Rab 6 mediates membrane organization and determinant localization during Drosophila oogenesis

INTRODUCTION In Drosophila melanogaster, the embryonic body axes are defined by the localization and restriction of determinants as mRNAs and proteins in the oocyte (Riechmann and Ephrussi, 2001). This asymmetry is the result of a succession of processes that take place during oogenesis. The ovary consists of ovarioles, which are composed of egg chambers of increasing developmental stages (Fig. 1B). In the germarium, germline stem cells divide asymmetrically, generating a new stem cell and a cystoblast (Huynh and St Johnston, 2004). The cystoblast undergoes four rounds of mitosis with incomplete cytokinesis, producing a germline cyst of 16 cells interconnected via 15 actin-rich cytoplasmic bridges, the ring canals. Each cystocyte displays a specific number of ring canals reflecting the mitosis at which it was generated: the two first-born cystocytes possess four ring canals, and the eight last-born possess one. Oocyte specification occurs via the progressive and selective concentration of cell fate determinants to the two four-branched cells and, finally, to just one of these cells, the oocyte. This process requires an organized and polarized microtubule (MT) network spanning the germline cyst and connecting the cystocytes (Grieder et al., 2000; Theurkauf et al., 1993; Vaccari and Ephrussi, 2002). The remaining 15 cells adopt the nurse cell fate and provide molecules required for oocyte development. The germarium also contains somatic stem cells. Some of these differentiate into follicular epithelial cells that encapsulate the germline cyst, thus forming the stage-1 egg chamber, which exits the germarium. Oogenesis has been subdivided into 14 stages (reviewed in King, 1970; Spradling, 1993). During the first six stages, the 16 cystocytes grow at roughly identical rates and contribute almost equally to the increase in size of the egg chamber. At stage 8, vitellogenesis begins and the increase in egg chamber size mainly reflects the major growth of the oocyte. Antero-posterior axis specification occurs at stage 7, with the secretion by the oocyte of the Gurken protein, a Drosophila TGF homolog and EGF-receptor ligand (reviewed in Nilson and Schupbach, 1999). This signal induces a group of overlying follicle cells to adopt the posterior fate (Gonzalez-Reyes et al., 1995; Roth et al., 1995). In return, these cells emit an unknown signal, causing the disassembly of the unique MT-organizing center (MTOC) of the germline cyst. This MTOC is located at the posterior of the oocyte until stage 7. New MT then nucleate from the anterior and lateral cortex of the oocyte, whose nucleus moves in a MT-dependent manner from its posterior location to an anterior corner of the cell. Here, a second Gurken signal causes the overlying follicle cells to adopt a dorsal fate, thus specifying the dorsoventral axis. Concomitantly, MT-dependent localization of bicoid and oskar mRNAs to the anterior and posterior of the oocyte, respectively, establishes the antero-posterior axis of the future embryo (reviewed in Riechmann and Ephrussi, 2001). Genetic analysis has revealed the involvement of proteins such as Bicaudal-D (BicD) and Dynein, a minus-end-directed MT motor, together with the MT network, in germline cyst development, oocyte specification, asymmetric mRNA localization within the oocyte and egg-chamber formation (Navarro et al., 2004; Oh and Steward, 2001; Suter and Steward, 1991; Swan et al., 1999; Vaccari and Ephrussi, 2002). Thus, in BicD mutant germaria, the MT cytoskeleton fails to maintain its polarization and none of the cystocytes enter meiosis, resulting in a cyst of 16 nurse cells (Huynh and St Johnston, 2000; Oh and Steward, 2001). It has been shown that the mammalian homologs of BicD (BICD1 and BICD2) physically interact with Dynamitin, a component of the Dynactin complex (the regulatory and cargo-loading subunit of Dynein), and with Dynein intermediate chain (Hoogenraad et al., 2001) to control Dynein-based motility (Hoogenraad et al., 2003). In addition, BICD1 and BICD2 bind RAB6a and RAB6a (Matanis et al., 2002; Short et al., 2002), members of the Rab family of small monomeric GTPases, which are among the major regulators of vesicular trafficking. Rab proteins segregate over the various organelle membranes in the cell, and their specific interactions with effectors promote asymmetric and polarized vesicle transport (van Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis