Neuropeptide visualization using split GFP in live C. elegans.

Abstract

Neuropeptides play essential roles as signaling molecules in the nervous system of animals. Visualization of neuropeptides in experimental settings has advanced through two main approaches: using antibody-based methods and fluorescent protein tagging. These conventional approaches have inherent drawbacks, such as poor antibody specificity and potential functional perturbations due to fusion with bulky, full-length fluorescent proteins. The split GFP system is a versatile tool with broad applications, yet it has been underutilized for neuropeptide labeling. We demonstrate the utility of the split GFP system in the in vivo visualization of two neuropeptides, INS-1 and NLP-40, released from and targeted to neuronal and non-neuronal cells and tissues in C. elegans. Released neuropeptides were successfully visualized by inserting the 16-amino acid GFP11 tag to the sequence of neuropeptides of interest and complementing it with extracellular GFP1-10 fused to a membrane anchor protein, CD4. We show that fusion with the GFP11 tag does not perturb INS-1 function. Furthermore, we explore the in vivo regulation of neuropeptide release using genetic approaches. Our findings uncover a new application of the split GFP system which offers advantages over conventional methods for labeling neuropeptides and new insights into the regulation of neuropeptide secretion.