New imaging tools for real-time monitoring of the C. elegans CaMK-CREB pathway in vivo

Nociception is the process of detecting and encoding harmful stimuli in the nervous system and underlies responses such as pain, an important medical concern. Calcium/Calmodulin-dependent protein kinases result to be involved in nociception, but their functions are not completely understood.

CaMKs pathway in mammals is complex and involves numerous molecular players, so we are studying it in a simpler animal model, the nematode C. elegans. We are focusing on CMK-1, the nematode homolog of the mammalian CaMKI and CaMKIV.

Previous findings in our lab show that the basal cytoplasmic activity of CMK-1 in thermal nociceptors acts to maintain their sensitivity to noxious heat, whereas upon prolonged stimulation and desensitization, CMK-1 translocates to the nucleus, where it acts to reduce nociception. These informations can be further enriched investigating the activation state of CMK-1, also because the relationship between protein localization in sub-cellular compartments and its functional state is not known yet.

For this reason, we are developing FRET sensors based on CMK-1 activation mechanism to be tested in living worms. These sensors are composed by the full length CMK-1 sequence flanked by two fluorophores, mTFP1 and Venus. CMK-1 in the inactive state maintains mTFP1 near to Venus determining FRET, while conformational changes in active CMK-1 determine an increased distance between the two fluorescent proteins, preventing energy transfer from mTFP1 to Venus and resulting in no/low FRET.

This approach will provide extremely detailed informations about a specific functional state of CMK-1 in a specific neuronal localization in C. elegans in vivo during nociceptive signal processing and adaptation. Moreover, considering the involvement of CMK-1 in nociception and its functional conservation from nematodes to humans, this project can be useful to provide cues for novel therapeutic strategies and analgesia production.