Characterization of the phosphoinositide composition of mast cells secretory granules and its regulation by Rab5 during the granule biogenesis and exocytosis

Mast cells (MCs) are highly engineered cells that perform critical functions in allergic reactions. Upon activation, MCs secrete inflammatory mediators that are preformed and stored in cytoplasmic secretory granules (SGs). The mechanisms underlying the biogenesis of SGs and their exocytosis remain largely unresolved. We have recently identified the small GTPase Rab5 as a key regulator of SG fusion during their biogenesis and compound exocytosis. Rab5 regulates endocytic trafficking by activating phosphoinositide 3-kinases (PI3Ks) and modifying the membrane lipid composition. To decipher the possible role of phosphoinositide remodeling in controlling SG fusion in MCs, we expressed phosphoinositide specific PH domains, fused to GFP, and visualized, by confocal microscopy, their distribution in resting and FcεRI-triggered cells. Here we demonstrate that the PH domains derived from Akt and FAPP1, that respectively bind to PI(3,4,5)P₃ and PI(4)P, display increased association with the SGs in cells that express constitutively active (CA) Rab5. In contrast, neither the PI(4,5)P₂ binding PLCδ-derived PH domain, nor the PI(3)P binding FYVE domain associate with the SGs. These results demonstrate that Rab5-stimulated SG fusion is accompanied by an increase in SG membrane levels of PI(3,4,5)P₃ and PI(4)P. Further we show that LY294002, a pan inhibitor of PI3Ks, disrupts Akt-PH association with the SGs, inhibits their CA Rab5 mediated fusion and stimulates their fission. Together our results implicate phosphoinositide conversion and PI3K(s) as critical for both homotypic SG fusion and SG fission that occur during SG biogenesis.