculin and resulting in vinculin binding. This provided the first evidence that application of force to single molecules can result in mechanotransduction (del Rio et al., 2009, Science 323:638 - 641).
We next studied how mechanotransduction is integrated at the cellular level by using magnetic tweezers at a higher nN force range, thus probing the force regime experienced by cell adhesion sites. Our study focused on talin and its connection to fibronectin through integrins alpha-5-beta1 and alpha-v-beta3. We found that the functions of maintaining cell adhesive strength to the extracellular matrix and of transducing mechanical signals were differently regulated. That is, while talin and integrin alpha-v-beta3 were necessary for mechanotransduction, it was integrin alpha-5-beta1 that withheld applied forces and maintained adhesion strength (Roca-Cusachs et al., 2009, PNAS 106: 16245 - 16250). Thus, we have observed that talin is capable of mechanotransduction at the single molecule level, and that this ability is required at the cellular level.
To further understand the molecular interplay that takes place during force sensing, we next studied the role of alpha-actinin. This study combined several techniques to analyse the role of alpha-actinin in detecting and responding to forces. We found that talin is responsible to establish initial links between integrins and actin, enabling the formation of nascent adhesions. However, talin then competes with alpha-actinin for binding to integrins, progressively incorporating into adhesions. Then, the force transmitted by the new connection that alpha-actinin establishes between actin and integrins enables adhesion maturation, and the formation of actin stress fibers (Roca-Cusachs et al., submitted).
This project has thus characterised how key molecules in the communication of cells with their environment carry the functions of detecting, transmitting, resisting, and responding to forces. The understanding of the molecular events that dictate cell response to forces has significantly improved. This provides an excellent departure point to begin understanding the molecular mechanisms behind all the different processes and pathologies that are affected by mechanical stimuli and the cellular mechanical environment, such as development, cancer, or wound healing. In the case of cancer, for example, some therapies have already begun to target integrin alpha-v-beta3 due to its prevalent role in certain types of tumours. Our finding that it is required for mechanotransduction points to a key role of force detection in cancer.