A Rab6 to Rab11 transition is required for dense-core granule and exosome biogenesis in Drosophila secondary cells

Abstract

Secretory cells in glands and the nervous system frequently package and store proteins destined for regulated secretion in dense-core granules (DCGs), which disperse when released from the cell surface. Despite the relevance of this dynamic process to diseases such as diabetes and human neurodegenerative disorders, our mechanistic understanding is relatively limited, because of the lack of good cell models to follow the nanoscale events involved. Here, we employ the prostate-like secondary cells (SCs) of the Drosophila male accessory gland to dissect the cell biology and genetics of DCG biogenesis. These cells contain unusually enlarged DCGs, which are assembled in compartments that also form secreted nanovesicles called exosomes. We demonstrate that known conserved regulators of DCG biogenesis, including the small G-protein Arf1 and the coatomer complex AP-1, play key roles in making SC DCGs. Using real-time imaging, we find that the aggregation events driving DCG biogenesis are accompanied by a change in the membrane-associated small Rab GTPases which are major regulators of membrane and protein trafficking in the secretory and endosomal systems. Indeed, a transition from trans-Golgi Rab6 to recycling endosomal protein Rab11, which requires conserved DCG regulators like AP-1, is essential for DCG and exosome biogenesis. Our data allow us to develop a model for DCG biogenesis that brings together several previously disparate observations concerning this process and highlights the importance of communication between the secretory and endosomal systems in controlling regulated secretion.

APP and β-amyloid modulate protein aggregation and dissociation from recycling endosomal and exosomal membranes

“…The number of compartments marked by Rab11 was unaltered following mfas knockdown when compared to control cells expressing an RNAi targeting the xanthine dehydrogenase gene, rosy , which did not affect DCG biogenesis (Figures 1D, 1F and S1B; Marie et al, 2023). Furthermore, the number of compartments labelled with Rab6, which marks precursor compartments that lack DCGs as well as less mature DCG compartments, was also unaffected (Figures S1C and S1E; Wells et al, 2023). Rab11 and Rab6 label a fraction of the ILVs present in DCG compartments (Fan et al, 2020; Wells et al, 2023).…”

“…We previously showed that GAPDH plays a conserved, so-called ‘moonlighting’, role in humans and flies in clustering Rab11-exosomes and other secreted extracellular vesicles (EVs; Dar et al, 2021). In SCs, DCGs are partially coated with strings of ILVs that extend in chains to the limiting membrane of the DCG compartment (Figure 1A; Fan et al, 2020; Wells et al, 2023). Following GAPDH2 knockdown, most mini-cores formed are located near the compartment boundary and are frequently surrounded by ILVs, which are therefore also located peripherally (Dar et al, 2021).…”

“…Accessory glands were dissected and prepared as described in Fan et al, 2020 and Wells et al, 2023. For live-cell imaging, six-day-old adult male virgin flies were anaesthetised using CO 2 .…”

“…Based on Wells et al, 2023, live SCs were imaged at room temperature using a DeltaVision Elite wide-field fluorescence deconvolution microscope (GE Healthcare Life Sciences) at 100x (Olympus UPlanSApo NA 1.4; oil objective), using immersion oil with refractive index of 1.514 (Cargille labs) and a manual auxiliary magnification of 1.6x. An EMCCD Evolve-512 camera was used to capture images.…”

“…Recently, we showed that SC DCG biogenesis requires Arf1 and AP-1, and using real time imaging, demonstrated that Rab6-labelled DCG precursor compartments must receive a Rab11-mediated input to initiate a Rab6 to Rab11 transition that rapidly triggers Rab11-exosome and DCG biogenesis (Figure 1A; Wells et al, 2023; Loh et al, 2023). The ILVs produced surround the DCG and form chains that link the DCG to the compartment’s limiting membrane (Figure 1A; Fan et al, 2020).…”