speaker: Mathieu Coppey
position: Physics and chemistry departments Curie Institute Paris
author: Teun Huijben
Mathieu Coppey works in the Physics and Chemistry Departments Curie Institute in Paris. He is interested in cell polarity and biophysical methods to manipulate this polarity. In his talk he enlightens us about the CRY2/CIBN technique; what it is and how it is used to study cell polarity.
A cell has polarity when one end of the cell is different from the other end. This can be a different shape, for example in a neuron, different protein concentrations or organel distributions. Mathieu Coppey and his group are interested in this polarity and questions they ask are like, how is this polarity established, maintained, and most important, can we manipulate or create this polarity by using biophysical methods like optogenetics? Using this optogenetics they try to develop a quantitative biophycisal approach to manipulate protein distributions at the subcellular scale.
The optogenetics method Coppey uses is the CRY2/CIBN dimerizer. CIBN is together with GFP and CAAX bound to the membrane as CIBN-GFP-CAAX, using CAAX as anchor that is embedded in the membrane. Cytoplasmic CRY2-mCherry undergoes a conformational change when illuminated with blue light. This conformational change enables CRY2 to bind CIBN (see figure 1A below). By binding to CIBN CRY2 moves from the cytoplasm to the plasma membrane, resulting in a higher concentration near the membrane and a lower concentration in the cytosol. This can be seen in figure 1B, before illumination CRY2 is equally distributed over the cell, but after illumination with blue light CRY2 gathers near the plasma membrane.
figure 1: (A) in this optogenetic technique CIBN-GFP is bound to a CAAX-anchor that is embedded in the membrane and cytoplasmatic CRY2 is bound to mCherry. When illuminated with blue light, CRY2 undergoes a conformational change so it can bind CIBN. (B) before illumination all the CRY2-mCherry is equally distributed throughout the cytoplasm, (C) but after blue illumination it is localized against the plasma membrane.
Now we have seen that it is possible to translocate CRY2 to the plasma membrane, we can use this technique to manipulate local subcellular protein concentrations. One way Coppey and his colleagues did this, is by only illuminating a small part of the cell. As can be seen in figure 2 below, when the area indicated with the red square is illuminated with blue light, after some time the CRY2-mCherry concentration in that area increases noticable. And when the blue illumination is moved to the green square, also the CRY2-mCherry moved to this area. These images are made with a TIRF microscopy, so only the membrane bound CRY2-mCherry is visible.
figure 2: TIRF images. (A) When only the area indicated with the red square is illuminated with blue light the CRY2-mCherry concentration increases in this area (see B). When later the area indicated with the green square is illuminated the mCherry level on the membrane on the other side of the cell increases (see C)
With localizing the illumination on only a small part of the cell, it is possible to get a high concentration on CRY2-mCherry at that location at the plasma membrane. When they combined this with Rho GTPases it gave them the possibility to activate other proteins only at this location, and this is exactly what Coppey and his colleagues did. They fused the catalytic domain (DHPH) of the Intersectin (ITSN) guanine exchange factor (GEF) to CRY2-mCherry. This catalytic domain triggers the RHO GTPase Cdc42. This is done by exchanging the GDP for a GTP and Cdc42 is inactive when GDP is bound and active when GTP is bound. So when a part of the cell is illuminated with blue light, ITSN-DHPH-CRY2-mCherry binds to membrane bound CIBN and Cdc42 is only activated in this region. Cdc42 has an influence on the cell movement, as can be seen in figure 3 below. Because after 2 minutes of illumination the part of the cell that was illuminated starts expanding, and a little later, the other side of the cell starts retracting. The local activation of Cdc42 is causing this. What once was a cell without polarity had changed into a cell with a clear polarity between front and back.
figure 3: (A) Scheme of the cell movement after illumination with blue light, the dashed blue rectangle indicates the area that is illuminated, the grey area (front of the cell) responds by expanding and the blue area (rear) responds by retracting. (B and C) The displacement and area of the front (black), rear (blue) and illuminated region (green) over time after illumination.
I think in the future this invention will make it possible to study intracellular signaling pathways better than we can now. By only activating a specific protein in a small part of the cell, the local effect of that activation will be become clear. Also the time it takes before the intracellular signaling or diffusion to transfer the signal to the other side of the cell can be studied.