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Myosin VI Functions in Actin Dynamics 4857 Research Article 4855 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 *Author for correspondence (e-mail: [email protected]) Accepted 4 September 2002 Journal of Cell Science 115, 4855-4865 © 2002 The Company of Biologists Ltd doi:10.1242/jcs.00149 Summary Myosin VI has been implicated in membrane dynamics in when dynamin and myosin VI function are both impaired, several organisms. The mechanism of its participation major defects in actin structures are observed. We conclude in membrane events is not clear. We have used that during spermatogenesis myosin VI and dynamin spermatogenesis in Drosophila to investigate myosin VI’s function in parallel pathways that regulate actin dynamics in vivo role. We demonstrate that myosin VI colocalizes and that cortactin and arp2/3 complex may be important with and is required for the accumulation of the actin for these functions. Regions of myosin VI accumulation are polymerization regulatory proteins, cortactin and arp2/3 proposed as sites where actin assembly is coupled to complex, on actin structures that mediate membrane membrane dynamics. remodeling during spermatogenesis. In addition, we show that dynamin localizes to these actin structures and that Key words: Myosin VI, Actin dynamics, Cortactin, Arp2/3, Dynamin Introduction of mitochondria and ER/Golgi-derived organelles during Myosin VI is a conserved unconventional myosin found in spermatogenesis, suggesting defects in organelle trafficking or several organisms including worms, flies, mice and humans localization. Consistent with a role in trafficking, myosin VI (Avraham et al., 1997; Avraham et al., 1995; Hasson et al., in vertebrate tissue culture cells localizes to trafficking 1996; Kelleher et al., 2000; Kellerman and Miller, 1992). Like membranes such as the trans-Golgi network (Buss et al., 1998) all members of the myosin superfamily, myosin VI contains an and clathrin-coated pits and vesicles (Buss et al., 2001a), and N-terminal motor domain with conserved actin- and ATP- overexpression of the tail fragment of myosin VI reduces binding motifs. However, unlike most other myosins that have transferrin uptake (Buss et al., 2001a). From these studies and been tested for direction of motility, myosin VI moves towards those in Drosophila (described below) it has been proposed the slow growing, pointed end of an actin filament (Homma et that myosin VI has a role in organizing and/or trafficking al., 2001; Wells et al., 1999). Only one other myosin so far membrane. However, the details of how myosin VI participates studied, myosin IX, moves towards the pointed end (Inoue et in these functions remain unclear. al., 2002). Recently, a heritable form of human deafness has In Drosophila there is one myosin VI gene. The proposed been linked to mutations in myosin VI (Melchionda et al., function for myosin IV is in membrane organization and 2001). Given the unusual direction of motility of myosin VI trafficking. In Drosophila syncytial blastoderm embryos, and the clinical relevance of myosin VI, there is much interest myosin VI protein localizes to cortical actin in transient mitotic in understanding its cellular and molecular functions. membrane invaginations (pseudocleavage furrows). Inhibition Some clues as to the cellular functions of myosin VI have of myosin VI function in the Drosophila embryo causes defects come from phenotypic analysis of mutations in Mus musculus, in formation of these transient mitotic membrane invaginations Caenorhabditis elegans and Drosophila melanogaster. The (Mermall and Miller, 1995). Thus, myosin VI is required for defects seen in the deaf Snell’s Waltzer mutant mouse are membrane remodeling during embryogenesis. caused by a loss-of-function mutation in the myosin VI gene. Myosin VI function is also required for membrane These mutants display disruptions in the organization and remodeling during the individualization step of morphogenesis of the stereocilia in the inner ear (Avraham spermatogenesis (Hicks et al., 1999). During individualization, et al., 1995; Self et al., 1999). The hair cell stereocilia in a syncytial membrane encasing a bundle of 64 spermatids is these mutants are disorganized, and the membrane between remodeled so that an individual membrane encases each of the neighboring stereocilia is uprooted, suggesting a defect in the 64 mature sperm. Myosin VI localizes to an actin complex, the anchoring of stereocilia and the apical membrane (Self et al., individualization complex, which assembles at the spermatid 1999). In C. elegans, loss-of-function mutations in one of two heads at the start of individualization (Hicks et al., 1999). This isoforms of myosin VI cause male sterility (Kelleher et al., complex progresses from the spermatid heads to the tips of 2000). These mutants display defects in the asymmetric sorting tails, remodeling membrane as it moves. Loss of myosin VI in 4856 Journal of Cell Science 115 (24) the testis leads to the disruption of these actin complexes as antibodies to dynamin-1 show the identical localization in the testis. they progress and, consequently, individualization is not The rat anti-Drosophila cortactin antibody (1:250) was a gift from completed (Hicks et al., 1999). Manabu Takahisa (Katsube et al., 1998). The rabbit anti-Drosophila The precise function of myosin VI in individualization Arp3 (1:50) was a gift from Bill Theurkauf and the rabbit anti-human remains unclear. Does myosin VI catalyze transport of ARPC2/p34 (1:100) was a gift from Matt Welch (Welch et al., 1997). membrane trafficking components at the actin The rabbit anti-Drosophila amphiphysin (1:100) and anti-Drosophila alpha adaptin (1:25) were gifts from Andrew Zelhof (Zelhof et al., individualization complex? Is it involved in anchoring 2001) and Nick Gay (Dornan et al., 1997; Zelhof et al., 2001). The membrane to actin and/or organizing actin polymerization sites rabbit and rat anti-Drosophila capping protein β (CP-β) antibodies at the membrane? To obtain a clearer understanding of myosin (1:10) were described previously (Hopmann et al., 1996). VI’s role in the individualization complex, we compared the localization of myosin VI in spermatids with that of proteins known to have roles in regulating actin dynamics or membrane Immunofluorescence staining and imaging trafficking. We also tested for genetic interactions between Fixed testes were blocked in PBST + 3% BSA for at least 30 minutes mutations in myosin VI and mutations in the membrane at room temperature or overnight at 4°C. All antibodies and dyes were trafficking gene, dynamin. We report that myosin VI localizes diluted in PBST + 3% BSA. Antibodies were incubated with tissue with regulators of actin dynamics and is required for the samples for 2 hours at room temperature or overnight at 4°C. After incubation with primary antibodies in a humid chamber, fixed testes localization of these proteins. We also show that myosin VI were washed four times in PBST + 3% BSA for 10 min at room mutations interact genetically with dynamin mutations to affect temperature. Then, secondary antibodies were incubated for 2 hours actin dynamics. We propose that myosin VI is important for in a humid chamber. Goat anti-mouse Alexa Fluor 488 and goat anti- actin assembly at sites of membrane remodeling. rabbit Alexa Fluor 488 secondary antibodies were diluted 1:500. Goat anti-mouse Alexa Fluor 568 and goat anti-rabbit Alexa Fluor 568 secondaries were diluted 1:1000. Alexa Fluor 488 phalloidin (1:125) Materials and Methods and the DNA dyes TOTO-3 (1:3000) and DAPI (1 µg/ml) were Fly culture and fly stocks included in incubations with secondary antibodies for 2 hours at room All fly stocks were raised and maintained on standard Drosophila temperature. All Alexa-conjugated secondary antibodies, phalloidin cornmeal, agar, sucrose medium at 22°C (except as indicated; see and TOTO-3 dyes were from Molecular Probes. After staining, testes below). The jaguar (jar1) mutant allele (Castrillon et al., 1993) and were washed four times in containers filled with PBST for 10 minutes shibire (shi1) mutant allele were obtained from the Bloomington at room temperature and finally mounted in mounting media Drosophila Stock Center (Bloomington, IN). [500 mg/ml glycerol, 17 mM Tris pH 8.5, 200 mg/ml Mowiole (Calbiochem)] All imaging of fluorescent stains was performed on a Leica laser Temperature shifts to inactivate shibire function scanning spectral confocal microscope (model TCS SP2). Images Newly eclosed adult flies (0-12 hrs after eclosure) were shifted to the shown are either single planes or, when noted, multiple planes non-permissive temperature of 30°C in a water bath for the times collapsed into a single images (a projection). Projections were indicated in each experiment. At the end of the temperature shift, flies obtained by collecting consecutive planes at 0.5 µm intervals in were immediately dissected in prewarmed (30°C) dissection buffer on volume samples that include most of the actin individualization prewarmed dissection slides. A temperature-controlled rubber mat complex. (controller and heating mat from Thermolyne) set at low temperature was placed under the dissection slide to maintain the non-permissive temperature during dissection. Testes dissected in this manner were Results immediately fixed as described below. Myosin VI colocalizes with cortactin and other actin- binding
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