PP2A-B55γ Counteracts Cdk1 and Regulates Proper Spindle Orientation Through the Cortical Dynein Adaptor Numa Riya Keshri, Ashwathi Rajeevan and Sachin Kotak*

RESEARCH ARTICLE PP2A-B55γ counteracts Cdk1 and regulates proper spindle orientation through the cortical dynein adaptor NuMA Riya Keshri, Ashwathi Rajeevan and Sachin Kotak*

ABSTRACT activity of such cortically anchored dynein–dynactin is assumed to Proper orientation of the mitotic spindle is critical for accurate generate pulling forces on the astral microtubules and thus on the development and morphogenesis. In human cells, spindle spindle apparatus to control mitotic spindle behavior in time and orientation is regulated by the evolutionarily conserved protein space (Nguyen-Ngoc et al., 2007; Redemann et al., 2010; Laan NuMA, which interacts with dynein and enriches it at the cell cortex. et al., 2012; Kotak et al., 2012, 2013; Collins et al., 2012; Schmidt Pulling forces generated by cortical dynein orient the mitotic spindle. et al., 2017; Okumura et al., 2018; Fielmich et al., 2018). Cdk1-mediated phosphorylation of NuMA at threonine 2055 (T2055) Other than its function in spindle orientation and elongation, negatively regulates its cortical localization. Thus, only NuMA not NuMA is required for proper assembly as well as maintenance of the – phosphorylated at T2055 localizes at the cell cortex. However, the mitotic spindle with the help of dynein dynactin (Yang and Snyder, identity and the mechanism of action of the phosphatase complex 1992; Compton and Cleveland, 1993; Merdes et al., 1996, 2000; involved in T2055 dephosphorylation remains elusive. Here, we Hueschen et al., 2019). NuMA is a large protein of 2115 amino characterized the PPP2CA-B55γ (PPP2R2C)–PPP2R1B complex acids, and it comprises two globular domains separated by a coiled- that counteracts Cdk1 to orchestrate cortical NuMA for proper spindle coil domain in the middle (Yang et al., 1992). NuMA interacts with orientation. In vitro reconstitution experiments revealed that this dynein through its N-terminus (Kotak et al., 2012), and its complex is sufficient for T2055 dephosphorylation. Importantly, we C-terminus contains domains that mediate its interaction with identified polybasic residues in NuMA that are critical for T2055 LGN (also known as GPSM2), ERM4.1 (EBP41), microtubules, α dephosphorylation, and for maintaining appropriate cortical NuMA importin- , and phosphoinositides (Mattagajasingh et al., 1999; Du levels for accurate spindle elongation. Furthermore, we found that Cdk1- et al., 2002; Du and Macara, 2004; Woodard et al., 2010; Seldin mediated phosphorylation and PP2A-B55γ-mediated dephosphorylation et al., 2013, 2016; Kiyomitsu and Cheeseman, 2013; Zheng et al., at T2055 are reversible events. Altogether, this study uncovers a novel 2014; Kotak et al., 2014; Gallini et al., 2016; Chang et al., 2017). mechanism by which Cdk1 and its counteracting PP2A-B55γ complex Interestingly, recent studies have highlighted the presence of a orchestrate spatiotemporal levels of cortical force generators for conserved region in the C-terminus of NuMA that is necessary for flawless mitosis. cluster formation, and possibly for generating strong pulling forces at the cell cortex (Okumura et al., 2018; Pirovano et al., 2019). KEY WORDS: Cdk1, NuMA, PP2A, Dynein, Mitosis, Spindle Because NuMA acts as an essential adaptor for cortical dynein– orientation dynactin in mitosis, its localization is tightly coupled with the mitotic phases. NuMA cortical levels are low in metaphase and INTRODUCTION substantially increased during anaphase (Kiyomitsu and Proper orientation and elongation of the mitotic spindle are critical Cheeseman, 2013; Kotak et al., 2014; Seldin et al., 2013; Zheng events for defining the accurate positioning of the cleavage furrow et al., 2014). These localization patterns ensure proper spindle and for faithful segregation of the genetic material (Gönczy, 2008; orientation and elongation events in metaphase and anaphase, Siller and Doe, 2009; Morin and Bellaiche, 2011). This process respectively (Kotak et al., 2012, 2014; Collins et al., 2012; Seldin further ensures that cell fate determinants are accurately segregated et al., 2013; Zheng et al., 2014). Notably, this tight regulation is in the resulting daughter cells during asymmetric cell division, achieved with the help of several mitotic kinases including Cdk1– including in stem cells (Knoblich, 2008). In animal cells, one of the cyclinB (hereafter referred to as Cdk1), Aurora A and Plk1 critical determinants of the control of spindle orientation and (Kiyomitsu and Cheeseman, 2012, 2013; Kotak et al., 2013; Seldin elongation is an evolutionarily conserved cortically anchored et al., 2013; Zheng et al., 2014; Gallini et al., 2016; Kotak et al., protein NuMA (LIN-5 in Caenorhabditis elegans; Mud in 2016; Connell et al., 2017; Sana et al., 2018). We have shown Drosophila; Siller and Doe, 2009; di Pietro et al., 2016; previously that NuMA is directly phosphorylated at threonine 2055 Bergstralh et al., 2017). Cortical NuMA serves to anchor the (T2055) by Cdk1 (Kotak et al., 2013). This phosphorylation microtubule-dependent minus-end-directed motor protein complex negatively regulates cortical accumulation of NuMA in metaphase, dynein and its associated dynactin complex (Kotak, 2019). Motor where cortical NuMA localization is LGN-dependent, as well as in anaphase, where NuMA relies on membrane phosphoinositides for its cortical accumulation (Kiyomitsu and Cheeseman, 2013; Kotak Department of Microbiology and Cell Biology, Indian Institute of Science, et al., 2014; Seldin et al., 2013; Zheng et al., 2014). Interestingly, 560012 Bangalore, India. loss of the catalytic subunit of PP2A, PPP2CA, abolishes cortical *Author for correspondence ([email protected]) NuMA and dynein distributions (Kotak et al., 2013). However, whether PPP2CA regulates cortical NuMA by directly acting at the S.K., 0000-0003-0219-7970 Cdk1-phosphorylated residue T2055 remains unknown. PP2A

Handling Editor: David Glover forms a trimeric holoenzyme complex that consists of a catalytic

Received 9 January 2020; Accepted 16 June 2020 subunit, a scaffold subunit, and variable regulatory subunits of four Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs243857. doi:10.1242/jcs.243857 different families: B55 (also known as PR55 or B), B56 (PR61 or B′), localization in HeLa cell line that stably expresses GFP fused B72 (PR72 or B″)andstriatin(PR93orB‴) (Barr et al., 2011; Moura to the dynein heavy chain DHC1 (also known as DYNC1H1) and Conde, 2019). It is the regulatory subunit that provides the (Fig. S2E,F). Next, we decided to identify the nature of the regulatory substrate specificity, and thus even if PPP2CA counteracts Cdk1- and scaffold subunits whose loss stabilizes pT2055 upon acute Cdk1 mediated phosphorylation of T2055, the identity of the regulatory inactivation. Remarkably, in this assay as well, only B55γ and subunit, and the mechanism of its association with NuMA, remains PPP2R1B siRNA-treated cells showed significant stabilization of elusive. In general, in stark contrast to mitotic kinases, our knowledge pT2055 (Fig. 1E–I; Fig. S1G–I). Analogous observations were made regarding how mitotic phosphatases regulate spindle orientation and in non-transformed hTERT-RPE1 cells (data not shown). Overall, spindle elongation remains limited. these data suggest that PPP2CA–B55γ–PPP2R1B is a bona fide In this study, using a combination of cell biology and biochemical tripartite phosphatase holoenzyme complex involved in NuMA experiments, we uncovered that a PP2A complex containing B55γ dephosphorylation at T2055, and which thereby orchestrates cortical (also known as PPP2R2C) is involved in T2055 dephosphorylation, NuMA levels during mitosis. and thus for maintaining the cortical pool of NuMA and dynein– dynactin in mitosis. Because cortical NuMA is critical for proper The PP2A-B55γ phosphatase complex dephosphorylates spindle orientation, loss of B55γ caused spindle orientation defects pT2055 during metaphase. Moreover, we discovered that conserved Next, we sought to biochemically determine whether the B55γ- polybasic residues in the vicinity of the Cdk1 phosphorylation containing PPP2CA-based tripartite complex can dephosphorylate a site T2055 are the key for robust spatiotemporal NuMA localization NuMA peptide sequence that is phosphorylated by Cdk1. To this end, in mitosis. Importantly, data obtained from chemically induced we generated a stable HeLa cell line constitutively expressing low mitotic exit followed by mitotic entry reveal that phosphorylation levels of B55γ–AcGFP (Fig. 2A,B). Immunoprecipitates (IP) from and dephosphorylation at T2055 are reversible events. In summary, nocodazole-synchronized mitotic cell extract made from B55γ– our work identifies a novel B55γ-containing PP2A complex that AcGFP-expressing cells, but not those from control cells expressing counteracts Cdk1 and spatiotemporally regulates cortical levels of AcGFP, interacted with endogenous NuMA (Fig. 2C; Fig. S3A). This NuMA and dynein to ensure unperturbed spindle behavior during data suggests that B55γ specifically interacts with NuMA. As mitosis. expected, the IP fraction from B55γ–AcGFP cells also showed interaction with the catalytic subunit PPP2CA, and the regulatory RESULTS subunit PPP2R1B, but not with p150Glued (Fig. 2C). Interestingly, B55γ regulates cortical levels of NuMA and dynein by ectopic expression of B55γ was sufficient to significantly enrich dephosphorylating T2055 of NuMA cortical NuMA and concomitantly decrease the levels of pT2055 Cdk1-mediated phosphorylation of NuMA at T2055 (pT2055), (Fig. 2D–G). Next, we attempted to test whether the IP fraction prevents its accumulation at the cell cortex in metaphase (Kotak et al., obtained from B55γ–AcGFP cells could dephosphorylate a hexa-His- 2013; Seldin et al., 2013). Because RNAi-mediated depletion of tagged NuMA(1876–2115) [6His–NuMA(1876–2115)] fragment that had PPP2CA negatively impacts cortical localization of NuMA in been phosphorylated by recombinant Cdk1 (Fig. 2H). Notably, the metaphase (Kotak et al., 2013), one possibility is that PPP2CA B55γ–AcGFP IP fraction could dephosphorylate pT2055 with high counteracts Cdk1-mediated phosphorylation of T2055. Therefore, we specificity, whereas IP fractions that were either made from cells sought to investigate whether PPP2CA dephosphorylates NuMA at stably expressing AcGFP fused with a non-specific protein (CAAX– T2055. As shown previously, we found that acute treatment with the FLAG–AcGFP) or another B55 subunit (B55α, also known as Cdk1 inhibitor RO-3306 led to robust dephosphorylation of T2055, PPP2R2A) did not show significant dephosphorylation of pT2055 and thus loss of pT2055 signal at the spindle pole (Fig. S1A,B; Kotak (Fig. 2I,J; Fig. S3B–E). Furthermore, to ensure the specificity of et al., 2013). However, T2055 dephosphorylation upon RO-3306 B55γ in these dephosphorylation experiments, we incubated the treatment was significantly reduced in cells that were depleted for B55γ–AcGFP IP fraction with an antibody against GFP to mask PPP2CA (Fig. S1B,C). Importantly, among all phosphoprotein the AcGFP-tagged B55γ regulatory subunit prior to the phosphatase (PPP) family members that are generally involved in the dephosphorylation reaction (Fig. 2K). Interestingly, the IP-fraction dephosphorylation of the mitotic substrates (Barr et al., 2011; Moura incubated with the GFP antibody had a substantially weakened and Conde, 2019), PPP2CA appeared the only catalytic subunit that dephosphorylation capability (Fig. 2L). Moreover, the B55γ–AcGFP was responsible for T2055 dephosphorylation (Fig. S1D). IP fraction from cells depleted of the catalytic subunit PPP2CA did PPP2CA interacts with a regulatory subunit and a scaffold not support efficient pT2055 dephosphorylation (Fig. 2M; Fig. S3F). subunit (Fig. 1A), and it is the regulatory subunit that provides Overall, these data strongly support the notion that the B55γ- substrate specificity to the PPP2CA-containing trimeric complex containing PP2A complex is sufficient to dephosphorylate NuMA at (Barr et al., 2011; Cundell et al., 2013; Moura and Conde, 2019). To the evolutionarily conserved Cdk1 residue, T2055. establish which regulatory and scaffold subunits are part of PPP2CA-containing phosphatase holoenzyme involved in pT2055 MASTL and its substrate ENSA are dispensable for dephosphorylation, we depleted all 13 regulatory subunits and the dephosphorylation of pT2055 two scaffold subunits that could be part of a PPP2CA-based trimeric In metazoans, during mitotic entry B55 is inhibited by endosulfine- complex. Interestingly, siRNA-mediated depletion of B55γ and alpha (ENSA) and cAMP-regulated phosphoprotein-19 (ARPP19), PPP2R1B using multiple siRNAs caused the loss of cortical NuMA two related proteins activated by the protein kinase and dynein-interacting dynactin subunit p150Glued (also known as microtubule-associated serine/threonine kinase-like enzyme DCTN1) (Fig. 1B–D; Fig. S1E,F; data not shown). This impact of (MASTL) (Gharbi-Ayachi et al., 2010; Mochida et al., 2010). B55γ depletion on cortical NuMA was also observed in HeLa Kyoto Depletion of MASTL, ENSA, or ARPP19 leads to constitutive B55 cells that stably express NuMA with AcGFP (Aequorea activity that causes mitotic catastrophe (Cundell et al., 2013; coerulescens GFP) and a mono-FLAG epitope (AcGFP–NuMA; Manchado et al., 2010; Voets and Wolthuis, 2010). Thus, we

Fig. S2A–D). Analogous results were obtained for dynein scrutinized whether RNAi-mediated loss of MASTL impacts cortical Journal of Cell Science

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Fig. 1. PP2A-B55γ is required for NuMA dephosphorylation at Cdk1 site T2055. (A) Schematic representation of a PPP phosphatase complex containing a catalytic subunit, a regulatory (B) subunit, and a scaffold subunit. (B) HeLa cells, transfected with control siRNAs or siRNAs against all thirteen different PP2A regulatory subunits, as indicated, were assayed for NuMA localization in metaphase. Cells were fixed 72 h after siRNA transfection and stained for NuMA. Stacked columns show the extent of cortical NuMA that was visually quantified under the epifluorescence microscope, and categorized as either absent (violet bars; example cell is shown in D) or weak (green bars; example cell is shown in C). More than 50 cells were analyzed in each condition, and the experiments were repeated three times. Data are mean±s.d. calculated from the percentage of cells in each category (weak or absent) from three independent experiments. Two or three different siRNAs were tested for each gene (see Table S1). Also, note that there could be potential false negatives in the dataset as we have not assayed the knockdown efficiency for each siRNA. (C,D) Metaphase HeLa cells transfected with control siRNAs (C) or siRNAs against B55γ (D). Cells were fixed 72 h after siRNA transfection and stained for NuMA (green) and DNA (blue). Arrows indicate cortical localization. Quantification of cortical enrichment was performed in an area of size 1.8 μm×4 μm (yellow box; see Materials and Methods for detail). Quantification of mean±s.d. cortical enrichment is shown for ten metaphase cells in each condition. (E) HeLa cells, transfected with control siRNAs or siRNAs against the thirteen regulatory PP2A subunits, and treated with Cdk1 inhibitor RO-3306 (10 μM; Vassilev et al., 2006) for 5 min, were assayed for pT2055 in metaphase. Cells were fixed 72 h after siRNA transfection and stained with an antibody against pT2055. Stacked columns show the extent of pT2055 signal at the spindle poles, as quantified visually under the epifluorescence microscope, and categorized as either weak (comparable to control cells upon RO-3306 treatment, green bars; example cell shown in H), strong (blue bars; example untreated control cell shown in F), or moderate (signal between weak and strong, red bars; example cell shown in I). More than 50 cells were analyzed in each condition, and the experiment was repeated three times. Data are mean±s.d. calculated from the percentage of cells in each category (weak, moderate or strong) from three independent experiments. (F–I) Metaphase HeLa cells transfected with control siRNAs (F), siRNAs against B55γ (G), control cells that were treated with RO-3305 for 5 min (H), or B55γ siRNA- treated cells that were treated with RO-3306 for 5 min (I). pT2055 signal is shown in gray and DNA is shown in blue. Quantification of spindle pole enrichment was performed for ten representative cells using a 16 µm2 region (yellow) and adjusted for background signal (see Materials and Methods for detail). Data are mean ±s.d. P=0.156 between control and B55γ siRNA-treated metaphase cells for the spindle pole, P<0.0001 between control untreated and RO-3306-treated cells, and P=0.0048 between control RO-3306-treated and B55γ siRNA RO-3306-treated cells. **P<0.01, ***P<0.0001 (two-tailed Student’s t-test).

NuMA. As reported previously, we observed that RNAi-mediated 2010). However, MASTL depletion did not affect cortical NuMA in depletion of MASTL led to cytokinesis defects as well as metaphase (Fig. S4D,E). Similarly, we did not observe any change in chromosome congression defects (Fig. S4A,C; Voets and Wolthuis, cortical NuMA localization in cells depleted for ENSA (Fig. S4B,F). Journal of Cell Science

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Fig. 2. See next page for legend.

Next, we analyzed the impact of MASTL and ENSA depletion on Kotak et al., 2012, 2013). Because B55γ loss markedly reduced cortical pT2055 levels at the spindle poles in metaphase. Interestingly, despite NuMA and dynein, we monitored x-y spindle orientation by analyzing substantial depletion of MASTL or ENSA, as observed by fixed cells grown on coverslips with an L-shaped fibronectin micro- immunoblotting (Fig. S4A,B), their loss did not affect pT2055 pattern (Thery et al., 2005), which were treated with control siRNAs or levels (Fig. S4G–I). Although the reason why MASTL and ENSA do siRNAs against B55γ. We found that spindle orientation was perturbed not regulate the B55γ-containing PP2A complex will be of interest for in cells depleted of B55γ in contrast to the spindle orientation observed future work, at this stage our data suggest that, unlike other B55 in control cells (Fig. 3A–D), indicating that loss of cortical NuMA and regulatory subunits such as B55α (Cundell et al., 2013; 2016), the dynein upon B55γ depletion disrupts proper spindle orientation. B55γ-containing PP2A complex is not regulated by the MASTL or its substrate ENSA. PP2A-B55γ controls cortical NuMA levels upon Cdk1 inactivation during anaphase onset B55γ regulates proper spindle orientation RNAi-mediated loss of B55γ and PPP2CA causes chromosome Cortical NuMA-mediated dynein localization is essential for proper instability (data not shown; Kotak et al., 2013) and, presumably, this spindle orientation in metaphase (Kiyomitsu and Cheeseman, 2012; provokes the spindle assembly checkpoint. This observation precluded Journal of Cell Science

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Fig. 2. PP2A-B55γ dephosphorylates pT2055. (A) Schematic of B55γ polybasic recognition site flanking a Cdk1 phosphorylation site construct with FLAG (FL) and AcGFP tags at the C-terminus (B55γ–AcGFP). (Cundell et al., 2016). Importantly, we found that T2055 is γ– (B) Characterization of HeLa Kyoto cell line stably expressing B55 AcGFP surrounded by a few lysine or arginine residues, and these amino using western blot analysis. β-actin is shown as a loading control. (C) Co-immunoprecipitation (IP) by GFP-Trap from lysates of nocodazole- acids are evolutionarily conserved (Fig. 5A). To evaluate the arrested mitotic HeLa cells stably expressing B55γ–AcGFP. Resulting blots were function of these polybasic residues for cortical NuMA localization, probed for NuMA, PPP2CA, PPP2R1B, p150Glued, and GFP, as indicated. IN, we mutated either two or four lysine or arginine residues input (1% of total); IP, 50% of the total immunoprecipitate; FT, flow-through. For downstream of the Cdk1 phosphorylation site in the GFP-tagged GFP detection in the IP fraction, only 10% of the IP fraction was loaded. Note, NuMA C-terminal fragment [GFP–NuMA – ; Fig. 5B; Fig. γ (1411 2115) B55 interacts with NuMA, but not with the dynein-interacting dynactin subunit S5A], which localizes at the cell cortex, similar to the wild-type p150Glued. (D,E) Metaphase HeLa Kyoto control cells (D), or cells stably expressing B55γ–AcGFP (E) were fixed and stained for NuMA (red) and DNA protein (Kotak et al., 2014; Sana et al., 2018). Mutation of either two (blue). Arrows indicate cortical localization, box indicates example region used for lysine, two arginine, or all the four polybasic residues had a significant quantification. Note enrichment in cortical NuMA in cells expressing B55γ– impact on the cortical localization of GFP–NuMA(1411–2115) (Fig. 5C– AcGFP. Data are mean±s.d. cortical NuMA levels of 12 cells. P=0.0061 between E,G; Fig. S5B,D). Notably, we also found that an Escherichia coli- control and B55γ–AcGFP-expressing cells (two-tailed Student’s t-test). (F,G) generated hexa-His-tagged mutant of NuMA, where all four basic Control metaphase HeLa cells (F), or cells stably expressing B55γ–AcGFP residues are mutated to alanine was substantially weaker in its (G) fixed and stained for pT2055 (gray) and DNA (blue). Circle indicates example interaction with B55γ–AcGFP (Fig. S5E,F). This data indicates that region used for quantification. Note significantly diminished levels of spindle pole pT2055 in cells expressing B55γ–AcGFP. Data are mean±s.d. spindle pole these polybasic residues present in NuMA are critical for efficient pT2055 levels of 20 cells. P<0.0001 between control and B55γ–AcGFP- interaction with B55γ. The impact on cortical GFP signal in cells expressing cells (two-tailed Student’s t-test). (H) Schematic representation of the expressing polybasic NuMA mutant appeared not because of their in vitro – dephosphorylation assay whereby 6His NuMA(1876–2115) is inability to localize at the membrane, as the introduction of a phosphorylated by Cdk1/cyclinB and detected by anti-pT2055 antibody. This phospho-dead mutation at T2055 in GFP–NuMA(1411–2115)2RR>A phosphorylated substrate was utilized in the dephosphorylation reaction using IP rescued the cortical signal (Fig. S5B–D). We further established that fraction from B55γ–AcGFP-expressing cells. (I) Detection of phosphorylated the impact on cortical NuMA signal in cells expressing GFP– 6His–NuMA(1876–2115) with anti-pT2055 antibody. Please note that 6His– – NuMA(1876–2115) is unstable, therefore two species are observed in the NuMA(1411 2115)4KR>A was not due to impairment of T2055 Coomassie-stained gel and western blots. (J) Dephosphorylation reaction with phosphorylation by Cdk1 at T2055 (Fig. S5G–K). Taken together, – γ– Cdk1/cyclinB-phosphorylated 6His NuMA(1876–2115) and the B55 AcGFP IP these results strengthen our model that B55γ recognizes polybasic fraction at a different times, as indicated. Note that in this and other amino acids in the vicinity of the Cdk1 phosphorylation site for dephosphorylation experiments, values below the pT2055 western blot represent efficient localization of NuMA at the cell cortex. the band intensity, normalized to the intensity value from the control sample. HIS, blot using anti-His-tag antibody. (K) Schematic representation of the in vitro Because cortical NuMA promotes proper spindle elongation in dephosphorylation assay as mentioned above, however, the dephosphorylation anaphase cells (Kotak et al., 2013), and the quadruple mutant of reaction is performed by either incubating the B55γ–AcGFP IP fraction with anti- NuMA is unable to localize at the cell cortex, presumably because GFP antibody (GFP-Ab) to mask AcGFP–B55γ, or with a B55γ–AcGFP IP of the lack of dephosphorylation, we attempted to characterize the fraction from cells depleted of PPP2CA catalytic subunit. (L) Dephosphorylation impact of this mutant on spindle elongation during anaphase. For reaction with the B55γ–AcGFP IP that was incubated with GFP-Ab before the this purpose, we utilized a stable HeLa Kyoto cell line co-expressing dephosphorylation reaction. (M) Dephosphorylation reaction with the B55γ– AcGFP–NuMA4KR>A and mCherry–H2B. This engineered line AcGFP IP fraction from the cells depleted for PPP2CA by RNAi. Please note a – – substantial decrease in dephosphorylation potential was observed for B55γ– expresses transgenic AcGFP NuMA4KR>A, analogous to AcGFP AcGFP IP fractions that were either incubated with GFP-Ab (L) or from cells NuMA, and its endogenous counterpart (Fig. S2A,B). In contrast to that were depleted for PPP2CA (M). **P<0.01, ***P<0.0001 (two-tailed the wild-type AcGFP–NuMA-expressing cells, we did not observe Student’s t-test). significant cortical accumulation of mutated NuMA in cells expressing AcGFP–NuMA4KR>A either in metaphase, anaphase or us from studying the function of the B55γ-based tripartite complex in in cells that mimic anaphase-like conditions (i.e. STLC- and RO- cortical NuMA and dynein distributions during anaphase. To 3306-treated cells) (Fig. 4E,F; Fig. S2G–I). Because loss of NuMA circumvent this experimental limitation, we sought to investigate the impacts bipolar spindle formation (Kotak et al., 2013; Hueschen role of B55γ as well as the whole tripartite PPP2CA–B55γ–PPP2R1B et al., 2019), we established an experimental regimen where we complex in anaphase-like conditions. To this end, HeLa Kyoto cells could study proper spindle elongation in a relatively weak NuMA stably co-expressing AcGFP–NuMA and mCherry–H2B were treated (RNAi) background without impacting spindle bipolarity and with kinesin 5 (KIF11) inhibitor (STLC) to block the cells in anaphase onset (data not shown). In such a setting, loss of prometaphase stage, and subsequently these cells were forced to exit endogenous NuMA significantly affected inter-chromatid distance mitosis by the addition of the Cdk1 inhibitor RO-3306 (Fig. 4A). As during anaphase in HeLa cells stably expressing the DNA marker expected, Cdk1 inactivation for 10 min in STLC-treated cells led to mCherry–H2B alone ( Fig. 5I,J,M,N). However, this phenotype was robust cortical NuMA enrichment (Fig. 4B,F). However, this cortical fully rescued in cells ectopically expressing RNAi-resistant enrichment was highly impaired in cells depleted of either B55γ or AcGFP–NuMA, but not in cells expressing AcGFP–NuMA4KR>A PPP2CA–B55γ–PPP2R1B by RNAi (Fig. 4C,D,F). These data (Fig. 5K,L,N). support that B55γ,aswellastheB55γ-containing tripartite complex, Serine and threonine residues phosphorylated by Cdk1 display is essential for proper cortical localization of NuMA, not only in differential dephosphorylation kinetics upon mitotic exit (McCloy metaphase but also in conditions that mimic anaphase. et al., 2015; Godfrey et al., 2017; Hein et al., 2017). Also, PP2A complexes prefer phospho-threonine over phospho-serine (Pinna Polybasic amino acid residues in the vicinity of the Cdk1 et al., 1976; Deana et al., 1982; Deana and Pinna, 1988). Besides, it phosphorylation site T2055 are critical for cortical NuMA is further revealed that the catalytic efficiency of PP2A-B55 is localization in mitosis almost 20-fold higher for phospho-threonine in comparison with How does B55γ recognize NuMA phosphorylated by Cdk1 at phospho-serine (Hein et al., 2017). Because Cdk1 inactivation

T2055? B55 substrates were recently shown to possess a bipartite robustly dephosphorylates pT2055 through B55γ, and therefore Journal of Cell Science

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Fig. 3. Loss of B55γ by RNAi misorients the mitotic spindle. (A) Schematic representation of the mitotic spindle on the L-shape micropattern. The mitotic spindle and the centrosomes are shown in cyan, and chromosomes are in magenta. Spindle orientation angles (α°) were determined as depicted, with spindle angle measured relative to the hypotenuse of the micropattern (0°), as shown. (B,C) HeLa cells on an L-shape fibronectin micropattern that were either treated with control siRNAs (B) or RNAi against B55γ (C). Cells were stained for γ-tubulin (green) and DNA (blue). (D) Frequency of angular distribution of spindle positioning from 0°–15°, 16°–45°, and more than 45°. Note the change in the axis of spindle orientation in cells depleted of B55γ. Data are mean±s.d. of n>50 cells. ***P<0.0001 (two-tailed Student’s t-test). substantially enriches cortical NuMA in metaphase, we wondered conditions. As expected, pT2055 levels were substantially whether substitution of the conserved threonine with serine would reduced in cells treated with MG132 and RO-3306 (Fig. 6J,K). delay the dephosphorylation of pT2055 upon Cdk1 inactivation in However, timely activation of Cdk1 upon release from RO-3306- anaphase. To this end, we analyzed the cortical localization of GFP– mediated inhibition in the presence of MG132 restored pT2055 NuMA(1411–2115)T>S in cells expressing H2B–mCherry during levels (Fig. 6K–M). Overall, these data suggest that anaphase progression. Remarkably, HeLa cells expressing the phosphorylation and dephosphorylation of NuMA at T2055 is serine substitution allele showed slow kinetics of membrane GFP dynamic and interconvertible. localization in early anaphase compared to cells expressing GFP– NuMA(1411–2115) (Fig. 5C,F,H). Overall, these data suggest that DISCUSSION polybasic residues in the vicinity of T2055 are the key for cortical Protein phosphorylation and dephosphorylation events are NuMA generation, and therefore for robust spindle elongation. critical for regulating several biological processes, such as the Also, a threonine amino acid residue is a preferred over serine to cell cycle, proliferation and development, signal transduction ensure strong cortical NuMA enrichment upon Cdk1 inactivation pathways, and mitotic progression (Hunter, 1995; Lechward during early anaphase. et al., 2001; Virshup, 2000; Bollen et al., 2009; Moura and Conde, 2019). Recently, studies have linked phosphatases with NuMA phosphorylation at T2055 is a reversible event mitotic spindle orientation and elongation (Afshar et al., 2010; Cdk1 inactivation by RO-3306 promotes cortical NuMA Kotak et al., 2013, 2016; Xie et al., 2013). However, the enrichment and loss of pT2055 at the spindle poles. Thus, we mechanistic understanding relating to these phosphatases and their wondered whether NuMA dephosphorylation initiated by acute substrates in orchestrating spindle behavior remained poorly Cdk1 inactivation is reversible. To investigate this, we first characterized. In this study, we revealed that the existence of a incubated mitotically synchronized HeLa cells with the biochemical cross-talk between Cdk1 and a B55γ-based PP2A proteasome inhibitor MG132 (to block cyclinB1 degradation), complex is vital for temporal regulation of cortical NuMA and dynein and subsequently we treated these cells with the Cdk1 inhibitor for proper spindle orientation and elongation (Fig. 6P). Our model RO-3306 (to promote anaphase entry) (Fig. 6A). MG132- and RO- suggests that the gradient of Cdk1 activity emanating from the 3306-treated cells showed robust CYK4 (also known as centrosomes that is counteracted by the uniformly weak activity of MgcRacGAP or RACGAP1) accumulation at the spindle mid- PP2A-B55γ controls cortical levels of the dynein adaptor NuMA in zone and cortical NuMA accumulation, indicating onset of the metaphase. These low levels of cortical NuMA and dynein–dynactin anaphase program (Fig. 6B,C,F,G,). Importantly, when the Cdk1 are critical for proper spindle orientation in metaphase. At anaphase inhibitor was subsequently washed away in the presence of onset, Cdk1 inactivation causes robust dephosphorylation of NuMA MG132 for 10 and 20 min, we observed time-dependent at T2055, and this leads to substantial cortical enrichment of NuMA diminution of CYK4 accumulation at the spindle mid-zone, and and the dynein–dynactin complex in anaphase, which promotes reduction in cortical NuMA levels (Fig. 6D,E,H,I). Next, we spindle elongation (Fig. 6P). attempted to study cortical NuMA reversibility by live-recording cells co-expressing AcGFP–NuMA and mCherry–H2B (Fig. 6N). AB55γ-based PP2A complex involved in NuMA Such MG132-incubated cells, when treated with RO-3306, dephosphorylation at T2055 showed substantial enrichment of AcGFP–NuMA at the cell Previous work has uncovered the involvement of PP2A-B55 in cortex (Fig. 6O). Importantly, as demonstrated for endogenous several essential processes such as cytokinesis and reassembly of NuMA, the excess cortical levels of AcGFP–NuMA were the Golgi apparatus and nuclear envelope during mitotic exit dramatically reduced in a time-dependent manner when the (Schmitz et al., 2010; Cundell et al., 2013). Herein, by conducting a Cdk1 inhibitor was washed away. Next, we tested the candidate-based chemical genetics screen, we discovered the reversibility of phosphorylated pT2055 in our experimental function of B55γ in cortical accumulation of NuMA during Journal of Cell Science

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Fig. 4. See next page for legend. Journal of Cell Science

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Fig. 4. B55γ-PP2A is necessary for NuMA cortical localization in cells more basic than substrates with a slower dephosphorylation rate. It mimicking anaphase. (A) Experimental protocol for chemically induced was further postulated that electrostatic interactions between anaphase onset by acute Cdk1 inactivation using RO-3306 treatment in the negatively charged residues of PP2A-B55 and positively charged presence of the kinesin-5 inhibitor STLC for cells stably co-expressing amino acids in the substrates might determine the dephosphorylation AcGFP–NuMA and mCherry–H2B, or AcGFP–NuMA4KR>A and mCherry– H2B. (B–D) Time-lapse confocal images of HeLa cells stably co-expressing rate (Xu et al., 2008; Cundell et al., 2016). Herein, we identified a few AcGFP–NuMA (green) and mCherry–H2B (magenta) that were either polybasic residues in the vicinity of T2055 of NuMA. Notably, the transfected with control siRNAs and treated with STLC and RO-3306 (B, replacement of four evolutionarily conserved positively charged Control), transfected with B55γ siRNAs and treated with STLC and RO-3306 amino acids (K or R) to a neutral amino acid (A) significantly γ (C), or co-transfected with PPP2CA, B55 and PPP2R1B siRNAs then treated reduced NuMA cortical localization during mitosis. Our data with STLC and RO-3306 (D). Note the absence of cortical GFP signal in cells γ γ further revealed that this quadruple mutant of NuMA weakly that were either transfected with B55 siRNAs or with PPP2CA, B55 and γ PPP2R1B siRNAs, in contrast to control cells. Arrows indicate cortical interacted with B55 . Also, cells expressing this quadruple NuMA localization of NuMA. (E) Time-lapse confocal images of HeLa cells stably co- mutant were considerably impaired in spindle elongation during expressing AcGFP–NuMA4KR>A (green) and mCherry–H2B (magenta) and anaphase. Thus, our data suggest that a complementary acidic were treated with STLC and RO-3306. Note the substantially weaker cortical surface on B55γ could recognize these basic residues on NuMA, GFP signal in these cells upon RO-3306 treatment. (F) Quantification of GFP similar to what has been shown in the context of B55-mediated – cortical enrichment in cells as described for B E. Note significantly reduced regulation of Tau and PRC1 dephosphorylation (Xu et al., 2008; cortical signal in cells that were either transfected with B55γ siRNAs, PPP2CA, Cundell et al., 2016). B55γ and PPP2R1B siRNAs, or expressing AcGFP–NuMA4KR>A upon treatment with RO-3306 for 10 min as indicated. Data show mean±s.d. for Interestingly, our work suggested that the phospho-threonine more than 10 cells, with individual data points plotted. ***P<0.0001 (two-tailed preference of PP2A-B55γ is critical for cortical NuMA localization Student’s t-test). A.U., arbitrary units. in a temporal manner. Previously, it was shown that the PP2A-B55 complex shows an intrinsic choice for phospho-threonine (Cundell et al., 2016; Hein et al., 2017). This preference is not limited to mitosis. B55 (mainly α and δ isoforms) are inhibited in mitosis by PP2A-B55 complexes; PP2A-B56 complexes also show this bias ENSA and ARPP19, which are activated by MASTL (also known (Pinna et al., 1976; Deana et al., 1982). However, the molecular as Greatwall) (Gharbi-Ayachi et al., 2010; Mochida et al., 2010; details of this preference are unclear. It could well be that the Cundell et al., 2013). To our surprise, we did not observe any presence of an additional methyl side group present in threonine has significant impact upon depletion of MASTL or ENSA on T2055 a better binding affinity for PP2A-B55 and PP2A-B56. dephosphorylation in our experimental setting. This data suggests Overall, our work identifies a novel PP2A-B55γ as an essential that weak PP2A-B55γ activity independent of MASTL or its trimeric complex involved in a biochemical tug-of-war with Cdk1 substrate ENSA maintains cortical NuMA for proper spindle to orchestrate cortical levels of NuMA and dynein for proper spindle orientation. Also, it is intriguing that cells with siRNA-mediated behavior. Interestingly, reduction in the levels of B55γ is associated depletion of B55γ in anaphase showed cortical NuMA levels with the growth of prostate cancer (Bluemn et al., 2013), and comparable to that of control cells. However, we noticed that cells therefore, because of the existing link between spindle orientation that are presumably strongly depleted of B55γ display chromosomal and tumorigenesis (Caussinus and Gonzalez, 2005; Quyn et al., instability, which triggers the spindle checkpoint, thereby making 2010; Hehnly et al., 2015; Noatynska et al., 2012), it would be such cells difficult to follow during anaphase (data not shown). interesting to evaluate whether low levels of expression of B55γ in Therefore, the fact that there is no apparent change in the anaphase prostate cancer cells drive cancer progression through spindle cortical levels of NuMA could be due to partial depletion of B55γ. misorientation. In such a scenario, Cdk1 inactivation in anaphase would lead to robust activation of the residual PP2A-B55γ complex. This MATERIALS AND METHODS assumption is supported by the finding that acute inactivation of Cell culture, cell synchronization, and transfection Cdk1 caused rapid T2055 dephosphorylation kinetics. Therefore, it HeLa cells stably expressing GFP–Centrin-1, mCherry–H2B (a kind gift may well be that Cdk1-mediated phosphorylation on B55γ from Arnaud Echard, Institut Pasteur, Paris, France), HeLa Kyoto contributes to its temporal regulation between metaphase and (generously provided by Daniel Gerlich, IMBA, Vienna, Austria), HeLa anaphase. This hypothesis aligns with the observation that a Kyoto stably expressing mCherry–H2B, AcGFP–NuMA, AcGFP– γ– – phospho-mimicking substitution at the conserved serine (S167) in NuMA4KR>A,B55 AcGFP and GFP DHC1 (kindly provided by B55α affects binding of the regulatory (B55α) and catalytic Anthony Hyman, MPI, Dresden, Germany) as well as hTERT1-RPE1 cells, were maintained in high-glucose DMEM with GlutaMAX media subunits (Schmitz et al., 2010). This regulation could also act at supplemented with 10% fetal calf serum (FCS) in a humidified 5% CO2 the level of the catalytic subunit PPP2CA, as phosphorylation of incubator at 37°C. For monitoring spindle positioning in fixed specimens, T304 (a Cdk1 consensus site) in PPP2CA prevents its association HeLa cells were synchronized with 2 mM thymidine (Sigma-Aldrich, with B55 (Longin et al., 2007). Nonetheless, our data reveal that T1895) for 20 h, released for 9 h, trypsinized and plated on fibronectin L- B55γ and the PPP2CA–B55γ–PPP2R1B complex are necessary to shape micropatterns (CYTOO SA), as described previously by Sana et al. regulate cortical NuMA accumulation upon Cdk1 inactivation in the (2018). In brief, ∼80,000 cells were placed on a CYTOO chip in a 35 mm induced anaphase-like scenario. culture dish. After 1 h, cells that had not been attached to the micropatterns were removed by gently washing with medium. Cells were then fixed, 9 h Polybasic residues close to the conserved T2055 residue are after the release, with cold methanol and stained with antibodies against γ-tubulin (GTU88, Sigma-Aldrich). crucial for spatiotemporal regulation of NuMA localization For siRNA experiments, ∼100,000 cells were seeded in 6-well plates. Recently, bipartite polybasic amino acid residues upstream and 20 μM siRNAs in 100 μl RNase-free water and 4 μl of Lipofectamine downstream of the Cdk1 phosphorylation site consisting of [S/T]P RNAiMAX (Invitrogen; 13778150) in 100 μl RNase-free water (with the preference of threonine) were shown to be key to temporal were incubated in parallel for 5 min, mixed for 20 min and then added regulation of dephosphorylation events (Cundell et al., 2016). to 2.5 ml medium per 35 mm dish. siRNA sequences are shown in

Notably, substrates that have fast dephosphorylation kinetics are Table S1. Journal of Cell Science

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Fig. 5. See next page for legend. For transient transfections, cells were transfected at 80% confluency with For the generation of stable human cells, cells were transfected at 80% 4 μg of plasmid DNA in 400 μl serum-free medium either with 6 μl confluency with 6 μg of plasmid DNA in 500 μl of JetPRIME buffer with Turbofect (ThermoScientific, R0531) or 4 μl Lipofectamine 2000 (Life 12 μl of JetPRIME reagent (Polyplus Transfection SA), incubated for

Technologies, 11668019) incubated for 15–20 min and added to each well. 15 min and added to a 10 cm dish. Journal of Cell Science

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Fig. 5. Polybasic residues in the vicinity of T2055 are necessary for for 1 min. Blocking was performed using 1% bovine serum albumin (BSA; cortical NuMA enrichment. (A) Sequence alignments of NuMA amino acid HiMedia, RM3159) in PBST for 1 h, followed by primary antibody sequence near T2055 from various NuMA orthologs. A red asterisk marks the incubation for 4 h. Washes were done three times with 1× PBST for 5 min Cdk1 phosphorylation site, and blue asterisks show basic amino acids. each; cells were then incubated with secondary antibodies for 1 h, (B) Schematic representation of wild-type fragment of NuMA(1411–2115) tagged counterstained with 1 μg/ml Hoechst 33342 (Sigma-Aldrich, B2261), with GFP at the N-terminus, and several mutant forms where either two or four washed three times for 5 min in PBST and mounted using Fluoromount-G basic residues were mutated to alanine (2KK>A, 4KR>A), or where the (Southern Biotech, 0100-01). Primary antibodies were used at the following conserved threonine residue was mutated to serine (T>S). Asterisks indicate dilutions: 1:200 rabbit anti-NuMA (Santa Cruz, sc-48773), 1:200 mouse T2055; MTs, region mediating interaction with microtubules; NLS, nuclear anti-p150Glued (Transduction Laboratories, 612709), 1:100 rabbit anti- localization signal. (C–F) Images from time-lapse microscopy of HeLa cells stably p2055 NuMA (Kotak et al., 2013), 1:2000 mouse anti-α-tubulin (T6199; expressing mCherry–H2B and were transfected with GFP–NuMA – (C), (1411 2115) Sigma Aldrich) and 1:300 rabbit CYK-4, (Bethyl Laboratories, A302- GFP–NuMA – (D), GFP–NuMA – (E), and GFP– (1411 2115)2KK>A (1411 2115)4KR>A 797A), Secondary antibodies used were 1:500 Alexa Fluor 488 goat anti- NuMA(1411–2115)T>S (F). The GFP signal is shown in green and mCherry signal is in magenta. Note the significant loss of cortical NuMA in cells expressing GFP– mouse (Invitrogen, A11001), 1:500 Alexa Fluor 488 goat anti-rabbit (Invitrogen, A11008), 1:500 Alexa Fluor 568 goat anti-mouse (Invitrogen, NuMA(1411–2115)2KK>A or GFP–NuMA(1411–2115)4KR>A. Also, note the dramatically weaker cortical GFP signal in cells expressing GFP–NuMA(1411–2115)T>S.Arrows A11004) and 1:500 Alexa Fluor 568 goat anti-rabbit (Invitrogen, A11011). indicate cortical GFP localization and boxes indicate examples of regions used for Confocal images were acquired on an Olympus FV 3000 confocal laser quantification. (G) Quantification of cortical enrichment for cells that underwent scanning microscope using a 60× NA 1.4 oil objective, and were processed metaphase to anaphase transition, as shown in C–E (see Materials and Methods in ImageJ and Adobe Photoshop, maintaining relative image intensities. for detail). Data show mean±s.d. for n>5 cells, with individual data points plotted. Time-lapse microscopy was conducted on an Olympus FV 3000 confocal GFP–NuMA(1411–2115) versus GFP–NuMA(1411–2115)2KK>A; P=0.28, 0.83, 0.032, laser scanning microscope using a 40× NA 1.3 oil objective (Olympus 0.006, 0.023 and 0.068 for 0, 3, 6, 9, 12 and 15 min, respectively. GFP– Corporation, Japan) to image cells in an imaging dish (Eppendorf, – P P NuMA(1411–2115) versus GFP NuMA(1411–2115)4KR>A; =0.0003 at 0 min, <0.0001 0030740017) at 5% CO2, 37°C, 90% humidity. Precise CO2 and at 3–15 min timepoints (two-tailed Student’s t-test). (H) Quantification of cortical humidity were maintained using Tokai Hit STR stage top incubator enrichment for cells that underwent metaphase to anaphase transition, as shown (Tokai Hit, Japan). Images were acquired every 3 min or 2 min, capturing 8– ≥ in C and F. Data show mean±s.d. for 18 cells, with individual data points plotted. 10 sections, 3 μm apart, at each time point. Figures containing time-lapse – – P GFP NuMA(1411–2115) versus GFP NuMA(1411–2115)T>S; =0.89, 0.04, 0.002, images were made using a single confocal section of the z-stack. <0.0001, 0.18 and 0.35 for 0, 3, 6, 9, 12 and15 min, respectively. (I–L) Images from time-lapse microscopy of HeLa Kyoto cells stably expressing mCherry–H2B (I), stably expressing mCherry–H2B and depleted of endogenous NuMA (J), stably Plasmids and generation of stable cell lines co-expressing AcGFP–NuMA and mCherry–H2B and depleted of endogenous All NuMA clones were constructed using full-length NuMA as a template γ NuMA (K), or stably co-expressing AcGFP–NuMA4KR>A and mCherry–H2B and with appropriate PCR primer pairs. B55 was obtained from Transomic depleted of endogenous NuMA (L). The GFP signal is shown in green, the technologies (Huntsville, USA) as a glycerol stock (BC032954), and it was mCherry signal is in magenta. (M) Schematic representation for the calculation of amplified from pBluescript (Stratagene, BC032954). the distance [d] between inter-chromatids in cells that underwent anaphase For transient transfection, various NuMA fragments were cloned into progression. (N) Quantification of inter-chromatid distance in cells as shown in I–L. pcDNA3-GFP (Merdes et al., 2000; kindly provided by Andreas Merdes, Data are mean±s.d. inter-chromatid distances during anaphase progression for Paul Sabatier, Toulouse). For bacterial expression, NuMA – and P (1876 2115) more than 15 cells in each condition (see Materials and Methods). ** =0.0002 at NuMA – were cloned in the pET28a plasmid and expressed in P – – (1876 2115)4KR>A 2 min, *** <0.0001 for 4 10 min timepoints between AcGFP NuMA- and E. coli as described in Kotak et al. (2013). B55α was amplified from cDNA – AcGFP NuMA4K>R-expressing cells upon depletion of endogenous NuMA (two- prepared from HeLa cells and cloned with GFP. ’ t tailed Student s -test). To create an AcGFP- (A. coerulescens GFP; Clonetech) and single FLAG (DYKDDDDK)-tagged version of wild type, mutated NuMA or B55γ in HeLa stable lines, the amplified products were sub-cloned into the pIRES-AcGFP- Drug treatments FLAG plasmid (kindly provided by Mark Patronczki, Boehringer Ingelheim, Cdk1 inhibition was performed by incubating cells with 10 μM of RO-3306 Vienna, Austria). HeLa Kyoto cells were transfected with AcGFP–NuMA (Selleckchem, S7747) for either 3, 5, or 10 min as specified in the figure (wild-type or mutant) or B55γ–AcGFP expression constructs. The medium legends. Proteasome inhibitor MG132 (Sigma-Aldrich, M8699) was used at was supplemented with 0.4 μg/ml puromycin (Life Technologies, A1113803) the concentration of 20 μM for 2 h. to select for and maintan cell lines expressing pIRESpuro3-based AcGFP- To investigate the cortical enrichment of AcGFP-tagged proteins in a tagged NuMA or B55γ genes. Cells co-expressing mCherry–H2B with monopolar setting, HeLa Kyoto cells stably co-expressing AcGFP–NuMA AcGFP–NuMA wild-type or mutated protein were selected in a medium (wild type or 4KR>A mutant) and mCherry–H2B were synchronized using containing 500 μg/ml G418 (Life Technologies, 10131035) in addition to 7.5 μM Kinesin-5 inhibitor STLC (Sigma-Aldrich, 164739) for 17 h, and puromycin. After 2–3 weeks of antibiotic selection, cells were characterized by thereafter imaged by live recordings for every 2 min, either with or without immunoblotting and live imaging. 10 μM RO-3306. For the reversal experiment, HeLa cells were synchronized with 2 mM thymidine (Sigma-Aldrich, T1895) for 23 h and then released for 6 h, Visual quantification of NuMA and pT2055 followed by 5 h of nocodazole (Sigma-Aldrich, M1404) treatment. Cells For visual quantification of cortical NuMA (Fig. 1B), cells were analyzed under were washed with the regular medium twice, followed by incubation with an epifluorescence microscope, and these cells were categorized as either ‘weak’ 20 μM of MG132 for 2 h to arrest the cells in metaphase. These cells were (as in Fig. 1C) or absent (as in Fig. 1D). For pT2055 quantification at the spindle then treated with 10 μM RO-3306 for 10 min, and subsequently these cells poles (Fig. 1E; Fig. S1D), metaphase cells were categorized as either ‘strong’ (as were released in regular medium with MG132 for 0 min, 10 min, and in Fig. 1F), ‘moderate’ (as in Fig. 1I), or ‘weak’ (as in Fig. 1H). 20 min before fixation and staining. For live-imaging under these – conditions, HeLa Kyoto cells co-expressing AcGFP NuMA and Quantification of cortical intensity – μ mCherry H2B were treated with 20 M of MG132 for 2 h, before Quantification of either cortical NuMA or cortical GFP signal in cells μ treatment with 10 M RO-3306 for 10 min, followed by RO-3306 release expressing AcGFP or GFP-tagged NuMA protein was determined by for the next 20 min and live imaging. calculating the ratio of cortical mean intensity (of an area 1.8 µm ×4 µm, as shown in each figure) divided by the mean intensity value in the cytoplasm Indirect immunofluorescence and time-lapse imaging of HeLa cells (equal area). The raw values for the region of interest (ROI) that were used to For immunofluorescence, cells were fixed with −20°C methanol for 7 min. measure cortical intensity (ICortex) and cytoplasmic intensity (Icytosol) were

Cells were permeabilized in PBS containing 0.05% Triton X-100 (1× PBST) subtracted from the background intensity values (Ibg). Ibg values were Journal of Cell Science

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Fig. 6. See next page for legend. Journal of Cell Science

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Fig. 6. T2055 phosphorylation is dynamic and reversible. (A) Experimental Immunoprecipitation and western-blotting procedure for chemically induced anaphase onset by acute Cdk1 inactivation For immunoprecipitation, 3 mg of cell lysates procured from the nocodazole using RO-3306 in the presence of proteasome inhibitor MG132, followed by (100 nM)-arrested mitotic HeLa Kyoto cells expressing either AcGFP- the entry of cells into a metaphase-like state after washing off Cdk1 inhibitor for tagged or GFP-tagged proteins were incubated with 30 μl GFP-Trap agarose various time duration as indicated. Please note the color scheme, used beads (Chromotek, ACT-CM-GFA0050) in lysis buffer [50 mM Tris-HCl, throughout this figure, where orange represents cells (fixed or live) that were pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM sodium treated with MG132; cyan represents cells that were treated with MG132 and fluoride, 0.1 mM sodium orthovanadate (Sigma-Aldrich, S6508), 0.1 mM RO-3306, and brown shows cells that were washed free of RO-3306 using PMSF (Calbiochem, 7110), 0.2% Triton-X100, 0.3% NP-40, 100 nM media containing MG132. (B–E) HeLa cells treated with MG132 (B), MG132 okadaic acid and Complete EDTA-free protease inhibitor (Merck, 539134)] and RO-3306 (C), released from Cdk1 inhibition for 10 min (D), or released from Cdk1 inhibition for 20 min (E). Cells were fixed and stained for CYK4 (red), for 4 h at 4°C. The beads were washed three times with wash buffer (50 mM α-tubulin (green) and DNA (blue). Bars show mean±s.d. CYK4 intensity at the Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM central spindle for n>11 cells. Please note the increase in CYK4 intensity in sodium fluoride, 0.1 mM sodium orthovanadate, 0.1 mM PMSF, 0.2% cells treated with RO-3306 and MG132, which was significantly reduced when Triton-X100, 0.3% NP-40, 100 nM okadaic acid and complete EDTA-free cells were released from Cdk1 inhibition ( MG132 versus MG132+RO, protease inhibitor) at 4°C. The bead-bound complex was denatured at 99°C P<0.0001; MG132 versus 10-min release, P=0.0066; MG132 versus 20-min in 2× SDS buffer and was analyzed by SDS–PAGE and western blotting. release from RO-3306, P=0.86; two-tailed Student’s t-test). (F–I) HeLa cells For pulldown experiments with B55γ, each mg of mitotic cell lysate treated as described for B–E. Cells were fixed and stained for NuMA (green), prepared from cells expressing B55γ–AcGFP was incubated with 25 μgof Glued p150 (red) and DNA (blue). Bars show mean±s.d. cortical NuMA intensity 6His–NuMA(1876–2115) and 6His–NuMA(1876–2115)4KR>A for 30 min for n>10 cells. Please note the increase in NuMA cortical intensity in cells followed by pulldown with GFP-trap, as described above. treated with RO-3306 and MG132, which was significantly reduced when cells For western blotting analysis, HeLa cells synchronized with 100 nM were released from Cdk1 inhibition. (MG132 versus MG132+RO, P<0.0001; nocadazole for 16–20 h were lysed in lysis buffer (as decribed above) for 2 h MG132 versus 20-min release, P=0.3536). (J–M) HeLa cells treated as on ice. Protein concentration estimation was done using Bradford reagent described in B–E. Cells were fixed and stained for pT2055 (gray) and DNA (Biorad; 500-0001). Cell lysates were denatured at 99°C in 2× SDS buffer n (blue). Bars show mean±s.d. spindle pole NuMA intensity for >10 cells. and analyzed by SDS–PAGE followed by immunoblotting. For Please note the significant decrease in pT2055 signal at the spindle poles in immunoblotting, 1:1000 rabbit anti-NuMA (Santa Cruz, sc-48773), cells that were treated with RO-3306 and MG132. Also, note that pT2055 1:1000 rabbit anti-p2055 NuMA (Kotak et al., 2013), 1:5000 rabbit anti- intensity was rescued after 10- or 20-min release from Cdk1 inhibition. (MG132 GFP (Santa Cruz, sc-8334), 1:5000 anti-β-actin (Santa Cruz, 58673), versus MG132+RO, P<0.0001; MG132 versus 10-min release, P=0.24; 1:1000 rabbit anti-ENSA (Cell Signaling Technology, 8770S), 1:1000 MG132 versus 20-min release, P=0.13; two-tailed Student’s t-test). (N) Experimental protocol for chemically induced anaphase onset followed by rabbit anti-MASTL (Cell Signaling Technology, 12069S), 1:2000 rabbit mitotic entry, as described above, for live imaging. (O) HeLa cells stably anti-PPP1CA (Bethyl Laboratories, A300-904A), 1:2000 rabbit anti- expressing AcGFP–NuMA were treated with RO-3306 in live-imaging PPP1CB (Bethyl Laboratories, A300-905A), 1:2000 goat anti-PPP1CC conditions for 0–10 min and then released from Cdk1 inhibition for 20 min. (Santa Cruz, sc6108), 1:2000 rabbit anti-PPP2CA (CST, 2038S), 1:2000 Quantification of cortical enrichment is shown on the right for a HeLa cell first rabbit PPP4C (Bethyl Laboratories, A300-835A), 1:2000 rabbit PPP5C treated with the Cdk1 inhibitor RO-3306 for 10 min (top) and then released from (Bethyl Laboratories, A300-909A), 1:2000 rabbit anti-PPP6C (Bethyl this inhibition for the next 20 min in the presence of the proteasome inhibitor Laboratories,A300-844A), 1:2000 rabbit anti-PPP2R1B (Abcam, MG132 (bottom). More than five videos were recorded, and a representative EPR10158), 1:1000 mouse anti-p150Glued (Transduction Laboratories, cell is shown here. A.U, arbitrary units. (P) Model for cortical localization of 612709), and 1:5000 rabbit anti-His (Sigma, SAB4301134) were used. NuMA and dynein during metaphase and anaphase in human cells. The PPP2CA–B55γ–PPP2R1B phosphatase complex counteracts Cdk1 In vitro phosphorylation experiment phosphorylation of T2055 and maintains weak cortical localization of NuMA 1 μg of recombinant 6His–NuMA(1876–2115) was incubated with 0.125 ng of and the dynein–dynactin complex. This localization is critical for proper spindle Cdk1/cyclinB (Merck Millipore, 14-450) in 10μl of kinase buffer (50 mM orientation in metaphase. However, in anaphase, Cdk1-inactivation causes HEPES, pH 7.8, 1 M MgCl2, 1 M KCl, 100 mM EGTA, 200 mg/ml BSA, robust enrichment of cortical NuMA and dynein–dynactin at the cell cortex in a 0.2 mM ATP) at 30°C for 30 min. The reaction was stopped by adding – γ– manner dependent on PPP2CA B55 PPP2R1B, and this promotes proper 20 μM of RO-3306 (Selleckchem, S7747), followed by snap-freezing the spindle elongation in anaphase. In B–M,O, arrows indicate cortical localization, reactions in liquid N2. The phosphorylation status of recombinant NuMA boxes and circles indicate examples of regions used for quantification. was confirmed by western blotting with 1:1000 rabbit anti-p2055 NuMA **P<0.01, ***P<0.0001. (Kotak et al., 2013).

In vitro dephosphorylation experiment − obtained from a similar area outside the cell. The equation (ICortex Ibg)/ HeLa Kyoto cells stably expressing, B55γ–AcGFP or CIBN–CAAX– − (Icytosol Ibg) was used for the calculation of cortical intensity. The brightest FLAG–AcGFP or GFP–B55α, and arrested in prometaphase by treatment polar cortical region was used as a selection criterion in control as well as in with 100 nM of nocodazole for 17 h, were lysed in buffer [50 mM Tris-HCl, a given experimental condition. Significance was determined using a two- pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton-X100, tailed Student’s t-test for each condition in GraphPad Prism 8. 0.3% NP-40, 0.1 mM PMSF (Calbiochem, 7110), and Complete EDTA- free protease inhibitor (Merck, 539134)] on ice for 2 h. After protein Quantification of spindle pole intensity concentration estimation, each mg of cell lysate was incubated with 10 μlof Quantification of the spindle pole intensity of phosphorylated NuMA at GFP-Trap agarose beads (Chromotek, ACT-CM-GFA0050) for 4 h at 4°C. T2055 was calculated by determining the mean spindle pole intensity from a After extensive washing with wash buffer [50 mM Tris-HCl, pH 7.4, maximum intensity projection image of an area 16 µm2, as shown in each 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton-X100, 0.3% NP- figure, and correcting for the background signal of a similar area. 40, 0.1 mM PMSF and Complete EDTA-free protease inhibitor (Merck, Significance was determined using a two-tailed Student’s t-test for each 539134)] at 4°C, GFP-bound beads were collected and stored on ice. condition in GraphPad Prism 8. For each dephosphorylation reaction, GFP-bound beads equivalent to 1 mg of cell lysate were suspended in 25 μl of dephosphorylation buffer Spindle elongation and chromosome separation [50 mM Tris-HCl, pH 7.35, 150 mM NaCl, 0.1 mM MnCl2, 1 mM MgCl2, The distance between the separating chromosomes in cells expressing 0.1% NP-40, 0.2 mg/ml BSA, 20μM RO-3306] and was incubated with mCherry–H2B, was quantified every 2 min after anaphase onset using 50–100 ng of Cdk1/cyclinB-phosphorylated recombinant 6His– Imaris (Bitplane Inc.). Significance was determined using a two-tailed NuMA(1876–2115) at 30°C. The reaction was stopped by adding 2× SDS

Student’s t-test in GraphPad Prism 8. buffer, and the samples were boiled at 99°C. The supernatant was obtained Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs243857. doi:10.1242/jcs.243857 following centrifugation at 13,000 rpm (17,000 g) for 5 min, and was Cundell, M. J., Bastos, R. N., Zhang, T., Holder, J., Gruneberg, U., Novak, B. and utilized for further analysis by SDS–PAGE and western blotting. Barr, F. A. (2013). The BEG (PP2A-B55/ENSA/Greatwall) pathway ensures Mol. Cell For GFP antibody-mediated inhibition of dephosphorylation, 1 mg cytokinesis follows chromosome separation. 52, 393-405. doi:10.1016/j. γ– molcel.2013.09.005 equivalent of B55 AcGFP-bound beads were incubated with 330 ng of Connell, M., Chen, H., Jiang, J., Kuan, C. W., Fotovati, A., Chu, T. L., He, Z., mouse anti-GFP (DSHB-GFP-8H11) at 22°C for 30 min prior to the addition Lengyell, T. C., Li, H., Kroll, T. et al. (2017). HMMR acts in the PLK1-dependent of Cdk1/cyclinB-phosphorylated recombinant 6His–NuMA(1876–2115). spindle positioning pathway and supports neural development. Elife 6, e28672. doi:10.7554/eLife.28672.027 Acknowledgements Deana, A. D. and Pinna, L. A. (1988). Identification of pseudo ‘phosphothreonyl- We thank Andreas Merdes, Daniel Gerlich, Arnaud Echard, Mark Petronczki, and specific’ protein phosphatase T with a fraction of polycation-stimulated protein Biochim. Biophys. Acta Anthony Hyman for providing us plasmids and cell lines. We are grateful to Jon phosphatase 2A. 968, 179-185. doi:10.1016/0167- Pines, Iain Hagan, Phong Tran, Xavier Morin, Helder Maiato and Carlos Conde for 4889(88)90006-7 providing us critical comments on this manuscript. We thank DST-FIST, UGC Centre Deana, A. D., Marchiori, F., Meggio, F. and Pinna, L. A. (1982). for the Advanced Study, DBT-IISc Partnership Program and IISc for the Dephosphorylation of synthetic phosphopeptides by protein phosphatase-T, a phosphothreonyl protein phosphatase. J. Biol. Chem. 257, 8565-8568. infrastructure support. di Pietro, F., Echard, A. and Morin, X. (2016). Regulation of mitotic spindle orientation: an integrated view. EMBO Rep. 17, 1106-1130. doi:10.15252/embr. Competing interests 201642292 The authors declare no competing or financial interests. Du, Q. and Macara, I. G. (2004). Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins. Cell 119, 503-516. doi:10.1016/j.cell. Author contributions 2004.10.028 Conceptualization: S.K.; Methodology: R.K., S.K.; Validation: R.K., A.R., S.K.; Du, Q., Taylor, L., Compton, D. A. and Macara, I. G. (2002). LGN blocks the ability Formal analysis: R.K., A.R., S.K.; Investigation: R.K., S.K.; Resources: S.K.; Data of NuMA to bind and stabilize microtubules. A mechanism for mitotic spindle curation: R.K., S.K.; Writing - original draft: S.K.; Writing - review & editing: R.K., assembly regulation. Curr. Biol. 12, 1928-1933. doi:10.1016/S0960- A.R., S.K.; Visualization: S.K.; Supervision: S.K.; Project administration: S.K.; 9822(02)01298-8 Funding acquisition: S.K. 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