The high degree of functional conservation of genes involved in the cell cycle combined with the superb genetics and cytology of Drosophila melanogaster make it an ideal model organism for studying cell-cycle regulation in a developmental context. Spermatogenesis utilizes mitotic and meiotic cell cycles coordinated with growth and differentiation programs to generate functional sperm. By mutational analysis, we have identified asunder (asun), which encodes an evolutionarily conserved protein, as an essential regulator of Drosophila 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. Dynein is a minus end-directed microtubule motor complex that is required for diverse biological processes, from transport of intracellular cargo to cell migration. Dynein is controlled at multiple levels, including regulation of its subcellular localization;the mechanisms underlying the targeting of dynein to various sites within cells, however, are not well understood. Our current model is that asun coordinates spermatogenesis by promoting dynein recruitment to the nuclear surface, a critical step that is required for nucleus-centrosome coupling at M-phase entry and fidelity of meiotic divisions. ASUN exhibits a dynamic localization pattern during Drosophila male meiosis, and the timing of its release from the nucleus to the cytoplasm correlates with the appearance of dynein on the nuclear surface in G2 spermatocytes. We will assess whether this regulated movement of ASUN within spermatocytes is critical for controlling the activity of dynein. We propose experiments that will allow us to gain a more detailed understanding of the mechanism by which dynein is recruited to the nuclear surface with a focus on elucidating the role of ASUN in this process. Our preliminary data suggest that dynein anchored on the nuclear surface of spermatocytes may preferentially bind to microtubules that are post-translationally modified by acetylation. We will test our hypothesis that this pool of acetylated microtubules mediates key events of Drosophila male meiosis, including nucleus-centrosome coupling. The proposed experiments have the potential to illuminate the mechanism of action of ASUN, to identify additional factors required for recruitment of the dynein motor complex to the nuclear surface, and to define the role of microtubule acetylation during spermatogenesis.
The mechanisms controlling localization of dynein motors within cells are not well understood. We have shown that ASUN is required for recruitment of dynein to the nuclear surface of Drosophila spermatocytes, a critical event for fidelity of meiotic divisions. Because the vertebrate homolog of ASUN also controls dynein localization in cultured human cells during mitosis, elucidation of the mechanism of action of ASUN may have important implications for human diseases such as cancer.
|Sitaram, Poojitha; Merkle, Julie A; Lee, Ethan et al. (2014) asunder is required for dynein localization and dorsal fate determination during Drosophila oogenesis. Dev Biol 386:42-52|
|Sitaram, Poojitha; Hainline, Sarah Grace; Lee, Laura Anne (2014) Cytological analysis of spermatogenesis: live and fixed preparations of Drosophila testes. J Vis Exp :e51058|
|Wallace, Heather A; Merkle, Julie A; Yu, Michael C et al. (2014) TRIP/NOPO E3 ubiquitin ligase promotes ubiquitylation of DNA polymerase ?. Development 141:1332-41|
|Jodoin, Jeanne N; Sitaram, Poojitha; Albrecht, Todd R et al. (2013) Nuclear-localized Asunder regulates cytoplasmic dynein localization via its role in the integrator complex. Mol Biol Cell 24:2954-65|
|Merkle, Julie A; Rickmyre, Jamie L; Garg, Aprajita et al. (2009) no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila. Development 136:449-59|
|Anderson, Michael A; Jodoin, Jeanne N; Lee, Ethan et al. (2009) Asunder is a critical regulator of dynein-dynactin localization during Drosophila spermatogenesis. Mol Biol Cell 20:2709-21|
|Von Stetina, Jessica R; Tranguch, Susanne; Dey, Sudhansu K et al. (2008) alpha-Endosulfine is a conserved protein required for oocyte meiotic maturation in Drosophila. Development 135:3697-706|
|Rickmyre, Jamie L; Dasgupta, Shamik; Ooi, Danny Liang-Yee et al. (2007) The Drosophila homolog of MCPH1, a human microcephaly gene, is required for genomic stability in the early embryo. J Cell Sci 120:3565-77|