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doi: 10.1242/10.1242/jcs.00149

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A role for myosin VI in actin dynamics at sites of membrane remodeling during Drosophila spermatogenesis

Aaron D. Rogat and Kathryn G. Miller*

Department of Biology, Washington University, One Brookings Drive, St Louis, MO 63130, USA



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  		Fig. 1. (A) Schematic representation of three spermatids undergoing individualization. Spermatogenesis produces 64 elongated spermatids interconnected by cytoplasmic bridges. During individualization, syncytial membrane (black) of the bundled spermatids (far left) is remodeled to individual membranes encasing each of the spermatids separately (far right). The individualization complex is responsible for membrane remodeling, and its central constituent is actin (green; one actin cone is associated with each spermatid in the complex) (Fabrizio et al., 1998Go). At the beginning of individualization (far left) the actin cones assemble around the spermatid nuclei (blue), and myosin VI (red dots) coats the actin cones in a particulate fashion. Later, the actin cones progress away from the nuclei and down the length of the spermatid axonemes (orange), extruding the cytoplasm between the spermatid tails, resolving the cytoplasmic bridges (area between ovals of membrane between the spermatid axonemes) and remodeling the syncytial membrane into individual membranes that encase each spermatid (middle). As soon as the actin cones move away from the nuclei, myosin VI localizes to an intense band at the front of the actin cones (red band). A bulge of plasma membrane, syncytial cytoplasm, vesicles and organelles (the cystic bulge) develops. When the actin cones reach the apical end of the testis, membrane and cytoplasm collected are pinched off, leaving the spermatids completely encased in their own membranes. The length of the spermatids is approximately 2 mM, and the cones take an estimated 18 hours to travel this distance. (B-M) Laser scanning confocal images of myosin VI staining at different stages of individualization complex progression. Early (B-E) in the process of individualization, actin cones (B) have assembled around the nuclei (D) of spermatids near the basal end of the testis, and myosin VI (C) coated the surface of actin cones in a particulate fashion. A little later (F-I), the actin cones (F) have moved away from the nuclei (H), myosin VI (G) was concentrated at the front of the actin cones. Much later (J-M), myosin VI accumulated and concentrated in a tight band (K) at the front of the actin cones as the individualization complex moves down the testis. Each image shows a single plane through one group of 64 bundled spermatids with 64 associated actin cones.

 


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  		Fig. 2. Myosin VI colocalizes with cortactin, arp2/3 complex and capping protein at the front of individualization complexes. (A-L) Confocal images of spermatids double labeled with Alexa Fluor 488 phalloidin (A,D,G,J) and antibodies to cortactin (B), arp3 (E), ARPC2/p34 (H) or capping protein (K). (M-U) Projections of images of spermatids double labeled with anti-myosin VI antibodies (M,P,S) and antibodies to cortactin (N), arp3 (Q) and capping protein (T).

 


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  		Fig. 3. Cortactin and arp2/3 complex accumulation were abnormal on individualization complexes in myosin VI mutants. (A-L) Low magnification of a confocal image of wild-type (A-C, G-I) or myosin VI mutant (jar/jar; D-F, J-L) individualization complexes stained with Alexa Fluor 488 phalloidin (A,D,G,J) and anti-cortactin antibodies (B,E) or anti-arp3 antibodies (H,K). (M-X) Zoomed confocal images of a few actin cones from the individualization complexes in A-L. Cortactin localized to the fronts of the actin cones (blue arrow; B,N) and on the cyst membrane (yellow arrow; B). Cortactin localized to cyst membranes (yellow arrow, E) and actin cones in mutants but was not concentrated at the front of the actin cones (white arrow; E,Q). Cortactin localized normally to the cyst membrane in the myosin VI mutant (yellow arrow, E). Arp3 localized to the front of actin cones (blue arrowhead; H,T) and the cytoplasm of the cystic bulge (H). Arp3 was present on actin cones in mutants but was not well concentrated at the front of actin cones (white arrowhead; K,W). Images of wild-type and myosin VI mutant testes were collected at the same gain.

 


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  		Fig. 4. Localization of proteins involved in membrane events on actin individualization complexes. Confocal images of individualizing spermatids double-labeled with Alexa Fluor 488 phalloidin (A,G,J) and anti-dynamin antibody (B), anti-{alpha} adaptin antibody (H) or anti-amphiphysin antibody (K). In double labeled samples, myosin VI (E) was concentrated at the front of dynamin-stained complexes (D). Dynamin and amphiphysin colocalized with actin in the individualization complexes (C,L); however, unlike dynamin, amphiphysin also was concentrated at the front of the actin complexes.

 


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  		Fig. 5. Actin stability defects in dynamin myosin VI double mutants. Confocal projections of mutant testes stained with phalloidin. Volume samples were taken near the basal end of testis where all the actin individualization complexes are associated with nuclei. Within each testis, several groups of 64 bundled spermatids are shown. (A) All actin individualization complexes in dynamin (shi1/Y) single mutants exposed to the non-permissive temperature for 6 hours stained strongly with phalloidin (white arrowhead). (B) Most actin complexes in myosin VI single mutants (jar/jar) exposed to the non-permissive temperature for 6 hours stained brightly with phalloidin, although a few disrupted complexes stained weakly with phalloidin (asterisk). (C) Few actin complexes in dynamin myosin VI double mutants (shi1/Y; jar/jar) exposed to the non-permissive temperature for 6 hours were visible, and those that were visible stained weakly with phalloidin (white arrow). All images are projections of multiple planes. Images of single and double mutants were taken at the same gain and scale.

 


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  		Fig. 6. Quantification of number of actin complexes in dynamin myosin VI double mutants. Wild-type (OreR), dynamin mutants (shi1/Y), myosin VI mutants (jar/jar) or dynamin myosin VI double mutants (shi1/Y; jar/jar) were exposed to the non-permissive temperature for the times indicated. (A) The average number of early actin individualization complexes per testis is shown for each genotype and condition. (B) The average proportion of early actin complexes that stained weakly with phalloidin are shown.

 

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© The Company of Biologists Ltd 2002