Separating from the pack: Molecular mechanisms of Drosophila spermatid individualization.

Successful completion of gametogenesis is critical for perpetuation of the species. In addition to the inherent interest, studies of gamete development, in particular spermatogenesis, have yielded insight into diverse biological processes, including actin and microtubule organization, mitochondrial dynamics, plasma membrane remodeling, lipid signaling, apoptosis, and many others. Mammalian sperm are formed from germline stem cells that reside near the basal surface of the seminiferous tubules. Spermatogonia produced from these stem cells undergo amplifying mitotic divisions with incomplete cytokinesis to eventually produce interconnected chains of spermatocytes that synchronously transition into meiosis. Cytokinesis of the meiotic divisions also is incomplete, such that cytoplasmic channels remain between sister spermatids after each division. This allows for the sharing of cytoplasm between sister spematids, which synchronizes their development and protects them from the genetic effects of haploidy. Following meiosis, the haploid spermatids undergo spermiogenesis, the terminal differentiation process wherein acrosomes are formed from Golgi, chromatin compacts, the nuclei are reshaped, and the flagella elongate. After terminal differentiation, the cytoplasmic contents are removed and the cytoplasmic bridges connecting sister spermatozoa are dissolved. 10 This last process is dependent on the actin cytoskeleton and is essential for proper sperm function. The spermatozoa are released from the testis into the epididymis, where their plasma membranes undergo molecular changes. Epididymal activation is required for motility and fertilization. Spermatogenesis is strikingly similar in the fruit fly, and many molecular players are conserved between mammals and Drosophila. A single Drosophila gonialblast, formed by division of a germline stem cell, undergoes four mitotic divisions and two meiotic divisions to produce 64 interconnected sister spermatids in a germline cyst. As in mammals, incomplete cytokinesis leads to cytoplasmic sharing between sister spermatids, via intercellular bridges called ring canals. Following nuclear compaction and formation of the flagella, the interspermatid bridges are dissolved concurrently with cytoplasm removal in an actin-dependent process called spermatid individualization. Much has been discovered about this process in the 21 century. Individualization is carried out by the individualization complex (IC), which first forms at the rostral end of the cyst, around the spermatid nuclei (Figure 1). The IC is composed of 64 actin cones, one for each germ nucleus of the cyst. Actin filaments form a meshwork at the leading edge of the cones and are organized into parallel bundles at the rear of the cones. The meshwork is formed by the Arp2/3 actin nucleating complex. The actin motor Myosin VI works with unknown binding partners to localize Arp2/3 and to stabilize the meshwork at the front of the cones. Other factors at the cone fronts include Actin Capping Protein and Cortactin, and the membrane binding protein Amphiphysin. At the rear of the cones, the actin bundling proteins Quail/Villin, Chickadee/Profilin, and Singed/Fascin localize. As individualization proceeds, the actin cones of the IC move synchronously away from the nuclei toward the caudal end of the cyst, traversing the spermatid flagella at an average speed of 3 mm/minute and finishing the 1.8 mm journey in 10 hours. As it travels, the IC removes the cyst cytoplasmic contents and individualizes each spermatozoon in its own plasma membrane (Figure 1). The cones accumulate actin during this process, especially at their front edges, and proper accumulation of actin filaments in the leading edge meshwork is required for cytoplasmic extrusion. Extruded cytoplasmic contents are collected in a cystic bulge that forms around the IC. When the IC and cystic bulge reach the end of the flagella, the actin cones and cytoplasmic contents find themselves in a waste bag, the contents of which are degraded. It is not yet known what generates the force for IC movement. Although Myosins V and VI are important for this process, motor activity does not seem to power migration of the