PgmNr D1019: Differential Subcellular Trafficking of Membrane Proteins in Secondary Cells of the Drosophila male Accessory Glands.

Authors:
Felix Castellanos; Benjamin Kroeger; Shih-Jung Fan; Mark Wainwright; Siamak Redhai; Carina Gandy; Clive Wilson


Institutes
University of Oxford, Oxford, GB.


Keyword: endocytosis and membrane dynamics

Abstract:

The male accessory glands of D. melanogaster are paired structures whose secretory products constitute an essential fraction of the seminal fluid and play a critical role in reproduction. Two morphologically different populations of secretory cells form a monolayered epithelium in the accessory glands. The less abundant (~5%) and larger spherical cells, collectively known as secondary cells (SCs), localise at the distal ends of each lobe.

A number of different subcellular compartments can be identified in SCs by assessing acidity and other biochemical properties such as the differential Rab GTPase localisation. For example, Rab7 localises to the surface of large acidic late endosomes and lysosomes, while Rab11 localises to the limiting membrane of non-acidic secretory vacuoles that contain dense-core granules (DCGs).

We are currently investigating how secretory compartments and different populations of intraluminal vesicles (ILVs) are formed in adult SCs. Many cells secrete vesicles that are thought to be formed in the late endosomal multivesicular body (MVB) and are ultimately released by compartment fusion to the cell surface. These so-called exosomes can fuse with target cells, transferring a broad range of biomolecules.

Using state-of-the art imaging and pulse-chase analysis of membrane proteins tagged with fluorescent markers, we have characterised the selective trafficking routes taken by different fusion proteins to reach their final subcellular destinations. These proteins localise to the plasma membrane, in addition to non-acidic vacuolar limiting membranes, their DCGs and ILVs. Using a fusion protein that traffics to DCGs, we are currently testing several molecules for putative roles in DCG biogenesis (e.g. lipid modifying enzymes, as well as the ESCRT genes). Based on these experiments, we have started to develop a mechanistic model to explain cargo trafficking to DCGs, which unexpectedly implicates ILVs. Our results contrast with other data using a previously characterised human CD63-GFP exosome marker, which traffics to other compartments and is loaded on to a different subset of ILVs.

This work sheds new light on the mechanisms regulating DCG compartment biogenesis and the different trafficking routes taken by cargos to reach these compartments. Ultimately, it may inform our understanding of DCG regulation in processes controlling homeostasis, such as glucose-dependent insulin secretion by pancreatic beta cells.