Rab6 and the secretory pathway affect oocyte polarity in Drosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFalpha homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.     

Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis

The Drosophila melanogaster body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. Here, we investigate the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. We show that exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. We show that, in vivo, Rab6 is found in a complex with Bicaudal-D, and that Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila.     

MOESIN crosslinks actin and cell membrane in Drosophila oocytes and is required for OSKAR anchoring

In Drosophila, development of the embryonic germ cells depends on posterior transport and site-specific translation of oskar (osk) mRNA and on interdependent anchoring of the osk mRNA and protein within the posterior subcortical region of the oocyte. Transport of the osk mRNA is mediated by microtubules, while anchoring of the osk gene products at the posterior pole of the oocyte is suggested to be microfilament dependent. To date, only a single actin binding protein (TropomyosinII) has been identified with a putative role in osk mRNA and protein anchoring. This communication demonstrates that mutations in the Drosophila moesin (Dmoe) gene that encodes another actin binding protein result in delocalization of osk mRNA and protein from the posterior subcortical region and, as a consequence, in failure of embryonic germ cell development. In Dmoe mutant oocytes, the subcortical actin network is detached from the cell membrane, while the polarized microtubule cytoskeleton is unaffected. In line with the earlier observations, colocalization of ectopic actin and OSK protein in Dmoe mutants suggests that the actin cytoskeleton anchors OSK protein to the subcortical cytoplasmic area of the Drosophila oocyte.     

An interaction type of genetic screen reveals a role of the Rab11 gene in oskar mRNA localization in the developing Drosophila melanogaster oocyte.

Abdomen and germ cell development of Drosophila melanogaster embryo requires proper localization of oskar mRNA to the posterior pole of the developing oocyte. oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, we developed a novel interaction type of genetic screen by which we isolated 14 dominant enhancers of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. Here we describe the detailed analysis of two allelic modifiers that identify Drosophila Rab11, a gene encoding small monomeric GTPase. We demonstrate that mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of oskar mRNA, whereas localization of gurken and bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. We show that the ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. Our results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in oskar RNA localization.     

The Drosophila hnRNPA/B homolog, Hrp48, is specifically required for a distinct step in osk mRNA localization

The Staufen-dependent localization of oskar mRNA to the posterior of the Drosophila oocyte induces the formation of the pole plasm, which contains the abdominal and germline determinants. In a germline clone screen for mutations that disrupt the posterior localization of GFP-Staufen, we isolated three missense alleles in the hnRNPA/B homolog, Hrp48. These mutants specifically abolish osk mRNA localization, without affecting its translational control or splicing, or the localization of bicoid and gurken mRNAs and the organization of the microtubule cytoskeleton. Hrp48 colocalizes with osk mRNA throughout oogenesis, and interacts with its 5' and 3' regulatory regions, suggesting that it binds directly to oskar mRNA to mediate its posterior transport. The hrp48 alleles cause a different oskar mRNA localization defect from other mutants, and disrupt the formation of GFP-Staufen particles. This suggests a new step in the localization pathway, which may correspond to the assembly of Staufen/oskar mRNA transport particles.     

An oskar-dependent positive feedback loop maintains the polarity of the Drosophila oocyte

The localization of oskar mRNA to the posterior of the Drosophila oocyte defines the site of assembly of the pole plasm, which contains the abdominal and germline determinants. oskar mRNA localization requires the polarization of the microtubule cytoskeleton, which depends on the recruitment of PAR-1 to the posterior cortex in response to a signal from the follicle cells, where it induces an enrichment of microtubule plus ends. Here, we show that overexpressed oskar mRNA localizes to the middle of the oocyte, as well as the posterior. This ectopic localization depends on the premature translation of Oskar protein, which recruits PAR-1 and microtubule-plus-end markers to the oocyte center instead of the posterior pole, indicating that Oskar regulates the polarity of the cytoskeleton. Oskar also plays a role in the normal polarization of the oocyte; mutants that disrupt oskar mRNA localization or translation strongly reduce the posterior recruitment of microtubule plus ends. Thus, oskar mRNA localization is required to stabilize and amplify microtubule polarity, generating a positive feedback loop in which Oskar recruits PAR-1 to the posterior to increase the microtubule cytoskeleton's polarization, which in turn directs the localization of more oskar mRNA.     

The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly

Cell fate is often determined by the intracellular localization of RNAs and proteins. In Drosophila oocytes, oskar (osk) RNA localization and the subsequent Osk synthesis at the posterior pole direct the assembly of the pole plasm, where factors for the germline and abdomen formation accumulate. osk RNA produces two isoforms, long and short Osk, which have distinct functions in pole plasm assembly. Short Osk recruits downstream components of the pole plasm, whose anchoring to the posterior cortex requires long Osk. The anchoring of pole plasm components also requires actin cytoskeleton, and Osk promotes long F-actin projections in the oocyte posterior cytoplasm. However, the mechanism by which Osk mediates F-actin reorganization remains elusive. Furthermore, although long Osk is known to associate with endosomes under immuno-electron microscopy, it was not known whether this association is functionally significant. Here we show that Rabenosyn-5 (Rbsn-5), a Rab5 effector protein required for the early endocytic pathway, is crucial for pole plasm assembly. rbsn-5(-) oocytes fail to maintain microtubule polarity, which secondarily disrupts osk RNA localization. Nevertheless, anteriorly misexpressed Osk, particularly long Osk, recruits endosomal proteins, including Rbsn-5, and stimulates endocytosis. In oocytes lacking rbsn-5, the ectopic Osk induces aberrant F-actin aggregates, which diffuse into the cytoplasm along with pole plasm components. We propose that Osk stimulates endosomal cycling, which in turn promotes F-actin reorganization to anchor the pole plasm components to the oocyte cortex.     

Drosophila Mon2 couples Oskar-induced endocytosis with actin remodeling for cortical anchorage of the germ plasm

Drosophila pole (germ) plasm contains germline and abdominal determinants. Its assembly begins with the localization and translation of oskar (osk) RNA at the oocyte posterior, to which the pole plasm must be restricted for proper embryonic development. Osk stimulates endocytosis, which in turn promotes actin remodeling to form long F-actin projections at the oocyte posterior pole. Although the endocytosis-coupled actin remodeling appears to be crucial for the pole plasm anchoring, the mechanism linking Osk-induced endocytic activity and actin remodeling is unknown. Here, we report that a Golgi-endosomal protein, Mon2, acts downstream of Osk to remodel cortical actin and to anchor the pole plasm. Mon2 interacts with two actin nucleators known to be involved in osk RNA localization in the oocyte, Cappuccino (Capu) and Spire (Spir), and promotes the accumulation of the small GTPase Rho1 at the oocyte posterior. We also found that these actin regulators are required for Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. We propose that, in response to the Osk-mediated endocytic activation, vesicle-localized Mon2 acts as a scaffold that instructs the actin-remodeling complex to form long F-actin projections. This Mon2-mediated coupling event is crucial to restrict the pole plasm to the oocyte posterior cortex.     

A functional link between localized Oskar, dynamic microtubules, and endocytosis

Many cell types including developing oocytes, fibroblasts, epithelia and neurons use mRNA localization as a means to establish polarity. The Drosophila oocyte has served as a useful model in dissecting the mechanism of mRNA localization. The polarity of the oocyte is established by the specific localization of three critical mRNAs-oskar, bicoid and gurken. The localization of these mRNAs requires microtubule integrity, and the activity of microtubule motors. However, the precise organization of the oocyte microtubule cytoskeleton remains an open question. In order to examine the polarity of oocyte microtubules, we visualized the localization of canonical microtubule plus end binding proteins, EB1 and CLIP-190. Both proteins were enriched at the posterior of the oocyte, with additional foci detected within the oocyte cytoplasm and along the cortex. Surprisingly, however, we found that this asymmetric distribution of EB1 and CLIP-190 was not essential for oskar mRNA localization. However, Oskar protein was required for recruiting the plus end binding proteins to the oocyte posterior. Lastly, our results suggest that the enrichment of growing microtubules at the posterior pole functions to promote high levels of endocytosis in this region of the cell. Thus, multiple polarity-determining pathways are functionally linked in the Drosophila oocytes.     

Drosophila Ge-1 promotes P body formation and oskar mRNA localization

mRNA localization coupled with translational control is a widespread and conserved strategy that allows the localized production of proteins within eukaryotic cells. In Drosophila, oskar (osk) mRNA localization and translation at the posterior pole of the oocyte are essential for proper patterning of the embryo. Several P body components are involved in osk mRNA localization and translational repression, suggesting a link between P bodies and osk RNPs. In cultured mammalian cells, Ge-1 protein is required for P body formation. Combining genetic, biochemical and immunohistochemical approaches, we show that, in vivo, Drosophila Ge-1 (dGe-1) is an essential gene encoding a P body component that promotes formation of these structures in the germline. dGe-1 partially colocalizes with osk mRNA and is required for osk RNP integrity. Our analysis reveals that although under normal conditions dGe-1 function is not essential for osk mRNA localization, it becomes critical when other components of the localization machinery, such as staufen, Drosophila decapping protein 1 and barentsz are limiting. Our findings suggest an important role of dGe-1 in optimization of the osk mRNA localization process required for patterning the Drosophila embryo.     

Dusky-like functions as a Rab11 effector for the deposition of cuticle during Drosophila bristle development

The morphogenesis of Drosophila sensory bristles is dependent on the function of their actin and microtubule cytoskeleton. Actin filaments are important for bristle shape and elongation, while microtubules are thought to mediate protein and membrane trafficking to promote growth. We have identified an essential role for the bristle cuticle in the maintenance of bristle structure and shape at late stages of bristle development. We show that the small GTPase Rab11 mediates the organized deposition of chitin, a major cuticle component in bristles, and disrupting Rab11 function leads to phenotypes that result from bristle collapse rather than a failure to elongate. We further establish that Rab11 is required for the plasma membrane localization of the ZP domain-containing Dusky-like (Dyl) protein and that Dyl is also required for cuticle formation in bristles. Our data argue that Dyl functions as a Rab11 effector for mediating the attachment of the bristle cell membrane to chitin to establish a stable cuticle. Our studies also implicate the exocyst as a Rab11 effector in this process and that Rab11 trafficking along the bristle shaft is mediated by microtubules.     

Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11

Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homologue of arfophilin-2, an ADP ribosylation factor effector that binds Rab11 and influences recycling endosome (RE) organization. Here, we show that Nuf is an important component of the RE, and that these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component. GST pull-downs and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation.     

Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes

Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu-Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.     

Cup-ling oskar RNA localization and translational control

RNA localization and spatially restricted translational control can serve to deploy specific proteins to particular places within a cell. oskar (osk) RNA is a key initiatior of posterior patterning and germ cell specification in Drosophila, and its localization and translation are under elaborate control. In this issue, Wilhelm et al. (2003) show that the protein Cup both promotes osk localization and participates in repressing translation of unlocalized osk.     

Drosophila anterior-posterior polarity requires actin-dependent PAR-1 recruitment to the oocyte posterior

The Drosophila anterior-posterior axis is established at stage 7 of oogenesis when the posterior follicle cells signal to polarize the oocyte microtubule cytoskeleton. This requires the conserved PAR-1 kinase, which can be detected at the posterior of the oocyte in immunostainings from stage 9. However, this localization depends on Oskar localization, which requires the earlier PAR-1-dependent microtubule reorganization, indicating that Oskar-associated PAR-1 cannot establish oocyte polarity. Here we analyze the function of the different PAR-1 isoforms and find that only PAR-1 N1 isoforms can completely rescue the oocyte polarity phenotype. Furthermore, PAR-1 N1 is recruited to the posterior cortex of the oocyte at stage 7 in response to the polarizing follicle cell signal, and this requires actin, but not microtubules. This suggests that posterior PAR-1 N1 polarizes the microtubule cytoskeleton. PAR-1 N1 localization is mediated by a cortical targeting domain and a conserved anterior-lateral exclusion signal in its C-terminal linker domain. PAR-1 is also required for the polarization of the C. elegans zygote and is recruited to the posterior cortex in an actin-dependent manner. Our results therefore identify a molecular parallel between axis formation in Drosophila and C. elegans and make Drosophila PAR-1 N1 the earliest known marker for the polarization of the oocyte.     

Drosophila decapping protein 1, dDcp1, is a component of the oskar mRNP complex and directs its posterior localization in the oocyte

In Drosophila, posterior deposition of oskar (osk) mRNA in oocytes is critical for both pole cell and abdomen formation. Exon junction complex components, translational regulation factors, and other proteins form an RNP complex that is essential for directing osk mRNA to the posterior of the oocyte. Until now, it has not been clear whether the mRNA degradation machinery is involved in regulating osk mRNA deposition. Here we show that Drosophila decapping protein 1, dDcp1, is a posterior group gene required for the transport of osk mRNA. In oocytes, dDcp1 is localized posteriorly in an osk mRNA position- and dosage-dependent manner. In nurse cells, dDcp1 colocalizes with dDcp2 and Me31B in discrete foci that may be related to processing bodies (P bodies), which are sites of active mRNA degradation. Thus, as well as being a general factor required for mRNA decay, dDcp1 is an essential component of the osk mRNP localization complex.     

Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis

BACKGROUND: Embryonic cleavage leads to the formation of an epithelial layer during development. In Drosophila, the process is specialized and called cellularization. The trafficking pathways that underlie this process and that are responsible for the mobilization of membrane pools, however, remain poorly understood. RESULTS: We provide functional evidence for the role of endocytic trafficking through Rab11 endosomes in remobilizing vesicular membrane pools to ensure lateral membrane growth. Part of the membrane stems from endocytosed apical material. Mutants in the endocytic regulators rab5 and shibire/dynamin inhibit basal-lateral membrane growth, and apical endocytosis is blocked in shibire mutants. In addition, shibire controls vesicular trafficking through Rab11-positive endosomes. In shibire mutants, the transmembrane protein Neurotactin follows the secretory pathway normally but is not properly inserted in the plasma membrane and accumulates instead in Rab11 subapical endosomes. Consistent with a direct role of shibire in vesicular trafficking through Rab11 endosomes, Shibire is enriched in this compartment. Moreover, we show by electron microscopy the large accumulation of intracellular coated pits on subapical endocytic structures in shibire mutants. Finally, we show that Rab11 is essential for membrane growth and invagination during cellularization. CONCLUSION: Together, the data show that endocytic trafficking is required for basal-lateral membrane growth during cellularization. We identify Rab11 endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. This pathway may be required in other morphogenetic processes characterized by the growth of a membrane domain.     

Drosophila par-1 is required for oocyte differentiation and microtubule organization

BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.