Signal transduction cascades play essential roles for various organisms to adapt environmental changes and consist of various signaling molecules. Therefore, to analyze mechanisms of signal transduction pathways, it is important to observe dynamics of signaling molecules in vivo. For example, intracellular ion level has been observed using in vivo imaging analyses such as calcium imaging1. However, signal transduction pathways are also composed of various signaling proteins. Signaling proteins alter their status in response to continuous environmental stimuli. Even though observation of specific protein activity in vivo is crucial for understanding the signal transduction pathways, temporal dynamics of signaling proteins in living animals are largely unknown. To answer this question, we tried to monitor the activities of Ras protein which is one of the key regulators for cell growth, differentiation, olfactory reception and other biological processes2-4. Thus, we observed Ras activity in olfactory neuron in a living nematode worm Caenorhabditis elegans5. We used Raichu-Ras, a FRET-based sensor for Ras activity6. Raichu-Ras was expressed in AWC olfactory neurons in C. elegans and we monitored Ras protein activity in vivo after stimulation of isoamyl alcohol which is an odorant sensed by the AWC neurons. Here we describe a detailed protocol for imaging of specific protein’s activity in intact animals.
In vivo imaging of protein activity may be more difficult than calcium imaging since its dynamics of activity change is weaker than that of intracellular ion level in many cases. Therefore it is important to restrain animal’s movement by gluing the animal on the agarose pad to minimize noise of fluorescent changes from the animal motion.