The Fusome Mediates Intercellular Endoplasmic Reticulum Connectivity inDrosophilaOvarian Cysts

Abstract

Drosophila ovarian cysts arise through a series of four synchronous incomplete mitotic divisions. After each round of mitosis, a membranous organelle, the fusome, grows along the cleavage furrow and the remnants of the mitotic spindle to connect all cystocytes in a cyst. The fusome is essential for the pattern and synchrony of the mitotic cyst divisions as well as oocyte differentiation. Using live cell imaging, greenfluorescent protein-tagged proteins, and photobleaching techniques, we demonstrate that fusomal endomembranes are part of a single continuous endoplasmic reticulum (ER) that is shared by all cystocytes in dividing ovarian cysts. Membrane and lumenal proteins of the common ER freely and rapidly diffuse between cystocytes. The fusomal ER mediates intercellular ER connectivity by linking the cytoplasmic ER membranes of all cystocytes within a cyst. Before entry into meiosis and onset of oocyte differentiation (between region 1 and region 2A), ER continuity between cystocytes is lost. Furthermore, analyses of hts and Dhc64c mutants indicate that intercellular ER continuity within dividing ovarian cysts requires the fusome cytoskeletal component and suggest a possible role for the common ER in synchronizing mitotic cyst divisions. INTRODUCTIONGametogenesis takes place within germline cysts in a wide array of organisms (Telfer, 1975;Bü ning, 1994;de Cuevas et al., 1997). In Drosophila a single oocyte develops within the environment of a 16-cell cyst. The development of this cyst occurs in a series of morphologically defined regions (see Figure 1A). Cyst production begins when a germline stem cell divides asymmetrically to produce a cystoblast. The cystoblast undergoes four synchronous mitotic divisions (regions 1 and 2a), with incomplete cytokinesis, to produce a cluster of 16 cells that are connected via arrested mitotic cleavage furrows (reviewed in de Cuevas et al., 1997). Later in oogenesis, the arrested cleavage furrows mature into stable ring canals and are reinforced with an actin cytoskeleton (reviewed in Mahajan-Miklos and Cooley, 1994). Initially, all 16 cystocytes develop in synchrony. However, as development proceeds, differentiation within the cyst becomes asynchronous. Ultimately, only one cell commits to meiosis and differentiates as the oocyte, whereas the other 15 cystocytes enter the endocycle and develop as trophic nurse cells.A unique membranous organelle, the fusome, grows and branches along the spindle equators after each mitotic division to physically connect all cells within a cyst. The fusome is found in the germline cysts of Drosophila as well as other insects (reviewed in Telfer, 1975;McKearin, 1997). The Drosophila fusome is comprised of cytoplasmic endomembranes, membrane skeletal proteins and polarized microtubules (reviewed in de Cuevas et al., 1997;McKearin, 1997).Molecular genetic studies over the last ten years indicate the fusome mediates several essential steps in germline cyst development (reviewed in McKearin, 1997). The differentiation of the oocyte depends on the pr...

The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental Studies

“…A general and extremely useful application of the collection is to identify new proteins that are associated with such structures and analyze the effect of mutations. For example, the preferential accumulation of Sec61 in the fusome observed here has been validated in recent studies (Snapp et al 2004). Proof of principle experiments of this type that focus on the fusome will be described elsewhere.…”

Rtnl1 is enriched in a specialized germline ER that associates with ribonucleoprotein granule components

“…Only one integral membrane protein has been described in a recent study that localizes to fusomal membranes: the ER translocon channel protein Sec61␣. A GFP-fusion protein of Sec61␣ marks equally the fusome and all of the rough ER at some stages of cyst development in the germarium (Snapp et al, 2004). The same study showed that ectopic expression of a GFP-fusion protein of lysozyme with an engineered ER-retention signal (KDEL), LysGFP-KDEL, also localized to the fusome lumen in germaria.…”

The mushroom body defect Gene Product Is an Essential Component of the Meiosis II Spindle Apparatus in Drosophila Oocytes

“…In addition, in germaria Mud is found strikingly colocalized with spectrosomes and the fusomes of region 1 ( Figure 10A). These structures are not only rich in cytoskeletal proteins like Hts (Lin et al 1994) but also rich in ER-derived membranes (McKearin 1997;Snapp et al 2004). Thus, their association with Mud could depend on its carboxy-terminal transmembrane-spanning domain.…”