Dissection back-to-front cell polarity with optogenetics

Speaker: Mathieu Coppey
Department: Physics and Chemistry Department, Marie Curie Institute Paris                       Subject: Optogenetics
Location: TU Delft

Date: 16 September 2016

Author: Gabriele Kockelkoren

Mathieu Copey works in the Physics and Chemistry department of the Marie Curie Institute in Paris, France. The general motivation and driving force in his research is understanding the various scales that govern life. By understanding them all, insights can be gained in the behaviour and mechanisms of cells. One of the main interests of the his lab is cell polarity and ways to manipulate cell polarity. Cell polarity is the asymmetric organisation of several cellular components, including its plasma membrane, cytoskeleton or organelles. This asymmetry can be used for specialised functions, such as maintaining a barrier within an epithelium or transmitting signals in neurons. Polarity occurs at many scales in biology. The two main projects in his lab are: 1. Manipulation of protein distribution: light-gated dimerize for plasma membrane localization. 2. RhoA signalling perturbation and back to front polarity.

In order to manipulate cell polarity, Mathieu Coppey uses optogenetics.  The optogenetics method the lab applies for manipulation makes use of the CRY2/CIBN dimerizer. In this technique the CRY2 protein is labelled with mCherry and CIBN is a protein labelled with GFP. By illuminating the sample with blue light CRY2-mCherry will bind to CIBN-GFP which is attached to the plasma membrane (Figure 1). Excitation with blue light is done using TIRF microscopy and only a small part of the cell can be excited.

 

Figure2

 

Figure 1: By excitation with blue light CRY2 binds to CIBN. 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)

By succeeding in the use of light to manipulate the cell polarization, they introduced Rho GTPase to the system. Rho GTPase signalling controls cell morphology and migration. Cdc42  is a Rho GTPase. Rho GTPases gives the possibility to activate other proteins at the location of excitation. The Rho GTPase used in the experiments is Cdc42, which works as a switch that is active when bound to GTP and inactive when bound to GDP. Its activation occurs via ITSN (intersectin), which has the catalytic domain DHPH. This domain is fused to the CRY2-mCherry construct and the CIBN-GFP construct. Through this construction excitation by blue light causes the DHPH-CRY2-mCherry construct to bind the DHPH-CIBN-GFP at the plasma membrane. Cdc42 influences movements within the cell and as a result only the Cdc42 which is nearby the plasma membrane is activated. This results in the polarization of the cells and controlled migration by blue light excitation.

I particularly enjoyed this talk as it combines innovation of techniques with the field of biology and physics. In other words, a perfect talk for a Nanobiology student.

 

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