Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA
*Author for correspondence (e-mail: rcohen{at}ku.edu)
Accepted 22 October 2001
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SUMMARY |
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Key words: Oogenesis, Cell polarity, Membrane trafficking, Membrane recycling, oskar, mRNA localization, Microtubule plus ends, Drosophila, Rab11
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INTRODUCTION |
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To date, the best evidence that the plasma membrane of the oocyte and membrane trafficking pathways are polarized comes from the expression pattern of human transferrin receptor (Htr) in transgenic flies (Bretscher, 1996). During stages 8-10 [see Spradling (Spradling, 1993) for a complete description of the 14 stages of oogenesis], the posterior pole of the oocyte becomes enriched with Htr, both along the plasma membrane and in vesicles. Htr rapidly disappears from vesicles upon inhibition of endocytosis (Bretscher, 1996), indicating that it is actively internalized and recycled in Drosophila oocytes as it is in many other examined cells (Mukherjee et al., 1997). Together with the observation that Htr is restricted to the posterior plasma membrane, its active internalization and recycling strongly suggests that the membrane recycling pathway of the oocyte is polarized towards the posterior pole.
We now identify Rab11 and -adaptin as endogenous markers of oocyte membrane polarity. These proteins are localized to distinct compartments at the posterior end of the oocyte. Based on the reported roles of Rab11 and
-adaptin in other examined cells (Mukherjee et al., 1997; Novick and Zerial, 1997; Rodman and Wandinger-Ness, 2000) and on findings we present that Rab11 is required for the recycling of internalized transferrin, we propose that their expression patterns in mid-stage oocytes reflect polarized membrane recycling directed towards the posterior pole. Additional findings presented here indicate that Rab11-mediated receptor recycling plays a critical role in the polarization of the cytoplasm of the oocyte through its specification of membrane domains that organize microtubule plus ends and support osk mRNA translation and anchoring. Finally, we demonstrate the existence of a positive feedback loop whereby Osk amplifies its own synthesis and localization through its maintenance/enhancement of Rab11 localization.
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MATERIALS AND METHODS |
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Generation of rab11 deletion mutants
The rab11 deletion alleles were generated by imprecise excision. w1118; FRT82B, rab11P2148/TM3, Sb females were mated to TM2, ry, P[2,3]/MKRS P[
2,3] males. w/Y; FRT82B, rab11P2148/MKRS[
2,3] male progeny were mated to w; TM3, Sb/TM6, Tb females. Progeny with excised P elements were identified by searching vials for white-eyed flies with normal (Sb+) bristles. These progeny were pair-mated to TM6, Tb flies to generate w; FRT82B, rab11(ex)/TM6, Tb stocks, where rab11(ex) indicates excision of w+. Several of the resulting stocks failed to produce Tb+ progeny, indicating the presence of a rab11 mutation, which was subsequently verified by complementation tests with rab11P2148. Two non-complementing mutations were shown by Southern blot analysis to carry small deletions. Physical maps of corresponding alleles (rab11ex1 and rab11ex2) are shown in Fig. 2.
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Cloning and molecular characterization of rab11
rab11 genomic DNA was cloned from w1118; FRT82B, rab11P2148/TM3, Sb flies by plasmid rescue after cleavage of genomic DNA with XbaI and ligation under dilute conditions. The presence of the P element in recovered plasmids was verified by restriction mapping and partial DNA sequencing. Flanking chromosomal DNA was subcloned from positive clones and used to screen a Drosophila genomic DNA library (kindly provided by Richard Mann). Two overlapping genomic clones spanning 22 kb were isolated. Partial DNA sequencing revealed that the P element was in the second intron of the rab11 gene. A 6.3 kb Asp718I-XhoI genomic fragment was subsequently identified that restores viability and fertility to rab11P2148 homozygotes. This fragment includes the entire rab11 transcription unit and about 800 bp each of 5' and 3' flanking DNA (see Fig. 2A). Sequencing of the entire 6.3 kb rescuing fragment revealed excellent correspondence with previous cDNA sequencing (Sasamura et al., 1997; Satoh et al., 1997) and with the BDGP genomic sequence. Northern blot analyses were carried out as described by Frank et al. (Frank et al., 1994), using a partial rab11 cDNA as a probe.
For the rescue experiment, the 6.3 kb genomic fragment was subcloned into the CaSpeR4 transformation vector (Pirrotta, 1988) and microinjected into w1118 flies according to standard procedures (Rubin and Spradling, 1982; Spradling and Rubin, 1982). Two independent transformed lines were established, one containing a single copy of the transgene and another containing two linked copies. Standard genetic crosses were used to introduce the transgenes into the w1118; FRT82B, rab11P2148/TM3, Sb stock. Rescue was scored by the recovery of Sb+ flies. Strong rescue was observed with two copies of the rab11 transgene. Partial rescue was observed with a single copy of the transgene.
The Rab11-GFP construct was made within the context of the rab11 rescuing fragment by substituting an EcoRI-BamHI polylinker for the rab11 translation stop codon. An EcoRI-BamHI, GFP-containing fragment was subsequently inserted into the polylinker in-frame with Rab11. Transformation was as described above and resulted in several lines.
Generation of Rab11 antisera
The entire protein-coding region of rab11 was amplified from a partially purified rab11 cDNA clone using Taq DNA polymerase. The amplified sequence was fused to a 6x His tag and expressed in bacteria from the Pet14b expression vector (Novagen). The His-tagged protein was purified by passage through a Nickel-column and SDS-PAGE. Rats were immunized with 100 µg of gel-purified protein in Fruends complete adjuvant and then given six booster shots consisting of 100 µg of fusion protein in Fruends incomplete adjuvant at 2 week intervals. All of the data shown were obtained with crude sera.
Enzyme-linked in situ hybridization, immunolocalization and western blotting
Enzyme-linked in situ hybridization to whole-mount ovaries was carried out according to Tautz and Pfeifle (Tautz and Pfeifle, 1989) with modifications described by Cheung et al. (Cheung et al., 1992). Digoxigenin-labeled DNA probes were made by the random priming method (Feinberg and Vogelstein, 1983). The grk and K10 probes were as described in Serano et al. (Serano et al., 1995). The osk probe was as described by Saunders and Cohen (Saunders and Cohen, 1999). The bcd probe corresponds to a 2368 nucleotide BamHI-XbaI fragment, which contains the entire bcd transcription unit plus a small genomic region adjacent to the 3' end of the transcript. Immunocytochemistry was carried out as described by Serano et al. (Serano et al., 1995), using primary antibodies at the following concentrations: Stau (1/5000), Vas and Osk (1/1000). Western blotting was carried out according to standard procedures (Maniatis et al., 1982) using the Rab11 antisera at 1/2000 and a biotinylated secondary antibody-DAB detection scheme (Vector laboratories) according to procedures recommended by the manufacturer.
Immunofluorescence
Ovaries were fixed and incubated with antibodies as described above for enzyme-linked immunolocalization. Following incubation with secondary antibody, ovaries were washed in the dark for 4x15 minutes in TNBTT (Serano et al., 1995), 2x20 minutes in PBT (phosphate-buffered saline (PBS) with 0.1% Tween20), mounted in Pro-long antifade (Molecular probes), and visualized by confocal microscopy. Primary antibodies were used at the following concentrations: Rab11 (1/2000), D-clip190 (1/2000), -adaptin (1/300), ß-gal (1/700; ICN/Cappel) and Mab078
-tubulin (1/10; Harlan Sera-lab). Secondary antibodies were FITC-conjugated AffiniPure donkey anti-rRabbit (Jackson Labs, used at 1/200) and Cy3-conjugated AffiniPure donkey anti-mouse (Jackson Labs, used at 1/100). For preservation of microtubules, ovaries were dissected at room temperature in Graces Media (Gibco), and viewed under halocarbon oil 700 (Sigma; for live ovaries carrying a tau-GFP transgene) or put immediately into fix for immunofluorescence. For lectin labeling, ovaries were dissected in PBS, washed five times for 3 minutes, once for 10 minutes in PBT and twice for 5 minutes in lectin buffer (10 mM Hepes, pH 7.5, 0.15M NaCl), and incubated in the dark overnight at 4°C with fluorescein-Datura stramonium lectin and fluorescein-Lycopersicon esculentum (tomato) lectin (Vector labs) (20 µg/ml in lectin buffer) for labeling of the vitelline and plasma membrane (Bretscher, 1996). After incubation with lectins, ovaries were washed in three times of five minutes each in lectin buffer, then mounted and visualized as above.
Receptor recycling assay
Ovaries were dissected at room temperature in Graces Media, incubated for 1 minute in Graces Media containing 5 µg/ml Texas Red-conjugated human transferrin (Molecular Probes), washed in fresh Graces Media for 20 minutes to allow for recycling, mounted in Graces Media and visualized by confocal microscopy.
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RESULTS |
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Rab11 accumulates at the posterior pole of the oocyte during mid-oogenesis and is required for its own localization
To gain insight into the role of Rab11 in the generation of oocyte polarity, we determined its subcellular distribution in developing oocytes by staining fixed ovaries with Rab11 antisera (see Materials and Methods). We also examined the distribution of green fluorescent protein (GFP)-tagged Rab11 in living tissues (see Materials and Methods). Both approaches revealed a similar Rab11 expression pattern.
Rab11 was abundant in stage 1-10 oocytes. Detection of the protein in later stage oocytes was problematic due to the deposition of the chorion and extrachorionic membranes. The protein was also expressed in follicle cells and nurse cells, but at reduced levels compared with oocytes. In stage 1-7 oocytes, Rab11 accumulated in a distinct perinuclear compartment and was abundantly distributed in a thick crescent along the lateral and posterior cortexes (Fig. 2A). In stage 8-10 oocytes, Rab11 continued to accumulate in the perinuclear compartment, but the thick cortical crescent was gradually replaced by a small cap of protein at the extreme posterior pole of the oocyte (Fig. 2B,F). Double label experiments showed near perfect colocalization of Rab11 with Osk in stage 9 and 10 oocytes (Fig. 2C).
In situ hybridization for Rab11 mRNA revealed no specific accumulation of the transcript at the posterior pole of the oocyte. Rather, the mRNA was uniformly dispersed throughout the oocyte through at least stage 9. Thus, in contrast to Osk, Rab11 localization would appear to be mediated by a protein-based localization machinery.
We also used the Rab11 antisera to stain rab112148 GLCs. Protein was detected in stage 1-10 oocytes. Localization to the perinuclear compartment and to the lateral and posterior cortex of stage 1-7 oocytes was normal (Fig. 2D). However, no localization to the posterior pole was observed in stage 8-10 oocytes. Instead the protein was diffusely dispersed throughout the oocyte, with slight enrichment along the plasma membrane (Fig. 2E). To address the possibility that the rab11P2148 allele produces an altered form of the protein that is not competent for localization in stage 8-10 oocytes, we immunostained heterozygous (rab11P2148/rab11+) oocytes. Only normally localized protein was detected in these oocytes (data not shown), indicating that the rab11P2148 allele produces normal protein. Consistent with this idea, northern and western blots of rab11P2148/rab11+ ovaries revealed single RNA and protein products of the expected sizes (Fig. 1D and data not shown). We conclude from these data that Rab11P2148 GLCs produce abnormal amounts of otherwise normal protein and thus that Rab11 is required for its own localization.
Rab11 is required for polarized endocytic recycling
To determine if Drosophila Rab11, like its vertebrate counterparts (Rodman and Wandinger-Ness, 2000; Prekeris et al., 2000), mediates endocytic recycling, we followed the recycling of transferrin receptor by monitoring the distribution of its ligand, iron-conjugated transferrin, in cultured ovaries (see Materials and Methods). The transferrin receptor is expressed on the plasma membranes of most cells, where it is actively internalized and recycled (Mukherjee et al., 1997). Upon ligand binding, the receptor-ligand complex is internalized and delivered to endosomes where iron atoms are released (Klausner et al., 1983). The transferrin-transferrin receptor complex is then recycled [often in a Rab11-dependent fashion (Ren et al., 1996)] to the plasma membrane, where transferrin dissociates from the receptor to scavenge more iron (Klausner et al., 1983). When added to cultured ovaries, transferrin-iron conjugates were rapidly transcytosed through the follicle cell epithelium and accumulated at the posterior pole of the oocyte and in the perivitelline compartment between the follicle cell epithelium and the oocyte (Fig. 3B). In wild-type ovaries, significantly more transferrin accumulated in the perivitelline space than in the oocyte (Fig. 3A,B). By contrast, in rab11P2148 GLCs, most of the transferrin accumulated in the oocyte (Fig. 3C). These patterns of accumulation indicate that wild-type oocytes and rab11P2148 GLCs both internalize transferrin, but that only wild-type oocytes are able to recycle it to the plasma membrane for release into the perivitelline compartment. To address the possibility that the altered distribution of transferrin in rab11P2148 GLCs reflects a general defect in membrane integrity rather than a specific defect in receptor recycling, we stained ovaries with fluorescein-conjugated lectins, which preferentially label the perivitelline and plasma membranes (Bretscher, 1996). No gaps or other indications of gross defects in the structure of either membrane in the rab11P2148 GLCs were observed (Fig. 3D-G). We conclude that Drosophila rab11 is required for endocytic recycling of transferrin and, presumably, other molecules, to the posterior plasma membrane of the oocyte.
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Rab11 is required for the sharp focusing of microtubule plus ends onto the posterior pole of stage 8-10 oocytes
Given recent findings in other systems that microtubule ends may be organized through attachment to specific membrane compartments (Schuyler and Pellman, 2001), we investigated the organization of the microtubule cytoskeleton in rab11P2148 GLCs. Tubulin immunolocalization experiments revealed normal organization of microtubule minus ends in rab11P2148 GLCs. The highest levels of tubulin staining occurred at the posterior end of the oocyte during stages 1-6 (data not shown) and at the anterior cortex during stages 7-10 (Fig. 6A,D). Similar labeling patterns were observed in non-fixed oocytes following expression of a GFP-tagged version of the microtubule associated Tau protein (Fig. 6B,E) (Micklem et al., 2000). Additional evidence that rab11 is not required for the organization of microtubule minus ends comes from observations that bicoid and K10 mRNA are correctly localized to the anterior cortex of rab11 oocytes and that gurken (grk) mRNA is correctly localized to the dorsal anterior corner of rab11 oocytes (Fig. 6C,F, respectively). Similar results have also recently been reported (Janovics et al., 2001).
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Rab11 is required for efficient transport of osk mRNA to the posterior pole of the oocyte and for its subsequent translation
To investigate further the role of Rab11 in the organization of the posterior pole of the oocyte, we examined the distribution of osk mRNA and protein in rab11P2148 GLCs. In wild-type oocytes, osk mRNA is transported to the posterior pole during stages 8 and 9 (Kim-Ha, 1991; Karlin-McGinness et al., 1996; Brendza et al., 2000), coincident with the polarization of the microtubule cytoskeleton. During transport, and in the initial hours following transport, osk mRNA is seen as a large ball (Fig. 7A). By the end of stage 9, the ball resolves into a thin cap along the posterior cortex, which persists through the end of oogenesis (Kim-Ha et al., 1991) (Fig. 7B). The nature of the transition from the ball to the cap is not clear, but coincides with the activation of osk translation (Rongo et al., 1995) (Fig. 7C). The cap structure is much more resistant to disruption with colchicine than is the ball (Pokrywka and Stephenson, 1995) (G. D., E. S., J. M. and R. S. C., unpublished) and appears, then, to represent the binding of the mRNA to an anchor, which might be Osk (Rongo et al., 1997). We found that rab11P2148 GLCs were defective in the transport of osk mRNA to the posterior pole, and in its subsequent translation and anchoring. The transport defect was temporal in nature. Thus, while most osk transcripts reached the posterior pole of wild-type oocytes during stage 8 (Fig. 7A), only a small fraction of transcripts reached the posterior pole of rab11 oocytes during stage 8 (Fig. 7D). Delayed transport to the posterior pole of the oocyte has also recently been reported by Jancovics et al. (Jancovics et al., 2000). Typically, the lagging transcripts were aggregated into a mass near the center of the cell, possibly representing stalled transport at an intermediate step (Fig. 7D). Although most osk transcripts eventually reached the posterior pole of rab11 oocytes (Fig. 7E) two observations suggest that they are never anchored. First, no Osk was ever detected in rab11 oocytes (Fig. 7F). Second, the osk transcripts of rab11P2148 GLCs never formed the characteristic cap at the posterior pole, but instead remained as a ball (Fig. 7E). Moreover, during late stages of oogenesis (e.g. when microtubules are bundled along the entire egg cortex), the ball of osk mRNA appeared to drift away from the posterior pole and was often fragmented into several smaller balls (see Fig. 7G). We conclude from these studies that Rab11 is required for the efficient transport of osk mRNA to the posterior pole of the oocyte and for its subsequent translation and anchoring.
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DISCUSSION |
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The Drosophila oocyte plasma membrane has a unique posterior domain defined by rab11
The expression pattern of Htr in transgenic flies shows clearly that the oocyte establishes a posterior plasma membrane domain (PMD) (Bretscher, 1996). Further evidence for such a domain comes from our finding that Rab11 localizes to the posterior pole of wild-type oocytes. Interestingly, the PMD is established independently of microtubule polarity or grk signaling, as Rab11 is localized normally in grk null mutants. Given the rapid rate at which Htr is internalized and recycled (Bretscher, 1996; Hanover et al., 1984), it is likely that the maintenance, if not also the initial specification, of the PMD requires polarized endocytic recycling directed towards the posterior pole. The data we present indicate that Rab11 is responsible for such recycling: Rab11 is localized to the posterior pole of the oocyte and is required for the recycling of internalized transferrin (the ligand for transferrin receptor) to the plasma membrane of cultured oocytes. Independent evidence that Rab11 mediates polarized endocytic recycling comes from studies with vertebrates, where Rab11 recycles internalized molecules to the apical surface of polarized epithelial cells (Prekeris et al., 2000; Wang et al., 2000).
What polarizes endocytic recycling to the posterior pole of the oocyte? The polarization of the endocytic pathways of other cells is triggered by Rho GTPase family members (i.e. Rho, Cdc42 and Rac) (Kroschewski et al., 1999; Ellis and Mellor, 2000; Garrett et al., 2000; Garred et al., 2001), which are activated at specific regions of the cell cortex by a variety of intrinsic and extrinsic cues (Drubin and Nelson, 1996). Rho GTPases have also been strongly implicated in the polarization of exocytosis (Adamo et al., 1999; Guo et al., 1999; Guo et al., 2001). Specifically, they have been shown to recruit the exocyst to specific sites of the plasma membrane. The exocyst is a conserved complex of proteins to which vesicles of the secretory pathway fuse (Grindstaff et al., 1998; Adamo et al., 1999; Guo et al., 2001). Thus, through local activation of Rho GTPases, secretory vesicles are targeted to specific regions of the plasma membrane. By analogy, the Rho GTPases could localize Rab11 and polarize receptor recycling through local recruitment of an exocyst-like complex for Rab11-containing vesicles. Because Drosophila Rho GTPases are required for progression through early oogenesis (Genova et al., 2000; Murphy and Montell, 1996), the analysis of their role in Rab11 localization and other aspects of oocyte polarization must await the identification of conditional mutants.
The role of Rab11 in microtubule plus end organization and osk mRNA transport
The Kin:ß-gal expression studies indicate that microtubule plus ends are not sharply focused onto the posterior pole of the oocyte in rab11P2148 mutant oocytes. The simplest interpretation of this finding is that microtubule plus ends are attached to the PMD, or to a neighboring membrane domain whose identity is established and/or maintained by Rab11. In wild-type oocytes, this domain is tightly defined such that Kin:ß-gal is concentrated at the posterior tip of the oocyte, while in rab11P2148 mutant oocytes, the domain is poorly defined and the Kin:ß-gal expression pattern is expanded. The slight enrichment of Kin:ß-gal at the posterior tip of rab11P2148 oocytes could reflect partial Rab11 activity and/or the polarizing activities of membrane trafficking pathways that may not rely on Rab11 (e.g. the secretory pathway) (Rodman and Wandinger-Ness, 2000), which targets newly synthesized molecules from the Golgi to the plasma membrane. Recent studies have identified two types of protein-protein interactions (CLIP-CLASP and APC-EB1) responsible for the stable association of microtubule plus ends with membranes (Akhmanova et al., 2000; Nakamura et al., 2001; Lu et al., 2001; Schuyler and Pellman, 2001). While the CLIP, CLASP, APC and EB1 protein families are all well-represented in the Drosophila genome, their role in the establishment of oocyte polarity has not yet been investigated.
Apart from Rab11, the only protein known to play a specific role in microtubule plus end organization in Drosophila oocytes is Par-1, a kinase, whose suspected targets include the microtubule associated protein Tau (Shulman et al., 2000). In strong par-1 mutants, microtubule plus ends, as revealed by Kin:ß-gal expression patterns, are not enriched at the posterior pole of the oocyte, but instead are concentrated tightly as a dot at the center of the cell. In weak par-1 mutants, a small amount of Kin:ß-gal is also found at the posterior pole. This small amount of Kin:ß-gal is always tightly localized to the cell tip, suggesting that Par-1 is not required for the specification of the PMD, but rather only for the efficient movement of already focused microtubule plus ends from the cell center to the PMD. Consistent with the idea that microtubule plus ends initially focus to a sharp point at the center of the cell and then move to the posterior pole, Kin:ß-gal and oskar mRNA show transient concentration at the center of the cell in wild-type oocytes (Clark et al., 1994; Clark et al., 1997) (G. D., E. S., J. M. and R. S. C., unpublished). How Par-1 might promote the movement of microtubule plus ends from the cell center to the posterior pole is not clear. One possibility is that it promotes attachment of microtubule plus ends to a structure that is then moved to the posterior pole. Alternatively, Par-1 might stimulate a burst of microtubule growth, forcing growth toward the posterior end of the cell.
The observation that osk mRNA transport to the posterior pole is delayed in rab11P2148 mutant oocytes suggests that Rab11 might also have a role in the movement of microtubule plus ends from the cell center to the posterior pole, and therefore, that such movement is membrane dependent. For example, microtubule plus ends could become attached to membrane compartments or vesicles at the cell center, and the vesicles may then be targeted to the posterior pole in a Rab11-dependent manner. Because osk mRNA arrives at the posterior pole as a fairly well-defined ball in rab11P2148 oocytes, Rab11 does not appear to be required for focusing microtubule plus ends at the cell center, but rather only for their timely movement and attachment to the posterior membrane domain.
The role of Rab11 and microtubule plus ends in osk mRNA anchoring and translation
Although most osk transcripts are eventually transported to the posterior pole in rab11P2148 oocytes, they are not translated. As Osk is required to anchor oskar mRNA at the posterior pole (Rongo et al., 1995), the lack of oskar translation in rab11P2148 GLCs could explain the inability of the oskar mRNA ball to resolve into the thin posterior crescent. The nature of the osk translation block in rab11P2148 oocytes is not clear. One possibility is that key osk translation factors are localized to the posterior membrane domain established by Rab11. In rab11P2148 oocytes, this domain may be too poorly defined to support assembly of such factors into an active translation complex.
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ACKNOWLEDGMENTS |
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