Student Publication: Asunder promotes dynein recruitment
Jodoin JN, Shboul M, Sitaram P, Zein-Sabatto H, Reversade B, Lee E, Lee LA.
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232-8240, USA.
Abstract
Recruitment of dynein motors to the nuclear surface is an essential step for nucleus-centrosome coupling in prophase. In cultured human cells, this dynein pool is anchored to nuclear pore complexes through RanBP2-Bicaudal D2 (BICD2) and Nup133- centromere protein F (CENP-F) networks. We previously reported that the asunder (asun) gene is required in Drosophila spermatocytes for perinuclear dynein localization and nucleus-centrosome coupling at G2/M of male meiosis. We show here that male germline expression of mammalian Asunder (ASUN) protein rescues asun flies, demonstrating evolutionary conservation of function. In cultured human cells, we find that ASUN down-regulation causes reduction of perinuclear dynein in prophase of mitosis. Additional defects after loss of ASUN include nucleus-centrosome uncoupling, abnormal spindles, and multinucleation. Coimmunoprecipitation and overlapping localization patterns of ASUN and lissencephaly 1 (LIS1), a dynein adaptor, suggest that ASUN interacts with dynein in the cytoplasm via LIS1. Our data indicate that ASUN controls dynein localization via a mechanism distinct from that of either BICD2 or CENP-F. We present a model in which ASUN promotes perinuclear enrichment of dynein at G2/M that facilitates BICD2- and CENP-F-mediated anchoring of dynein to nuclear pore complexes.
mASUN partially rescues spermatogenesis defects of Drosophila asun mutants. (A) Anti-CHY immunoblot of testes extracts from Drosophila wild-type (WT) males with or without germline expression of CHY-dASUN or CHY-mASUN; a relatively low expression level of a fusion protein of the expected size was observed for the latter. Tubulin was used as loading control. (B, C) Male fertility assays. “Rescue” indicates asun males with germline expression of CHY-mASUN, which increased the percentage of asun males producing progeny (B) and the average number of progeny per fertile male (C). (D–H) Germline CHY-mASUN expression restored perinuclear dynein in asun primary spermatocytes and spermatids. (D–F) Representative G2 spermatocytes stained for DHC and DNA are shown (D) with bar graphs depicting percentages of spermatocytes exhibiting perinuclear DHC (E) and average ratios of perinuclear to diffusely cytoplasmic DHC signal intensities (F). (G, H) Representative immature spermatids stained for DHC and DNA are shown (G) with bar graph depicting percentages of spermatids exhibiting perinuclear DHC (H). (I, J) Germline CHY-mASUN expression restored nucleus–centrosome coupling in asun primary spermatocytes. (I) Prophase I spermatocytes stained for β-tubulin and DNA. (J) Quantification of nucleus–centrosome coupling in prophase spermatocytes. **p < 0.0001, *p < 0.001. Scale bars, 50 μm.