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doi: 10.1242/10.1242/dev.00406

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A role for actin dynamics in individualization during spermatogenesis in Drosophila melanogaster

Department of Biology, Washington University in St Louis, Campus Box 1229, 1 Brookings Drive, St Louis, MO 63130, USA

View larger version (21K): [in a new window]   Fig. 1. An individualizing cyst. Individualization takes place at the cystic bulge. The membrane reorganization occurs as the actin cone (red triangle) moves down along the sperm axoneme from its head to the end of the tail. Arrow indicates the direction of movement. Cytoplasm and membranous organelles are squeezed out and accumulate in the cystic bulge as the actin cones move down the cyst. At the end of individualization all cytoplasm and organelles are discarded as a waste bag and 64 thin individual sperm are formed.

View larger version (64K): [in a new window]   Fig. 2. Primary culture of isolated cyst from Drosophila testis. (A) DIC image of dissected testis. Large arrowhead indicates basal (seminal vesicle) end of testis. Fibers (arrows) are elongated cysts. At the apical end (small arrowhead), a number of round cells are seen which are spermatocytes and spermatids at early stages. (B) Isolated individualizing cyst. Large white arrowhead indicates a moving cystic bulge. Large arrow indicates the direction of movement. The cystic bulge starts from the end with sperm nuclei (small white arrowhead) and moves to the apical end (end of sperm tail, small arrow). Small black arrowhead indicates the nuclei of somatic cyst cells, which surround the cyst. (C-F) Cystic bulge at various stages of individualization. The arrow at the bottom indicates the direction of movement of the cystic bulge. (C) Early cystic bulge right after onset of movement (arrow). (D) Cystic bulge in the middle of cyst. (E) Cystic bulge at tail end of cyst. Round waste bag can be seen (arrow). (F) Individualizing spermatids with cyst cells removed. The individualized region of a number of thin sperm tails (small arrowheads) are visible adjacent to the cystic bulge (arrow). (G) An isolated cyst undergoing individualization from head to tail. (H) Representative plot of movement of the cystic bulge. Scale bars: 100 µm in A,B; in F, 50 µm for C-F.

View larger version (31K): [in a new window]   Fig. 3. Immunofluorescence localization of microtubules (green), F-actin (red) and sperm nuclei (blue) in cysts at various stages after elongation. (Column A) Elongated cyst before DNA condensation. There were a number of microtubules around sperm nuclei and microtubule bundles along sperm tails. Nuclei were elongated but not yet condensed (inset). Note that at this stage, no accumulation of actin was seen. (Column B) DNA condensation and F-actin accumulation into an actin cone. Sperm nuclei became thinner (inset in DNA) and actin began to accumulate at the apical side of nuclei. Actin cones were still thin (arrowheads in B-D indicate a single actin cone shown in each inset at higher magnification). (Column C) As the actin cones grew and became thicker, microtubules were excluded from the region but still remained in the cytoplasm. Condensation of sperm nuclei was completed. (Column D) After the onset of actin cone movement, microtubules disappeared. Actin cones became triangular in shape and moved away from the group of sperm nuclei. (E) Actin cones (red) in a cystic bulge in the middle of a cyst. Outline of the individualizing cyst is traced with a white line. The front of the actin cones are wider and the cones are more polarized than at earlier stages. In a non-individualizing cyst (arrowhead), a strong signal of -tubulin staining (green) was observed. However, the individualizing cyst (arrow) had no -tubulin signal. Scale bar: 20 µm.

View larger version (16K): [in a new window]   Fig. 4. Effects of pharmacological reagents on cystic bulge movement. Speed before treatment is the average speed of cystic bulge movement for 40 minutes before treatment. Speed during treatment is the average speed from 40 minutes to 80 minutes after application of drug in the same cyst. In each column, average speeds of 5-10 cysts are shown. (A) Effects of drugs that alter actin dynamics. Both LTA and jasplakinolide (JSP) inhibited cystic bulge movement in a dose-dependent manner. LTA (6 µM) completely stopped movement 20 minutes after application. (B) Effects of drugs that alter microtubule dynamics. Nocodazole (noc), colchicine (col) and taxol show minimal effect on cystic bulge movement. (C) Effect of inhibitors of motor proteins. Kinesin inhibitor monastrol and myosin inhibitor BDM did not inhibit movement of the cystic bulge.

View larger version (21K): [in a new window]   Fig. 5. Actin cones are stable structures. (A) A series of F-actin stainings after LTA treatments of different time duration. Even after 2 hours treatment, some actin cones remained visible. Scale bar: 10 µm. (B) Quantification of fluorescence intensity of individualization complex (IC; a cluster of 64 actin cones in a cystic bulge) after LTA treatment. In order to quantify the effect of treatments, fluorescence intensity of each IC was measured. In each of three experiments and at each time period of treatment, fluorescence intensities of 40 to 50 ICs were measured and average intensities were calculated.

View larger version (103K): [in a new window]   Fig. 6. FRAP of F-actin in cones to examine actin dynamics. (A,B) FRAP experiment 1. Line bleaching across actin cones in a moving cystic bulge. (A) Live image of a moving cystic bulge from a GFP-actin expressing cyst. Arrowhead indicates position of the bleached actin cones. (B) Time-lapse recording of fluorescence recovery after photobleach across a single actin cone. The cystic bulge moved from the bottom to the top of the photograph. Arrowhead indicates position of photobleach. The numbers indicate time after photobleach. Fluorescence recovered completely between 7 minutes 21 seconds and 9 minutes 48 seconds time-points. The bleached spot moved forward as the actin cone moved forward. (C,D) FRAP experiment 2. Bleaching and recovery in a whole single actin cone. (C) Actin cones in moving cystic bulge. Arrowhead indicates actin cone to be bleached. (D) Time series of photobleach and recovery of a whole single actin cone. Recovery takes place relatively uniformly along the cone, and plateaued after 17 minutes 9 seconds, although the fluorescence intensity did not recover completely. (E) FRAP experiment 3. Time series of bleaching and recovery in very early actin cones before onset of movement. Half of the actin cone cluster was bleached. The fluorescence intensity plateaued after 43 minutes 36 seconds. Scale bars: 10 µm for A,C,E; 2.5 µm for B,D.

View larger version (46K): [in a new window]   Fig. 7. Plot of fluorescence intensities after photobleach. (A) Raw images of actin cones in a line bleach experiment. The data were used in the plot (B). Numbers in the upper-left corner indicate time after line bleach. Distance A (between green arrowheads) indicates distance from the front of actin cone to the front of bleached line. Distance B (between yellow arrowheads) is the distance of actin cone movement during the time indicated. Short yellow lines indicate the position of front of actin cone in each image. (B) Distances A and B of cone 1 and 2 in A are plotted. Distance A in cone 1 (black circle) and cone 2 (black square) did not change through the recording time suggesting no movement of bleached line relative to the front of the actin cone. However, distance B of cone 1 (white circle) and cone 2 (white square) increased linearly, suggesting constant movement of both actin cones. (C) Representative image of moving actin cones. Fluorescence intensity in the region indicated by rectangles (red:front and yellow:back) were measured and plotted in D. (D) Relative fluorescence intensities of GFP-actin in moving actin cones after photobleaching are plotted. A whole actin cone was photobleached and fluorescence recovery in front (black square) and back (black circle) were plotted separately. Relative intensity was calculated as fluorescence intensity at each time point/fluorescence intensity before photobleach in the region of interest. Six actin cones were examined for each plot. Bulk fluorescence of the actin cone recovers in about 12 minutes in both parts of the cone; however, the front of the cone recovers slightly more quickly than the back. (E) Representative plot of FRAP in actin cones before onset of movement. The fluorescence recovery plataued at 43.6 minutes after photobleach.

View larger version (114K): [in a new window]   Fig. 8. (A) Live imaging of cell membrane of the cystic bulge with FM1-43. Some open mouths of the membrane tunnels (arrowheads) are indicated. However, around the neck of individualizing sperm tail (arrows), no membrane invaginations or up-take of membrane vesicles was observed. (B-E) Double labeling of membrane with FM 1-43 (B) and F-actin cones with GFP-actin (C). Merged image (D) and higher magnification image (E) demonstrate that F-actin occupied the space of the sperm tail neck that is directly adjacent to the cystic bulge membrane. Scale bars: 6 µm in A,E; in B, 12 µm for B-D.

View larger version (53K): [in a new window]   Fig. 9. Model of cystic bulge movement. (A) An actin cone has two domains. The first domain is actin cone itself, which is highly crosslinked and moves forward as a unit. The second domain is cortical actin, which is dynamic and responsible for pushing actin cone forward, as indicated by purple arrows. As the actin cone moves forward (blue arrows), cytoplasm (green) is squeezed out by the thick network in the actin cone. The membrane and axoneme (green bar) become directly connected just behind the actin cone (see the Movie 9 at http://dev.biologists.org/supplemental/). (B) Enlarged diagram of the region of the actin cones and a speculative model of actin dynamics. Actin polymerization occurs near the membrane. The actin filaments elongate and are crosslinked into an actin cone. New filaments branch out from the side the of old filaments due to the activity of Arp2/3 complex. This branching is more active at the front of the actin cone because of the concentration of Arp2/3 complex, and more pushing force should be generated there. Probably because actin depolymerization is faster at the back (and/or there is less assembly), the actin cone becomes thinner toward the back and eventually disappears.