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. 2024 Feb 5;20(2):e1011152. doi: 10.1371/journal.pgen.1011152

Endolysosomal trafficking controls yolk granule biogenesis in vitellogenic Drosophila oocytes

Yue Yu 1,2,#, Dongsheng Chen 1,3,#, Stephen M Farmer 1,2,4,#, Shiyu Xu 1, Beatriz Rios 1,2, Amanda Solbach 1,2,5, Xin Ye 1, Lili Ye 1, Sheng Zhang 1,2,5,6,*
Editor: Pablo Wappner7
PMCID: PMC10898735  PMID: 38315726

Abstract

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.

Introduction

Endocytosis and the ensuing cascade of endolysosomal trafficking are membrane-based mechanisms to interact with extracellular environment and coordinate intracellular maintenance and responses [15]. Essential for almost all physiological functions of eukaryotes from signaling to morphogenesis and defense, their dysfunctions are being linked to a growing number of diseases from cancer to ageing and neurodegeneration [613]. In case of the clathrin-dependent endocytosis, a main pathway for receptor mediated endocytosis, the cascade is a continuous and highly dynamic vesicular events involving cargo recognition and sequestration by specific receptors, formation of clathrin-coated pits (CCPs), followed by membrane fission that generates clathrin coated vesicles (CCVs) [11]. After de-coating to shed the clathrin and associated factors from CCVs, the naked endocytic vesicles fuse with each other and with early endosomes through heterotypic and homotypic fusion to grow while being transported along cytoskeleton network, gradually maturate into late endosomes accompanied by characteristic morphological changes, and eventually fuse with lysosomes for the final degradation of enclosed cargoes [4,5]. This sequential maturation process is marked by progressive modification of the external membrane composition, and gradually acidification of internal endosomal lumen that promotes the separation of cargoes from their cognate receptors followed by their active sorting into discrete degradative or recycling routes. Such a highly dynamic and heterogeneous process is orchestrated in a spatially and temporally controlled manner by a group of small Rab GTPases, including Rab5 that controls early endocytosis, Rab4 and Rab11 that mediate fast- and slow-recycling pathways, respectively, and Rab7 that promotes endosomal maturation into late endosome and their transportation and eventual fusion with the lysosome [1419]. For example, as the master regulator of early endocytosis, active Rab5 on endocytic vesicles and early endosomes recruits a diverse set of downstream effectors, including phosphatases for the turnover of plasma membrane-enriched phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), the class III PI3 kinase complex to locally produce phosphatidylinositol (3)-phosphate (PI(3)P) that acts as a landmark for early endosomes to enlist additional effectors such as EEA1 and Hrs that promote endosomal tethering, fusion, intralumenal vesicle formation and trafficking [4,2026]. CCZ1/Mon1 complex, which has been shown to interact with both active Rab5 and PI(3)P, in turn acts as a guanine nucleotide exchange factor (GEF) to recruit Rab7 onto early endosomes to promote their maturation into late endosomes [27,28]. Thus, endocytosis and endosomal maturation are characterized by the gradual conversions of phosphatidylinositol (PI) species and associated Rab proteins, from Rab5 on endocytic vesicles and early endosomes to Rab7 on late endosomes, and from plasma membrane PI(4,5)P2 to PI(3)P on early endosomes as its identity marker [4,14,19,22].

Most of our understanding of these complex membrane trafficking events are from studies in yeast and cultured mammalian cells, but how they operate in multicellular organisms under physiological setting remains largely unknown, as the significant heterogeneity of endosome morphology and miniature sizes of endosomal intermediates further complicate such studies [29]. Just as clathrin-dependent endocytosis was first observed in the oocyte of insect mosquito Aedes aegypti L [30], one potentially valuable metazoan model for studing endosomal trafficking is vitellogenic stage oocyte in Drosophila, a classic model organism that has been invaluable in discovering the conserved signaling pathways and dissecting the myriad of developmental and cellular processes in metazoans, including endocytosis and endolysosomal trafficking [3133]. Newly deposited fly eggs are filled with large yolk granules, which are specialized storage lysosomes containing densely packed vitellogenins (yolk proteins) to support later embryogenesis [3437]. Vitellogenins are lipoproteins synthesized exclusively outside the oocyte, by fat bodies and surrounding follicle cells [3841]. The biogenesis of yolk granules is controlled solely by receptor-mediated endocytosis [4244]. Vitellogenesis occurs during stages 8–10 of egg chamber development, when oocytes undergo a dramatic, more than 200-fold increase in volume within a ~16 hour window [45,46], primarily due to the massive uptake of yolk proteins from circulating hemolymphs into the oocytes by Yolkless (Yl), a low-density lipoprotein receptor (LDLR) for Yolk proteins [47,48]. After Yl-mediated internalization of Yolk proteins and their dissociation, Yl is quickly recycled back to the plasma membrane for numerous more rounds of endocytosis [4851]. The final mature granules are large spheres with highly acidic lumen packed with crystalized yolk proteins surrounded by a single layer of limiting membrane with characteristic smooth surface and rounded shape [5257]. Importantly, as the vitellogenic stage oocyte is a single cell of huge sizes and dedicated almost exclusively to endocytosis, it is filled with large number of easily detectable endosomal intermediates of different maturation stages, many with unusually large sizes [5257]. Therefore, even a mild disruption of an endosomal trafficking step can have a dramatic amplifying effect, leading to easily identifiable phenotypes, which make the oocyte a highly sensitive in vivo model to study receptor-mediated endocytosis and endosomal trafficking.

Despite these unique features, there are few systematic studies on the molecular and cellular mechanisms that control yolk granule biogenesis in Drosophila. In this study, to facilitate the analyses of receptor-mediated endocytosis in oocytes, we created genome-tagging fly lines that express a functional Yl receptor with HA and fluorescent eGFP tags. We next analyzed the expression patterns and functional requirements of four major Rab GTPases as well as PI(4,5)P2 and PI(3)P phosphoinositides in vitellogenic oocytes. Focusing on a set of Rab5 effectors identified in a recent proteomic study [58], we carried out RNA interference (RNAi) studies to interrogate their roles in yolk granule biogenesis. Our results provide new insights into endocytosis and endolysosomal trafficking processes under this physiological setting and demonstrate vitellogenic oocyte as an excellent in vivo model for dissecting these highly complex yet important biological processes.

Results

Dynamic Yl distribution in vitellogenesis oocytes

To minimize artifacts often associated with ectopically expressed transgenes, we engineered a Yl genome-tagging construct by cloning a ~12kb genomic DNA that contains all the introns and exons as well as 5’ and 3’ UTR regions of the native yl gene together with an in-frame insertion of eGFP and 3 copies of HA epitope (eGFP-3xHA) near the C-terminal short cytoplasmic tail of encoded Yl receptor, just in front of one of the predicted endocytic signal sequences (Fig 1A and 1B) [47]. The ~5.4kb 5’ UTR within the construct included the known regulatory region of yl gene that controls its specific expression in female oocytes [47]. Western blot analyses confirmed the expression of Yl-eGFP-3xHA fusion at the predicted ~240 kDa size [48] only in female transgenic flies (Fig 1C). Immunofluorescent staining and confocal analyses further demonstrated that the Yl-eGFP-3xHA fusion is expressed in a pattern and with characteristic subcellular distribution similar to that of endogenous Yl (Fig 1D–1G) [48]. For example, in stage 8 egg chamber when vitellogenesis started, Yl-eGFP-3xHA became mobilized from intracellular granules to the plasma membrane to initiate the extracellular upake of Yolk proteins, as revealed by their co-localization on small puncta along the cortex and on small vesicular structures inside the oocyte (insets in Fig 1D). Further interior, Yl-eGFP-3xHA was largely absent from larger Yolk-positive granules (Fig 1D). By stage 10, when vitellogenesis reaches its peak and the oocyte becomes engaged in intensive Yl-mediated Yolk uptake, Yl-eGFP-3xHA showed an almost exclusive localization to the plasma membrane (Fig 1E). Closer inspection revealed numerous Yl-positive puncta, likely representing individual endocytosis units, on the surface of the plasma membrane (Fig 1F and 1G). This is consistent with the ultra-structures revealed by transmission electron microscope (TEM) (Fig 1H–1J) [46,55], which showed the presence of extensive invaginating CCPs and CCVs on or near the plasma membrane (green arrows in Fig 1J) and larger, irregular shaped tubular and vesicular structures nearby that contained electron-dense materials immersed in the middle of electron-lucent spaces, which likely corresponded to developing endosomes (red arrows in Fig 1I and 1J). Further interior were progressively larger and spherically shaped yolk spheres with irregular boundaries that were filled with electron-dense materials but largely devoid of electron-lucent materials, which likely corresponded to growing late endosomes as they reach their final maturation stages (labeled with “i” for immature granules in Fig 1I). The mature yolk granules were easily recognized for their characteristic spherical shape, smooth membrane boundary and darker electron-dense core with crystalized Yolk protein condensates (labeled with “m” for mature granules in Fig 1I) [57]. Importantly, homozygous adults for yl13, a strong loss of function allele (also known as k621) of yl gene, were females sterile [44,59]. In the presence of Yl-eGFP-3xHA transgene, this sterile phenotype was fully rescued and the homozygous yl13 flies can be maintained as stand-alone stock. Together, these findings support that the genome-tagged Yl-eGFP-3xHA is functional and trafficked similarly as endogenous Yl. We thus used this Yl genome-tagging construct in subsequent studies.

Fig 1. Genome-tagging of Yl receptor and Yl-mediated Yolk endocytosis in vitellogenic oocyte.

Fig 1

(A) Schematics of Yl genome-tagging construct. The amino acid sequences of the encoded Yl protein that flank the eGFP-3xHA insertion is annotated on top of the illustration. Black lines: introns and 5’ and 3’ untranslated regions (UTR) of yl gene. Rectangular boxes: exons of yl gene with the coding regions shaded in gray. (B) Schematics of Yl-eGFP-3xHA fusion protein (left) and closeup view of the C-terminal cytoplasmic tail of Yl protein with the eGFP-3xHA insertion (right). TM, transmembrane domain. (C) Western blot analysis of homogenates from adult female flies probed independently with anti-HA or anti-GFP antibodies, as indicated. A single ~240kDa band positive for both GFP and HA was present in the flies transgenic for Yl-eGFP-3xHA genome-tagging construct (YL) but absent in w1118 non-transgenic (WT) control. (D) Stage 8 oocyte double-labeled by anti-GFP for Yl-eGFP-3xHA (green) and anti-Yolk (red) proteins, as annotated. Inserts are zoom-in view of the region highlighted in white box, showing enrichment of Yl along the oocyte cortex and the differential distribution of Yl and Yolk on granules of different sizes inside the oocyte. (E-G) Expression and subcellular localization of Yl-eGFP-3xHA in stage 10 oocytes imaged for Yl-GFP-3xHA by anti-GFP (green) and yolk granules (gray) by differential interference contrast (DIC). (E) Low-magnification view of whole egg chamber, showing (bottom) the localization of Yl along the oocyte plasma membrane, (middle) yolk granules inside the oocytes and (top) their overlaying view. (F, G) Surface (F) and cross-section (G) views of a plasma membrane area at high-magnifications. (H-J) TEM of stage 9 egg chambers. (H) Low-magnifications overview, showing an oocyte filled with many electron-dense yolk granules surrounded by follicle cells (FC) and nurse cells (NC), as annotated. (I) Closer-up view of the cortex region of an oocyte. Notice that along the plasma membrane, the presence of many small granules (red arrows) with characteristic electron-dense core in the middle of translucent space surrounded by a single layer of membrane, larger granules of growing sizes with irregular boundaries (marked with “i”) and large mature granules (marked with “m”). (J) High-magnification view of plasma membrane area, showing the presence of CCPs and CCVs (green arrows) and nearby early endosomes (red arrows). Genotypes for (D-G): w1118; p{mini-W+, yl-eGFP-3xHA}. (H-J) w1118. The sizes of the scale bars as annotated inside images.

Expression and subcellular distribution of Rab4, 5, 7 and 11 in stage 10 oocytes

Among all the Rab GTPase proteins, Rab4, Rab5, Rab7, and Rab11 are known for their well-established roles in different steps of endosomal trafficking. However, except for Rab5, which was shown to be essential for Yl-mediated endocytosis and granule biogenesis [49], the expression and functions of the other Rab proteins in vitellogenic oocytes have not been fully examined in detail. Focusing on wildtype stage 10 oocytes, when endocytosis reaches its peak, we examined the expression and subcellular distribution of these Rab proteins, using Yl as an endocytosis marker. Yl showed a significant co-localization with Rab7 along the oocyte cortex, as revealed by co-staining with a specific anti-Rab7 antibody [60] (Fig 2A). The two also partially overlapped on small vesicles immediately adjacent to the plasma membrane (Fig 2A1–2A3). A little further inside, larger granules strongly positive for Rab7 but negative for Yl were present. Rab7 was notably absent on large granules that located further interior, indicating a tight spatial regulation of Rab7 recruitment along the granule biogenesis route. Interestingly, co-labeling with lysotracker, a marker for highly acidic cellular compartments, revealed that in the oocyte cortex, small-sized lysotracker-positive granules, but not the larger ones, were often surrounded by weak YL-GFP signal (S7A–S7C Fig).

Fig 2. Expression and subcellular localization of Rab4, Rab5, Rab7 and Rab11 in vitellogenic oocytes.

Fig 2

Confocal images of stage 10 egg chambers double-labeled for endogenous Rab7 (red) and eGFP or YFP tag (green) for (A) Yl-eGFP-3xHA, (B) YFP-Rab5, (C) YFP-Rab11 and (D) YFP-Rab4, respectively, from the corresponding endogenous tagging fly lines. Yolk granules within the same oocytes were visualized by DIC (gray). (A-D) Overview of Rab7 (A), YFP-Rab5 (B), YFP-Rab11 (C) and YFP-Rab4 (D) in the imaged oocytes, respectively. (A1-D4) High-magnification view of the corresponding cortex regions highlighted in (A-D), respectively, as indicated. Endogenous Rab7 used as reference for endosomes in all the images. Images were shown in individual channels in gray or as overlaying images in color, as indicated. Genotypes: (A) w1118; p{w(+mC), yl-eGFP-3xHA}. (B) w1118; TI{TI} EYFP-Rab5 /CyO (BDSC #62543). (C) w1118; TI{TI} EYFP-Rab11 (BDSC #62549). (D). y1, w1118; TI{TI} EYFP-Rab4 (BDSC #62542). The sizes of the scale bars as labeled.

In oocytes from YFP-Rab5 endogenous tagging line [61], YFP-Rab5 showed a predominant enrichment on the cortex, similar as that reported in a previous study using an anti-Rab5 antibody (Fig 2B) [49]. YFP-Rab5 partially co-localized with Rab7 on puncta-like structures along the cortex (Fig 2B1). Similar to Yl, YFP-Rab5 also co-localized with Rab7 on granules immediately adjacent to the plasma membrane, but its signal faded rapidly on Rab7-positive granules located further interior (Fig 2B1–2B3). Noticeably, on granules where both Rab5 and Rab7 co-existed, the two showed an uneven and non-identical distribution along the granule membrane, consistent with the models of Rab subdomains and Rab5-to-Rab7 conversion that govern endosomal maturation [17,18,25].

Examination in both YFP-Rab11 endogenous tagging line [61] and by an anti-Rab11 antibody revealed similar enrichment of Rab11 along the whole cortex region in stage 10 oocytes (Figs 2C and S1C–S1F), a pattern that is different from a previous report showing a specific localization of Rab11 at the extreme posterior pole of the oocyte [62]. Unlike Rab5 and Rab7, Rab11 showed no clear association with vesicular structures (Figs 2C1–2C3 and S1C–S1F).

Surprisingly, despite its well-documented role in fast recycling, in YFP-Rab4 endogenous tagging flies [61], no clear enrichment of YFP-Rab4 was observed along the oocyte cortex. Instead, YFP-Rab4 showed a weak and scattered distribution as puncta inside the oocytes (Fig 2D) and intriguingly, partially co-localized with Rab7-positive granules (highlighted by asterisks in Fig 2D1–2D4).

Importantly, GFP signals from the YFP-Rab5, -Rab11 and -Rab4 genome tagging lines were specific, as when processed in parallel with the same anti-GFP antibody, only low background signals were detected in controls that did not carry YFP-tagging lines (S1A–S1D Fig).

Actin cytoskeleton dynamics and phosphatidylinositols in vitellogenic oocytes

Considering the importance of actin dynamics in endocytosis and endosomal trafficking [63], we next examined the distribution of actin cytoskeleton in vitellogenic oocytes. In stage 10 oocytes, phalloidin staining revealed a dense layer of filamentous actin (F-Actin) that largely overlapped with the Rab7-positive layer and the top half of Yl-positive band along the plasma membrane (Fig 3A and 3B). Additionally, a thin web of F-Actin filaments projected down from the dense F-Actin layer, extending about ~4 um depth into the cytoplasm, where smaller granules positive for Yl and Rab7 were located (Fig 3B). Further below this F-Actin web, where larger Rab7-positive and Yl-negative granules existed, small F-Actin puncta were scattered in the cytoplasm (arrows in Fig 3B). Interestingly, each Rab7-positive granule was always decorated by one or more of these F-Actin puncta that were often on the side of the granules proximal to the plasma membrane, a pattern that was especially apparent on smaller Rab7 granules (arrows in Fig 3B, see also Fig 3H and arrows in 3I, illustrated in 3J).

Fig 3. Actin cytoskeleton dynamics and phosphatidylinositol phospholipids in vitellogenic oocytes.

Fig 3

Confocal microscopy of stage 10 egg chambers co-labeled with phalloidin for F-Actin and anti-GFP antibody for (A, B) Yl-eGFP-3xHA (blue), (C, D, F, G) PI(4,5)P2 reporter PLCδ1(PH)-GFP (green) and (H,I) PI(3)P reporter 2xFYVE-GFP (green), respectively, (A, B, F, G) together with endogenous Rab7, from flies with the corresponding reporter lines, as indicated. (A, C, F, H) Overview of the distribution of (A) Yl-GFP-3xHA, (C, F) PLCδ1(PH)-GFP and (H) 2xFYVE-GFP, respectively, in the oocytes, as indicated. (B, D, G, I) High-magnification view of the corresponding cortex regions highlighted in (A, C, F, H), respectively, as annotated. (E) Cartoon illustration of the results from Fig 2 and Fig 3(A-D), showing the spatial relationship between Yl, PI(4,5)P2, F-Actin, Rab5 and Rab7 during endocytosis. (J) Cartoon illustration of the results from Fig 3, highlighting the association of F-Actin puncta with FYVE- and Rab7-positive granules. Genotypes: (A, B) w1118; p{mini-W+, yl-eGFP-3xHA}. Note that the oocyte in Fig 3(A,B) is the same oocyte imaged in Fig 2A. Other images were from females flies heterozygous for both matalpha4-GAL-VP16 driver (BDSC #7062) and the following UAS-transgenic lines: (C, D) UASp-PLCδ1(PH).Cerulean.6 (BDSC #31421). (F, G) UASp-PLC δ11(PH).Cerulean.2 (BDSC #30895); (H, I) UAS-GFP-myc-2xFYVE (BDSC #42712); The sizes of the scale bars as labeled.

During endocytosis, a central regulator of actin dynamics is PI(4,5)P2, which is essential for the initiation of endocytosis, including the recruitment of AP-2 adaptor complex and Rab5 GTPase as well as other effectors that lead to the formation of CCPs and CCVs [20,21]. Similar as reported before [49], when detected by an ectopically expressed PLCδ1(PH)-GFP, a PI(4,5)P2 reporter composed of the Pleckstrin homology (PH) domain of the phospholipase-Cδ fused to GFP [64,65], it revealed a strong enrichment of PI(4,5)P2 on the plasma membrane of stage 10 oocytes, in a pattern similar to that of F-Actin, although the two did not completely overlap (Fig 3C, 3D, illustrated in 3E). Notably, even at relatively low expression levels, small patches of ectopically expressed PLCδ1(PH)-GFP could be found inside the oocytes that were always accompanied by aberrant F-Actin aggregation, a phenotype that was not observed in wildtype controls (arrows in Fig 3D1–3D2). The PLCδ1(PH)-GFP reporter can exert a dominant-negative effect by sequestering PI(4,5)P2 from its endogenous binding partners [6669]. Indeed, at higher expression levels, larger PLCδ1(PH)-GFP-positive aggregates of different sizes accumulated together with F-Actin and Rab7 and strikingly long F-Actin filaments inside the oocytes (Fig 3F and 3G). Notably, similar phenotypes were observed in yeast defective for inositol(5)phosphatase synaptojanin, which controls the metabolism of PI(4,5)P2 [70]. Therefore, elevated levels of PLCδ1(PH)-GFP reporter sequester endogenous PI(4,5)P2 and blocks its efficient conversion, resulting in an abnormal buildup of PI(4,5)P2-associated actin regulators and stalled endosomal intermediates, including Rab7-positive late endosomes, inside the oocyte (Fig 3F and 3G). Together, these phenotypes support the potent role of PI(4,5)P2 on actin cytoskeleton dynamics and the importance of efficient phosphoinositide conversion in endosomal trafficking [20,21].

In contrast to the strong effect associated with ectopically expressed PI(4,5)P2 reporter, no apparent defects were observed in oocytes expressing 2xFYVE-GFP, the reporter for the early endosome marker PI(3)P [71,72]. Similar as Rab7, 2xFYVE-GFP was observed as numerous small puncta along the F-Actin-rich plasma membrane and also on the surfaces of larger granules near the cortex, but was absent on mature granules located deeper inside the oocyte (Figs 3H, 3I and S2A–S2C). Further, almost all these 2xFYVE-GFP-positive vesicles were also positive for Rab7 (S2A–S2C Fig), and comparable to Rab7-positive granules, were always decorated by one or more small F-Actin puncta that were located on the side proximal to the plasma membrane (arrows in Fig 3I, illustrated in 3J).

Rab5 and Rab11 but not Rab4 and Rab7 are essential for yolk granule biogenesis

Using the established transgenic lines for normal and mutated forms of Rab proteins [73], we next examined the functional requirement of main Rab GTPases in yolk granules biogenesis. Consistent with the essential role of Rab5 in this process [49], oocytes overexpressing a YFP-tagged dominant negative (DN) Rab5 (YFP-Rab5-DN) contained only small vesicles visible under DIC imaging, with occasional presence of small Rab7-positive granules below the cortex (S3E and S3F Fig). Further, only granules at the very posterior, where the endocytosis is most active [46], were lysotracker-positive (S3G and S3H Fig, compared to wildtype S3C, S3D). The phenotypes induced by YFP-Rab5-DN were notably weaker than those reported for rab5-null flies [49], for example both F-Actin and Rab7-positive puncta were still enriched along the cortex. This is likely due to the presence of endogenous Rab5 that remains active in the oocytes over-expressing YFP-Rab5-DN. Nevertheless, they support the critical role of Rab5 in both endosomal fusion and maturation during granule formation. Interestingly, although the ectopically expressed YFP-Rab5-DN was diffusive throughout the cytoplasm, it still showed a relatively mild enrichment along the cortex (S3E1 and S3F1 Fig), indicating a local recruitment mechanism for Rab5 independent of its activity. Opposite of the phenotypes induced by YFP-Rab5-DN, constitutive active (CA) YFP-Rab5-CA induced large and abnormally shaped granules in the middle of the oocytes (S3I–S3L Fig), as reported in multiple previous studies [25,74]. These large granules were hybrid of early and late endosomes, as they were positive for lysotracker (S3K and S3L Fig compared to wildtype control in S3C, S3D) and their surface were decorated by both YFP-Rab5-CA, Rab7 and associated small F-Actin puncta (S3J1–S3J3 -Fig). Notably, YFP-Rab5-CA and Rab7 also accumulated on many smaller aggregates inside the oocytes, but their levels along the cortex were significantly reduced (S3I and S3J Fig). In contrast, the pattern of F-Actin was not apparently affected, still showing normal enrichment along the cortex. As a control, oocytes over-expressing wildtype YFP-Rab5 showed no apparent defect, with both YFP-Rab5 and endogenous Rab7 enriched strongly along the cortex (S3M–S3N Fig). Together, they support that proper regulation of Rab5 is essential for granule biogenesis.

Oocytes expressing YFP-Rab11-DN showed severely disrupted vitellogenesis, as no recognizable yolk granules and hardly any recognizable Rab7-positive granules were present in these oocytes, which also appeared thinner at their posterior end, supporting a critical role of Rab11 in yolk granule biogenesis (Fig 4A and 4B). YFP-Rab11-DN was diffusively distributed in the cytoplasm, but still with a relatively stronger enrichment along the cortex (Fig 4B1). In addition, there was a reduced enrichment of Rab7 along the cortex, which also became more diffusive at the posterior, no longer anchored along the cortex (Fig 4A2, compare with Fig 2A). Lastly, although F-Actin showed relatively normal enrichment along the cortex, aberrant F-Actin aggregates were present inside the oocytes. But unlike the abnormal endosomal structures induced by ectopic PLCδ1(PH)-GFP(Fig 3F and 3G), these F-Actin aggregates were negative for Rab7 and not associated with long actin filaments. In contrast to overexpressed YFP-Rab11-DN, neither constitutive active nor wildtype (WT) YFP-Rab11 disrupted granule biogenesis when overexpressed (S4 Fig). The ectopically expressed WT YFP-Rab11 was sharply concentrated in small puncta along the cortex, in contrast to the more diffusive presence of YFP-Rab11-CA. Neither YFP-Rab11-CA nor WT YFP-Rab11 showed apparent association with Rab7-positive granules.

Fig 4. Rab4, Rab5, Rab7 and Rab11 in yolk granule biogenesis.

Fig 4

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of dominant negative (A-B4) YFP-Rab11-DN, (C-F) YFP-Rab7-DN and (G-J) YFP-Rab4-DN, respectively, that were co-stained by anti-GFP antibody (green), anti-Rab7 (red) and phalloidin for F-Actin (blue), presented as overlaying images in color or as individual channels in gray, or (E, F, I, J) by lysotracker stain alone (red), as annotated. (B-B4, D-D4, H-H4) High-magnification views of the cortex regions highlighted in (A, C, G), respectively, as indicated. Genotypes: The samples were from adult females flies heterozygous for both matalpha4-GAL-VP16 driver (BDSC #7062) and the following UAS-transgenic lines: (A, B) Rab11-DN: UASp-YFP.Rab11.S25N (BDSC #9792). (C-F). Rab7-DN: UASp-YFP.Rab7.T22N (BDSC #9778). (G-J). Rab4-DN: UASp-YFP.Rab4.S22N (#9768). The sizes of the scale bars as annotated inside images.

Surprisingly, despite Rab7’s proposed role as a major regulator of endosomal growth and maturation, vitellogenesis did not appear to be significantly affected in stage 10 oocytes overexpressing either DN-, WT- or CA-YFP-Rab7, as large numbers of lysotracker-positive yolk granules were present in these oocytes (Figs 4C–4F and S5), echoing the results of a recent study [74]. As expected, YFP-Rab7-WT was enriched near the cortex region and notably decorated the surface of some small- and large-sized lysotracker-positive granules (S5C and S5D Fig). Similarly, YFP-Rab7-CA was enriched as puncta along the cortex and on the surface of larger granules nearby (S5E and S5F Fig), while YFP-Rab7-DN was diffusive in the cytoplasm and not located on the surface of the granules (Fig 4D). Unexpectedly, in oocytes expressing YFP-Rab7-DN, some large granules were shrouded in an F-Actin layer, with some granules in irregular shape while others had F-Actin extensions projecting from the granule surface (Fig 4D3), indicating a role for Rab7 in actin regulation during endosomal maturation.

Lastly, Rab4 is well-documented for its role as a major regulator of fast recycling in endosomal trafficking [75]. However, perturbation of Rab4 in oocytes by overexpressing either DN-, CA- or WT- Rab4 did not appear to affect vitellogenesis, as F-Actin and Rab7 distribution, granule formation and acidification all proceeded normally (Figs 4G–4J and S6). These observations are in line with the relatively low levels of endogenous Rab4 inside the oocyte and its lack of enrichment at the cortex (Fig 2D), and consistent with the recent findings that rab4 knockout flies are homozygous viable and fertile, in contrast to the essential roles of Rab5, Rab7 and Rab11 in fly development [76], supporting its dispensable role in vitellogenesis. Surprisingly, while both the ectopically expressed DN- and CA-YFP-Rab4 were distributed diffusively throughout the cytoplasm, ectopic YFP-Rab4-WT overlapped significantly with endogenous Rab7 (Figs 4G–4H and S6), reminiscent of the association of endogenous YFP-Rab4 with Rab7-positive endosomes (Fig 2D).

Together, these results, summarized in Table 1, support the critical roles of Rab5 and Rab11 in yolk granule biogenesis, and imply a potential role for Rab7 in regulating actin dynamics.

Table 1. Summary of phenotypes in oocytes overexpressing YFP-tagged Rab4, Rab5, Rab7 and Rab11 proteins.

UASp-YFP-Rab Lines BDSC stock # Phenotypes of stage 10 oocytes overexpressing YFP-Rab proteins driven by matalpha4-GAL-VP16 driver
Subcellular localization of YFP-Rab protein Rab7 Granule Size F-Actin Lysotracker
Rab4-WT 9767 Co-localize with Rab7-positive granules, also diffusive in cytoplasm Normal Normal Normal Normal
Rab4-CA 23268 Diffusive, with enrichment near oocyte cortex Normal Normal Normal Normal
Rab4-DN 9768 Diffusive, with enrichment near oocyte cortex Normal Normal Normal Normal
Rab5-WT 24616 Strongly enriched as puncta at cortex Normal Normal Normal Normal
Rab5-CA 9774 Enriched in puncta and on the membrane surface of large granules. Reduced enrichment at oocyte cortex. Enriched in puncta and on the membrane surface of large granules. Reduced enrichment at oocyte cortex. Very large Normal near the cortex, more smaller F-actin puncta associated with large YFP-Rab5-CA granules inside the oocytes Large granules are lysotracker-positive.
Rab5-DN 9778 Diffusive, with enrichment near oocyte cortex Enrichment at oocyte cortex. More diffusive Rab7 in the cytoplasm. Few large Rab7-positive granules exist Small granules Normal Existence of few very small lysotracker-positive granules near the posterior of oocytes
Rab7-WT 23641 Enriched at oocyte cortex and on the surface of Rab7-positive granules Normal Normal Normal Normal
Rab7-CA 24103 Enriched at oocyte cortex and on the surface of Rab7-positive granules Normal Normal Normal Normal
Rab7-DN 9778 Diffusive, with enrichment near cortex Diffusive Large Aberrant "rings" on some Rab7-positive granules Normal
Rab11-WT 50782 Enriched as small puncta at oocyte cortex. Normal Normal Normal Normal
Rab11-CA 23260 Diffusive, with enrichment near oocyte cortex Normal Normal Normal Normal
Rab11-DN 9792 Diffusive, with enrichment near oocyte cortex Diffusive, reduced enrichment at oocyte cortex, increased levels near the posterior of the oocytes Few visible and very small-sized granules Aberrant small F-actin aggregates inside the oocytes Existence of few small lysotracker-positive granules near the posterior of oocytes

An in vivo RNAi screen to evaluate Rab5 effectors in yolk granule biogenesis

The above results establish the vitellogenic stage oocyte as a potential in vivo model for studying endocytosis and endolysosomal trafficking. Taking advantage of the existing collections of genome-wide transgenic RNAi lines [7779], we analyzed a selected group of Rab5 effectors identified in a recent proteomic study [58] for their roles in yolk granule biogenesis. To bypass the animal lethality associated with global knockdown of essential genes, we chose matalpha4-GAL-VP16, a maternal-specific GAL4 line that expresses a strong transcriptional activator GAL4-VP16 fusion only in germline oocyte and nurse cells under the control of alphaTub67C promoter [80]. Since this matalpha4-GAL line initiates dsRNA expression around stage 5 of egg chamber development [74], it should avoid potential disruption on earlier oocyte development or complication of animal lethality while might still afford sufficient time for RNAi-mediated knockdown of target genes by stage 8, when vitellogenesis starts.

Among the 36 RNAi lines we tested that targeted 22 unique reported Rab5 effectors (Table S1) [58], only a few caused distinct phenotypes. Among them, oocytes expressing dsRNA against PI3K59F contained hardly any visible granules (Figs 5A4 and 5B4), and lysotracker staining revealed the presence of only very few and small-sized lysotracker-positive structures, which were also surrounded by weak Yl-GFP signal, similar as that observed in wildtype control (compared Supplemental S7D–S7F Fig to control S7A–S7C). PI3K59F encodes the fly homolog of VPS34, the class III PI3-kinase that is required for the endosomal production of PI(3)P [24]. Consistently, these oocytes contained few small FYVE-GFP-positive granules, which only partially co-localized with, but not completely overlapped, with Rab7 (Figs 5C, 5D and S2D–S2F, compare to WT controls in Figs 3H, 3I and S2A–S2C). Moreover, Yl-GFP was no longer concentrated on the plasma membrane but became dispersed in membranous structures throughout the oocyte (Figs 5A, 5B, S7D–S7F, S7I and S7J). Rab7 was similarly disrupted, showing no enrichment along the plasma membrane and no visible Rab7-positive granules, but became similarly sequestered with Yl inside the oocyte, including an abnormal enrichment near the posterior (Fig 5A, 5B, and Supplemental S7I, S7J). Further, in regions with ectopic enrichment of Yl-GFP and Rab7, there was a similar increased accumulation of small F-Actin puncta, although F-Actin enrichment on plasma membrane was largely normal (Fig 5B1–5B3). Closer inspection revealed that the internally sequestered Yl-GFP did not overlap with the remaining lysotracker-positive structures (S7D–S7F Fig), and although Yl and Rab7 showed a similar pattern of sequestration inside the oocyte, they only partially overlapped (S7J Fig).

Fig 5. Essential roles of VPS34/VPS15 PI3 kinase complex in Yl recycling and yolk granule biogenesis.

Fig 5

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of dsRNA again (A-D1) VPS34/PI3K59F or (E, F) Vps15, from flies (A-B4, E-F4) carrying genome-tagging Yl-eGFP-3xHA, co-labeled with antibodies against GFP (green), endogenous Rab7 (red) and phalloidin for F-Actin (blue), or (C-D1) expressing 2xFYVE-GFP, co-labeled for F-Actin (red), presented as overlaying images in color or as individual channels in gray, as annotated. (B, D, F) High-magnification view of the cortex regions highlighted in (A, C, E), respectively, as indicated. Genotypes: The samples were from adult females flies heterozygous for both matalpha4-GAL-VP16 driver (BDSC #7062) and the following UAS-transgenic RNAi and Yl- or FYVE-reporter lines: (A,B) P{TRiP.HMJ30324}attP40 (#64011); p{mini-W+, yl-eGFP-3xHA}. (C,D) P{w(+mC) = UAS-GFP-myc-2xFYVE}2 (#42172); P{TRiP.HMS00261}attP2 (#33384); (E,F). P{TRiP.GL00085}attP2 (#35209)/ p{mini-W+, yl-eGFP-3xHA}. The sizes of the scales as annotated inside images.

Importantly, PI3K59F forms an active class III PI3K complex by dimerizing with its obligate partner VPS15 [81,82], and oocytes with RNAi-mediated depletion of VPS15 manifested almost identical phenotypes as those seen in PI3K59F knockdown, including a lack of yolk granules and the abnormal accumulations of Yl-GFP, Rab7 and F-Actin inside the oocytes (Fig 5E and 5F). Given that cortical F-Actin was largely normal in PI3K59F- and VPS15-knockdown oocytes (Fig 5A3, 5B3 compared to 5E3, 5F3), similar as oocytes expressing YFP-Rab5-DN (S3E and S3F Fig), they are consistent with the model that the VPS34/VPS15 kinase complex functions downstream of PI(4,5)P2 conversion and is critical for endosomal PI(3)P production, which is essential for the subsequent endosomal fusion, sorting and recycling. Diminished levels of PI(3)P compromises the sorting and trafficking in early endosomes, resulting in blocked Yl recycling and its abnormal accumulation along with F-Actin and Rab7 within the stalled endosomal structures.

CCZ1 is a Rab5 effector critical for the recruitment of Rab7 onto Rab5-positive endosomes [27,28]. Consistently, in oocytes expressing dsRNA against CCZ1, Rab7 showed significantly reduced enrichment along the cortex and most strikingly, was no longer associated with the granule membrane but became diffusive in the cytoplasm (Fig 6, compared with Rab7 in wildtype Figs 2A, S2A–S2C, and S3A, S3B). However, CCZ1-depleted oocytes showed a normal distribution of both F-Actin (Fig 6A2, 6B2) and Yl-GFP (Fig 6A3, 6B3), with normal presence of PI(3)P-positive granules (Fig 6C and 6D), and numerous granules (Fig 6A4 and 6B4). These phenotypes are consistent with the role of CCZ1 in the endosomal recruitment of Rab7 and interestingly, also with the observation that perturbation of Rab7 did not significantly disrupt granule biogenesis (Fig 4C–4F).

Fig 6. The roles of CCZ1 in Yl recycling and yolk granule biogenesis.

Fig 6

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of dsRNA against CCZ1from flies (A-B4) carrying genome-tagging Yl-eGFP-3xHA reporter, co-labeled with phalloidin (red) and antibodies against endogenous Rab7 (green) and GFP for Yl (blue), or (C, D) with ectopic expression of 2xFYVE-GFP, co-labeled with antibody against GFP (green) and phalloidin (red), presented as overlaying images in color or as individual channels in gray, as annotated. (B-B4) and (D) are high-magnification views of the cortex regions highlighted in (A) and (C), respectively. Genotypes: The samples were from adult females flies heterozygous for both matalpha4-GAL-VP16 (BDSC #7062) and the following PI(3)P or Yl and UAS-transgenic RNAi lines: (A,B) P{TRiP.HMJ24129}attP40 (#62889); p{mini-W+, yl-eGFP-3xHA}. (C,D) P{w(+mC) = UAS-GFP-myc-2xFYVE}2 (#42172), P{TRiP.HMJ24129}attP40 (#62889). The sizes of the scale bars as annotated.

RAVE complex and V-ATPase vacuolar proton pump control the early endosome formation

Rabconnectin-3A (Rbcn-3A) is another Rab5 effector isolated from the proteomic study [58]. In oocytes expressing dsRNA against Rbcn-3A, there were few granules visible under DIC (Fig 7A3, 7B3) and few Rab7-positive granules detectable under confocal microscope. Instead, an abnormally high level of Rab7 became accumulated near the posterior region of the egg chamber (Fig 7A). Similarly, there were few large FYVE-GFP-positive granules (Fig 7C compared to control Figs 3H and S2A–S2C). Instead, FYVE-GFP accumulated as small puncta inside the cytoplasm (Fig 7C1). However, F-Actin, Rab7 and FYVE-GFP still showed enrichment along the cortex. Further examinations revealed a severe disruption of Yl distribution, as Yl was no longer enriched in puncta along the cortex but was trapped in membranous structures inside the oocyte (Fig 7D). The observed phenotypes in Rbcn-3A knockdown bore some similarity with those observed in oocytes depleted of VPS34/VPS15 PI3K kinase complex (Fig 5), including the absence of recognizable yolk granules and trapped Yl receptor, although Rab7 and F-Actin were not as severely affected, and 2xFYVE-GFP signals were still present but accumulated in small puncta (Fig 7 compared to Figs 5 and S2D–S2F).

Fig 7. Rbcn-3A is essential for Yl recycling and yolk granule biogenesis.

Fig 7

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of dsRNA against Rbcn-3A, double-labeled with phalloidin (red) and antibody against (A, B) endogenous Rab7 (green) or (C, D) GFP (green) from flies (C) with ectopic expression of GFP-2xFYVE or (D) carrying a genome-tagging Yl-eGFP-3xHA. Images are presented as overlaying images in color or in individual channels in gray, as annotated. (B-B3, C1, D1) High-magnification views of the cortex regions highlighted in (A, C, D), respectively, as annotated. Genotypes: The samples were from adult females flies heterozygous for both matalpha4-GAL-VP16 driver (BDSC #7062) and the following UAS-transgenic RNAi and PI(3)P or Yl reporter lines: (A,B) P{TRiP.HMS01287}attP2 (#34612). (C) P{w(+mC) = UAS-GFP-myc-2xFYVE}2 (#42172); P{TRiP. HMS01287}attP2 (#34612). (D). P{TRiP.HMS01287}attP2 (#34612); p{mini-W+, yl-eGFP-3xHA}. The sizes of the scale bars as annotated.

Rbcn-3A is a component of the evolutionarily conserved RAVE (Regulator of H+-ATPase of Vacuolar and Endosomal membranes) complex, which functions to assist the proper assembly of integral membrane V0 multi-subunit subcomplex and cytosolic V1 ATPase subcomplexes into an active vacuolar proton-translocating ATPase (V-ATPase) holo-enzyme on the vesicular membrane [83]. As V-ATPase is required for the proton uptake into the lumens of the residing organelles and their acidification [84,85], the strong effects of Rbcn-3A depletion would predict a similar functional requirement of the V1 and V0 ATPase subcomplexes in yolk granule biogenesis. To test this, we examined RNAi lines targeting eight different subunits in V0 and V1 subcomplexes (Vha68-2, Vha36-3, Vha26, Vha55, Vha100-1, Vha100-2, VhaAC45RP, VhaAC39-2). Among them, oocytes expressing dsRNA against Vha26 (Fig 8A and 8B), an E subunit of the V1 subcomplex, or Vha68-2 (Fig 8C–8F), subunit 2 of the V1 subcomplex, exhibited phenotypes similar to those induced by Rbcn-3A knockdown, including the absence of yolk granules, trapped Yl receptor and loss of Rab7- and FYVE-positive endosomes inside the oocytes (Fig 8). Together, these findings support that the recruitment of RAVE complex and the subsequent assembly of active V-ATPase complex are essential early steps in endosomal trafficking events that lead to the biogenesis of mature yolk granules.

Fig 8. V-ATPase is essential for Yl recycling and yolk granule biogenesis.

Fig 8

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of dsRNA against (A, B) Vha26a or (C-F) Vha68-2 double-labeled for F-Actin (red) and (A, B, E, F) endogenous Rab7 (green) or (C, D) GFP (green) from flies (C) with ectopic expression of 2x-FYVE-GFP or (D) carrying genome-tagging Yl-eGFP-3xHA reporter. (B, C1, D1, F) High-magnification views of the cortex regions highlighted in (A, C, D, E), respectively, as annotated. Genotypes: The samples were from adult females flies heterozygous for both matalpha4-GAL-VP16 driver (BDSC #7062) and the following UAS-transgenic RNAi and PI(3)P or Yl reporter lines: (A, B) P{TRiP.HMS01912}attP2 (#38996). (C) P{w(+mC) = UAS-GFP-myc-2xFYVE}2 (#42172); P{TRiP.HMS01056}attP2 (#34582). (D). P{TRiP.HMS01056}attP2 (#34582);p{mini-W+, yl-eGFP-3xHA}. (E, F) P{TRiP.HMS01056}attP2 (#34582). The sizes of the scale bars as annotated.

Discussion

Our results support that yolk granule biogenesis largely follows the canonical endolysosomal trafficking processes, regulated through cascades of effectors that coordinate and execute different steps of endosomal dynamics, including Rab5 and Rab11 in endocytosis and recycling, the VPS34/VPS15 PI3K kinase complex and PI(3)P production in endosomal biogenesis, and the Rab5 to Rab7 conversion regulated through the CCZ1/Mon1 complex. Together, they demonstrate that the vitellogenic oocyte is an excellent in vivo model for studying receptor-mediated endocytosis and subsequent endolysosomal trafficking in a multicellular system under physiological conditions.

Roles of Rab4 and Rab7 in endolysosomal trafficking

One surprising finding from the study is the dispensable roles of Rab4 and Rab7 in yolk granule biogenesis. It is generally accepted, mainly from studies in mammalian cells, that Rab4 mediates the fast recycling directly from early endosomes back to the cell surface, while Rab11 controls the slow recycling that often transits through peri-nuclear endocytic compartment to the plasma membrane [1419]. Given the heavy intensity of Yl recycling that happen primarily near the plasma membrane during vitellogenesis (Fig 1), it is unexpected that disturbances of Rab4 had no apparent effect on Yl recycling and yolk granule biogenesis (Figs 4G–4J and S6). More intriguingly, YFP-Rab4 is distributed diffusively as small puncta and co-localized with Rab7-positive granules (Fig 2D). Importantly, such localization patterns are unlikely to be artifacts, as endogenous Rab4 in surrounding follicle cells also clearly co-localized with Rab7 on the same endosomal structures (Fig 2D). Lastly, ectopically expressed wildtype YFP-Rab4 prominently localized on Rab7-positive endosomes (S6B Fig). These observations are rather surprising, as in mammalian cells, Rab4 and Rab7 were shown to locate on non-overlapping endosomal compartments and with different endosomal functions [86]. However, a later study reported that Rab4 was involved in both recycling and degradative endosomal trafficking [87]. Altogether, they raise an intriguing possibility that Rab4 and Rab7 overlap spatially and functionally during endolysosomal trafficking and that similar mechanisms control their endosomal recruitment. More studies are needed to determine whether the Rab4/Rab7 colocalization is cell type-specific and exclusive to Drosophila, and to clarify their spatial and functional relationships.

Similarly, Rab7 has been accepted as a key regulator of endosomal maturation, controlling various aspects of endosomal dynamics, such as fusion among late endosomes and with lysosomes, cargo retrieval by retromer and transport by microtubule-based motors [4,14,88,89]. However, manipulations of Rab7 did not significantly affect yolk granule biogenesis, as vitellogenic oocytes were filled with acidified granules of relatively normal sizes (Figs 4C–4F and S5). Consistently, in CCZ1-knockdown oocytes, which blocked the recruitment of Rab7 to the endosomal membrane, yolk granule biogenesis also appeared to proceed normally (Fig 6). Together, they raise a tantalizing possibility that, at least under this physiological setting, Rab7 might be dispensable for endosomal maturation. Notably, in an earlier study in HeLa cells, Rab7 depletion similarly did not affect late endosome biogenesis but blocked the fusion of late endosome with the lysosome [90]. Therefore, one possible scenario is that during yolk granule biogenesis, although Rab7 is recruited early onto endosomal membrane, it is primarily required at much later stages, after the growth and maturation of late endosomes, to promote their fusion with lysosomes. In this scenario, it is important to note that, although yolk granules are assumed to be specialized latent lysosomes containing inactive lysosomal enzymes [3437], we still lack the molecular tools to definitively define whether the yolk granules we observed under confocal microscope are lysosomes or just late endosomes. Alternatively, the dispensable role of Rab7 in granule biogenesis could be due to redundant mechanisms involving Rab7 and other GTPases such as Arl8b and Rab2 as well as the HOPS complex, which together coordinate the endosomal maturation and their fusion with lysosomes [14].

Interestingly, in oocytes expressing dominant negative Rab7, large granules with a dense F-Actin coating were observed (Fig 4C and 4D), suggesting a role of Rab7 in preventing F-Actin accumulation on late endosomal membrane. In both yeast and mammalian cells, membrane assembly of actin filaments is required for the fusion between mature endosomes and lysosomes [9194]. Further, in yeast, endosomal actin polymerization is catalyzed by Rho GTPase Rho1p and Cdc42, which in turn are activated by Ypt7p, the yeast Rab7 homologue [95]. It remains to be clarified whether Rab7 engages in similar conserved mechanisms to regulate actin dynamics.

Dynamics of actin cytoskeleton in endocytosis

Actin dynamics are involved in various aspects of endocytosis and endolysosomal trafficking, including the internalization of endocytic vesicles, endosome motility and potentially the fusion between late endosomes and lysosomes [4,63,96]. Consistently, distinct actin structures that correlate with different stages of endosomal trafficking exist in the vitellogenic oocyte, including a dense F-Actin layer that overlaps significantly with Yl receptor on the plasma membrane, an adjacent thin web of F-Actin projecting down into the cortex, and scattered small F-Actin puncta decorating the FYVE- and Rab7-positive granules that emerge from the cortex area (Fig 3). Notably, these small F-Actin puncta were mostly localized on the side of the granules proximal to the cortex (Fig 3B and 3I), reminiscent of the actin tails on motile endosomes in mammalian cells [97101], suggesting their potential role in propelling endosomes into the interior of the oocytes, in a mechanism similar to the proposed “actin-rocketing” model that drives the motility of invading pathogens in infected cells [102].

The cortical F-Actin dynamics are likely regulated by phospholipid PI(4,5)P2, which has well-established roles in recruiting actin regulators such as WASP-family proteins and Arp2/3 complex [103107], and is enriched on oocyte membrane where it overlaps with F-Actin (Fig 3C and 3D). Consistently, higher levels of ectopic PLCδ1(PH)-GFP reporter, which can exert a dominant negative effect by sequestering PI(4,5)P2 [6669], severely disrupted endocytosis and caused the abnormal congregation of endosomal structures with Rab7 and long F-actin filaments (Fig 3F and 3G).

RAVE and V-ATPase complexes are essential during early endosomal formation

The highly conserved V-ATPase complex controls endosomal acidification, which is essential for cargo sorting, membrane trafficking and endosomal maturation [4,108]. The reversible disassembly and regulated re-assembly of functional V-ATPase is controlled by RAVE complex in yeast and Rabconnectin-3 complexes in metazoan [83]. Consistently, oocyte-specific depletion of either Rbcn-3A (Fig 7) or subunits of V-ATPase complex (Fig 8) caused similar granule biogenesis defects, in particular a blocked Yl receptor recycling and endosome formation, in line with their critical role for efficient endocytosis and endosomal trafficking. Notably, Rbcn-3A was isolated as a Rab5 effector in a proteomic study from Drosophila S2 cells [58], suggesting that Rab5 recruits V-ATPase onto earlier endosomes via the RAVE complex, which in turn leads to the acidification of early endosomes and the subsequent release of Yolk cargo from Yl receptor. In the absence of functional RAVE and V-ATPase complexes, Yolk cargo can not be efficiently freed from Yl receptor in a PH-dependent manner, resulting in blocked Yl recycling and aborted yolk granule formation.

A model for yolk granule biogenesis

In line with the current models of endocytosis and endosomal trafficking [15,14], our results support the following events that control yolk granule biogenesis in Drosophila oocytes (Fig 9). During vitellogenesis, Rab5 promotes the extensive rounds of clathrin-mediated endocytosis of Yolk proteins by Yl receptor, a process that is facilitated by dynamic actin cytoskeleton networks regulated by PI(4,5)P2-associated actin regulators. After the removal of clathrin and depletion of PI(4,5)P2 and associated factors from the endocytosed vesicles, membrane-associated Rab5 recruits a collection of effectors that set off a cascade of downstream events, including the VPS34/VPS15 PI3 kinase complex for local production of PI(3)P and RAVE/Rabconnectin-3 complex that facilitates the stable assembly of V-ATPase complex on early endosomes. The conversion of PI(4,5)P2 to PI(3)P on endosomal membrane is accompanied by the replacement of branched actin filaments with small actin nucleations on the distal side of granule surface to propel their inward movement. Simultaneously, Rab5 synergizes with PI(3)P on the early endosomal membranes to recruit PI(3)P-binding effectors such as Hrs and other ESCRT components and CCZ1/Mon1 complexes to catalyze endosomal fusion, sorting, Rab5 to Rab7 conversion and maturation, while acidification of endosomal lumen by V-ATPase leads to the separation of Yl from Yolk cargo. The ligand-free Yl is subsequently segregated away and enriched in the tubular membrane extension where it is quickly recycled back to the plasma membrane by Rab11 for new rounds of endocytosis, while Yolk proteins are retained in Rab7-positive vacuolar domain. Through concerted action by Rab7 and other functionally redundant cytoskeleton and membrane regulators to coordinate actin dynamics with membrane fusion, Yolk-containing endosomes undergoes continuous fusion and maturation processes while being transported into the interior of the oocyte through actin- and/or microtubule-based mechanisms, and eventually fuse with the lysosome to complete their maturation.

Fig 9. Model of yolk granule biogenesis.

Fig 9

See text in the Discussion for details.

Many questions arise from this study, such as the exact spatial and functional relationship between Rab4 and Rab7 in endolysosomal trafficking, and when and which GTPase-activating proteins (GAPs) control the release of Rab7 from mature granules [109,110], among others. Many factors would affect the interpretation of our observations, such as the limited resolution of subcellular structures under conventional confocal microscope. Notably, most of the genes tested in our RNAi screen did not show significant effect on granule biogenesis (Table S1), which could be due to inefficient depletion of the targeted protein products in stage 10 oocytes by RNAi, either because of long perdurance of the studied proteins or due to ineffective RNAi constructs, or both, especially considering the relatively short duration between stage 5 when matalpha4-GAL-VP16 becomes active to drive dsRNA expression and stage 10 when oocytes are analyzed. In support of this, using the same driver, we found that RNAi against Rab4 did not have apparent effect on granule biogenesis, as expected, RNAi against Rab5 induced robust phenotypes similar as or even more severe than that by Rab5-DN, but a RNAi line against Rab7 only partially knocked down endogenous Rab7 protein (S8 Fig). Together, they suggest variable knockdown efficiency by different RNAi lines, which also implies that only a fraction of the RNAi lines tested in our pilot screen induced significant loss of function phenotypes. Nevertheless, several of them, including PI3K59/VPS15, Rbcn3A and Rab5, showed distinct granule biogenesis defects when driven by matalpha4-GAL-VP16 (Figs 58). When directed by ubiquitous tubulin-GAL4, we found that the same RNAi lines for these essential genes caused animal lethality, preventing their functional evaluation in the oocytes. Thus, although not 100% effective in depleting the target proteins in stage 10 oocytes, dsRNA expression driven by matalpha4-GAL-VP16 can still be a valuable option to quickly survey large number of essential genes for their roles in yolk granule biogenesis. Development of more potent and oocyte-specific GAL4 drivers will greatly facilitate similar studies in the future.

Our simplified model will require further validation and revision at molecular and subcellular levels by incorporating new imaging tools such as live- and super-resolution microscopy and by employing additional markers specific for different endosomal trafficking steps, such as lysosomal markers that can distinguish between maturating and matured granules. Moreover, as our studies primarily focused on stage 10 oocytes, it will be important to test to what extent the findings from this unique time point can be applied to membrane trafficking in Drosophila oocytes at other developmental stages, to other cell types and tissues such as neurons, and especially to other organisms such as mammals that have more redundant genes (e.g., three Rab5-like genes in human genome as comparted to a single Rab5 gene in the fly) [76] and likely more complicated regulatory circuitries on endosomal trafficking to meet their more complex physiological demands.

Materials and methods

Drosophila husbandry and genetics

Fly stocks were maintained at room temperature following standard culture conditions. All fly crosses were performed at 25°C following standard genetic procedure unless otherwise specified.

For RNAi-mediated knockdown of target genes in oocytes, virgin females of the matalpha4-GAL-VP16 line were crossed with males of UAS-dsRNA lines, and their female progenies of right genotypes were collected and aged over yeast paste overnight, their egg chambers dissected for immunofluorescent staining. Phenotypes were evaluated based on yolk granule size, observed by DIC imaging, and also using a panel of markers, including Yl-eGFP for Yolk endocytosis and receptor recycling, PLCδ1(PH)-GFP for early endocytosis, 2xFYVE-GFP for early and late endosomes, Rab7 for late endosomes, Rab11 for recycling endosomes, and F-Actin for actin cytoskeleton dynamics.

The following fly lines were from Bloomington Drosophila Stock Center (BDSC):

yl13 v24/FM3 (#4320). matalpha4-GAL-VP16 (#7062). w*;UAS-GFP-myc-2xFYVE (#42712)

Endogenous tagging lines for Rab4, Rab5 and Rab11: y1, w1118; TI{TI} EYFP-Rab4 (#62542). w1118; TI{TI} EYFP-Rab5 /CyO (#62543). w1118; TI{TI} EYFP-Rab11 (#62549).

UAS- based transgenic lines for YFP-tagged WT, DN and CA Rab proteins:

Rab4-WT (#9767), Rab4-CA (# 23268), Rab4-DN (#9768), Rab5-WT (#24616), Rab5-CA (#9774), Rab5-DN (#9771), Rab7-WT (#23641), Rab7-CA (#24103), Rab7-DN (#9778), Rab11-WT (#50782), Rab11-CA (#23260), Rab11-DN (#9792). Control of dsRNA for RNAi of Firefly Luciferase (FBgn0014448) under UAS control in the VALIUM1 vector (#31603).

dsRNA transgenic lines for RNAi-mediated knockdown of the following target genes: sktl (#27715), HK (#28330), CG7800 (#28922), Rabex-5 (#29357), mtm (#31552), mtm (#38339), mtm (#57298), Pi3K59F (#36056), Pi3K59F (#33384), VPS15 (#57011), VPS15 (#34092), VPS15 (#35209), Ocrl (#34722), Tbc1d15-17 (#34859), CG41099/ Rabankyrin-5, RIPK4, ANKK1 (#34883), TBCK (#35332), CG17471 / PIP4K (#35338), CG17471 / PIP4K (#35660), Vps45 (#38252), YKT6 v-SNARE CG1515 (#50937), YKT6 v-SNARE CG1515 (#38314), Hip1 (#38377), Tbc1d15-17 (#43409), Rab4 (#33757), Rab5 (#67877), Rab7 (#27051), Rab11 (#27730), Rabex-5 (#50573), RUFY1 (#51494), Rbpn-5 (#52996), TBCK (#57223), Rbsn-5 (#57459), RUFY1 (#60496), NADSYN1 (#62265), Ccz1 (#62889), Bulli (regulator of MON1-CCZ1) (#63531), Pi3K59F (#64011), CG17471 / PIP4K (#65891), Hip1 (#77315), Rbcn-3A (#34612), Vha36-3 (#65075), Vha26 (#38996), Vha55 (#40884), Vha100-1 (#57860), Vha100-2 (#64859), VhaAC45RP (#64549), VhaAC39-2 (#67809), Vha68-2 (#34582).

Genome tagging of yl

yl genome tagging constructs with eGFP and three copies of human influenza hemagglutinin (HA) tags were engineered as the following. BacPac clone CH321-59C03 (BACPAC Resource Center (BPRC), Children’s Hospital Oakland Research Institute in Oakland, California), which covers all the genome coding regions of yl was selected as starting template for the tagging constructs. A 4.3kb SpeI and ApaI fragment covering N-terminal one third of yl genome region and an 8.0kb ApaI and HpaI fragment containing the remaining part of yl genome region were cloned separately from BacPac CH321-59C03 into PHSX cloning vector. KpnI and AscI restriction enzyme recognition sites were introduced in the middle of a 550 bp intermediate fragment encoding the C-terminal ST-rich region of Yl protein, and subsequently cloned into the 8.0kb ApaI/HpaI fragment in the PHSX vector. Separately, a DNA sequence encoding eGFP and 3 copies of HA tags flanked by KpnI and AscI restriction enzyme recognition sites were engineered and amplified by PCR, and then cloned into the modified 8.0kb ApaI/HpaI fragment in PHSX vector using the KpnI and AscI restriction enzyme sites. The 4.3kb N-terminal SpeI/ApaI yl fragment and the modified 8.0kb Apa1-Hpa1 yl fragment with eGFP-3xHA insertion were subsequently ligated together and cloned into the pCaspeR4 transgenic vector to assembly the ~14kb genome-tagging construct encoding full-length Yl protein with an eGFP and 3xHA tags near the C-terminus of encoded Yl protein. After DNA sequencing verification of the cloned construct, the purified DNA for the pCaspeR4-Yl-eGFP-3xHA tagging construct was injected into w1118 embryos together with pπ25.7wc helper plasmid, and transgenes were selected and established following standard protocol [111]. The expression of tagged transgenes was validated by Western blots on protein extracts and by immunofluorescent stating by anti-eGFP and anti-HA antibodies from adult transgenic female flies (Figs 14 and 69).

Dissection and immunofluorescent staining and imaging of egg chambers

Female flies of proper genotypes, aged 3 to 5 days, were fattened over yeast paste overnight, then egg chamber was were dissected in Drosophila S2 media containing 15% fetal bovine serum, fixed in 1xPBS with 4% paraformaldehyde for 20 minutes, follow by brief rinse with 1xPBS twice, wash with 1xPBT (1xPBS, plus 0.3% Triton X-100) for four times, 5 mins each, and then block with 5% normal goat or donkey serum in 1xPBT for 30 minutes. Incubate with primary antibodies at proper dilution in 1X PBT overnight at 4°C. Next day, remove the primary antibodies, wash the samples 5 times with 1X PBT. Leave the samples for 5–10 min in 1X PBT solution in between each wash. Incubate the washed samples in appropriate secondary antibodies as indicated dilution at room temperatures for 2 hours or at 4C overnight. Rinse 3 times with 1X PBS. Wash 6 times with 1X PBT with 10 min incubation between each wash. Incubate in 1:10,000 dilution of 1mg/mL 4’, 6’-diamidino-2-phenylindole (DAPI) in 1XPBT solution for 10 min. Rinse 3 times with 1X PBS. Transfer egg chambers to slide with anti-fade mounting medium (H-1900, Vector Laboratory) for imaging by a confocal microscope (Leica TCS SP5; Leica Microsystems, Wetzlar, Germany).

Each experiments were repeated independently at least once. In each experiment, at least 5 adult females of appropriate genotypes were dissected for immunofluorescent staining and microscopy imaging, 20 or more stage 10 egg chambers were inspected under immunofluorescent microscope, and five or more independent egg chambers were imaged through Z-serial section by confocal microscope.

Antibodies

Antibodies were from the following sources: rat anti-Yl and rabbit anti-Yolk antibodies were from Dr. Mahowald [48]. mouse anti-Rab7 (1:10, Developmental Studies Hybridoma Bank (DSHB)), mouse anti-Rab11 (1:200, BD Transduction Laboratories (#610657)), mouse anti-actin (1:10000, MAB1501, Chemicon, and #ab-6276, Abcam); mouse anti-αTubulin (1:10000, DM1A, Sigma); mouse anti-HA (12CA5, Roche); chicken anti-GFP (Aves. 1:10,000 for Western blot and 1:2,000 for immunofluorescent staining). Alexa Fluor 488 AffiniPure Donkey Anti-Chicken IgY (1:1000, 703-545-155) and Rhodamine Red AffiniPure Donkey Anti-Mouse IgG (1:1000, 715-295-151) from Jackson ImmunoResearch Inc. TRITC-Phalloidin (Phalloidin–Tetramethylrhodamine B isothiocyanate) (1:200, P1951, Sigma). Alexa Fluor 488-Phalloidin (1:200, A-12379), Alexa Fluor 594-Phalloidin (1:200, A-12381), Alexa Fluor 647-Phalloidin (1:200, A-30107), Alexa Fluor 680-(A-21076) were from Molecular Probes (Invitrogen). Alexa Fluor 680-(A-21076) and Alexa Fluor 800-(926–32212) conjugated secondary antibodies for immunoblotting (1:10,000) were from Molecular Probes (Invitrogen) and LI-COR, respectively.

Lysotracker staining

LysoTracker Red DND-99 (L7528, ThermoFisher Scientific) was used to detect acidic compartment in egg chambers following the manufacturer’s instruction. Briefly, egg chambers were incubated with 200 μL of the 1:100 dilution of 1mM LysoTracker working solution in DMSO (final at 10uM) per tube of ovarioles for 5 minutes, fixed by adding 200 μL of 4% paraformaldehyde, followed by a 10-minute agitation at room temperature. This was succeeded by three wash cycles using 400 μL 1xPBST to permeabilize the tissue, 10 minutes each wash. The stained egg chambers were then visualized under a confocal microscope.

Western blotting

Standard 8% SDS-PAGE gels were used for separation of Yl-eGFP-3xHA protein. The boiled samples were separated on SDS-PAGE and transferred to nitrocellulose membranes from Millipore. After blocking with 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour, membranes were incubated with primary antibodies. Secondary antibodies conjugated with Alexa-800 or Alexa-680 (Invitrogen) were used and the signals were detected by the Odyssey Infrared Imaging System and quantified by Odyssey Application Software 3.0 or by densitometry of the digital images using ImageJ software (NIH).

Supporting information

S1 Fig. Controls for anti-GFP and anti-Rab11 antibody staining in wildtype egg chambers.

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with immunofluorescent staining by anti-GFP antibody (green) on (A-D) w1118 wild type control or (E, F) YFP-Rab11 endogenous tagging flies, co-labeled with (A-D) phalloidin for F-Actin and (C-F) mouse anti-Rab11 antibody, as annotated. (B, D and F) High magnification views of the regions highlighted in (A, C and E), respectively. Images are presented as overlaying in color or individual channels in gray, as annotated. Genotypes: (A-D) w1118; (E,F), w1118; TI{TI} EYFP-Rab11 (BDSC #62549). The sizes of the scale bars as annotated.

(TIF)

pgen.1011152.s001.tif (9.6MB, tif)
S2 Fig. Subcellular localization of 2xFYVE-GFP reporter in wildtype and VPS34/PI3K59F-RNAi oocytes.

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of 2xFYVE-GFP reporter from (A-C) wild type control or (D-F) oocyte co-expressing dsRNA again VPS34/PI3K59F, triple-labeled for GFP (green), Rab7 (red) and F-Actin (blue). Images are presented as overlaying in color or individual channels in gray, as annotated. Genotypes: (A-C). w*/ w1118; matalpha4-GAL-VP16 (BDSC #7062), UAS-GFP-myc-2xFYVE (BDSC #42712)/+; (D-F). w*/ w1118; matalpha4-GAL-VP16 (BDSC #7062), UAS-GFP-myc-2xFYVE (BDSC #42712)/+; P{TRiP.HMJ30324}attP40 (BDSC #64011)/+. The sizes of the scale bars as annotated.

(TIF)

pgen.1011152.s002.tif (9.6MB, tif)
S3 Fig. The roles of Rab5 on yolk granule biogenesis in vitellogenic oocytes.

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers from oocytes of (A-D) wildtype control or (E-N) overexpressing (E-H) dominant-negative YFP-Rab5-DN, (I-L) constitutive-active YFP-Rab5-CA and (M, N) wildtype YFP-Rab5, that were (A, B, E, F, I, J, M, N) triple-labeled for GFP (green), endogenous Rab7 (red) and phalloidin for F-Actin (blue), or (C, D, G, H, K, L) stained with lysotracker alone (red), with data presented as overlaying images in color or individual channels in gray, as annotated. (B, F, J and N) High-magnification views of the cortex regions highlighted in (A, E, I and M), respectively, as annotated. The sizes of the scale bars as annotated. Genotypes: The samples were from adult female flies heterozygous for both matalpha4-GAL-VP16 (#7062) driver and the following UAS-transgenic lines: (A-D) w1118. (E-H) Rab5-DN: P{UASp-YFP.Rab5.S43N}01 (#9771). (I-L). Rab5-CA: P{UASp-YFP.Rab5.Q88L} (#9774). (M, N). Rab5-WT: P{UASp-YFP.Rab5}02 (#24616).

(TIF)

pgen.1011152.s003.tif (9.6MB, tif)
S4 Fig. The roles of Rab11 on yolk granule biogenesis in vitellogenic oocytes.

Confocal fluorescent microscopy and DIC imaging of a stage 10 egg chambers with oocyte-specific expression of (A, B) constitutive active YFP-Rab11-CA or (C, D) wildtype YFP-Rab11 triple-labeled for YFP (green), endogenous Rab7 (red) and phalloidin for F-Actin (blue), presented as overlaying image of all channels in color or individual channels in gray, as annotated. (B, D) High-magnification views of the cortex regions highlighted in (A, C), respectively, as annotated. Genotypes: The samples were from adult female flies heterozygous for both matalpha4-GAL-VP16 driver (#7062) and the following UAS-transgenic lines: (A, B) Rab11-CA: P{UASp-YFP.Rab11.Q70L} (#23260). (C, D). Rab11-WT: P{UASp-YFP.Rab11} (#50782). The sizes of the scale bars as annotated.

(TIF)

pgen.1011152.s004.tif (9.6MB, tif)
S5 Fig. The roles of Rab7 on yolk granule biogenesis in vitellogenic oocytes.

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with ectopic expression of (A-D) wildtype YFP-Rab7 or (E-H) constitutive active (CA) YFP-Rab7-CA in oocytes that are (A-F) co-labeled for anti-GFP (green), F-Actin and (A, B) anti-Rab7 or (C, D) lysotracker (red), or (G, H) by lysotracker (red) alone, shown as overlaying images of all the channels in color or in individual channels in gray, as annotated. (B, D, F) High-magnification view of the cortex regions highlighted in (A, C, E), respectively, as annotated. Genotypes: The samples were from adult female flies heterozygous for both matalpha4-GAL-VP16 driver (#7062) and (A-D) wildtype YFP-Rab7: y[1 w*; P{w(+mC) = UASp-YFP.Rab7}21/SM5 (#23641): (E, F) constitutive active YFP-Rab7-CA: P{UASp-YFP.Rab7.Q67L} (#24103).The sizes of the scale bars as annotated.

(TIF)

pgen.1011152.s005.tif (9.7MB, tif)
S6 Fig. The roles of Rab4 on yolk granule biogenesis in vitellogenic oocytes.

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of (A, B) wildtype YFP-Rab4 or (C-F) constitutive active YFP-Rab4-CA (A-D) triple-labeled for YFP (green), endogenous Rab7 (red) and F-Actin (blue), shown in overlaying images in color or individual channels in gray, or (E, F) by lysotracker staining (red) alone, as annotated. (B, D) High-magnification views of the cortex regions highlighted in (A, C), respectively, as annotated. Genotypes: The samples were from adult female flies heterozygous for both matalpha4-GAL-VP16 driver (#7062) and the following UAS-transgenic lines: (A, B) wildtype Rab4: P{UASp-YFP.Rab4} (#9767). (C-F) Rab4-CA: P{UASp-YFP.Rab4.Q67L} (#9770). The sizes of the scale bars as annotated.

(TIF)

pgen.1011152.s006.tif (9.4MB, tif)
S7 Fig. Essential roles of VPS34/VPS15 PI3 kinase complex in Yl recycling and yolk granule biogenesis.

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers from flies carrying genome-tagging Yl-eGFP-3xHA with oocyte-specific expression of (A-C, G, H) control firefly Luciferase RNAi or (D-F, I, J) dsRNA again VPS34/PI3K59F, co-labeled with antibodies against GFP (green) and (A-F) lysotracker (red), or (G-J) endogenous Rab7 (red) and F-Actin (blue), as annotated. (B, E, H, J) High-magnification view of the cortex regions highlighted in (A, D, G, I), respectively. (C, F) Zoom-in view of the areas highlighted in (B, E), respectively. Images are presented as overlaying images in color or as individual channels in gray, as annotated. Genotypes: The samples were from adult females flies heterozygous for Yl-eGFP-3xHA reporter (p{mini-W+, yl-eGFP-3xHA}) and matalpha4-GAL-VP16 (BDSC #7062) driver together with (A-C, G, H). P{TRiP.JF01355}attP2 (BDSC#31603) or (D-F, I, J) P{TRiP.HMJ30324}attP40 (BDSC #64011). The sizes of the scales as annotated inside images.

(TIF)

pgen.1011152.s007.tif (9.7MB, tif)
S8 Fig. Variable knockdown efficiency by different RNAi lines in stage 10 oocytes.

Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of dsRNA against (A, B) Rab5, (C, D) Rab4 and (E, F) Rab7, co-stained for endogenous Rab7 (green) and F-Actin (red), as annotated. (B, D, F) High-magnification view of the cortex regions highlighted in (A, C, E), respectively. Images are presented as overlaying images in color or as individual channels in gray, as annotated. Genotypes: The samples were from adult females flies heterozygous for matalpha4-GAL-VP16 (BDSC #7062) driver together with (A, B) P{TRiP.GL01872}attP40 (BL# 67877). (C, D) P{TRiP.HMS01100}attP2P (BDSC #33757). (E,F) P{y(+t7.7] v(+t1.8) = TRiP.JF02377}attP2 (BDSC#27051). The sizes of the scales as annotated inside images.

(TIF)

pgen.1011152.s008.tif (9.7MB, tif)
S1 Table. Summary of the pilot RNAi screen.

(XLSX)

pgen.1011152.s009.xlsx (13.8KB, xlsx)

Acknowledgments

We are grateful to Bloomington Drosophila Stock Center for Drosophila lines and Developmental Studies Hybridoma Bank (DSHB) and contributor Dr. Sean Munro for mouse anti-Rab7 antibody[60] used in the study. Dr. Anthony Mahowald for rat anti-Yl and rabbit anti-Yolk antibodies. Dr. Zhenmei Mao at IMM Microscopy Core of UTHealth for confocal microscopy and imaging analyses.

Data Availability

All data are in the manuscript and its supporting information files.

Funding Statement

This work is supported by National Institutes of Health (NIH) grant R01 NS110943 (to S.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Gregory P Copenhaver, Pablo Wappner

12 Sep 2023

Dear Dr  Zhang,

Thank you very much for submitting your Research Article entitled 'Endolysosomal trafficking controls yolk granule biogenesis in vitellogenic Drosophila oocytes' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a revised version. We cannot, of course, promise publication at that time.

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PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In this manuscript, Yu et al. shed light on the significance of Rab5, Rab11, Rab7, dynein motor, and the RAVE/V-ATPase complex in endocytosis, endosomal trafficking, and yolk granule biogenesis. The authors generated a tagged version of Yolkless (YI) and studied its distribution in vitellogenic oocytes. They observed a connection between different endosomal structures, phosphoinositides, and actin cytoskeleton dynamics. The study identified Rab5 and Rab11 as crucial players in yolk granule biogenesis, while Rab4 and Rab7 role was dispensable. The authors carried out a small RNAi screen on Rab5 effector molecules identified in a previous proteomic study and proposed the involvement of the dynein motor and the RAV/V-ATPase complexes in the early stages of endosomal trafficking.

The data presented in this manuscript offers novel insights into endolysosomal pathway, and highlight the vitellogenic oocyte in Drosophila as a valuable in vivo system to study endocytosis and endolysosomal trafficking. Such insights are of broad interest to researchers studying complex membrane trafficking events.

This is an exciting and clearly-written manuscript. The results are well organized, and the data support the interpretation and conclusions drawn. However, some revisions are recommended to improve the manuscript.

1. To assess the functionality of the transgenic construct Yl-eGFP-3xHA, was it placed in yl mutant background?

2. For readability, it would be beneficial to combine Figure-1 and Figure-2 (Inserting Figure-2 panel between Fig.1D and Fig.1E)

3. In Fig.1G, please provide an inset of invaginating clathrin-coated pits/vesicles if available.

4. Please cite this article “Crystalline yolk spheroids in Drosophila melanogaster oocyte: Freeze fracture and two-dimensional reconstruction analysis” (https://doi.org/10.1016/j.jinsphys.2006.12.011) when referring to the condensed and crystalized yolk protein in mature granules.

5. Briefly describe how the transgenic construct Yl-eGFP-3xHA was used to rescue the sterility of yl mutant females.

6. It would be beneficial to place an arrow in the figures when referring to F-actin puncta (E.g. Fig.4B and 4I)

7. What were the selection criteria for selecting specific Rab5 effectors for the RNAi screens?

8. RNAi lines might have a variable knockdown efficiency and potential off-targets, since most of the tested lines did not show any effect on granule biogenesis in the RNAi screen, was qPCR performed to the test the knockdown efficiency?

9. It would be great if the authors can clarify the number of replicates performed to validate the observed phenotype in the RNAi screens, either in the figure legend or the methods section.

10. The study focuses on stage 10 oocytes, and the findings might be specific to this developmental stage. Independent of the context and potential redundancy it would help readers to discuss the general applicability of the findings in other cell types, tissues, or organisms.

Reviewer #2: In their manuscript Yu and colleagues are showing a detailed microscopic analysis of the endocytic pathway that is essential for the generation of the yolk granules in the Drosophila oocyte. The authors use multiple Rab transgenes and Rab5 effector RNAis to genetically dissect the sequence of the events of this pathway. Although the study is comprehensive and aims to give a more complete view about this phenomenon, it still has multiple flaws, like it relies almost entirely on one technique (confocal microscopy) but lacking any quantifications and contains multiple formal mistakes. Thus, the manuscript requires substantial improvement before it may get considered for acceptance in this Journal.

Major comments:

1. The first and most important flaw is the complete lack of quantifications and statistics. As the novelty factor of the findings is moderate the comprehensiveness could the major advantage of the study. However, it is really hard to compare the effect of the different genetic modifications without any quantifications. For example, visually the phenotype upon loss of of Rbcn3 may seems similar to the silencing of the class III PI3K complex, but it is really hard to make any conclusions without statistics.

2. The authors are using the overexpression of DN forms of the Rab proteins to show their loss of function phenotype. However, RabDN-s are frequently cause weaker loss of function phenotype than mutant alleles or RNAi-s. So, it would be important to confirm the effect of DN lines by silencing of Rab4, Rab5, Rab7 and Rab11 by appropriate RNAi lines.

3. In the data from the small-scale screen on Rab5 effectors (Table S1) it is visible that in some cases the used RNAi lines that silence the same gene (like in the case of Vps15) showed different phenotypes. Was the effectivity of RNAi lines tested (like with qPCR)?

4. Authors sometimes claim interesting suggestions based on fluorescent mictoscopy data, but these are remaining as speculations without testing them by molecular methods. For example, based on the results with ctp RNAi Authors suggest that the dynein-dynactin complex would work as the effector of Rab5 instead of Rab7 in this tissue. Although this is an attractive idea it should be tested by pull down or immunoprecipitations.

5. Upon the knockdown of PI3K59F (Fig6C) it is clearly visible that remarkable amount of FYVE-GFP positive structures. Which is surprising, as loss of PI3K59F should diminish the formation of PI3P positive granules. Was the RNAi efficient enough? What can be the identity of these persistent FYVE-GFP granules? Are these persistent FYVE granules are colocalizing with the remaining Rab7 puncta?

6. Fig6A shows that upon the KD of PI3K59 YL-GFP is leaving the cortical region and delivered further inside into the oocyte. Does YL-GFP is reaching the lysosomes upon these conditions? Although the overlap between the diffuse signal of YL-GFP and Rab7 is apparent, but there are still some Rab7 and YL-GFP puncta remaining in these cells. Are these YL-GFP puncta colocalizing with Rab7 or lysosomal markers?

7. On Fig9 the Authors are using the silencing of two Vha genes to show the effect of loss of the V-Atpase on yolk granule formation. However, they are using different RNAi lines to show the effect on Rab7 (Vha26 RNAi), and FYVE-GFP (Vha68 RNAi). Although the phenotypes are consistent, to rule out any concern of pleiotropic effects it would be better to use the same RNAi for both markers (or use both RNAis for both).

Minor comments:

1. The Legends for main figures and supplementary figures and sometimes the figures itself contains multiple inconsistence and annoying mistakes:

- Fig1: What are the green circles that appear on panels E, F, G? In the legend the second reference for panel (F) actually refers to panel (G)

- Fig4: The UASp-PLCD1(PH) reporter is called as PLCD-eGFP in the legend, but PH-GFP is written on the image panel, this inconsistence should be resolved. The legend refers to panel (G) that shows FYVE-GFP, however on the image this panel shows PH-GFP

- FigS3-S5: The logic of the panel labels of FigS3-S5 is different from the logic that was followed by the rest of the Figures. This inconsistence should be resolved.

- FigS5: The order of panel labels restarts after the 10th panel (panel J followed by another A, B, C, etc.) and inconsistent with the legend. This should be corrected.

2. On the 18th page of the submitted PDF Authors are writing the following: “overexpressing either DN-, WT- or CA-YFP-Rab7, as large numbers of lysotracker-positive yolk granules were present in these oocytes (Fig 5C-F and Fig. S4)”, however there are no panel showing the YFP-Rab7WT on these figures.

3. There is no reference for Fig7E, F panels in the main text.

Reviewer #3: Endocytic recycling is a fundamental cellular process that plays a key role in all tissues, and yet it has mostly been studied in mammalian tissue culture cells or yeast. The Drosophila model system has a lot of potential as a means to investigate how endocytic processes vary between tissues and are regulated to meet their disparate needs.

The Rab GTPases are known to be master regulators of the endocytic pathway, and this paper reports an analysis of the role of key Rabs in the endocytosis of vitellogenin and yolk granule formation in developing oocytes. These maturing oocytes are very large and so amenable to imaging, and also accessible with tools such as ectopic protein expression and RNAi for following cellular processes and perturbing protein expression. The authors apply these methods to investigate the contributions that Rabs 4, 5, 7 and 11 make to the endocytosis and recycling of Yolkless (the vitellogenin receptor) and the formation of yolk granules. They also investigate the effect of knocking down a set of c 16 putative Rab5 effectors that were identified in a previous study based on affinity chromatography. Overall, the authors conclude that Rab5 and Rab11 play key roles in the process and identify several Rab5 effectors that are particularly important. They also find that Rab7 is not critical for Yolkless recycling and granule formation which is an unexpected finding but convincingly shown by both the Rab7 reagents and the knock-down of a Rab7 GEF. This raises interesting questions about what purpose Rab7 serves in such recycling pathways in tissues.

The paper describes a lot of work, and the data consists almost entirely of a large number of immunofluorescence images which are of a good quality and are clearly presented with both lower magnification overviews and higher magnification images to show details. The text is clear, although it would benefit from condensing and also some grammatical tidying. Overall, the paper makes a useful contribution to the endocytosis field as it shows the potential of the oocyte system, and in particular its value for studying Rab5 and Rab11. However, before publication the following relatively minor issues would need to be addressed:

a) The text is rather long in the Introduction, Results and Discussion. Being more succinct would encourage more people to read the paper. It would be good to get the input from someone with lots of paper writing experience.

b) For most of the figures the single IMF channels are shown as grey scale which makes them easier to see than colour. However, this is not the case for Figure 3, and so this should be altered to follow the better practice of the other figures.

c) The authors state in the Introduction that PI3P acts as a landmark to enlist Ccz1/Mon1, but as they state later it is actually Rab5 that does this. There are other PI3P binders that could be mentioned instead.

d) For figures that show a YFP-Rab, the authors should include the YPF in the labelling on the figure and not just the name of the Rab to make clear that it is a tagged protein (ie YFP-Rab5 rather than Rab5).

e) Did the authors look at the distribution of Rab11 in an oocyte that is not also expressing YFP-Rab11? If so, it should be shown.

f) In Figure S2 they could include a wild-type control showing Rab7 and actin so as to aid comparisons. Perhaps the figure could be split into two figures to facilitate this.

g) Figure 5D is labelled Rab-DN but it should be Rab7-DN.

h) The authors should mention that flies lacking Rab4 are viable and fertile (in contrast to the situation for the other Rabs that they investigate). See PubMed ID 33666175.

i) The section on cut up (ctp) could be deleted as it is not directly linked to the Rab work in the rest of the paper.

k) It would be helpful to have a summary table showing the effect on the various markers of all of the different Rab perturbing treatments tested.

**********

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: No

Reviewer #3: No

Decision Letter 1

Gregory P Copenhaver, Pablo Wappner

16 Jan 2024

Dear Dr Zhang,

Thank you very much for submitting the revised version of your Article entitled 'Endolysosomal trafficking controls yolk granule biogenesis in vitellogenic Drosophila oocytes' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by the same reviewers that evaluated the original version. The reviewers feel that all their concerns have been properly addressed, although they raised a few  minor points that you need to change before the manuscript can be accepted for publication

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have done an excellent job of addressing and responding to all the comments and concerns raised during the review process. They have diligently offered well-reasoned explanations and additional data where necessary to support their findings and conclusions.

Reviewer #2: The Authors addressed most of my concerns experimentally. I also approve their arguments regarding the difficulties of the quantification. This problem was further addressed by the use of a large number of samples (20< oocytes/genotype). Overall, the data and the manuscript's quality greatly improved.

I still have one minor comment: The texts "Yl" and (DIC) on Fig1 G1 and G2 have been mixed up. This should be corrected.

Reviewer #3: In revising this manuscript the authors have done an excellent job of addressing the points that I made. It was already a good quality study and I am now very happy to recommend that it be published. I have one minor suggestion which is that the newly added Table S1 seems a valuable summary of all the data which would help readers. Thus it might be good to incorporate it into the main paper rather that include it as a supplementary Excel file.

**********

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Decision Letter 2

Gregory P Copenhaver, Pablo Wappner

22 Jan 2024

Dear Dr Zhang,

We are pleased to inform you that your manuscript entitled "Endolysosomal trafficking controls yolk granule biogenesis in vitellogenic Drosophila oocytes" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Gregory P. Copenhaver

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----------------------------------------------------

Comments from the reviewers (if applicable):

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Acceptance letter

Gregory P Copenhaver, Pablo Wappner

29 Jan 2024

PGENETICS-D-23-00857R2

Endolysosomal trafficking controls yolk granule biogenesis in vitellogenic Drosophila oocytes

Dear Dr Zhang,

We are pleased to inform you that your manuscript entitled "Endolysosomal trafficking controls yolk granule biogenesis in vitellogenic Drosophila oocytes" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Controls for anti-GFP and anti-Rab11 antibody staining in wildtype egg chambers.

    Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with immunofluorescent staining by anti-GFP antibody (green) on (A-D) w1118 wild type control or (E, F) YFP-Rab11 endogenous tagging flies, co-labeled with (A-D) phalloidin for F-Actin and (C-F) mouse anti-Rab11 antibody, as annotated. (B, D and F) High magnification views of the regions highlighted in (A, C and E), respectively. Images are presented as overlaying in color or individual channels in gray, as annotated. Genotypes: (A-D) w1118; (E,F), w1118; TI{TI} EYFP-Rab11 (BDSC #62549). The sizes of the scale bars as annotated.

    (TIF)

    pgen.1011152.s001.tif (9.6MB, tif)
    S2 Fig. Subcellular localization of 2xFYVE-GFP reporter in wildtype and VPS34/PI3K59F-RNAi oocytes.

    Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of 2xFYVE-GFP reporter from (A-C) wild type control or (D-F) oocyte co-expressing dsRNA again VPS34/PI3K59F, triple-labeled for GFP (green), Rab7 (red) and F-Actin (blue). Images are presented as overlaying in color or individual channels in gray, as annotated. Genotypes: (A-C). w*/ w1118; matalpha4-GAL-VP16 (BDSC #7062), UAS-GFP-myc-2xFYVE (BDSC #42712)/+; (D-F). w*/ w1118; matalpha4-GAL-VP16 (BDSC #7062), UAS-GFP-myc-2xFYVE (BDSC #42712)/+; P{TRiP.HMJ30324}attP40 (BDSC #64011)/+. The sizes of the scale bars as annotated.

    (TIF)

    pgen.1011152.s002.tif (9.6MB, tif)
    S3 Fig. The roles of Rab5 on yolk granule biogenesis in vitellogenic oocytes.

    Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers from oocytes of (A-D) wildtype control or (E-N) overexpressing (E-H) dominant-negative YFP-Rab5-DN, (I-L) constitutive-active YFP-Rab5-CA and (M, N) wildtype YFP-Rab5, that were (A, B, E, F, I, J, M, N) triple-labeled for GFP (green), endogenous Rab7 (red) and phalloidin for F-Actin (blue), or (C, D, G, H, K, L) stained with lysotracker alone (red), with data presented as overlaying images in color or individual channels in gray, as annotated. (B, F, J and N) High-magnification views of the cortex regions highlighted in (A, E, I and M), respectively, as annotated. The sizes of the scale bars as annotated. Genotypes: The samples were from adult female flies heterozygous for both matalpha4-GAL-VP16 (#7062) driver and the following UAS-transgenic lines: (A-D) w1118. (E-H) Rab5-DN: P{UASp-YFP.Rab5.S43N}01 (#9771). (I-L). Rab5-CA: P{UASp-YFP.Rab5.Q88L} (#9774). (M, N). Rab5-WT: P{UASp-YFP.Rab5}02 (#24616).

    (TIF)

    pgen.1011152.s003.tif (9.6MB, tif)
    S4 Fig. The roles of Rab11 on yolk granule biogenesis in vitellogenic oocytes.

    Confocal fluorescent microscopy and DIC imaging of a stage 10 egg chambers with oocyte-specific expression of (A, B) constitutive active YFP-Rab11-CA or (C, D) wildtype YFP-Rab11 triple-labeled for YFP (green), endogenous Rab7 (red) and phalloidin for F-Actin (blue), presented as overlaying image of all channels in color or individual channels in gray, as annotated. (B, D) High-magnification views of the cortex regions highlighted in (A, C), respectively, as annotated. Genotypes: The samples were from adult female flies heterozygous for both matalpha4-GAL-VP16 driver (#7062) and the following UAS-transgenic lines: (A, B) Rab11-CA: P{UASp-YFP.Rab11.Q70L} (#23260). (C, D). Rab11-WT: P{UASp-YFP.Rab11} (#50782). The sizes of the scale bars as annotated.

    (TIF)

    pgen.1011152.s004.tif (9.6MB, tif)
    S5 Fig. The roles of Rab7 on yolk granule biogenesis in vitellogenic oocytes.

    Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with ectopic expression of (A-D) wildtype YFP-Rab7 or (E-H) constitutive active (CA) YFP-Rab7-CA in oocytes that are (A-F) co-labeled for anti-GFP (green), F-Actin and (A, B) anti-Rab7 or (C, D) lysotracker (red), or (G, H) by lysotracker (red) alone, shown as overlaying images of all the channels in color or in individual channels in gray, as annotated. (B, D, F) High-magnification view of the cortex regions highlighted in (A, C, E), respectively, as annotated. Genotypes: The samples were from adult female flies heterozygous for both matalpha4-GAL-VP16 driver (#7062) and (A-D) wildtype YFP-Rab7: y[1 w*; P{w(+mC) = UASp-YFP.Rab7}21/SM5 (#23641): (E, F) constitutive active YFP-Rab7-CA: P{UASp-YFP.Rab7.Q67L} (#24103).The sizes of the scale bars as annotated.

    (TIF)

    pgen.1011152.s005.tif (9.7MB, tif)
    S6 Fig. The roles of Rab4 on yolk granule biogenesis in vitellogenic oocytes.

    Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of (A, B) wildtype YFP-Rab4 or (C-F) constitutive active YFP-Rab4-CA (A-D) triple-labeled for YFP (green), endogenous Rab7 (red) and F-Actin (blue), shown in overlaying images in color or individual channels in gray, or (E, F) by lysotracker staining (red) alone, as annotated. (B, D) High-magnification views of the cortex regions highlighted in (A, C), respectively, as annotated. Genotypes: The samples were from adult female flies heterozygous for both matalpha4-GAL-VP16 driver (#7062) and the following UAS-transgenic lines: (A, B) wildtype Rab4: P{UASp-YFP.Rab4} (#9767). (C-F) Rab4-CA: P{UASp-YFP.Rab4.Q67L} (#9770). The sizes of the scale bars as annotated.

    (TIF)

    pgen.1011152.s006.tif (9.4MB, tif)
    S7 Fig. Essential roles of VPS34/VPS15 PI3 kinase complex in Yl recycling and yolk granule biogenesis.

    Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers from flies carrying genome-tagging Yl-eGFP-3xHA with oocyte-specific expression of (A-C, G, H) control firefly Luciferase RNAi or (D-F, I, J) dsRNA again VPS34/PI3K59F, co-labeled with antibodies against GFP (green) and (A-F) lysotracker (red), or (G-J) endogenous Rab7 (red) and F-Actin (blue), as annotated. (B, E, H, J) High-magnification view of the cortex regions highlighted in (A, D, G, I), respectively. (C, F) Zoom-in view of the areas highlighted in (B, E), respectively. Images are presented as overlaying images in color or as individual channels in gray, as annotated. Genotypes: The samples were from adult females flies heterozygous for Yl-eGFP-3xHA reporter (p{mini-W+, yl-eGFP-3xHA}) and matalpha4-GAL-VP16 (BDSC #7062) driver together with (A-C, G, H). P{TRiP.JF01355}attP2 (BDSC#31603) or (D-F, I, J) P{TRiP.HMJ30324}attP40 (BDSC #64011). The sizes of the scales as annotated inside images.

    (TIF)

    pgen.1011152.s007.tif (9.7MB, tif)
    S8 Fig. Variable knockdown efficiency by different RNAi lines in stage 10 oocytes.

    Confocal fluorescent microscopy and DIC imaging of stage 10 egg chambers with oocyte-specific expression of dsRNA against (A, B) Rab5, (C, D) Rab4 and (E, F) Rab7, co-stained for endogenous Rab7 (green) and F-Actin (red), as annotated. (B, D, F) High-magnification view of the cortex regions highlighted in (A, C, E), respectively. Images are presented as overlaying images in color or as individual channels in gray, as annotated. Genotypes: The samples were from adult females flies heterozygous for matalpha4-GAL-VP16 (BDSC #7062) driver together with (A, B) P{TRiP.GL01872}attP40 (BL# 67877). (C, D) P{TRiP.HMS01100}attP2P (BDSC #33757). (E,F) P{y(+t7.7] v(+t1.8) = TRiP.JF02377}attP2 (BDSC#27051). The sizes of the scales as annotated inside images.

    (TIF)

    pgen.1011152.s008.tif (9.7MB, tif)
    S1 Table. Summary of the pilot RNAi screen.

    (XLSX)

    pgen.1011152.s009.xlsx (13.8KB, xlsx)
    Attachment

    Submitted filename: Responses Letter -V2.docx

    pgen.1011152.s010.docx (47.8KB, docx)
    Attachment

    Submitted filename: Responses-PLoSG-Decision (PGENETICS-D-23-00857R1).docx

    pgen.1011152.s011.docx (23.5KB, docx)

    Data Availability Statement

    All data are in the manuscript and its supporting information files.


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