Physiologically, using a two-choice preference assay with apple cider vinegar, we found that Fas3G overexpression-facilitated non-lethal caspase activation in olfactory receptor neurons suppresses the innate olfactory attraction behavior of Drosophila (Figure 6).

Mechanistically, we found that the non-lethal caspase activation driven by Fas3G overexpression is mediated by an increase in the initiator caspase Dronc, which is also in close proximity to Fas3G, thereby enabling caspase activation near the plasma membrane (Figure 5).

Using MASCaT, we found that Fas3G overexpression induces caspase activation without killing olfactory receptor neurons. Moreover, the simultaneous expression of Drice and Fas3G markedly enhanced caspase activation highlighting the promoting effect of their proximity (Figure 4).

To ascertain whether Fas3G modulates caspase activity, we developed a Gal4-Manipulated Area Specific CaspaseTracker/CasExpress (MASCaT) system, which permits the monitoring of caspase activity near plasma membrane with high sensitivity (Figure 3).

Among the identified proximal proteins, we focused on Fasciclin 3, a transmembrane protein containing immunoglobulin-like domains. Notably, Drice is proximal to the specific alternative splicing isoforms of Fas3, Fas3 isoform G (Figure 2).

To investigate the regulatory mechanisms of non-lethal function of caspase, we conducted comprehensive analysis of proximal proteins of executioner caspase Drice in the adult brain using TurboID and found that Drice is primarily proximal to cell membrane proteins (Figure 1).