Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells

The biosensors developed in our laboratory have been based on different designs, each imparting specific strengths and weaknesses. Here we describe detailed protocols for the application of three biosensors exemplifying different designs -- first a design in which an environmentally sensitive dye is used to report the activation of endogenous Cdc42, followed by two biosensors based on FRET, one using intramolecular and the other intermolecular FRET. The design differences lead to the need for different approaches in imaging and image analysis. Figure 1 shows the design of the three biosensors. MeroCBD (Fig 1a) is a sensor for Cdc42 activation in which a fragment of Wiskott Aldrich Syndrome protein that binds only to active Cdc42 is derivatized with an environmentally sensitive dye1. In living cells, the dye undergoes a fluorescence change when the biosensor binds to endogenous, untagged Cdc42. This design is advantageous for several reasons: Cdc42 is not tagged with a fluorophore or other biosensor component, expression of exogenous Cdc42 is not needed, and the signal is substantially brighter than FRET because a bright dye is directly excited, rather than indirectly as in FRET. The major disadvantage is that the biosensor is not genetically encoded so must be loaded in the cells via microinjection, electroporation etc. Fig.1 Fluorescent biosensor designs. A. MeroCBD, biosensor of endogenous Cdc42 activation. Here a fragment of Wiskott Aldrich syndrome protein (WASP) that binds only to activated Cdc42 is covalently derivatized with an environmentally sensitive dye. When the ... The FRET biosensor for RhoA is shown in figure 1B. A fragment of Rhotekin is attached directly to RhoA, and fluorescent proteins undergoing FRET are built into the chain connecting the RhoA and Rhotekin fragment. The C terminus of the RhoA is purposefully left free of fluorescent protein or other modifications, to maintain intact the regulation of RhoA by GDI. Unlike the Cdc42 biosensor, this biosensor is completely genetically encoded, greatly simplifying loading into cells. Finally, the biosensor for Rac1 is shown in Fig.1C. Named Rac1 FLAIR, it is a modification of a design originally published using a covalently attached fluorescent dye for FRET. In the modification described here, the dye has been replaced with a fluorescent protein, rendering the biosensor fully genetically encoded. A fragment of p21 activated kinase (PAK) is used to bind specifically to activated Rac1. The main difference between this design and that of the RhoA biosensor is that the PAK fragment is not attached to Rac1. Rac1 FLAIR is an intermolecular FRET biosensor, frequently referred to as a dual chain sensor. The use of a dual chain design significantly enhances the sensitivity of the biosensor in comparison with single chain FRET biosensors. The latter usually show substantial FRET even when the biosensor is in the `off' state; the two fluorophores are never fully separated. With the Rac1 sensor and other single chain sensors the dynamic range, the difference between the on and off states, is enhanced. There is a downside to the dual chain approach. The two components of the biosensor distribute differently in the cell, necessitating additional steps in image processing. Both single chain and dual chain biosensors are potentially perturbed by competition from native protein ligands; the dual chain sensor may be more likely to produce `false negatives' as it may be more sensitive to ligands that compete for the GTPase binding fragment, while the steric bulk of the single chain sensor may prevent it from reaching all sites normally accessed by the GTPases. The issue of competition and its effect on biosensor readout is complex and beyond the scope of this article. We recently completed a careful study of the effects of varying the concentration of each component of the dual chain Rac1 biosensor. This did not appreciably alter the results of our cell protrusion studies (manuscript submitted).