HIGHLIGHTED ARTICLE | INVESTIGATION Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes Arunika Das,* Shital J. Shah,* Bensen Fan,* Daniel Paik,* Daniel J. DiSanto,* Anna Maria Hinman,* Jeffry M. Cesario,* Rachel A. Battaglia,* Nicole Demos,* and Kim S. McKim*,†,1 *Waksman Institute, Rutgers, The State University of New Jersey, New Jersey 08854, and †Department of Genetics, Rutgers, The State University of New Jersey, New Jersey 08854 ABSTRACT Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were charac- terized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in meta- phase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule– chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation. KEYWORDS meiosis; synthetic lethal mutation; homolog bi-orientation; spindle; chromosome segregation; Drosophila HROMOSOMES are segregated during cell division by the in oocytes of many animals occurs in the absence of centro- Cspindle, a bipolar array of microtubules. In somatic cells, somes. This may contribute to the high rates of segregation spindle assembly is guided by the presence of centrosomes at errors that are maternal in origin and are a leading cause of the poles. In this conventional spindle assembly model, the miscarriages, birth defects, and infertility (Herbert et al. kinetochores attach to microtubules from opposing centro- 2015). How a robust spindle assembles without guidance somes and tension is established. This satisfies the spindle from the centrosomes is not well understood. While it is clear assembly checkpoint, which then allows the cell to proceed to that the chromosomes can recruit microtubules and drive anaphase (Musacchio and Salmon 2007). Cell division is spindle assembly (Tseng et al. 2010; Dumont and Desai completed by recruiting proteins to a midzone of antiparallel 2012), how a bipolar spindle is organized and chromo- microtubules that forms between the segregated chromo- somes make the correct attachments to microtubules is somes, signaling furrow formation (Fededa and Gerlich not understood. 2012; D’Avino et al. 2015). However, spindle morphogenesis The Drosophila oocyte provides a genetically tractable sys- tem for the identification of genes involved in acentrosomal Copyright © 2016 by the Genetics Society of America spindle assembly.Substantial evidence in Drosophila suggests doi: 10.1534/genetics.115.181081 that the chromosomes direct microtubule assembly, subse- Manuscript received July 23, 2015; accepted for publication November 6, 2015; published Early Online November 11, 2015. quent elongation of the spindle, and establishment of spindle Supporting information is available online at www.genetics.org/lookup/suppl/ bipolarity (Theurkauf and Hawley 1992; Matthies et al. doi:10.1534/genetics.115.181081/-/DC1. 1Corresponding author: Rutgers, the State University of New Jersey, Waksman Institute, 1996; Doubilet and McKim 2007). We have also shown that 190 Frelinghuysen Rd., Piscataway, NJ 08854. E-mail: [email protected] the kinesin-6 protein Subito, a homolog of human MKLP2 Genetics, Vol. 202, 61–75 January 2016 61 with a role in cytokinesis (Neef et al. 2003), is also essential spindle, however, demonstrating meiosis-specific features of for organizing the meiotic spindle and the bi-orientation of this structure. For example, Polo kinase may be required only homologous chromosomes (Giunta et al. 2002; Jang et al. for kinetochore function while the RhoGEF Pebble was not 2005; Radford et al. 2012). Subito colocalizes with members required for meiosis. In summary,this is the first documentation of the chromosomal passenger complex (CPC), which is com- that proteins known to be required for anaphase/telophase posed of the scaffolding subunit INCENP, the kinase Aurora andcytokinesisinmitoticcellsarealsoessentialinmeiotic B, and the targeting subunits Survivin (Deterin) and Borealin acentrosomal spindle assembly and chromosome bi-orientation. (Dasra) (Ruchaud et al. 2007). The CPC has a critical role in assembling the acentrosomal spindle and segregating chro- Materials and Methods mosomes (Colombié et al. 2008; Radford et al. 2012). In fi addition, with Subito, the CPC localizes to the equatorial region De ciency and mutagenesis screens for synthetic lethality ofthemeioticmetaphaseIspindleandaremutuallydependent for their localization (Jang et al. 2005; Radford et al. 2012). To test synthetic lethality of third chromosome mutations and This equatorial region is composed of antiparallel microtubules deficiencies, cn sub bw/CyO; e/ TM3, Sb females were crossed and is a structure that includes a plethora of proteins (Jang to Df/TM3, Sb females (Supporting Information, Figure S1). et al. 2005). Assembling a central microtubule array may be The cn sub bw/ +; Df/TM3 males were then crossed to sub a conserved mechanism to organize a bipolar spindle in the bw/CyO or cn sub/CyO females to generate cn sub bw/sub bw; absence of centrosomes (Dumont and Desai 2012). Df/+ progeny. The frequency of these progeny was compared The meiotic central spindle, while assembling during meta- to cn sub bw/sub bw; TM3/+ siblings to measure the synthetic phase, has several features and proteins associated with the lethal phenotype as a percentage of relative survival. midzone present during anaphase in mitosis. Indeed, Subito is The mutagenesis screen was performed for the second required for the localization of the CPC to the midzone at chromosome using ethyl methanesulfonate (EMS). y/y+Y; anaphase (Cesario et al. 2006). The mitotic spindle midzone sub131 bw sp/SM6 males were exposed to 2.5 mM EMS in proteins function in anaphase and telophase to direct abscis- 1% sucrose overnight. About 25 mutagenized males were sion, furrow formation, and cytokinesis (Glotzer 2005; mated to 50 al dp b pr Sp bw/SM6 virgin females (Figure D’Avino et al. 2015). The role of these proteins in the Dro- S2). Single sub131 bw sp*/SM6 (asterisk denotes random sophila acentrosomal meiotic spindle assembly pathway is mutations) males were mated with virgin cn sub1 bw/SM6 unclear, however, since there is no cytokinetic function re- females, and the progeny were scored for the absence of quired at metaphase I and Drosophila does not extrude polar brown-eyed flies, which indicates a synthetic lethal interac- bodies (Callaini and Riparbelli 1996). It is possible that these tion between the heterozygous EMS-induced mutation and proteins are loaded in the central spindle at metaphase for a the sub mutant. Initially, 51 synthetic lethal lines were iso- function later in meiotic anaphase, as has been proposed for lated. Each line was retested twice by crossing sub131 bw sp*/ Centrosomin (Riparbelli and Callaini 2005). Alternatively, SM6a sibling male progeny to cn sub1bw/SM6a females and these central spindle proteins could be adapted for a new examining again for brown-eyed progeny. Eventually 19 lines role, like Subito, in spindle assembly and/or bi-orientation carrying a synthetic lethal mutation (sub131 bw sp*/SM6a) of homologous chromosomes. were established and used for complementation testing and To identify and study the function of meiotic central spindle mapping. proteins, we carried out screens for genes that interact with Genetics, mapping, and complementation testing subito (sub) and the CPC component Incenp. First, an en- hancer screen was performed for mutations that are synthet- To generate recombinant chromosomes for mapping or to ically lethal with sub. Second, since synthetic lethality is a remove the sub131 allele, we mated sub131 bw sp*/SM6a males mitotic phenotype, a screen was performed for enhancement to al dp b pr cn c px sp/CyO virgin females, collected sub131 of the meiotic nondisjunction phenotype caused by a trans- bw sp*/al d b pr cn c px sp virgin females, and mated them to gene overexpressing an epitope-tagged Incenp (Radford et al. al dp b pr Bl cn c px sp/CyO males. Recombinants that were 2012). In these screens we identified new mutations in CPC al2 and c+ were collected and, because these recombinants components (Incenp, aurB, borr), the Centralspindlin gene likely carried the sub mutant allele, were tested for synthetic tumbleweed (tum), and the transcription factor snail. Muta- lethality. In contrast, recombinants that retained the c muta- tions in at