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)
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.
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.