Projet de recherche PX/7/LP/05 (Action de recherche PX)
Our specific goal is to determine the participation of the small GTPases of the Rho family (RhoGTPases) in the reception and reaction of mesenchymal cells to microgravity. Two types of receptors are located on the cell membrane, the integrins for chemical and mechanical signals issued from the extracellular matrix and specific receptors for diffusible ligands (cytokines, hormones, growth factors). The RhoGTPases are GDP/GTP-regulated binary switches that integrate the messages transduced by these receptors and dispatch the information to effectors regulating the main cellular functions. These molecules are key-regulators in the organization and turn-over of the cytoskeleton (CSK) and the formation of cell-matrix adhesions (focal adhesions, FA), the transcriptional control of gene expression as well as cell survival and multiplication. An important function of the CSK and the FA is to transduce and counterbalance forces applied to, or generated by cells, to maintain the mechanical cell homeostasis and to control cell movements. It can be assumed that the loss of gravity as experienced during space flight will affect the control of cell architecture and the function of the RhoGTPases. The three main members of the RhoGTPases family (RhoA, Rac1 and Cdc42) have each specific functions. We have created cell lines in which each of these GTPases has been engineered to be always active (QL) or specifically knocked-down (KD) by using small interfering RNAs (siRNA).
The proof of concept of our space programme is to determine end-points under the control of the GTPases altered in microgravity in control cells (CO) and rescued or increased by activating or silencing the GTPases. A significant part of the basic biology of this programme and the related technology are available. Some should be completed. New knowledge and recent procedure for cell engineering and detection of effects mediated by the RhoGTPases will be applied to improve the significance of the information obtained during flight.
Two experiments have been selected to be performed in the Biolab (2nd batch of experiments, around 2007) on the ISS.
1. Evaluate the effect of microgravity during adhesion and spreading in absence of growth factors of human fibroblasts (WI26) and osteoblasts (MG63), either expressing the wild-type (CO) or the constitutively activated (QL) form of each RhoGTPase or transfected with siRNA to completely and selectively silence their expression (KD). A second part of this experiment 1 will be to measure the RhoGTPase activation and down-stream signaling pathways in response to known biological mediators. This experiment will be performed with cells sent and kept frozen on the ISS. This experiment needs preliminary observations on ISS to determine, for each cell line, their viability, attachment and spreading.
2. Evaluate the role played by the RhoGTPases in the microgravity-induced alterations of the integrin-mediated mechano-transduction by applying a dynamic mechanical tension to WI26 and MG63, CO, QL or KD cells. The cells will be adherent to an extensible membrane coated with matrix protein and progressively stretched in microgravity. The experimental containers have to be prepared on ground.
In both experiments, cells and conditioned mediums will be preserved or fixed in microgravity and kept under the best appropriate conditions. Multiple tasks need to be performed to successfully achieve our goals. Active collaborations with industrial partners and with scientists specialized in biopolymers are required for the development of the hardware. Our role is to validate them in our biological models for successful flight experiments.
Satellite(s) or flight opportunity(ies):
- BIOLAB on the International Space Station
Field of research:
Life Science: Cell Biology
