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«Human Asunder promotes dynein recruitment and centrosomal tethering to the nucleus at mitotic entry a b a a Jeanne N. Jodoin, Mohammad Shboul, ...»

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Human Asunder promotes dynein recruitment and

centrosomal tethering to the nucleus at mitotic entry

a b a a

Jeanne N. Jodoin, Mohammad Shboul, Poojitha Sitaram, Hala Zein-Sabatto, Bruno

b,c a a,1

Reversade, Ethan Lee, and Laura A. Lee

a

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

Nashville, TN 37232-8240, USA

b

Institute of Medical Biology, A*STAR, Singapore, Singapore c Department of Pediatrics, National University of Singapore, Singapore Address correspondence to: Laura Lee (laura.a.lee@vanderbilt.edu) RUNNING HEAD: hASUN regulates dynein at mitotic entry

ABBREVIATIONS

Ab, antibody asun, asunder ASUN, Asunder BICD2, Bicaudal D2 CENP-F, Centromere protein F CHY, mCherry DHC, Dynein heavy chain DIC, Dynein intermediate chain DMN, Dynamitin dASUN, Drosophila Asunder HA, hemagglutinin hASUN, human Asunder LIS1, Lissencephaly 1 mASUN, mouse Asunder NT, Non-targeting NE, Nuclear envelope NEBD, Nuclear envelope breakdown NPC, Nuclear pore complex PH3, phosphorylated histone H3 RPE cells, retinal pigment epithelial cells ABSTRACT Recruitment of dynein motors to the nuclear surface is an essential step for nucleuscentrosome coupling in prophase. In cultured human cells, this dynein pool is anchored to nuclear pore complexes through RanBP2-BICD2 and Nup133-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 herein 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 downregulation causes reduction of perinuclear dynein in prophase of mitosis. Additional

Supplemental Material can be found at:

http://www.molbiolcell.org/content/suppl/2012/10/22/mbc.E12-07-0558v1.DC1.html defects following loss of ASUN include nucleus-centrosome uncoupling, abnormal spindles, and multinucleation. Co-immunoprecipitation and overlapping localization patterns of ASUN and 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.

INTRODUCTION

Cytoplasmic dynein plays critical roles in many cellular processes by carrying out minusend-directed transport along microtubules (Holzbaur and Vallee, 1994). Dynein is a multimeric complex composed of heavy, intermediate, light intermediate, and light chains. Each heavy chain contains six ATPase domains that power the motor. Non-catalytic subunits regulate dynein by linking the complex to its cargo and adaptor proteins. Dynein complexes approach 2 MDa in size, making dynein the largest of all known motor complexes. The composition of dynein complexes and the cellular events requiring these complexes have been defined, although a comprehensive understanding of the mechanisms by which these complexes are regulated within cells is lacking.

Dynein complexes are subject to several modes of regulation, including phosphorylation, subunit composition, subcellular localization, and binding of accessory proteins. Dynactin, another large multimeric complex, was identified through in vitro studies as an activator of dynein; subsequent work suggested that dynein requires dynactin to perform its cellular functions (Schroer, 2004). A dynein adaptor protein, Lissencephaly 1 (LIS1), binds directly to dynein heavy chains and is essential for multiple dynein functions, including coupling of centrosomes to the nucleus during neuronal migration (Tanaka et al., 2004; Kardon and Vale, 2009).

A dynein subpopulation stably anchored to the nuclear envelope (NE) at the G2/M transition is essential for proper nucleus-centrosome coupling in multiple systems (Malone et al., 2003; Anderson et al., 2009; Splinter et al., 2010; Bolhy et al., 2011). Minus-end-directed movement of anchored dynein motors along astral microtubules has been hypothesized to draw centrosomes toward the nuclear surface to facilitate their attachment to the NE prior to nuclear envelope breakdown (NEBD) (Burgess and Knight, 2004). This anchored pool of dynein has also been implicated in the process of NEBD (Beaudouin et al., 2002; Salina et al., 2002).

Although promotion of nucleus-centrosome coupling is a dynein-dependent function that is evolutionarily conserved, the exact molecular mechanisms appear to vary (Salina et al., 2002).

Dynein is anchored to the nuclear surface via KASH and SUN domain-containing proteins in C.

elegans embryogenesis and during neuronal migration in mice (Malone et al., 2003; Zhang et al., 2009). In a cultured mammalian cell line (HeLa), at least two distinct pathways are required to ensure proper execution of this critical event. These pathways both employ nuclear pore complex (NPC) interactions to anchor dynein motors to the nuclear surface. The first pathway uses RanBP2, a nucleoporin that associates with the cytoplasmic face of NPCs, as the docking site for Bicaudal D2 (BICD2) (Splinter et al., 2010). BICD2, directly bound to dynein, moves in a minus-end direction toward the nuclear surface just prior to the G2/M transition, thereby anchoring dynein at the NE. The second pathway uses another nucleoporin, Nup133, as a docking site for Centromere protein F (CENP-F) (Bolhy et al., 2011). Prior to G2/M, CENP-F directly binds dynein-bound NudE/EL; CENP-F then simultaneously binds Nup133 to anchor dynein, NudE/EL, and ultimately the centrosomes to the NE.





We previously identified asunder (asun; formerly known as Mat89Bb) as an essential regulator of dynein localization during Drosophila spermatogenesis (Anderson et al., 2009).

Spermatocytes of asun mutant testes arrest at prophase of meiosis I with centrosomes unattached to the nucleus. asun spermatocytes that progress beyond this arrest exhibit defects in meiotic spindle assembly, chromosome segregation, and cytokinesis. The severe loss of perinuclear dynein in asun spermatocytes is the likely basis for this constellation of defects. Our studies revealed that Drosophila ASUN (dASUN) plays a key role in recruiting dynein to the nuclear surface at G2/M, a critical step to establish nucleus-centrosome coupling and fidelity of meiotic divisions.

The human homolog of asun (also known as GCT1 or C12ORF11) was originally identified in a screen for genes upregulated in testicular seminomas (Bourdon et al., 2002).

While expression of Drosophila asun is limited to male and female germline cells, transcripts of the mouse homolog were detected in both germ lines and all somatic cells surveyed (Stebbings et al., 1998; Bourdon et al., 2002). These findings suggested that Asunder (ASUN) might play a broader role in mammals.

To further investigate the mechanism by which ASUN regulates dynein, we examined the function of the human homolog of ASUN (hASUN) in this study. We find that, like BICD2 and CENP-F, hASUN is required in cultured cells for enrichment of dynein on the nuclear surface at the onset of mitosis. hASUN depletion leads to mitotic defects, which are likely secondary to failure of dynein localization. We present a model in which hASUN acts via a mechanism distinct from that of BICD2 and CENP-F to promote dynein recruitment to the nuclear surface at G2/M, a critical step to establish nucleus-centrosome coupling and fidelity of mitotic divisions.

RESULTS

Mammalian ASUN can functionally replace dASUN during spermatogenesis The predicted hASUN and mouse ASUN (mASUN) proteins are 95% identical and 97% similar to each other; comparison of the predicted mammalian and Drosophila ASUN proteins revealed that they are 43% identical and 64% similar. We sought to determine if a mammalian form of ASUN could functionally replace dASUN in vivo. Using the Drosophila model system, we established transgenic lines expressing mCherry-tagged mASUN (CHY-mASUN) exclusively in the male germ line (Figure 1A). We found that the presence of a single copy of the CHY-mASUN transgene significantly increased the percentage of asunf02815 males (hypomorphic allele) that produced adult progeny (Figure 1B) (Anderson et al., 2009). We also observed a significant increase in the number of adult progeny produced per fertile male, albeit not to the level of wild-type controls (Figure 1C). We previously reported that germline expression of CHY-tagged dASUN fully rescued the sterility of asun males; the partial rescue obtained by germline expression of the mouse homolog might be due to the relatively low level of expression of this fusion protein in the fly testes (Figure 1A) (Anderson et al., 2009).

We previously identified a critical role for dASUN in recruitment of dynein motors to the nuclear surface and nucleus-centrosome coupling in Drosophila male germline cells (Anderson et al., 2009). We next asked whether a mammalian form of ASUN could similarly regulate these events. The severe reduction of perinuclear dynein heavy chain (DHC) that is a hallmark feature of asun G2 spermatocytes and immature spermatids was almost fully restored to wild-type levels by transgenic CHY-mASUN expression (Figure 1, D-H). We also observed a significant increase in the degree of nucleus-centrosome coupling at prophase in asun spermatocytes harboring the CHY-mASUN transgene (Figure 1, I and J). Together, these findings reveal that mASUN can functionally replace its Drosophila homolog in vivo to regulate dynein localization during spermatogenesis and suggest that the molecular function of ASUN is conserved across phyla.

hASUN is required for dynein anchoring to the NE at prophase Based on the role of dASUN during male meiosis and the broader expression pattern of mASUN, we reasoned that vertebrate homologs of ASUN might promote dynein recruitment to the NE of somatic cells at the onset of mitosis. To test this hypothesis, we performed siRNAmediated knockdown of hASUN in HeLa cells. We confirmed that ASUN was efficiently depleted from cells by immunoblotting with anti-peptide antibodies (M-hASUN Ab and ChASUN Ab) directed against distinct epitopes in the C-terminal region of hASUN (Figure S1A).

siRNA-treated cells were briefly incubated with nocodazole to enhance perinuclear localization of dynein-dynactin complexes and accessory proteins prior to fixation and immunostaining for dynein intermediate chain (DIC) (Beswick et al., 2006; Hebbar et al., 2008; Splinter et al., 2010;

Bolhy et al., 2011). We found that ~91% of NT-siRNA (non-targeting control) prophase cells showed strong perinuclear dynein staining (Figure 2, A and D-F). After hASUN knockdown, however, only ~30% of cells exhibited this pattern; instead, the majority of prophase cells displayed diffuse localization of dynein in the cytoplasm (Figure 2, B and D-F). Using a second independent hASUN-siRNA sequence that efficiently depleted ASUN from cells, we observed a similar reduction in the percentage of prophase cells with perinuclear dynein (Figure S1, B-D).

We considered the possibility that hASUN might function to destabilize microtubules; in that case, downregulation of hASUN could inhibit nocodazole-induced depolymerization of microtubules, thereby blocking access of dynein to the NE. We performed immunostaining experiments to assess whether microtubules undergo a normal degree of nocodazole-induced depolymerization following loss of hASUN. We observed essentially identical Tubulin staining patterns for NT-siRNA versus hASUN-siRNA cells in response to nocodazole treatment, suggesting that the lack of perinuclear dynein observed in hASUN-siRNA cells is not secondary to gross alterations of the microtubule network (Figure S2).

To further confirm that loss of perinuclear dynein in hASUN-siRNA HeLa cells was due to depletion of endogenous hASUN, we performed a rescue experiment by transiently expressing CHY-tagged dASUN (refractory to hASUN siRNA). CHY-dASUN restored perinuclear dynein in hASUN-siRNA prophase cells to levels similar to that of control cells (Figure 2, C-F). These results confirmed that loss of perinuclear dynein in hASUN-siRNA cells was specifically caused by hASUN depletion and demonstrated that dASUN can replace its human homolog to promote dynein recruitment to the NE at prophase.

We considered an alternative possibility that hASUN is required for stability of dyneindynactin complexes rather than to promote their enrichment on the NE. Depletion of individual dynein-dynactin components can destabilize other subunits of these complexes (Schroer, 2004;

Mische et al., 2008). We immunoblotted for DIC and Dynamitin (DMN; dynactin subunit) in extracts of HeLa cells following hASUN knockdown and found no changes in their cellular levels (Figure S3A). We also performed sucrose density gradient analysis to assess whether dynein complexes remained intact after hASUN knockdown and found no change in migration profiles (Figure S3B). Together, these data suggest that hASUN is not required to maintain integrity of dynein-dynactin complexes.

hASUN is required for proper coupling of centrosomes to the NE at prophase Perinuclear dynein is essential for proper tethering of centrosomes to the NE at G2/M (Splinter et al., 2010; Tanenbaum et al., 2010; Bolhy et al., 2011). Based on the loss of perinuclear dynein we observed in hASUN-siRNA HeLa cells at prophase, we predicted that nucleus-centrosome coupling defects would ensue. We used the human osteosarcoma U2OS cell line for these studies. Due to the relatively decreased density of the microtubule network in these cells, centrosomes undergo a more dramatic migration from the nucleus to the cortex at interphase and back to the nuclear surface at G2/M (Akhmanova and Hammer, 2010).

We found that ~19% of hASUN-siRNA U2OS cells exhibited loss of coupling of one or both centrosomes to the NE at prophase compared to a baseline rate of ~4% in control cells (Figure 3, A-E; Figure S1A). We measured an average distance of ~8 µm between the nucleus and centrosomes in hASUN-siRNA cells compared to a baseline distance of ~3 µm in control cells (Figure 3F). These data indicate that hASUN is required for normal linkage of centrosomes to the NE during prophase, and they further demonstrate evolutionary conservation of function of ASUN homologs.



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