Endolysosomal trafficking controls yolk granule biogenesis in vitellogenic Drosophila oocytes.

Endocytosis and endolysosomal trafficking are essential for almost all aspects of physiological functions of eukaryotic cells. As our understanding on these membrane trafficking events are mostly from studies in yeast and cultured mammalian cells, one challenge is to systematically evaluate the findings from these cell-based studies in multicellular organisms under physiological settings. One potentially valuable in vivo system to address this challenge is the vitellogenic oocyte in Drosophila, which undergoes extensive endocytosis by Yolkless (Yl), a low-density lipoprotein receptor (LDLR), to uptake extracellular lipoproteins into oocytes and package them into a specialized lysosome, the yolk granule, for storage and usage during later development. However, by now there is still a lack of sufficient understanding on the molecular and cellular processes that control yolk granule biogenesis. Here, by creating genome-tagging lines for Yl receptor and analyzing its distribution in vitellogenic oocytes, we observed a close association of different endosomal structures with distinct phosphoinositides and actin cytoskeleton dynamics. We further showed that Rab5 and Rab11, but surprisingly not Rab4 and Rab7, are essential for yolk granules biogenesis. Instead, we uncovered evidence for a potential role of Rab7 in actin regulation and observed a notable overlap of Rab4 and Rab7, two Rab GTPases that have long been proposed to have distinct spatial distribution and functional roles during endolysosomal trafficking. Through a small-scale RNA interference (RNAi) screen on a set of reported Rab5 effectors, we showed that yolk granule biogenesis largely follows the canonical endolysosomal trafficking and maturation processes. Further, the data suggest that the RAVE/V-ATPase complexes function upstream of or in parallel with Rab7, and are involved in earlier stages of endosomal trafficking events. Together, our study provides s novel insights into endolysosomal pathways and establishes vitellogenic oocyte in Drosophila as an excellent in vivo model for dissecting the highly complex membrane trafficking events in metazoan. Author summary: Endocytosis and endolysosomal trafficking are membrane-based package and delivery systems in eukaryotes for material exchanges intracellularly in-between membrane-enclosed compartments and extracellular with surrounding milieu. Current understanding of these exchange mechanisms are mainly from studies in yeast and cultured mammalian cells, but exactly how they operate in multicellular organisms under physiological conditions remain unclear. Here we focus on vitellogenic oocytes in Drosophila, which uptake large quantity of extracellular lipoproteins by a low-density lipoprotein receptor called Yolkless into the oocyte and package them into large yolk granules, a specialized lysosome, for storage and usage in later development. Using novel fly lines that allows faithful detection and manipulation of endogenous Yolkless receptor and known endosomal regulators, we show that the formation and maturation of yolk granules largely follows the canonical endolysosomal trafficking pathways, including the critical involvement of small GTPases Rab5 and Rab11 as well as distinct phospholipid species and actin networks, although the results also raise questions on the roles of other regulators including Rab4 and Rab7 in granule biogenesis. Together, this study provides novel insights into the highly complex membrane trafficking events in multicellular organisms and supports Drosophila oocyte as a useful in vivo model for such studies in the future. [ABSTRACT FROM AUTHOR]