A role for actin dynamics in individualization during spermatogenesis inDrosophila melanogaster

Abstract

In order to better understand the mechanism of sperm individualization during spermatogenesis in Drosophila melanogaster, we have developed an in vitro culture system in which we can perform live observation of individualization in isolated cysts. The whole process of individualization,during which a bundle of 64 syncytial spermatids is separated into individual sperm, takes place in these cultures. Individualization complexes, which consist of 64 cones of actin that assemble around the sperm nuclei, move to the basal end of the tails, forming a characteristic `cystic bulge' that contains an accumulation of cytoplasm, syncytial membrane and vesicles. The cystic bulge is the site of membrane remodeling and its movement was used to follow the progress of individualization. The speed of cystic bulge movement is fairly constant along the length of the cyst. Actin drugs, but not microtubule drugs inhibit cystic bulge movement, suggesting that the movement requires proper actin dynamics but not microtubules. GFP-tagged actin was expressed in the cyst and fluorescence recovery after photobleaching was monitored using confocal microscopy to analyze actin dynamics in cones. Actin turns over throughout the cone, with that at the leading edge of the cones turning over with slightly faster kinetics. Actin does not treadmill from the front to the back of the cone. Actin in moving actin cones turns over in about 12 minutes, although prior to onset of movement, turnover is much slower. Visualization of membrane using FM1-43 reveals that the cystic bulge has an extremely complicated series of membrane invaginations and the transition from syncytial to individualized spermatids occurs at the front of the actin cones. We also suggest that endocytosis and exocytosis might not be important for membrane remodeling. This system should be suitable for analysis of defects in male sterile mutants and for investigating other steps of spermatogenesis.

Drosophila polypyrimidine tract-binding protein is necessary for spermatid individualization

“…To determine how loss of dmPTB affects spermatid individualization, we next examined whether loss of dmPTB affected the spermatid individualization complex (IC), which is composed of highly crosslinked actin cones that surround each individualizing spermatid (23,24). The IC moves from the basal end of the cyst to the apical end, severing links between spermatids and removing most of the cytoplasm while encasing each spermatid in its own membrane.…”

Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization inDrosophila

“…The characteristics of F-actin dynamics at the cones suggest that they are similar to the leading edges of lamellipodia (Noguchi and Miller, 2003) and they are compared with the actin comets found in mammalian cells (Bazinet and Rollins, 2003). Actin comets are dynamic actin-based structures that form on the endocytic vesicles and power their movement inside the cell.…”

“…The entire individualization process is estimated to take nearly 24 h at 25°C inside the testis (reviewed in Lindsley and Tokuyasu, 1980) and the ICs take 10 h to complete the 1.8-mm journey under specific culture conditions in vitro (Noguchi and Miller, 2003).…”

“…F-actin-associated proteins, such as capping protein, cortactin, Arp2/3 complex, and myosin VI, are enriched at the leading edges of F-actin cones, whereas dynamin is localized throughout (Rogat and Miller, 2002), indicating that F-actin assembly would be initiated at the leading edges. Although, a later study showed that the F-actin assembly occurs throughout the cone (Noguchi and Miller, 2003). The F-actin cone bundles appeared disrupted in myosin VI homozygous testes (Hicks et al, 1999) and genetic interaction studies showed that myosin VI (jar 1 ) and dynamin (shi ts1 ) could act in parallel pathways to maintain the F-actin levels in these cones (Rogat and Miller, 2002).…”

“…Pharmacological treatments of isolated cysts established that F-actin dynamics plays a critical role in membrane extraction and F-actin cone movement and that both these processes are microtubule independent (Noguchi and Miller, 2003). F-actin-associated proteins, such as capping protein, cortactin, Arp2/3 complex, and myosin VI, are enriched at the leading edges of F-actin cones, whereas dynamin is localized throughout (Rogat and Miller, 2002), indicating that F-actin assembly would be initiated at the leading edges.…”