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«asunder is a critical regulator of dynein-dynactin localization during Drosophila spermatogenesis Michael A. Anderson1, Jeanne N. Jodoin1, Ethan ...»

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asunder is a critical regulator of dynein-dynactin localization

during Drosophila spermatogenesis

Michael A. Anderson1, Jeanne N. Jodoin1, Ethan Lee1, Karen G. Hales3, Thomas S. Hays2, and

Laura A. Lee1

Department of Cell and Developmental Biology, Vanderbilt University Medical Center,

Nashville, TN, 37232-8240, USA

Department of Genetics, Cell Biology, and Development, University of Minnesota,

Minneapolis, MN, 55455, USA

Department of Biology, Davidson College, Davidson, NC 28035-7118, USA

To whom correspondence should be addressed:

Laura A. Lee Vanderbilt University Medical Center Department of Cell & Developmental Biology 465 21st Avenue South U-4227 MRBIII Nashville, TN 37232-8240 Phone: (615) 322-1331 Fax: (615) 936-5673 E-mail: laura.a.lee@vanderbilt.edu RUNNING TITLE: Drosophila spermatogenesis requires asun

Supplemental Material can be found at:

http://www.molbiolcell.org/content/suppl/2009/04/08/E08-12-1165.DC1.html ABSTRACT Spermatogenesis utilizes mitotic and meiotic cell cycles coordinated with growth and differentiation programs to generate functional sperm. Our analysis of a Drosophila mutant has revealed that asunder (asun), which encodes a conserved protein, is an essential regulator of spermatogenesis. asun spermatocytes arrest during prophase of meiosis I. Strikingly, arrested spermatocytes contain free centrosomes that fail to stably associate with the nucleus.

Spermatocytes that overcome arrest exhibit severe defects in meiotic spindle assembly, chromosome segregation, and cytokinesis. Furthermore, the centriole-derived basal body is detached from the nucleus in asun postmeiotic spermatids, resulting in abnormalities later in spermatogenesis. We find that asun spermatocytes and spermatids exhibit drastic reduction of perinuclear dynein-dynactin, a microtubule motor complex. We propose a model in which asun coordinates spermatogenesis by promoting dynein-dynactin recruitment to the nuclear surface, a poorly understood process required for nucleus-centrosome coupling at M-phase entry and fidelity of meiotic divisions.


Spermatogenesis is a dynamic process in which the cell cycle is coordinated with developmental events to produce haploid sperm capable of fertilizing eggs. Drosophila melanogaster has proven to be an excellent model system for the study of spermatogenesis. The relatively large size of meiotic spindles of Drosophila spermatocytes makes them well suited for cytological examination (Cenci et al., 1994). Due to relaxation of checkpoints, meiotic progression occurs even in the face of errors in spindle assembly (Rebollo and Gonzalez, 2000).

Immature spermatids, which are abundant in the testis, are highly regular inappearance;

deviations in their uniformity are diagnostic of earlier defects in meiotic divisions. These features, combined with genetic tools available in Drosophila, have facilitated mutational analysis of male meiosis (Wakimoto et al., 2004).

The stages of Drosophila spermatogenesis have been clearly defined (Fuller, 1993).

Germline stem cells at the apical tip of the testis give rise to spermatogonial cells that proceed through four synchronous rounds of mitosis with incomplete cytokinesis to form 16-cell cysts of primary spermatocytes. Following DNA replication, primary spermatocytes enter a prolonged G2 growth phase. Progression through meiosis I leads to formation of 32-cell cysts of secondary spermatocytes, and further division in meiosis II generates 64-cell cysts of spermatids. Immature, round spermatids differentiate into elongated and individualized sperm that move into the seminal vesicles.

Spermatogenesis is one of only a few aspects of Drosophila development that requires centrioles. While centrioles appear to be largely dispensable for mitosis, acentriolar spermatocytes form highly abnormal meiotic spindles and do not initiate cytokinesis (Bettencourt-Dias et al., 2005; Basto et al., 2006; Rodrigues-Martins et al., 2008). In addition, centrioles are required in postmeiotic spermatids to form the axoneme (Bettencourt-Dias et al., 2005).

The centrosome cycle during Drosophila male meiosis is unique in that centrosomes undergo dramatic changes in position and a reductive division (Fuller, 1993). After premeiotic S phase, centrosomes separate from the nucleus and move to the cortex. Cortical centrosomes nucleate astral microtubules in late G2 and move to the nucleus in early prophase to initiate spindle formation. Centrosomes divide without duplicating in meiosis II; thus, each postmeiotic spermatid contains one centriole, which converts to a basal body to direct axoneme formation.

Centrosome behavior has been shown in many systems to depend on dynein, a minusend-directed microtubule motor complex (Hook and Vallee, 2006). There are two general forms of dynein; axonemal dynein, which provides the force for movement of cilia and flagella, and cytoplasmic dynein. Both forms are composed of one to three heavy chain motor subunits, as well as multiple intermediate, light intermediate, and light chains. Another large multimeric complex, dynactin, enhances dynein’s motor activity and plays essential roles in regulating its subcellular localization and interactions with other proteins (Schroer, 2004). Cytoplasmic dynein plays key roles in a variety of biological processes such as nucleus-centrosome interactions, spindle assembly, chromosome segregation, nuclear migration, and cell movement. In Drosophila, dynein regulates many aspects of development, including both male and female gametogenesis (Gepner et al., 1996).

We report here that asunder (previously known as Mat89Bb) is required in Drosophila for male fertility. Primary spermatocytes of asunder testes undergo prophase arrest with defects in nucleus-centrosome coupling; cells that overcome arrest exhibit abnormal meiotic spindle assembly, chromosome segregation, and cytokinesis. Additionally, nucleus-basal body associations are disrupted during postmeiotic stages of differentiation. We show that this constellation of defects in germline cells of asunder males is likely due to reduced perinuclear localization of dynein-dynactin.


Drosophila stocks y w was used as the "wild-type" stock. -tubulin-GFP flies were a gift from H. Oda and Y. Akiyama-Oda. The Dmn-GFP stock has been previously described (Wojcik et al., 2001).

piggyBac insertion line f02815 was from the Exelixis Collection (Harvard Medical School). asp alleles were a gift from C. Gonzalez (Rebollo et al. 2004). The following lines were from Bloomington Stock Center: Df(3R)Exel7329, nanos-Gal4-VP16, piggyBac transposase, twe1, Tub85DDrv1, fwdneo1, Df(3L)7C, Dhc64C4-19, Dhc64C6-10, Df(3L)fz-GF3b, and Gl1.

DNA clones and transgenics cDNA clone LD33046 encoding ASUN was from the Drosophila Gene Collection. A cDNA clone encoding human GCT1 (ID 2989678) was from Open Biosystems. UASp-MycASUN was created by subcloning of PCR-amplified coding sequence from LD33046 into a modified version of UASp that confers an N-terminal Myc tag (Rorth, 1998). For male germline expression of fluorescently tagged ASUN, cDNA encoding ASUN with an N-terminal GFP or Cherry tag was subcloned into testis expression vector tv3 (gift from J. Brill) (Wong et al., 2005). Two independent transgenic lines with male germline expression of GFP-ASUN were used in this study: line 16 (chromosome III) to demonstrate perinuclear localization of GFPASUN (Figure 10) and line 11 (chromosome II) for all other experiments. Transgenic lines were generated by P-element-mediated transformation via embryo injection (Rubin and Spradling, 1982).

Cytological analyses of live and fixed testes Live testes cells were prepared for examination by phase contrast or fluorescent microscopy as described (Kemphues et al., 1980). Methanol fixation (for immunofluorescence and visualization of cells expressing -tubulin-GFP) was performed as follows: slides of squashed testes were snap-frozen, immersed in methanol for 10 minutes at -20˚C after coverslip removal, and washed twice in phosphate-buffered saline. Formaldehyde fixation (for actin staining and visualization of cells co-expressing Cherry-ASUN and DMN-GFP) was performed as described (Gunsalus et al., 1995).

Primary antibodies were used as follows: rat anti--tubulin (Mca77G, 1:500, Accurate Chemical & Scientific Corp.), mouse anti--tubulin (GTU-88, 1:250, Sigma), mouse anti-Cyclin A (A12, 1:50, Developmental Studies Hybridoma Bank), mouse anti-Cyclin B (F2F4, 1:50, Developmental Studies Hybridoma Bank), and mouse anti-dynein heavy chain (P1H4, 1:150) (McGrail and Hays, 1997).

The following antibodies were used to assess centrosome integrity:

rabbit anti-CNN (1:1000), rabbit anti-SPD-2 (1:200), rabbit anti-D-PLP (1:250), rabbit anti-SASand rabbit anti-TACC (1:500) (gifts from J. Raff except for anti-CNN, a gift from W.

Theurkauf). Cy2- and Cy3-conjugated secondary antibodies were used at 1:800. Actin individualization cones were stained with Alexa Fluor 594 phalloidin (1:100, Invitrogen). Fixed samples were mounted in phosphate-buffered saline with DAPI (0.2 g/ml) to visualize DNA.

Fluorescent images were obtained using one of two microscopes: Nikon Eclipse 80i with Plan-Apo 100x and Plan-Fluor 40x objectives or Zeiss Axioplan with Neo-Fluor Ph2 40x objective. Bright field images of whole testes were obtained using a Zeiss Stemi 2000-CS stereoscope. Phase contrast images were captured using one of three microscopes: Nikon Eclipse 80i with Plan 20x Ph1 objective, Zeiss Axiophot with Neo-Fluor Ph2 40x objective, or Zeiss Axioplan with Neo-Fluor Ph2 40x objective. The ratio of the intensity of perinuclear to cytoplasmic DMN-GFP signal and of cytoplasmic to nuclear Cherry-ASUN signal in individual G2 spermatocytes was determined using Adobe Photoshop.

Mammalian cell transfection, staining, and microscopy HeLa cells were maintained in Dulbecco’s modified Eagle Medium (DMEM) containing 10% fetal bovine serum. Plasmids for expression of N-terminally tagged versions of Drosophila ASUN and/or human GCT1 (GFP or Myc, respectively) generated by subcloning into pCS2 were transfected into cells using Lipofectamine 2000 (Invitrogen) according to manufacturer’s directions.

Cells were plated on fibronectin-coated coverslips 21 hours post-transfection and fixed three hours later. For direct GFP fluorescence alone or in combination with phospho-histone H3 or Myc immunostaining, cells were fixed 20 minutes with 4% formaldehyde in CBS (10 mM MES pH 6.1, 138 mM KCl, 3 mM MgCl2, 2 mM EGTA, 0.32 M sucrose). For direct GFP fluorescence in combination with dynein intermediate chain staining, cells were fixed 5 minutes at -20˚C in methanol. Cells were permeabilized 10 minutes with 0.5% Triton X-100 in Trisbuffered saline. For nocodazole treatment, cells were exposed to 5 g/ml nocodazole for 3 hours prior to fixation. Primary antibodies were used as follows: mouse anti-c-Myc (9E10, 1:1000), rabbit anti-phospho-histone H3 (Mitosis Marker, 1:200, Upstate Antibody), and mouse antidynein intermediate chain (74.1, 1:100, Santa Cruz). Fluorescently conjugated secondary antibodies were used at 1:5000. Slides were mounted in Vectashield with DAPI (Vector Labs).

Images were acquired using a Nikon Eclipse 80i microscope equipped with a CoolSNAP ES camera (Photometrics) and Plan-Apo 60X objective. For experiments involving quantification, at least 400 cells per condition were scored.

RT-PCR RNA was extracted from testes of newly eclosed males using RNA STAT-60 (Tel-Test, Inc.). RT-PCR was performed using Ready-To-Go RT-PCR Beads (GE Healthcare). 5’ and 3’

regions of asun (Products 1 and 2, respectively) were amplified using the following primer sets:

5’-gccgcgcattcccaacaagg-3’ (1F), 5’-gcggcatttccagcaagact-3’ (1R), 5’-actaaatgccaccacaatgc-3’ (2F), and 5’-gcgtcccgagaaatccaatc-3’ (2R). A region of Mst89B, which is expressed specifically in the testes, was amplified as a positive control using the following primer set: 5’tgcaacctcaagttcagtcg-3’ (Mst89B-F) and 5’-gcgtcccgagaaatccaatc-3’ (Mst89B-R) (Stebbings et al., 1998).

Immunoblots Protein extracts were prepared by homogenizing dissected testes from newly eclosed males in SDS sample buffer. The equivalent of two testes pairs was loaded per lane. Proteins were transferred to nitrocellulose for immunoblotting using standard techniques. Primary antibodies were used as follows: mouse anti-Cyclin A (A12, 1:50, Developmental Studies Hybridoma Bank), rabbit anti-Cyclin B (Rb271, 1:2000, gift from D. Glover), mouse anti-actin (pan Ab-5, 1:1000, NeoMarkers), mouse anti-dynein heavy chain (P1H4, 1:2000) (McGrail and Hays, 1997), mouse anti-dynein intermediate chain (74.1, 1:1000, Santa Cruz), mouse antiDynamitin (1:250, BD Biosciences), and mouse anti-tubulin (DM1, 1:7000). HRP-conjugated secondary antibodies and chemiluminescence were used to detect primary antibodies.

RESULTS asunder is required for male fertility Mat89Bb (Maternal transcript 89Bb) was originally identified by virtue of its rich expression in Drosophila ovaries (Stebbings et al., 1998). In a Drosophila genome-scale biochemical screen, we identified Mat89Bb as a substrate of the kinase encoded by pan gu (png) that is required in the early embryo (Lee et al., 2005). To assess Mat89Bb's developmental role, we obtained a candidate mutant (Figure 1A). f02815 is a piggyBac insertion in the second intron of Mat89Bb predicted to remove the C-terminal 64 residues from the full length (689-amino acid) protein (Thibault et al., 2004). Homozygous Mat89Bbf02815 adults are viable and appear normal. Unexpectedly, we found that Mat89Bbf02815 males are almost completely sterile (Table 1), whereas mutant females have only slightly decreased fertility (data not shown). We have changed the Mat89Bb gene name to “asunder” (“asun”) to better reflect its loss-of-function phenotype as described below.

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