Development of New and Updated Rho GTPase Biosensors:
Rho GTPases are molecular switches that serve as central integrators and regulators of a host of cellular functions downstream of extracellular receptors. Given that these GTPases very specifically control certain cellular functions in response to very specific cellular cues, it is clear that significant spatial and temporal control is exerted on these GTPases to regulate their functions. Understanding these controls has been largely inaccessible until the development of biosensors that could examine Rho GTPase activity in live cells (Kraynov et al. 2000b; Mochizuki et al. 2001; Yoshizaki et al. 2003; Nalbant et al. 2004a; Pertz et al. 2006b; Kitano et al. 2008). However, despite the value of these probes, there has been an endless drive to improve these sensors (Seth et al. 2003; Yoshizaki et al. 2003), much as has been done for other sensors (Miyawaki et al. 1997; Miyawaki et al. 1999; Truong et al. 2001; Palmer et al. 2006) to obtain improvements in brightness, in dynamic range, and in sensitivity without perturbation of cellular signaling. Additionally, as our microscopes and computational abilities improve, researchers seek to study the relationship between two GTPases simultaneously in the same cell (Schultz et al. 2005; Ai et al. 2008; Piljic and Schultz 2008; Machacek et al. 2009).
To this end, in the present study, we demonstrate the development of novel dual- chain FRET biosensors for the GTPases Cdc42, RhoG, RhoA, and Rac1, along with a new single-chain sensor for Rac1 which maintains GDI regulation. Through this development process, we illustrate some of the difficulties in building and testing biosensors, such as the choice of linker regions and proper positioning of binding domains and fluorophores, and the choice of assays performed for validation of the
sensors. Additionally, we show that by use of a tandem acceptor cassette, tdYPet, we can further enhance the dynamic ranges of dual-chain sensors, based on the reports of
anomalous surplus FRET energy transfer in the presence of multiple acceptors (Koushik et al. 2009). Additionally, we report the conversion of these sensors to red-shifted varieties that can be used simultaneously with CFP/YFP FRET pairs. Drawing upon our experience in the development of a tdYPet acceptor, we find that development of a tdmCherry acceptor similarly improves the dynamic range of dual-chain red-shifted FRET sensors.
Artefacts Inherent to Biosensor Imaging:
Many arguments have been made that favor the use of single-chain sensors for protein activity in live cells, particularly that single-chain sensors perturb intracellular signaling to a lesser degree, and that they exhibit higher amounts of FRET which are easier to detect in live-cell imaging. However, little data that supports these points is available for the Rho GTPase sensor field from a comparative standpoint with dual-chain sensors (Pertz and Hahn 2004). Because we consistently saw higher dynamic ranges for dual-chain sensors during probe development, we set out to compare the value of dual- chain and single-chain sensors in live cell imaging.
Through these comparative analyses, we find that dual-chain sensors indeed have larger dynamic ranges than single-chain sensors, and exhibit higher sensitivity, permitting lower expression levels of dual-chain probes compared to single-chain probes to observe the same intracellular signals. Further, both sensors display imaging artefacts that must be understood when using them. Single-chain sensors are prone to exhibit FRET where
none exists, or false positive signals. Dual-chain sensors are prone to exhibit false negative signals, or lack of FRET where activation of GTPase does occur. Thus, a careful understanding of these risks is required during their use.
Additionally we demonstrate that both sensor designs perturb downstream signaling to the same degree, presumably from effects not only on interactions with effectors, but also due to effects on GDI-1 binding and saturation. Lastly, we show that due to the design of the dual-chain probes, we can analyze FRET in low signal situations by the study of total FRET with certain caveats.
Lastly, we examine a number of modifications used to enhance cellular signals during imaging, such as the use of different C-terminal modifications, and their effects on FRET interpretation. As expected, loss of the C-terminal tail greatly disrupts patterns of activation seen in live cells. Modification by constitutive membrane-tagging using the K- Ras4b sequence is more subtle. Grossly, FRET patterns are intact, but the probes localize very differently, with loss of cytosolic and endomembrane localization for the Rac1- KRas4b sensor and placement of the sensor at locations different than where some of its regulators exist. Additionally, we show that Rac1 activity seen in these locations with the wild-type sensor is lost by use of the K-Ras modified version. Such modifications are useful in certain circumstances, but must be used with an understanding of their caveats. For example, for a number of GTPases it has been shown that vesicular trafficking is key to their regulation, and that in the case of Rac1, activation occurs on endosomes via the GEF Tiam-1 (Palamidessi et al. 2008; Osmani et al. 2010). Indeed, use of a K-Ras modified sensor would fail to detect such activation events. However, if utilized properly, these sensors can be targeted to specific subcellular regions to enhance the
contrast of FRET detected in those regions, such as on endomembranes or at focal adhesions.
Overcoming Hurdles to the Use of Dual-Chain Sensors:
As we have shown, dual-chain sensors have higher sensitivity and dynamic range than single-chain sensors, and can be extremely valuable for detecting small changes in intracellular signaling with low expression of the probe such that intracellular signaling is not perturbed. However, the two biggest hurdles to their use, practically, are the need for bleedthrough correction, and the uneven control of expression of the two sensor
components. To circumvent the issues with uneven expression of the two sensor components, an autocleavable cassette has been developed which normalizes the
expression of both components from a single plasmid, yielding roughly a 1:1 expression ratio for the two components. Our hope is that this will enable broader use of these probes in cells in tissue culture, and eventually in organisms. Achieving equal and even expression of two exogenously expressed proteins in living organisms is notoriously difficult, making the use of dual-chain sensors quite difficult. However, with the ability to express a dual-chain sensor from a single plasmid gene, the use of these sensors could be eased significantly. Thus, use of a more sensitive dual-chain sensor in live organisms could permit the study of much smaller changes in intracellular signaling.
Concluding Remarks:
In closing, we have attempted to further the field of genetically encodable FRET sensors for Rho GTPases by performing a quantitative comparison of biosensor
Our findings indicate that, while infrequently used in the literature, dual-chain FRET probes for the Rho GTPases are of significant value. Also, as is the case for any
experimental setup, a solid understanding of the advantages and disadvantages to the use of both single-chain and dual-chain FRET probes is needed when selecting a probe to use in the study of intracellular signaling pathways. Such an understanding is critical for the appropriate analysis and interpretation of FRET signals and Rho GTPase activation during cellular signaling.