Speaker: Mathieu Coppey
Department: Physics and Chemistry, Institut Curie Paris
Subject: Dissecting back-to-front cell polarity with optogenetics
Location: TU Delft
Date: 16 September 2016
Author: Carolien Bastiaanssen
One of the main interests of Mathieu Coppey is cell polarity. This term refers to the phenomenon that different sides of a cell can show different features. Some examples are a differently organized cytoskeleton, a different composition of proteins or a different distribution of organelles. Cell polarity is of crucial importance to processes such as cell migration, differentiation and morphogenesis. Through the manipulation of factors that influence cell polarity Mathieu Coppey and his colleagues try to establish a model for cell polarity.
In order to be able to influence cell polarity, Mathieu Coppey and his colleagues designed a method to control the distribution of certain proteins with light. They used a protein called CRY2 fused with mCherry and a protein called CIBN fused with GFP. Under the influence of blue light CRY2-mCherry will bind to CIBN-GFP which is attached to the plasma membrane (Figure 1). This changes the initially homogeneous distribution of CRY2-mCherry into a strong localization of this construct to the membrane of the part of the cell that has been illuminated. After three minutes CRY2-mCherry dissociates again. There are different constructs with different times until dissociation.
Figure 1: a. Under the influence of blue light the CRY2-mCherry dimer binds to CIBN-GFP on the plasma membrane. b. GFP and mCherry allow the visualization of CIBN and CRY2 respectively. The left picture shows CIBN on the membrane, the middle picture shows CRY2 in the cytoplasm before excitation and the right picture shows CRY2 at the membrane after excitation. Source: Kennedy, M.J., Hughes, R.M., Peteya, L.A., Schwartz, J.W., Ehlers, M.D. & Tucker, C.L. Nature Mehods 7, 973-975 (2010)
After Coppey and his colleagues succeeded in manipulating the polarization of a cell using light, they applied the technique to manipulate cell migration. Cdc42 is a Rho GTPase, which are molecular switches that are active when they are bound to GTP and inactive when they are bound to GDP. Cdc42 activation is triggered by ITSN. Therefore the catalytic domain of ITSN (DHPH) was fused to CRY2-mCherry and the same construct of CIBN-GFP was used as before. Under the influence of blue light the ITSN-DHPH-CRY2-mCherry complex binds to CIBN-GFP and is thus recruited to the plasma membrane. As a result the Cdc42 present near the plasma membrane is activated. By illuminating only one side of a cell, Coppey and his colleagues managed to polarize cells. They can control the migration direction of a cell using blue light (Figure 2).
Figure 2: a. Cdc42 is activated through illumination with blue light in the dashed blue box. b. The cell on the left has not yet been illuminated and the cell on the right has been illuminated 28 minutes before imaging. The red represents mCherry and it thus indirectly shows Cdc42. c. The lines represent outlines of a cell at different points in time. From blue to red, the time increases from the time of activation to 30 minutes after activation. The results show that the cell, which has been illuminated on the right side, migrates towards the right. Source: Valon, L., Etoc, F., Remorino, A., Pietro, F. di, Morin, X., Dahan, M. & Coppey, M. 2015. Predictive Spatiotemporal Manipulation of Signaling Perturbations Using Optogenetics .Biophysical Journal, 109, 1785-1797.
Coppey and his colleagues developed an elegant system with which they can manipulate protein distributions in a predictive and reproducible way using light. With this system they try to elucidate the mechanism behind cell polarization. This research really fits a nanobiologist, since the method requires knowledge of cell biology while the analysis and development of a model address mathematical skills.