Projet de recherche PX/8/LP/12 (Action de recherche PX)
The loss of gravity as experienced during space flights causes systemic alterations in Man and animals that might be due to perturbations of the mechano-signaling. The aim of our programme is to understand the cellular and molecular mechanisms operating in the perception and transduction of mechanical signals, including gravity. Our ultimate goal is to disclose key-pathways that might represent potential targets for pharmacological intervention to prevent microgravity-related health alterations and similar diseases on earth. This programme is the continuation of the research project PX/7/mG/73.
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 small G proteins of the Rho family (RhoA, Rac1 and Cdc42) 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. 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.
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 will be reached by observing a modulation of the effect of mechanical signals in the engineered cells in ground-based experiments and 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.
Two flight 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.
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.
Ground-based experiments using the same engineeered cells aiming at better understanding the mechano-signaling pathways and the mechanisms regulating the cellular reactions to the mechanical environment will also allow to define and validate adequate alternative procedures and relevant end-points for the flight experiments.
Furthermore we have been invited to participate in the Biobox 6 mission on the Foton M3 flight. A model of cell migration after "in vitro wounding" useing GFP- tagged WI26 wild type (WT) cells mixed with WI26 cells transfected with the QL forms or with small interfering RNA (siRNA) to knock-down each of the GTPases. The effect of microgravity and the participation of the GTPases will be demonstrated by a different rate of filling of the wound gap by the GFP/WT cells and the engineered cells in µg compared to the 1g centrifuge on board. This experiment represents an original and useful complement of information to the experiments of the programme RHOCYT on the ISS.
Satellite(s) or flight opportunity(ies):
- BIOLAB on the International Space Station
Field of research:
Life Science: Cell Biology
