New Advances in Live-cell Imaging: Multiplexed Protein Activity Measurements and Correlational Studies of Rho GTPase Regulation in Living Cells.

Abstract

Ability to measure directly protein posttranslational modifications or binding events in living cells and in real time has dramatically shifted the way in which we study the signal transduction processes in cell biology. Current state-of-the-art in this area is the multiplex imaging of fluorescent biosensors, directly reading out multiple signal node activities. Fluorescent biosensors are engineered protein constructs that can detect protein activations through sensing the binding or conformational change due to protein activation events [1]. Using complementary fluorescent signals, we measure two separate protein activation events in living cells at the same time [2,3]. Coupling this technology with the computational analysis of the leading edge evolutions [2, 4], we are able to determine a precise coordination of these protein activities at sub-micron resolution and in time scale of seconds in living cells. Using the computational multiplexing approach, we are able to now relate many protein activities measured in living cells by relating directly to the motion of the leading edge serving as the internal fiduciary. This vastly expands the potential signal nodes that can be measured and correlated to each other in live cell experiments [2]. We have developed a set of new biosensors which can detect the binding of the endogenous guanine nucleotide dissociation inhibitor to Rho family GTPases. This biosensor uses a new fluorescent resonance energy transfer (FRET) detection module consisting of monomeric Cerulean and circularly permutated monomeric Venus fluorescent proteins, wherein the binding of GDI to the GTPase target confers sterically-driven conformational change within the biosensor construct to affect the FRET emissions. By monitoring the ratio of the fluorescence emission changes between the cyan and the yellow emission channels, we can characterize the relative binding states of the GDI and the Rho GTPase target. Furthermore, we have re-engineered the previously published biosensor for measuring the activation dynamics of the endogenous Cdc42 [5] to be compatible and complementary to the GDI-binding biosensor, allowing for a simultaneous use of the two biosensor systems in a single living cell. This approach allows for the observation of the "on" versus the "off" kinetics of the Cdc42 GTPase at previously unprecedented spatial and temporal resolutions in live cell imaging. Together with the development and implementation of these biosensors, we have optimized the imaging platform to allow for 4-channel, full-field fluorescence acquisitions required to observe simultaneously the two biosensors in a single living cell [3]. 130