Dissecting back-to-front cell polarity with optogenetics

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.

cry2-cibn-system

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).

migrating-cells

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.

 

Dissecting back-to-front cell polarity with optogenetics

Speaker: Mathieu Coppey
Department: Physics and Chemistry Dept, Institut Curie Paris
Subject: Dissecting back-to-front cell polarity with optogenetics
Location: Room A1.100 TNW South (TU Delft)
Date: 16-09-2016

Author: Kristian Blom

At the 16th of September I visited a BN seminar given by Mathieu Coppey, a current postdoc in biophysics in the ‘single molecule imaging of cell dynamics laboratory’ at the école normale supérieure in Paris. Dr. Coppey mainly focuses on the development of modeling tools in accordance with the emergence of out of equilibrium processes. In this seminar dr. Coppey highlighted a quantitative framework, using the light gated CRY2/CIBN dimerizer, which allows a simple and predictive manipulation of protein distribution on the plasma membrane of mammalian cells.

The seminar started with a short introduction about cell polarity, which is the difference in shape, structure and function of cells. Almost all cell types exhibit some sort of polarity, e.g. migratory cells which have a defined front and rear to move in a specific direction. Cell polarization originates from a non-homogenous distribution of molecules in the cell and it can be categorized in three successive major events: First comes the initiation and formation of a polarity axis by a nonhomogeneous distribution of molecules, then the amplification and maintenance of polarity signals, and finally the propagation of these signals to the receptor(s). In the same sequence of events dr. Coppey guided us through his seminar.

First the initiation and formation of a polarity axis was established using the proteins CRY2 and CIBN (the N-terminus of CIB1) were used. The former is a flavoprotein which is sensitive to blue light. In the initial setup, CRY2-mCherry is homogeneously spread in the cytoplasm, while CIBN is localized at the plasma membrane. Under blue illumination, CRY2 changes conformation and binds to the immobile CIBN (fig 1.c). After excitation, it takes 3 minutes before the dimer is decoupled. Due to the fluorescent protein mCherry, it is possible to observe the spatial movement of CRY2 in its mobile and immobile state (pmCRY2). Data showed that there was an increase in the intensity of light at the illumination spots on the cell membrane, and a decrease in intensity in a neighborhood around that spot (fig 1.b). The exposure time in this experiment was 3 seconds at an area of 3 micrometers. Using the same set-up, dr. Coppey and his research group also looked at the steady state formation when the pulse frequency was changed. Data revealed that the higher the pulse frequency, the longer it took for the emergence of the steady state.

Optogenetics-quantitative-framework
Figure 1 – (a) CRY2-mCherry TIRF images before illumination (top) and after six local activations in the red box (middle) and after six local activations in the indicated green box (bottom). (b) Quantification of the relative increase in signal in the red and green region over time. (c) Scheme of the biophysical processes involved in CRY2-mCherrt localization at the plasma membrane. Source: Predictive spatiotemporal manipulation of signaling perturbations using optogenetics, Mathieu Coppey, Biophysical Journal (109), 1785-1797, November 2015.

 

Second, dr. Coppey and his group applied their quantitative optogenetic method to the regulation of the Rho GTPase Cdc42. The Intersectin (ITSN) guanine nucleotide exchanging factor (GEF) was fused to CRY2-mCherry, so that local recruitment of ITSN to the plasma membrane was possible. Cdc42 becomes active GTP-loaded when it binds to the catalytic domain of ITSN. Under blue light illumination, ITSN-DHPH-CRY2-mCherry was recruited at the plasma membrane and led to the activation of Cdc42. A couple of minutes after recruitment, the cell started to migrate in the direction of the active Cdc42 area. It was noticed that the rear side started to move later than the front side of the cell, which indicates that the cell was a little bit stretched along the axis of migration.

Finally, the same experiment was repeated with RhoA, a small GTPase protein, instead of Cdc24. When RhoA was locally activated at one side of the cell, migration was initiated opposite to the active RhoA area. But this time the rear and front side of the cell started moving at the same moment. The mechanism which caused this behavior is so far unknown. We can already suggest that there must be some kind of time delay mechanism involved, which causes a delay in migration at the active RhoA area, until a signal is transmitted from that area to the other side of the cell so that migration starts at the same time on both sides. Perhaps that in the future some nanobiologists will expose the underlying mechanism which causes this synchronous movement of cell poles.