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Human Microcephaly ASPM Protein Is a Spindle Pole-Focusing Factor That Functions Redundantly with CDK5RAP2

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Human Microcephaly ASPM Protein Is a Spindle Pole-Focusing Factor That Functions Redundantly with CDK5RAP2 bioRxiv preprint doi: https://doi.org/10.1101/102236; this version posted July 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Human microcephaly ASPM protein is a spindle pole-focusing factor that functions redundantly with CDK5RAP2 Elsa A. Tungadi*, Ami Ito*, Tomomi Kiyomitsu, and Gohta Goshima† Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan †E-mail: [email protected] *These authors contributed equally to this study Nonsense mutations in the ASPM gene have attributed to chromosome mis-segregation been most frequently identified among familial associated with unfocused spindle poles (Rujano et microcephaly patients. Depletion of the al., 2013). Drosophila orthologue causes spindle pole The cellular function of human ASPM has been unfocusing during mitosis in multiple cell types. evaluated using RNA interference (RNAi)-mediated However, it remains unknown whether human knockdown. Small interfering RNA (siRNA)-based ASPM has a similar function. Here, using knockdown in U2OS cells led to spindle CRISPR-based gene knockout (KO) and RNA misorientation, cytokinesis failure, reduction in interference combined with auxin-inducible mitotic cells, and apoptosis (Higgins et al., 2010), degron, we show that ASPM functions in possibly through interactions with the citron kinase spindle pole organisation during mitotic (Gai et al., 2016, Paramasivam et al., 2007). metaphase redundantly with another Another study observed downregulation of BRCA1 microcephaly protein CDK5RAP2 (also called protein upon ASPM knockdown (Zhong et al., 2005). CEP215) in human tissue culture cells. Deletion To the best of our knowledge, the pole-focusing of the ASPM gene alone did not affect spindle defect commonly observed upon Asp depletion in morphology or mitotic progression. However, Drosophila has not been reported in studies of when the pericentriolar material protein human ASPM knockdown. However, RNAi has a CDK5RAP2 was depleted in ASPM KO cells, general limitation in that residual protein expression spindle poles were unfocused during might suffice to fulfil the function of the target protein. prometaphase and anaphase onset was Moreover, none of the previous RNAi studies of significantly delayed. The phenotypic analysis ASPM involved a rescue experiment in which of CDK5RAP2-depleted cells suggested that the full-length ASPM was ectopically expressed after pole-focusing function of CDK5RAP2 is endogenous ASPM depletion, leaving the possibility independent of its known function to localise that some of the observed phenotypes were derived the kinesin-14 motor HSET or activate the from off-target effects of the siRNAs utilised. The γ-tubulin complex. Finally, a hypomorphic effect of mutations identified in microcephaly mutation identified in ASPM microcephaly patients has also not been assessed using a cell patients similarly caused spindle pole culture model. Nevertheless, the lack of the spindle unfocusing in the absence of CDK5RAP2, pole phenotype is surprising, given the result suggesting a possible link between spindle pole obtained in Drosophila. disorganisation and microcephaly. In this study, we used CRISPR/Cas9-based knockout (KO) as well as RNAi to decipher the Introduction mitotic function of ASPM in the human HCT116 cell line. Our data provide the first demonstration that The most common cause of autosomal recessive human ASPM is an important factor in spindle pole primary microcephaly is a homozygous mutation of organisation, working redundantly with the the abnormal spindle-like microcephaly-associated centrosomal protein CDK5RAP2. Furthermore, a (ASPM) gene (Bond et al., 2002, Tan et al., 2014, mutation identified in microcephaly patients similarly Bond et al., 2003, Abdel-Hamid et al., 2016). ASPM impaired this function of ASPM. was originally identified in Drosophila, as the orthologue Asp (abnormal spindle), whose mutation Results results in abnormal spindle formation (Ripoll et al., 1985). In the absence of Asp, centrosomes are No mitotic abnormality upon ASPM KO in the detached from the main body of the spindle, and human HCT116 cell line spindle microtubules (MTs) are unfocused at the To precisely assess the spindle pole-organising pole (Saunders et al., 1997, Wakefield et al., 2001, function of human ASPM, we utilised the human Morales-Mulia and Scholey, 2005, Ito and Goshima, colorectal cancer cell line HCT116, which has a 2015, Schoborg et al., 2015). Asp is concentrated at stable diploid karyotype, assembles well-focused the spindle pole, the area enriched with the spindle bipolar spindles, and is highly amenable to MT minus ends. The current model is that Asp binds CRISPR/Cas9-based genome editing and other directly to the spindle MT ends using the middle gene perturbation techniques such as RNAi and region containing calponin homology domains and auxin-inducible degron (AID) (Natsume et al., 2016). cross-links them each other using the C-terminal We aimed to delete the ASPM gene in HCT116 cells domain, thus postulating Asp as a critical using CRISPR/Cas9-based genome editing. Since it pole-focusing factor (Ito and Goshima, 2015). was reported that all ASPM isoforms are translated Recent studies have also shown that asp mutant from a common start codon in exon 1 (Kouprina et flies have reduced brain size (Rujano et al., 2013, al., 2005), we aimed to select homozygous KO lines Schoborg et al., 2015), which was at least partly by inserting a drug-resistant marker in exon 1 and 1 bioRxiv preprint doi: https://doi.org/10.1101/102236; this version posted July 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. deleting the start codon (Fig. S2A, B). (1.2 ± 0.0-fold increase, NEBD to anaphase onset, n Immunofluorescence microscopy using anti-ASPM = 3 experiments). In sharp contrast, when antibody indicated that ASPM was significantly CDK5RAP2 was depleted in the ASPM KOC depleted in the selected lines (termed KOExon1), background, >60% of the cells exhibited unfocused confirming successful editing (Fig. S2C). However, spindle poles; spindle MTs were not persistently in the course of this project, we noticed that some associated with the pole during metaphase (Fig. 2A, residual protein expression was still detected with B). In these cells, anaphase onset was drastically this antibody when the image contrast was further delayed (4.3 ± 1.5-fold increase in enhanced (Fig. S2C, quantification is presented in prometaphase/metaphase duration, n = 3 Fig. 4B). Therefore, we also selected the cell lines in experiments; Fig. 2C). which the entire open reading frame was replaced As another means to deplete CDK5RAP2, we by a marker gene cassette (Fig. 1A, S1). The polar introduced the auxin-inducible degron (AID) system, staining completely disappeared in the complete KO which allows the acute degradation of a target line (termed KOC; Fig. 1B, 4B). In either KO line, protein (Nishimura et al., 2009). We tagged time-lapse microscopy of MTs did not reveal any endogenous CDK5RAP2 with mCherry and abnormality in mitotic progression and spindle mini-AID (mAID) sequences in cells possessing the formation, suggesting that ASPM is not an essential gene encoding Oryza sativa (Os) TIR1, the auxin gene for mitosis in this cell line (Fig. 1C, D; S2D, E; responsive F-box protein that constitutes a and Movie 1). functional SCF ubiquitin ligase in human cells (Fig. S3A–C). In this cell line, OsTIR1 protein was Spindle poles were unfocused upon double induced by doxycycline hyclate (Dox) (Natsume et depletion of ASPM and CDK5RAP2 al., 2016), and CDK5RAP2-mAID-mCherry was The centrosome constitutes the focal point of subsequently degraded upon auxin (indole-3-acetic spindle MTs at the pole in animal somatic cells. acid; IAA) addition to the culture medium (Fig. S3D). However, in flies and mammals, acentrosomal We deleted the first exon of ASPM in this line and spindles with two focused poles can be assembled observed spindle dynamics after 24 h of auxin after centrosomal protein depletion/inhibition, treatment. We observed an identical phenotype to indicating that centrosomes are not a prerequisite that observed upon RNAi (Fig. 2D; unfocusing was for pole focusing (Wong et al., 2015, Megraw et al., observed in 82% (n = 68) or 86% (n = 72) of KOExon1 2001). Nevertheless, centrosomes play a supportive #3 and KOExon1 #20 lines, respectively). We role in spindle MT focusing, perhaps by supplying concluded that ASPM is required for pole focusing additional MTs for MT cross-linking motors and when the CDK5RAP2 function is compromised. MAPs for the interaction (Goshima et al., 2005, Ito To investigate if the synthetic polar phenotype and Goshima, 2015, Silk et al., 2009, Wakefield et is attributed to γ-tubulin delocalisation and reduction al., 2001, Mountain et al., 1999, Heald et al., 1997, of astral MTs (Fong et al., 2008) or HSET Baumbach et al., 2015, Chavali et al., 2016). We delocalisation (Chavali et al., 2016) in the absence hypothesised that lack of the pole unfocusing of CDK5RAP2, we immunostained for γ-tubulin, phenotype in the absence of ASPM may also be HSET, and MTs after CDK5RAP2 depletion (Fig. partly due to the presence of strong centrosomes in S4). Contrary to the result in U2OS cells, we clearly this cell line. observed γ-tubulin
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