M BoC | ARTICLE The Seckel syndrome and centrosomal protein Ninein localizes asymmetrically to stem cell centrosomes but is not required for normal development, behavior, or DNA damage response in Drosophila Yiming Zhenga,†, Vito Mennellab,c,†, Steven Marksa, Jill Wildongerd, Esraa Elnagdib,c, David A. Agarde, and Timothy L. Megrawa,* aDepartment of Biomedical Sciences, Florida State University, Tallahassee, FL 32306-4300; bDepartment of Biochemistry, University of Toronto, Toronto, M5G 0A4, Canada; cCell Biology Program, The Hospital for Sick Children, Toronto, M5G 1X8, Canada; dDepartment of Biochemistry, University of Wisconsin–Madison, Madison, WI 53706; eThe Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143-2240 ABSTRACT Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome Monitoring Editor (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cog- Yukiko Yamashita nitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem University of Michigan cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is Received: Sep 16, 2015 evolutionarily conserved, yet its role in cell division and development has not been investi- Revised: Mar 18, 2016 gated in a model organism. Here we characterize the single Nin orthologue in Drosophila. Accepted: Mar 28, 2016 Drosophila Nin localizes to the periphery of the centrosome but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian orthologue of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of stem cells. In wing epithelia and muscle, Nin localizes to noncentrosomal microtubule-organizing centers. Surprisingly, loss of nin expression from a nin mutant does not significantly affect embryonic and brain development, fertility, or locomotor performance of mutant flies or their survival upon exposure to DNA-damaging agents. Although it is not essential, our data sug- gest that Nin plays a supportive role in centrosomal and extracentrosomal microtubule orga- nization and asymmetric stem cell division. This article was published online ahead of print in MBoC in Press (http://www INTRODUCTION .molbiolcell.org/cgi/doi/10.1091/mbc.E15-09-0655) on April 6, 2016. Microcephalic primordial dwarfism (PD) is a spectrum of inherited †These authors contributed equally to this work. The authors declare no competing interests. recessive developmental disorders that cause fetal growth failure *Address correspondence to: Tim Megraw ([email protected]). resulting in severe dwarfism, microcephaly, and cognitive deficien- Abbreviations used: Cnn, centrosomin; GSC, germline stem cell; Mira, Miranda; cies (Majewski and Goecke, 1982; Klingseisen and Jackson, 2011; MTOC, microtubule-organizing center; NB, neuroblast; Nin, Ninein; PCM, Megraw et al., 2011; Chavali et al., 2014). The most prevalent PD pericentriolar material. disorders include Seckel syndrome, microcephalic osteodysplastic © 2016 Zheng, Mennella, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it PD (MOPD) types I and II, and Meier–Gorlin syndrome. Mutations of is available to the public under an Attribution–Noncommercial–Share Alike 3.0 at least 14 genes have been associated with PD disorders (Chavali Unported Creative Commons License (http://creativecommons.org/licenses/by -nc-sa/3.0). et al., 2014). The genes identified for Seckel syndrome encode “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of proteins that are fundamental to centrosome function (NIN, the Cell®” are registered trademarks of The American Society for Cell Biology. CEP63, CENPJ/CPAP/SAS-4, and CEP152; Al-Dosari et al., 2010; Supplemental Material can be found at: 1740 | Y. Zheng, V. Mennella, et al. http://www.molbiolcell.org/content/suppl/2016/04/03/mbc.E15-09-0655v1.DC1 Molecular Biology of the Cell Kalay et al., 2011; Sir et al., 2011; Dauber et al., 2012) and the DNA RESULTS damage response (ATR, ATR-interacting protein [ATRIP], DNA2, and A single Nin-family orthologue in Drosophila RBBP8/CTIP; O’Driscoll et al., 2003; Qvist et al., 2011; Ogi et al., To identify putative members of the Nin family in Drosophila, we 2012; Shaheen et al., 2014). However, whether the functions of all of performed a sequence alignment search using the National Center these genes are integrated into a common pathway responsible for for Biotechnology Information PSI-BLAST program with the N- Seckel syndrome is unclear (Arquint et al., 2014; Chavali et al., 2014; terminal region of human Nin as a query. This portion of Nin protein Antonczak et al., 2016). sequence contains two regions conserved in the Nin family in mam- The centrosome is the major microtubule-organizing center mals, and it was shown to associate with components of the γTurc (MTOC) in most animal cells. It is composed of a pair of centri- complex by immunoprecipitation (Casenghi et al., 2003; Delgehyr oles (a mother and its daughter), which organize a supramolecu- et al., 2005). In contrast to the rest of the Ninein primary sequence, lar protein assembly (Mennella et al., 2014; Woodruff et al., the N-terminal domain is also not predicted to be coiled-coil and is 2014), the pericentriolar material (PCM), where microtubule as- therefore more likely to be conserved throughout evolution. This sembly and anchoring is regulated. During late mitosis, the pair analysis identified the gene Bsg25D (CG14025) as the putative of centrioles inherited by each cell mature, allowing them to as- member of the ninein family in Drosophila (Figure 1). Subsequent semble PCM and become centrosomes. Then, after centriole du- phylogenetic analysis revealed that lower metazoan species possess plication in S phase, only the mother centriole is largely respon- a single nin ancestor gene that might have duplicated in the phylum sible for organizing the PCM at mitotic centrosomes (Wang et al., Chordata. In addition to the apparent homology ascertained from 2011). Although the centrosome is dispensable for proper cell sequence similarity, we also found Nin associated with other centro- division (Megraw et al., 1999; Khodjakov et al., 2000; Lecland some proteins (Gopalakrishnan et al., 2011). et al., 2013) and even for most of Drosophila development (Megraw et al., 2001; Basto et al., 2006; Debec et al., 2010), Nin can assemble microtubule-organizing centers mutations in the core centrosome machinery result in a spectrum To test whether Drosophila Nin shares the microtubule anchoring of diseases that cause primordial dwarfisms and impairment of and nucleation function of vertebrate Nin, we expressed Nin–green brain development (microcephaly; Megraw et al., 2011; Chavali fluorescent protein (GFP) in S2 cells, a Drosophila cell line of em- et al., 2015). NIN was recently identified as one of the genes that bryonic origin. For this and all experiments in which a nin transgene cause Seckel syndrome when mutated (Dauber et al., 2012). was expressed, the protein encoded by the nin-RB isoform was However, despite its importance to human health and mamma- used (see Figure 6A later in this paper and Materials and Methods). lian brain development, the functions of Ninein (Nin) at the In the majority of S2 cells, Nin accumulated in large cytoplasmic developmental, cellular, and molecular levels are not clearly assemblies and occasionally clustered in the proximity of the defined. plasma membrane. The Nin-GFP foci in S2 cells were sufficient to In mammals, Nin is enriched around the centriole wall and at establish a partial reorganization of the microtubule cytoskeleton the subdistal appendages, structures present only on the mother into a polarized microtubule array (Figure 2A and Supplemental centriole (Ou et al., 2002), which can anchor microtubules to Figure S1A). This is remarkable, considering that in interphase S2 the centrosome (Delgehyr et al., 2005). Nin and its paralogue cells, centrosomes normally do not act as major microtubule-orga- in vertebrates, Ninein-like protein (Nlp), are γ-tubulin complex– nizing centers and microtubules are nucleated from many regions associated proteins that regulate microtubule nucleation and in the cytoplasm, including Golgi membrane (Rogers et al., 2008). anchoring at centrosomes and noncentrosomal MTOCs Nin structures were not associated with Golgi markers, as shown by (Mogensen et al., 2000; Casenghi et al., 2003; Delgehyr et al., costaining with dGMAP, and do not appear to induce Golgi disper- 2005). sal, in contrast to mammalian cells (Casenghi et al., 2005; Figure In mouse embryonic neural progenitor cells, the older (mother) 2A). Nin colocalized partially with centrosomin (Cnn), a component centrosome is inherited by the self-renewing stem cells at each of the PCM, but was more concentrated in the space surrounding asymmetric division (Wang et al., 2009). RNA interference (RNAi)– the PCM (Figure 3, B and C). mediated Nin knockdown in embryonic mouse brains in utero dis- To better understand the mechanism of microtubule organiza- rupted asymmetric segregation of mother and daughter centro- tion by Nin, we observed individual microtubule nucleation events somes and also reduced the number of radial glia