Additional file 1:

A Three-dimensional rocking movie of the nuclei bundles and actin caps distribution at the base of the wild-type testes. A confocal Z-focus series of an immunostained specimen was collected by using a 40× NA 0.9 objective. The testes preparation was mounted with protective spacers to maintain the original spatial distribution in the tissue. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (blue) and F-actin was stained with fluorescein isothiocyanate (FITC):phalloidin (green). A corresponding Z-projection of the image stack is shown in Figure 1(A).

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Additional file 2:

Movie of the acrosome movements in the wild-type testis in phosphate buffered saline. The time-lapsed images of isolated y w; P [w ^+mc snky:GFP] testis collected using a 60× (oil) NA 1.42 objective attached to a laser scanning confocal microscope. Each frame was averaged for 2 scans (at around 1 second per scan) and collected every 3 minutes. A total of 20 frames are presented in this movie. The movie runs at two frames per second. The acrosomes of the elongating spermatids (red arrows) were found to move quite well. In comparison, the compacted acrosome bundles were mostly stationary (white arrows). A two-dimensional time projection of these frames is shown in Figure 5(A).

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Additional file 3:

The Gal4/UAS-reporter expression patterns in the testes. (A) Confocal sections show myosin VII-GFP (green) and myr-mRFP1 localizations in the testis from the w UAS-myosin VII-GFP pCOGGal4/Y; UAS-myr-mRFP1/+ adults. The expression was localized in the germ-line stem cells and primary gonial precursors (arrowheads) at an early stage. Later on, it was contained in the cyst cells (arrows). The UAS-myr-mRFP1 (myristoylated mRFP1) expression generally marks the cell membrane and highlights the cysts cell perimeter (arrows, A") around the spermatocytes in the testis. (B) The UAS-actin:GFP expression in the pCOGGal4/Y; UAS-actin:GFP/+ testis is increased in the head cyst cells (HC, dotted lines) at the final stages before the mature sperm release from the cysts. B-B" indicates different focus levels of the same testis. The rhodamine isothiocyanate (RITC):phalloidin (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue) staining are shown in appropriate false colors. (C) The head cyst cells (arrows) are prominently marked by the combined expression of UAS-GFP and UAS-actin:GFP in SG18.1Gal4 background during the sperm individualization stages.

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Additional file 4:

Movie of the acrosome movements in the wild type testis in phosphate buffered saline containing 5 μM vinblastine. The time-lapse images of isolated y w; P [w ^+mc snky:GFP] testis collected using a 60× (oil) NA 1.42 objective attached to a laser scanning confocal microscope as described for Additional file 5. The movie runs at two frames per second. The testes preparations were treated for 30 minutes in phosphate buffered saline containing 5 μM vinb before imaging. A total of 10 frames are presented in this movie with each frame having its own time stamp. The acrosomes of the elongating spermatids (red arrows) as well as the compacted acrosome bundles (white arrows) appeared to move very slowly. The two-dimensional time projection of these frames is shown in Figure 5(B).

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Additional file 5:

Transient disruption of the shibire function in the head cyst cell caused actin cap and NB disruptions. The UAS-shi ^ts1 expression in the head cyst cells due to the (A) pCOGGal4 and (B) SG18.1Gal4 drivers, disrupted the actin caps and nuclei bundles (NBs) at non-permissive temperature (29°C). Rhodamine isothiocyanate (RITC):phalloidin and 4',6-diamidino-2-phenylindole (DAPI) staining of these testes before (a), (b) and after (c), (d) the heat pulse showed visible disruptions of the actin caps and NBs (arrows). In addition, there was punctate accumulation of F-actin in the head cyst cell cytoplasm (arrowheads) after the heat pulse. Fine arrows indicate mature nuclei separated from the actin caps. This is not found in the wild-type controls treated in a similar manner. The actin cap and the NB morphology remained unaltered even after 30 minutes at 29°C in the pCOGGal4 UAS-GFPck/Y and SG18.1Gal4 UAS-GFP/UAS-actin:GFP testes (data not shown). (C) The UAS-WASP ^ΔCA expressions by using (a), (b) pCOGGal4 and (c)-(e) SG18.1Gal4 caused mild loss of F-actin staining from the actin caps but no detectable NB disruptions even after a 30 minute heat pulse.

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Additional file 6:

Table S1. Table listing Drosophila melanogaster stocks used in this study

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Additional file 7:

Table S2. Table listing the antibodies used in this study

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