Visualizing Fusome Morphology via Tubulin Immunofluorescence in Drosophila Ovarian Germ Cells.

In many species, oocytes are initially formed by the mitotic divisions of germline stem cells and their differentiating daughters. These progenitor cells are frequently interconnected in structures called cysts, which may function to safeguard oocyte quality. In Drosophila, an essential germline-specific organelle called the fusome helps maintain and coordinate the mitotic divisions of both germline stem cells and cyst cells. The fusome also serves as a useful experimental marker to identify germ cells during their mitotic divisions. Fusomes are cytoplasmic organelles composed of microtubules, endoplasmic reticulum-derived vesicles, and a meshwork of membrane skeleton proteins. The fusome branches as mitotic divisions progress, traversing the intercellular bridges of germline stem cell/cystoblast pairs and cysts. Here, we provide a protocol to visualize fusome morphology in fixed tissue by stabilizing microtubules and immunostaining for α-Tubulin and other protein constituents of the fusome. We identify a variety of fluorophore-tagged proteins that are useful for visualizing the fusome and describe how these might be combined experimentally. Taken together, these tools provide a valuable resource to interrogate the genetic control of germline stem cell function, oocyte selection, and asymmetric division.