Abstract:
Fluorescence or Förster resonance energy transfer (FRET) is a quantitative transfer of energy between two fluorescent moieties in proximal distance, between 10Å and 100Å. The energy transfer is useful in studying direct molecular interactions and activation. Hence, FRET led to the design of biosensors. Biosensors are two chromophores linked to 2 entities, joined together by a linker which determines their juxtaposition relative to each other, and thus the amount of energy transfer. By exciting one chromophore and observing the emission of the other, one can determine the activation and binding of certain proteins.
Using this technique, we are studying the crosstalk of Rho GTPases in different cell lines, with a focus on RhoA, RhoC, Cdc42 and Rac1. We first showed how RhoGTPase activity is concentrated at the cell periphery. We also found Cdc42 activation to be concentrated at invadopodia and membrane ruffles. Then, we investigated the interplay between Cdc42 and RhoA by knocking down one target and studying the activation of the other in brain cancer cells, and vice versa. The results indicate an antagonistic relationship between the two in one glioblastoma cell line and a cooperative relation in the other. As for the interplay of RhoA and RhoC with Rac1, we found out that RhoA regulates the expression of Rac1 in cells, whereas RhoC has no effect on Rac1 expression or activity levels. Additionally, we wanted to use FRET so test the regulation of RhoGTPases by upstream effectors such as STARD13. We checked for the effect of STARD13 knockdown on Cdc42 activation in lung cancer cells. In the A549 lung cancer cells, the activation of Cdc42 increased as we hypothesized. Altogether these experiments show the efficiency of using FRET as a tool for studying the activation kinetics and dynamics of Rho GTPases.
