Supplemental Material

This article contains the following supporting material:

• Movie 1 - A spermatogenic cyst from a wild type male undergoing individualization in vitro.
  The DIC time-lapse movie of cystic bulge movement was recorded at 4 minute intervals. Display rate is 6 frames / sec. Note that the cystic bulge moves smoothly from the top toward the bottom of the image. Bar = 30 μm.

• Movie 2 - A spermatogenic cyst from a myosin VI mutant male undergoing individualization in vitro.
  The time-lapse movie was recorded at 4 minutes intervals. Display rate is 6 frames / sec. At the beginning of the movie the cystic bulge moves relatively normally, but soon the bulge looks thinner and elongated. The bulge stops before reaching the end of the tail. The cysts often bend and twist as the cystic bulge slows down. This is the typical defect seen in myosin VI mutant cysts. We suspect this bending is caused by uneven pushing force because actin cones are not in register. Bar = 30 μm.

• Figure 1 - Schematic drawing of Drosophila permatogenesis
  Spermatogenesis initiates with division of germ line stem cells at the apical end of the testis. Germ line cells undergoing differentiation are enclosed by two somatic cells called cyst cells to form a cyst. Cysts move towards the basal end of the testis as differentiation proceeds. All the following processes take place in the cyst. The germ cell undergoes 4 rounds of mitosis without complete cytokinesis, generating 16 syncytial primary spermatocytes that grow and increase in volume significantly. The next step, meiotic division, generates 64 interconnected spermatids and immediately the cyst enters the onion stage. At the onion stage, the cyst contains 64 nuclei each paired with a mitochondrial derivative called the nebankern. The cyst elongates as axonemes grow, reaching 1.8 mm long. At the final stage of spermatogenesis, 64 syncytial spermatids are divided into 64 individual sperm. Before individualization, all spermatids are enclosed in a single plasma membrane (black lines) that connects all the spermatids using a complex array of cytoplasmic bridges. After condensation of DNA in the sperm nuclei (Blue), individualization process begins with formation of actin cones (red) surrounding each sperm nucleus. A group of 64 actin cones moves synchronously towards the tail end of the cyst, pushing out cytoplasm and attaching the cell membrane to the axoneme. The large accumulation of cytoplasm pushed forward by the actin cones is called cystic bulge. Myosin VI (orange) localizes to the front edge of each actin cone throughout the process. At the end of individualization, the cystic bulge detaches from the end of the tail and is discarded as a waste bag.

• Figure 2 - Actin dynamics of actin cone in myosin VI mutant
  Time-lapse images of FRAP of GFP-actin during cone movement in myosin VI mutant (a) and wild type (b). Region indicated by white lines in the ‘pre-bleach’ image was subsequently bleached (second panel) and recovery of fluorescence was recorded at 3 minute intervals. In some myosin VI mutant cysts, recovery was somewhat slower (a, upper row) than in wild type, but in most cases (a, lower row), recovery time was comparable to wild type, even when the actin cone morphology was abnormal. Bar in b = 10 μm. (c) Representative plot of fluorescence recovery of GFP-actin in myosin VI mutant. The lines fitted to the recovery of first 7 min are shown. (d) Graph that compares the GFP-actin turn-over rate in myosin VI mutant and wild type. Turnover rates during the first 7 minutes after photobleaching were measured. T-test was performed and there was no significant difference between wild type and myosin VI mutant (p < 0.5).

• Figure 3 - Fluorescence microscopy of live whole testes from GFP-FL, GFP-G-tail, and GFP-actin line.
  The testes were imaged using a fluorescence microscope with a 4 x lens. (exposure time: GFP-FL, 1 sec, GFP-G-tail 6 sec, GFP-actin 6 sec). GFP-FL has significantly higher fluorescence intensity compared with GFP-G-tail and GFP-actin. The intensity of fluorescence was adjusted identically in GFP-G-tail and GFP-actin images, to increase brightness in order to show the GFP-signals in elongated cysts. Although there is no apparent GFP localization to the cones in GFP-G-tail testis, actin cones were easily

• Figure 4 - Myosin VI dynamics before actin cone movement.
  (a) FRAP of GFP-myosin VI on actin cones before cone movement began. Region indicated by white lines in ‘prebleach’ image was bleached subsequently and recovery of the fluorescence was recorded at 40 seconds intervals. The recording times are indicated in the top left corner of each image. (b) A representative plot of relative fluorescence intensity of GFP-myosin VI during recovery on the cones before movement.