As discussed in the background materials, the Rho GTPases are biochemical switches that bridge the gap between extracellular stimuli and changes in intracellular signaling and cellular morphology. The GTPases couple a variety of extracellular receptors to a host of cellular processes, including gene transcription, phagocytosis, cell migration, cell-cycle progression, vesicular trafficking, and others. That these GTPases can selectively control so many responses indicates that these molecules are very tightly controlled both spatially and temporally. Thus, to tease apart these different signaling pathways, researchers have long sought to examine the activity of these GTPases in living cells.
Previous Methods for Assessing Rho GTPase Activity:
As molecular switches, Rho GTPases cycle between an “on” and “off” form, dictated by their GTP-loading status. When bound to GTP, Rho GTPases are “on,” and when bound to GDP, Rho GTPases are “off.” Guanine Exchange Factors (GEFs) are molecules that catalyze the exchange of GDP for GTP and activate the GTPases, while GTPase Activating Proteins (GAPs) catalyze the hydrolysis of GTP to GDP, turning the GTPases “off.” Only when the GTPases are in the “on” state do they interact with downstream effectors, and this state of the protein is the one that scientists are interested in, as it catalyzes the myriad cellular events controlled by the Rho GTPases. Accessing the activity status of Rho GTPases, however, has been a difficult process over the years. Early studies used proxy indicators for Rho GTPase activation, such as the presence of stress fibers, as an indicator of RhoA activation. Biochemical assays were also
developed to examine the activation status of the GTPases, first by adding 32Pi- orthophosphate to cell media, allowing its incorporation into GTP, immunoprecipitating the Rho GTPase, and then using thin layer chromatography to separate GDP and GTP (Gibbs 1995) as an indicator of Rho GTP loading status. Next, other researchers developed the “pull down” assay whereby the GTPases were immunoprecipitated by the use of a GST-fused binding domain that selectively bound the active form of the GTPase (Taylor and Shalloway 1996; de Rooij and Bos 1997).
Slowly, these concepts pushed their way into imaging studies, where fluorescently labeled forms of the GTPases were observed for changes in localization (Kranenburg et al. 1997; Michaelson et al. 2001). Others utilized fluorescently tagged versions of the binding domains used in the pull down studies as an indicator of where the active forms of the GTPases were located (Kim et al. 2000; Cannon et al. 2001; Srinivasan et al. 2003). While the biochemical assays showed excellent sensitivity and selectivity, they simply averaged what was going on in large populations of cells, in contrast to the ability of imaging to identify processes in single cells. However, the imaging studies were not very sensitive or specific. Thus, FRET-based sensors which were both sensitive and specific were developed to examine GTPase activity status in live cells (Kraynov et al. 2000b; Mochizuki et al. 2001). Over the course of development of these FRET-based sensors, a handful of basic designs have come to the front in terms of usefulness, but each sensor type must be used with a full understanding of the caveats of each.
Comparison of Basic Rho GTPase Biosensor Design:
The basic sensor designs that have arisen from studies attempting to discern Rho GTPase activation by FRET are: 1) intermolecular (dual-chain) and 2) intramolecular (single-chain) designs as illustrated in Figure 1.5. Within the intramolecular class of sensors, two basic flavors exist as well, the GTPase-effector fusion and effector domain- only designs (Itoh et al. 2002; Seth et al. 2003). These sensor designs have been used for a variety of other intracellular targets as well (Miyawaki 2003; Newman et al. 2011). Due to the lack of specificity and sensitivity inherent in the effector domain-only designs, these sensors are rarely used today, given the better characteristics of the other sensor formats. Basic differences in these sensor designs unfold in the acquisition and
processing of the imaging data. Intermolecular FRET sensors consist of two components which often distribute differentially within the cell at different expression levels, so bleedthrough correction must be performed to correct for contributions due to localized accumulation of one domain or the other. In contrast, bleedthrough correction has typically been ignored for single-chain sensors as both donor and acceptor fluors are present in the same place at the same time, reducing these artefacts. Such differences are largely responsible for the differences in their frequency of usage and popularity in scientific studies.
Comparison of Usage of Intramolecular and Intermolecular Rho GTPase FRET Biosensor Designs:
As mentioned above, intramolecular FRET sensors typically require less intensive image acquisition and processing techniques in order to obtain an informative FRET
fluorophores. Further, it has been argued that intramolecular FRET sensors typically interact less with other cellular components since it is likely that once the GTPase
becomes activated, it is immediately bound by the associated binding domain, preventing interaction with endogenous effectors and thus perturbing intracellular signaling to a smaller degree (at least as demonstrated for the cameleon sensors) (Miyawaki et al. 1999; Miyawaki 2003). These lines of reasoning have largely driven the use of intramolecular FRET sensors for the GTPases.
While there is some evidence that these concerns are true of the intramolecular Rho GTPase sensors (Pertz et al. 2006), these sensors could as a result behave as
dominant negative constructs, sequestering upstream regulators such as GAPs, GEFs, and GDI. Indeed, recent work suggests that overexpression of Rho GTPases alters
intracellular signaling by a mechanism different than expected: alterations in the expression of endogenous GTPases due to derangement of GTPase/GDI ratios in cells (Michaelson et al. 2001; Boulter et al. 2010). Interestingly, it has been shown recently that use of dual-chain and single-chain RhoA FRET sensors produced the same results in a thorough quantitative assessment of the role of RhoA activation at the leading edge of cells, suggesting that sensor design does not significantly influence the detection of GTPase activation dynamics in live cells (Machacek et al. 2009). Additionally, it is known that intermolecular FRET sensors, while requiring more extensive image processing, generate larger dynamic ranges due to lower FRET in the off state, perhaps permitting their expression at lower levels in cells (Pertz and Hahn 2004; Hodgson et al. 2008). This property could be exploited to detect smaller changes in intracellular signaling if the intermolecular sensors are used appropriately.
Thus, in light of the lack of comparative data on different Rho GTPase sensors and sensor designs, our aim was to generate new and improved versions of our Rho GTPase sensors utilizing dual-chain and single-chain sensor designs. Through
comparison of these designs, we are able to show that sensitivity and dynamic range of these GTPase sensors can be improved through several new strategies. Additionally, we show that both dual-chain and single-chain sensors suffer from artefacts that must be considered when selecting probes for imaging, that dual-chain sensors are more sensitive than single-chain sensors and can be analyzed by different methods compared to single- chain sensors, that both dual-chain and single-chain sensors perturb cellular signaling at high expression levels, that expression levels of dual-chain sensors can be normalized through utilization of a novel expression cassette, and that new red-shifted FRET pairs can be utilized in these sensors that are orthogonal to the traditional CFP/YFP pair so commonly used. This work thus provides a quantitative comparison of Rho GTPase sensor designs and enables researchers to better select and interpret data generated by these probes, in addition to providing new sensors for use.