Rab5 Is a Novel Regulator of Mast Cell Secretory Granules: Impact on Size, Cargo, and Exocytosis

Abstract

Secretion of inflammatory mediators prestored in mast cells secretory granules (SGs) enhances immune responses such as in allergy and host defense. However, the mechanisms underlying the biogenesis of the SGs remain largely unresolved. By combining high-resolution live cell imaging and quantitative morphometric analyses, we show that the small GTPase Rab5 controls the SG size and cargo composition by a VAMP8-dependent fusion mechanism. Knockdown of the endogenous Rab5, or expression of constitutively negative mutants, significantly reduces the size of SGs and increases their number. Conversely, expression of constitutively active Rab5 mutants induces few, but giant, SGs. Both the small and giant SGs maintain their exocytosis competence. Finally, we show that Rab5-mediated fusion between Golgi-derived SGs and early endosomes precedes the maturation of the SGs, as reflected by the recruitment of Rab27B, and allows the incorporation of cargo, such as CD63, that traffics through endosomes. Collectively, our results assign Rab5 a key role in mediating mast cell SG fusion during biogenesis, thereby controlling the amount and composition of the SGs content and maintaining the communication between new and pre-existing SGs.

Identification of a New Exo-Endocytic Mechanism Triggered by Corticotropin-Releasing Hormone in Mast Cells

“…This mechanism would provide reliable communication machinery for adaptation to environmental insults. Increases in Ca 2+ i and its sensor for vesicle fusion, synaptotagmin, are required for compound fusion (55) and other proteins such as Rab5 and VAMP8, are known to play a role in homotypic secretory granule fusion in MC (56).…”

Characterization of Mast Cell Secretory Granules and Their Cell Biology

“…The inverse relationship between the number and size of SGs suggested that Rab5-mediated homotypic fusion of the SGs. Indeed, high-resolution imaging of the giant SGs by confocal microscopy of living cells, which detected Rab5A decorating fusing SGs, and electron micrographs that depicted considerably larger SGs and homotypic fusion between two or more granules in CA Rab5A-expressing cells, have reinforced this conclusion (Azouz et al, 2014). The interaction of Rab5 with the SGs was transient and occurred shortly after their Golgi exit (Azouz et al, 2014).…”

“…Among the Rabs that displayed a remarkable phenotypic impact, we identified Rab5 as a regulator of SG fusion (Azouz et al, 2014). Coexpression of constitutively negative GDP-locked mutants of the endogenously expressed isoforms of Rab5 (Rab5A, Rab5B, and Rab5C) or expression of Rab5A/B/C targeting shRNAs, reduced significantly the SGs' size with a concomitant increase in their numbers (Azouz et al, 2014).…”

“…Coexpression of constitutively negative GDP-locked mutants of the endogenously expressed isoforms of Rab5 (Rab5A, Rab5B, and Rab5C) or expression of Rab5A/B/C targeting shRNAs, reduced significantly the SGs' size with a concomitant increase in their numbers (Azouz et al, 2014). Conversely, expression of a GTPlocked constitutively active (CA) Rab5A mutant (Rab5A Q79L, herein: CA Rab5A), which is known to facilitate homotypic fusion of early endosomes (Stenmark et al, 1994), resulted in the formation of giant Rab5A-decorated vesicles, which we identified as SGs based on their content of the secretory cargo NPY-mRFP and serotonin (Fig.…”

“…Conversely, expression of a GTPlocked constitutively active (CA) Rab5A mutant (Rab5A Q79L, herein: CA Rab5A), which is known to facilitate homotypic fusion of early endosomes (Stenmark et al, 1994), resulted in the formation of giant Rab5A-decorated vesicles, which we identified as SGs based on their content of the secretory cargo NPY-mRFP and serotonin (Fig. 2) and their capacity to exocytose in a regulated manner (Azouz et al, 2014). The inverse relationship between the number and size of SGs suggested that Rab5-mediated homotypic fusion of the SGs.…”