Nonsense mutations in the gene have been most frequently identified among familial microcephaly patients. Depletion of the orthologue () causes spindle pole unfocusing during mitosis in multiple cell types. However, it remains unknown whether human ASPM has a similar function. Here, by performing CRISPR-based gene knockout (KO) and RNA interference combined with auxin-inducible degron, we show that ASPM functions in spindle pole organisation during mitotic metaphase redundantly with another microcephaly protein, CDK5RAP2 (also called CEP215), in human tissue culture cells. Deletion of the gene alone did not affect spindle morphology or mitotic progression. However, when the pericentriolar material protein CDK5RAP2 was depleted in KO cells, spindle poles were unfocused during prometaphase, and anaphase onset was significantly delayed. The phenotypic analysis of CDK5RAP2-depleted cells suggested that the pole-focusing function of CDK5RAP2 is independent of its known function to localise the kinesin-14 motor HSET (also known as KIFC1) or activate the γ-tubulin complex. Finally, a hypomorphic mutation identified in ASPM microcephaly patients similarly caused spindle pole unfocusing in the absence of CDK5RAP2, suggesting a possible link between spindle pole disorganisation and microcephaly.

IntroductionThe most common cause of autosomal recessive primary microcephaly is a homozygous mutation of the abnormal spindle-like microcephaly-associated (ASPM) gene (Bond et al., 2002, Tan et al., 2014, Bond et al., 2003, Abdel-Hamid et al., 2016. ASPM was originally identified in Drosophila, as the orthologue Asp (abnormal spindle), whose mutation results in abnormal spindle formation (Ripoll et al., 1985). In the absence of Asp, centrosomes are detached from the main body of the spindle, and spindle microtubules (MTs) are unfocused at the pole (Saunders et al., 1997, Wakefield et al., 2001, Morales-Mulia and Scholey, 2005, Ito and Goshima, 2015, Schoborg et al., 2015. Asp is concentrated at the spindle pole, the area enriched with the spindle MT minus ends. The current model is that Asp binds directly to the spindle MT ends using the middle region containing calponin homology domains and cross-links them each other using the C-terminal domain, thus postulating Asp as a critical pole-focusing factor (Ito and Goshima, 2015). Recent studies have also shown that asp mutant flies have reduced brain size (Rujano et al., 2013, Schoborg et al., 2015, which was at least partly attributed to chromosome mis-segregation associated with unfocused spindle poles (Rujano et al., 2013).The cellular function of human ASPM has been evaluated using RNA interference (RNAi)-mediated knockdown. Small interfering RNA (siRNA)-based knockdown in U2OS cells led to spindle misorientation, cytokinesis failure, reduction in mitotic cells, and apoptosis (Higgins et al., 2010), possibly through interactions with the citron kinase (Gai et al., 2016, Paramasivam et al., 2007. Another study observed downregulation of BRCA1 protein upon ASPM knockdown (Zhong et al., 2005). To the best of our knowledge, the pole-focusing defect commonly observed upon Asp depletion in Drosophila has not been reported in studies of human ASPM knockdown. However, RNAi has a general limitation in that residual protein expression might suffice to fulfil the function of the target protein.Moreover, none of the previous RNAi studies of ASPM involved a rescue experiment in which full-length ASPM was ectopically expressed after endogenous ASPM depletion, leaving the possibility that some of the observed phenotypes were derived from off-target effects of the siRNAs utilised. The effect of mutations identified in microcephaly patients has also not been assessed using a cell culture model. Nevertheless, the lack of the spindle pole phenotype is surprising, given the result obtained in Drosophila.In this study, we used CRISPR/Cas9-based knockout (KO) as well as RNAi to decipher the mitotic function of ASPM in the human HCT116 cell line. Our data provide the first demonstration that human ASPM is an important factor in spindle pole organisation, working redundantly with the centrosomal protein CDK5RAP2. Furthermore, a mutation identified in microcephaly patients similarly impaired this function of ASPM. ResultsNo mitotic abnormality upon ASPM KO in the human HCT116 cell ...