Développement d'outils optogénétiques pour contrôler la signalisation cellulaire et étudier les mécanismes de la migration cellulaire

par Dwiria Wahyuni

Projet de thèse en Physique pour les Sciences du Vivant

Sous la direction de Antoine (phys) Delon et de Irène Wang.

Thèses en préparation à Grenoble Alpes , dans le cadre de Physique , en partenariat avec Laboratoire Interdisciplinaire de Physique (laboratoire) et de MOTIV : Matériaux, Optique et techniques instrumentales pour le vivant (equipe de recherche) depuis le 27-09-2016 .


  • Résumé

    Directional motility is a precisely-regulated process at the heart of many eukaryotic cellular events such as morphogenesis, leukocyte trafficking in immune surveillance, or tissue regeneration and repair. Cell migration is characterized by different actin structures at the front and the rear of the cell that are regulated by Rho family GTPases and lead to an asymmetry in traction force distribution. It is believed that this asymmetry results from different spatial distribution of GTPases (Rac1 and Cdc42 are the front, while RhoA is activated at the rear of a migrating cell). However, what triggers this symmetry breaking is still unclear. Optogenetics are recently developed tools, based on photosensitive proteins, i.e. proteins that can be either activated or inactivated upon illumination. This enables to control intracellular processes with light. One key benefit of optogenetics is the capability to induce signalling perturbations that are controlled in space and time, while classical genetic and pharmacologic approaches can only create permanent and global perturbations. Therefore, it is important to precisely characterize the physical processes that govern the spatial and temporal resolution that can be obtained with optogenetics. At LIPhy, we are using a cell line where the activity of RhoA GTPase can be tuned by light with the CRY2-CIBN optogenetic system. Upon blue light irradiation, CRY2 (associated with ArhGEF11, a specific activator of RhoA) will be recruited to the plasma membrane where CIBN is anchored, thereby inducing the activation of RhoA. The proposed project is divided in two steps. First, the optogenetic system CRY2/CIBN will be studied in order to optimize illumination conditions and improve the spatial resolution of light-induced effects. To understand which properties limit the spatial extent of optogenetic perturbations, we will measure the concentration, mobility and interactions of CRY2, CIBN and the photo-generated dimer as a function of irradiation intensity and duration. These measurements will be performed on a fluorescence microscope, confocal or TIRF (total internal reflection), and analysed using image correlation spectroscopy (ICS), a family of methods based on the analysis of the pixel intensity fluctuations in an image series. These fluctuations arise from changes in the occupation number of fluorophores in the focal volume. ICS provides informations on the concentration and diffusion constant of fluorescent molecules in subregions of the images, to yield maps of protein density and dynamics in living cells. The second step would focus on the onset of cell migration when the cell polarizes and an asymmetric distribution of signalling molecules appears. This event could be induced by photo-stimulation. Our initial data show that upon local RhoA photo-activation a retraction/contraction was observed at the site of photoactivation, accompanied by the appearance of protrusions at the other end of the cell, suggesting the existence of polarized flows of signalling molecules. Cell polarization is thought to be associated with increased RhoA activity at the rear of the cell while Rac1 and Cdc42 activities decrease. To correlate ICS concentration measurement with RhoA activation level, the latter will be quantified by Föster resonance energy transfer (FRET) using specific biosensors (Raïchu probes). In addition, a possible mechanism for the differential signalling activity might be the transport of signalling molecules by the flow of actin cytoskeleton. To test this hypothesis, actin flow velocity can be measured by ICS and correlated with RhoA flow velocity and spatial distribution.

  • Titre traduit

    Investigation of optogenetic tools to control cell signalling and unravel the mechanism of cell migration


  • Résumé

    Directional motility is a precisely-regulated process at the heart of many eukaryotic cellular events such as morphogenesis, leukocyte trafficking in immune surveillance, or tissue regeneration and repair. Cell migration is characterized by different actin structures at the front and the rear of the cell that are regulated by Rho family GTPases and lead to an asymmetry in traction force distribution. It is believed that this asymmetry results from different spatial distribution of GTPases (Rac1 and Cdc42 are the front, while RhoA is activated at the rear of a migrating cell). However, what triggers this symmetry breaking is still unclear. Optogenetics are recently developed tools, based on photosensitive proteins, i.e. proteins that can be either activated or inactivated upon illumination. This enables to control intracellular processes with light. One key benefit of optogenetics is the capability to induce signalling perturbations that are controlled in space and time, while classical genetic and pharmacologic approaches can only create permanent and global perturbations. Therefore, it is important to precisely characterize the physical processes that govern the spatial and temporal resolution that can be obtained with optogenetics. At LIPhy, we are using a cell line where the activity of RhoA GTPase can be tuned by light with the CRY2-CIBN optogenetic system. Upon blue light irradiation, CRY2 (associated with ArhGEF11, a specific activator of RhoA) will be recruited to the plasma membrane where CIBN is anchored, thereby inducing the activation of RhoA. The proposed project is divided in two steps. First, the optogenetic system CRY2/CIBN will be studied in order to optimize illumination conditions and improve the spatial resolution of light-induced effects. To understand which properties limit the spatial extent of optogenetic perturbations, we will measure the concentration, mobility and interactions of CRY2, CIBN and the photo-generated dimer as a function of irradiation intensity and duration. These measurements will be performed on a fluorescence microscope, confocal or TIRF (total internal reflection), and analysed using image correlation spectroscopy (ICS), a family of methods based on the analysis of the pixel intensity fluctuations in an image series. These fluctuations arise from changes in the occupation number of fluorophores in the focal volume. ICS provides informations on the concentration and diffusion constant of fluorescent molecules in subregions of the images, to yield maps of protein density and dynamics in living cells. The second step would focus on the onset of cell migration when the cell polarizes and an asymmetric distribution of signalling molecules appears. This event could be induced by photo-stimulation. Our initial data show that upon local RhoA photo-activation a retraction/contraction was observed at the site of photoactivation, accompanied by the appearance of protrusions at the other end of the cell, suggesting the existence of polarized flows of signalling molecules. Cell polarization is thought to be associated with increased RhoA activity at the rear of the cell while Rac1 and Cdc42 activities decrease. To correlate ICS concentration measurement with RhoA activation level, the latter will be quantified by Föster resonance energy transfer (FRET) using specific biosensors (Raïchu probes). In addition, a possible mechanism for the differential signalling activity might be the transport of signalling molecules by the flow of actin cytoskeleton. To test this hypothesis, actin flow velocity can be measured by ICS and correlated with RhoA flow velocity and spatial distribution.