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Impact on Spindle Pole Body Separation and Chromosome Capture Beryl Augustine, Cheen Fei Chin and Foong May Yeong*

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Impact on Spindle Pole Body Separation and Chromosome Capture Beryl Augustine, Cheen Fei Chin and Foong May Yeong* © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs211425. doi:10.1242/jcs.211425 RESEARCH ARTICLE Role of Kip2 during early mitosis – impact on spindle pole body separation and chromosome capture Beryl Augustine, Cheen Fei Chin and Foong May Yeong* ABSTRACT MTs (cMTs), which are present outside the nucleus. cMTs, Mitotic spindle dynamics are regulated during the cell cycle by otherwise known as astral MTs (aMTs), are an integral microtubule motor proteins. In Saccharomyces cerevisiae, one such component of the mitotic spindle and play an important role in protein is Kip2p, a plus-end motor that regulates the polymerization nuclear migration from the mother to daughter cell and in spindle and stability of cytoplasmic microtubules (cMTs). Kip2p levels are positioning (Shaw et al., 1997). regulated during the cell cycle, and its overexpression leads to the The dynamics of the mitotic spindle including its assembly, formation of hyper-elongated cMTs. To investigate the significance of disassembly and positioning, are mediated by force generation from varying Kip2p levels during the cell cycle and the hyper-elongated polymerization and depolymerization of MTs themselves, and from cMTs, we overexpressed KIP2 in the G1 phase and examined the MT-associated motor proteins (McCarthy and Goldstein, 2006). effects on the separation of spindle pole bodies (SPBs) and MTs undergo assembly and disassembly to generate pushing and chromosome segregation. Our results show that failure to regulate pulling forces, respectively (Dogterom et al., 2005). In vitro the cMT lengths during G1-S phase prevents the separation of SPBs. experiments have shown that MT assembly or disassembly by This, in turn, affects chromosome capture and leads to the activation themselves can create forces strong enough to transport organelles of spindle assembly checkpoint (SAC) and causes mitotic arrest. even in the absence of motor protein-based force generation (Inoué, These defects could be rescued by either the inactivation of 1996). Nevertheless, the force generated by MT polymerization and checkpoint components or by co-overexpression of CIN8, which depolymerization per se is insufficient to assemble a functional encodes a motor protein that elongates inter-polar microtubules bipolar spindle. (ipMTs). Hence, we propose that the maintenance of Kip2p level and The other major contributors to force generation are the motor cMT lengths during early cell division is important to ensure proteins. Motor proteins act through their intrinsic plus-end or coordination between SPB separation and chromosome capture by minus-end directed movement on the MTs (Yeh et al., 2000), and kinetochore microtubules (kMTs). play a vital role in the regulation of MT dynamics. A key motor protein involved in cMT dynamics is Kip2p, a plus-ended KEY WORDS: Cytoplasmic microtubules, Kip2, Chromosome kinesin. Kip2p acts as an exclusive MT polymerase that promotes capture, Spindle assembly checkpoint the growth of cMTs (Hibbel et al., 2015) and stabilizes the cMTs by transporting the yeast CLIP170 homolog, Bik1p, towards the INTRODUCTION plus ends of cMTs (Carvalho et al., 2004). In addition, Kip2p is The mitotic spindle is an elegant machine built by the cell to also essential for the maintenance of the normal number and segregate chromosomes during mitosis. In the budding yeast length of cMTs (Cottingham and Hoyt, 1997; Huyett et al., Saccharomyces cerevisiae, SPBs act as microtubule-organizing 1998). centers, in a manner that is analogous to the centrosomes in As previous studies have focused mostly on the role of cMTs mammalian cells (Jaspersen and Winey, 2004). The SPB is a during spindle positioning, not much is known about their dynamic organelle, with the daughter SPB assembling next to the function during early mitosis. Moreover, it is known that Kip2p is mother SPB in G1 phase (Winey and Byers, 1993). The duplicated subjected to cell cycle control (Carvalho et al., 2004), with side-by-side SPBs are connected by a bridge at the end of G1, relatively low levels during G1 and higher Kip2p level during M and this bridge is severed to allow SPB separation and phase, but the significance of its regulation is poorly understood. formation of the two spindle poles (Adams and Kilmartin, 1999). To understand the significance of the low Kip2p levels in G1, we Dynamic microtubules (MTs) from the SPBs capture duplicated sought to examine the effects of perturbing its abundance during chromosomes, with each SPB attached to one sister chromatid, G1 by using an overexpression system. Overexpression studies thereby forming a bipolar spindle (Huang and Huffaker, 2006). have a proven history of being utilized to determine the The budding yeast spindle is composed of three different classes physiological significance of a protein as a complementary of MTs, the kinetochore MTs (kMTs), which capture the approach to traditional loss-of-function studies (Prelich, 2012). chromosomes, the inter-polar MTs (ipMTs), which interdigitate We found that when KIP2 is overexpressed in G1, aberrant cMT with each other providing support to the spindle and the cytoplasmic elongation resulted in SPB separation defects, leading to failure in chromosome capture and mitotic arrest due to spindle assembly checkpoint (SAC) activation. These defects could be rescued by Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, MD4, 5 Science Drive 2, Singapore 117545. inactivation of genes IPL1 and MAD2, which encode SAC components and by inducing expression of the gene CIN8, which *Author for correspondence ([email protected]) encodes a motor protein that is involved in elongation of ipMTs. B.A., 0000-0002-2937-7798; F.M.Y., 0000-0002-5641-6588 We propose that low Kip2p level maintains short cMTs early in the cell cycle, allowing timely SPB separation and chromosome Received 5 October 2017; Accepted 30 April 2018 capture prior to mitosis. Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs211425. doi:10.1242/jcs.211425 RESULTS Wild-type) and FACS analysis (Fig. 1C, left panel) (all results in Kip2p level oscillates during the cell cycle and shows an main text given as mean±s.d. unless otherwise stated). The cMTs at inverse relationship with cMT length this time point were relatively short, and the SPBs had been The spindle undergoes various morphological changes during the segregated into the two daughter cells (Fig. 1A, left panel, pink cell cycle, with relatively long cMTs and short ipMTs during G1, S arrows). At 3 h after G1 release, the percentage of large-budded and early mitosis, and shorter cMTs and long ipMTs during late cells decreased to 41.8±3.5% (n=325) (Fig. 1B, Wild-type), while mitosis (Shaw et al., 1997). In relation to this, the interplay between the percentage of cells that did not have a bud (‘unbudded’) or those Kip2p levels and cMT length during the cell cycle has not been that only had a small bud was increased. Moreover, the FACS examined in depth. profile showed a mixed population of 1N and 2N cells (Fig. 1C, left To study this, we first examined spindle dynamics and Kip2p panel). In addition, western blot analysis showed that Pds1p levels levels in GFP-TUB1 MYO1-GFP SPC42-eqFP611 KIP2-6HA at 2 h were low (Fig. 1D). At 3 h, cells had progressed to the PDS1-9MYC cells released from an α-factor (α-F) arrest. Spc42p (a subsequent round of the cell cycle (Fig. 1B,C). central plaque component of SPBs) (Adams and Kilmartin, 1999; By contrast, cells overexpressing KIP2 arrested mostly as large- Jaspersen and Winey, 2004) fused to the red fluorescent protein budded cells (Fig. 1A, right panel) 3 h after α-F release (99±0.1%, eqFP611 (hereafter eqFP) was employed to visualize SPBs and n=347, Fig. 1B) and exhibited hyper-elongated MTs (Fig. 1A, right Myo1p–GFP was utilized to show the bud neck. In G1 phase, the panel, blue arrows). FACS analysis indicated that cells had 2N DNA arrested cells exhibited long cMTs (Fig. S1A, pink arrows). As the content (Fig. 1C), while western blots showed Pds1p and Clb2p cells progressed through mitosis, relatively short cMTs were levels remained high even 3 h after α-F release (Fig. 1D) suggesting observed in cells with elongated spindles at anaphase (Fig. S1A, that chromosomes remained unsegregated and cells were arrested in blue arrow), as seen from the peak of large-budded cells (Fig. S1B, M phase. Strikingly, at 3 h after release from α-F, more than 60% of 90 min) and fluorescence-activated cell sorting (FACS) analysis the cells displayed single SPB spots (Fig. 1A, right panel, pink (Fig. S1C, 90 min). arrows, Fig. 1E). We also used western blot analysis to examine the abundance of It was unclear whether the single SPB spots observed in KIP2- Kip2p over the cell division cycle. As markers for progression overexpressing cells were due to a problem with SPB duplication or through cell cycle, we detected Pds1p, the yeast homolog of securin, failure in SPB separation. To determine which of the two and the protein Clb2p. Pds1p prevents chromosome segregation by possibilities could explain the KIP2 overexpression phenotype, inhibiting the protease separase, which is required for the cleavage we performed time-lapse experiments with wild-type and GAL- of the cohesin Scc1p, which holds the sister chromatids together KIP2-TAP cells, to examine the dynamics of the SPBs. We assumed (Ciosk et al., 1998; Michaelis et al., 1997). Clb2p is the mitotic that if the SPBs failed to undergo duplication, only a single spot cyclin that activates the cyclin-dependent kinase Cdk1p to promote would be observed in the KIP2-overexpressing cells. However, if the transition from G2 to M phase (Fitch et al., 1992; Ghiara et al., the SPBs were duplicated but failed to separate completely, we 1991; Surana et al., 1991).
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