Abstract
Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Despite extensive efforts to visualize the spatiotemporal activity of these proteins, achieving the sensitivity and dynamic range necessary for in vivo application has been challenging. Here, we present highly sensitive intensiometric small GTPase biosensors visualizing the activity of multiple small GTPases in single cells in vivo. Red-shifted sensors combined with blue light-controllable optogenetic modules achieved simultaneous monitoring and manipulation of protein activities in a highly spatiotemporal manner. Our biosensors revealed spatial dynamics of Cdc42 and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. Thus, these intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.
Highlights
Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication
Compared with GA expression alone, co-expression of GA and each copy B increased basal fluorescence; the GA-B1 pair elicited a higher intensity than GA-B3. As both copies A and B were localized in the cytosol in this experiment, we examined whether spatial separation of GA and copy B could reduce fluorescence
We found that the detection limit for epidermal growth factor (EGF) by dependent fluorescent protein (ddFP) or Förster resonance energy transfer (FRET) sensor was 0.011 ng ml−1 or 24.946 ng ml−1, respectively (Supplementary Fig. 3, and see Methods for signal-to-noise ratio (SNR) analysis in detail)
Summary
Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Our biosensors revealed spatial dynamics of Cdc[42] and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. These intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals. The spectral overlap of cyan-yellow-paired FRET sensors with blue light-controllable optogenetic modules does not permit transient and local perturbation of target molecules under continuous monitoring of reporter activities[11] To address these issues, here we present an intensiometric small GTPase biosensor that utilizes the dimerization-dependent fluorescent protein (ddFP). Applying to the mouse brain, we identified spatially distinctive activity profiles of Ras and Cdc[42] in single dendritic spines and proved feasibility and effectiveness of our sensors for real-time monitoring of Ras activity in the brains of freely behaving mice at micron scale precision
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