Polo regulates Spindly to prevent premature stabilization of kinetochore–microtubule attachments
2019; Springer Nature; Volume: 39; Issue: 2 Linguagem: Inglês
10.15252/embj.2018100789
ISSN1460-2075
AutoresJoão Barbosa, Torcato Martins, Tanja Bange, Tao Li, Carlos Conde, Cláudio E. Sunkel,
Tópico(s)Cellular transport and secretion
ResumoArticle18 December 2019free access Polo regulates Spindly to prevent premature stabilization of kinetochore–microtubule attachments João Barbosa IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal i3S, Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Porto, Portugal Search for more papers by this author Torcato Martins Department of Genetics, University of Cambridge, Cambridge, UK Search for more papers by this author Tanja Bange MPI für molekulare Physiologie, Dortmund, Germany Search for more papers by this author Li Tao Department of Biology, University of Hawaii, Hilo, HI, USA Search for more papers by this author Carlos Conde Corresponding Author [email protected] IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal i3S, Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Porto, Portugal Search for more papers by this author Claudio Sunkel Corresponding Author [email protected] orcid.org/0000-0002-2963-9380 IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal i3S, Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Porto, Portugal ICBAS—Instituto de Ciência Biomédica de Abel Salazar, Universidade do Porto, Porto, Portugal Search for more papers by this author João Barbosa IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal i3S, Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Porto, Portugal Search for more papers by this author Torcato Martins Department of Genetics, University of Cambridge, Cambridge, UK Search for more papers by this author Tanja Bange MPI für molekulare Physiologie, Dortmund, Germany Search for more papers by this author Li Tao Department of Biology, University of Hawaii, Hilo, HI, USA Search for more papers by this author Carlos Conde Corresponding Author [email protected] IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal i3S, Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Porto, Portugal Search for more papers by this author Claudio Sunkel Corresponding Author [email protected] orcid.org/0000-0002-2963-9380 IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal i3S, Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Porto, Portugal ICBAS—Instituto de Ciência Biomédica de Abel Salazar, Universidade do Porto, Porto, Portugal Search for more papers by this author Author Information João Barbosa1,2, Torcato Martins3, Tanja Bange4, Li Tao5, Carlos Conde *,1,2,‡ and Claudio Sunkel *,1,2,6,‡ 1IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal 2i3S, Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Porto, Portugal 3Department of Genetics, University of Cambridge, Cambridge, UK 4MPI für molekulare Physiologie, Dortmund, Germany 5Department of Biology, University of Hawaii, Hilo, HI, USA 6ICBAS—Instituto de Ciência Biomédica de Abel Salazar, Universidade do Porto, Porto, Portugal ‡These authors contributed equally to this work *Corresponding author. Tel: +351 220 408 800; E-mail: [email protected] *Corresponding author. Tel: +351 220 408 800; E-mail: [email protected] EMBO J (2020)39:e100789https://doi.org/10.15252/embj.2018100789 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Accurate chromosome segregation in mitosis requires sister kinetochores to bind to microtubules from opposite spindle poles. The stability of kinetochore–microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing kinetochore–microtubule attachments. However, the mechanism underlying Polo–destabilizing activity remains elusive. Here, resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, we identified a strong genetic interaction between Polo and the Rod–ZW10–Zwilch (RZZ) complex, whose kinetochore accumulation has been shown to antagonize microtubule stability. We find that Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from kinetochores and consequently delays the formation of stable end-on attachments. We propose that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. Our findings demonstrate that Polo tightly regulates the RZZ–Spindly–dynein module during mitosis to ensure the fidelity of chromosome segregation. Synopsis Polo kinase is involved in both stabilizing and destabilizing kinetochore-microtubule attachments during mitosis. A Drosophila RNAi screen identifies the RZZ complex as mediator of Polo-dependent destabilization of early mitotic spindle mis-attachments. Constitutively-active Polo kinase delays formation of stable end-on kinetochore-microtubule attachments in an RZZ-dependent manner. Polo phosphorylates the dynein adaptor Spindly at prometaphase kinetochores, decreasing its affinity towards the RZZ complex. Dynein promptly strips phosphorylated Spindly from kinetochores, leaving the RZZ behind to reduce the stability of microtubule attachments. Decreasing Spindly phosphorylation as chromosomes congress enables efficient dynein-mediated removal of RZZ from kinetochores and consequent stabilization of microtubule attachments. Preventing Polo from phosphorylating Spindly during early mitosis leads to premature stabilization of merotelic attachments. Introduction To ensure the fidelity of chromosome segregation, sister kinetochores (KTs) mediate the attachment of chromosomes to microtubules (MTs) of opposite spindle poles (amphitelic attachments). However, the initial contact of KTs with MTs is stochastic and consequently erroneous attachments—syntelic (chromosome bound to MTs from the same spindle pole) or merotelic (same KT bound to MTs from opposite poles)—can be formed during early mitosis (Cimini et al, 2001; Thompson & Compton, 2011; reviewed Foley & Kapoor, 2013). Thus, accurate mitosis requires a tight regulation of KT-MT turnover so mistakes are prevented or corrected and amphitelic end-on interactions are stabilized (Bakhoum et al, 2009a,b; Zaytsev & Grishchuk, 2015). This relies heavily on the activity of two conserved mitotic kinases, Aurora B and Polo/Plk1. Aurora B promotes the destabilization of KT-MT interactions mainly through phosphorylation of proteins of the KMN network (KNL1/Spc105, Mis12 and Ndc80), which decreases their affinity for MTs (Lampson et al, 2004; Cheeseman et al, 2006; Cimini et al, 2006; DeLuca et al, 2006, 2011; Cimini, 2007; Welburn et al, 2010). Interestingly, it has been shown that the RZZ complex (Rod, ZW10 and Zwilch) is able to interact with Ndc80 N-terminal tail and prevent the adjacent calponin homology (CH) domain from binding to tubulin (Cheerambathur et al, 2013). This Aurora B-independent destabilizing mechanism is proposed to prevent Ndc80-mediated binding when KTs are laterally attached, hence reducing the potential for merotely during early mitosis. The RZZ additionally recruits Spindly and the minus end-directed motor dynein to KTs (Griffis et al, 2007; Gassmann et al, 2008, 2010; Chan et al, 2009; Gama et al, 2017; Mosalaganti et al, 2017), thus providing the means to relieve its inhibitory effect over KT-MT attachments, as well as to ensure the timely removal of spindle assembly checkpoint (SAC) proteins from KTs (Gassmann et al, 2010; Cheerambathur et al, 2013). However, it remains unclear how RZZ removal by Spindly–dynein is coordinated with end-on attachment formation. Polo/Plk1 activity is implicated in both stabilization (Elowe et al, 2007; Matsumura et al, 2007; Liu et al, 2012; Suijkerbuijk et al, 2012; Dumitru et al, 2017) and destabilization (Ahonen et al, 2005; Foley et al, 2011; Moutinho-Santos et al, 2011; Salimian et al, 2011; Hood et al, 2012; Paschal et al, 2012; Beck et al, 2013) of KT-MT attachments. While the contribution to the former function has been attributed to PP2A-B56 phosphatase recruitment through Plk1-dependent BubR1 phosphorylation (Elowe et al, 2007; Matsumura et al, 2007; Suijkerbuijk et al, 2012), the mechanism underlying Polo/Plk1 destabilizing activity remains unclear. Interestingly, Polo/Plk1 KT localization and activity decrease from early mitosis to metaphase (Ahonen et al, 2005; Conde et al, 2013), concurrent with an increase in KT-MT stability (Bakhoum et al, 2009a). Moreover, high Plk1 activity at KTs was shown to correlate with decreased stability of KT-MT attachments during prometaphase (Foley et al, 2011; Paschal et al, 2012; Beck et al, 2013), but the underlying molecular mechanisms have only been marginally addressed (Godek et al, 2015). Here, we describe the mitotic effect of expressing a constitutively active Polo-kinase mutant (PoloT182D) in Drosophila neuroblasts and cultured S2 cells. The expression of PoloT182D causes persistent KT-MT instability and congression defects, extends mitotic timing associated with SAC activation and increases chromosome mis-segregation. We designed a small-scale candidate-based RNAi screen to identify partners/pathways that are affected by constitutive Polo activity in the Drosophila eye epithelium. The screen revealed that downregulation of the RZZ subunit Rod rescues the defects resulting from PoloT182D expression. We show that PoloT182D causes permanent accumulation of the RZZ complex at KTs, which is associated with a delay in achieving stable biorientation. Accordingly, Rod depletion rescues the time required for establishing end-on KT-MT attachments and for chromosome congression. We further demonstrate that Polo phosphorylates the dynein-adaptor Spindly to decrease its affinity for the RZZ. This in turn prevents dynein-dependent stripping of RZZ from KTs, hence causing a delay in the formation of stable end-on attachments. Our findings provide a mechanism for the destabilizing action of Polo/Plk1 over KT-MT attachments. We propose a model in which Polo/Plk1 activity fine-tunes the RZZ–Spindly–dynein module throughout mitosis to ensure the fidelity of KT-MT attachments and chromosome segregation. Results Constitutively active Polo kinase leads to unstable KT-MT attachments Polo/Plk1 has been implicated in both stabilizing and destabilizing KT-MT interactions. To understand how these apparently opposing actions are coordinated to ensure proper chromosome segregation in mitosis, we first monitored the level of active Polo at KTs during different mitotic stages in Drosophila neuroblasts. Using a phosphospecific antibody for the activating T-loop phosphorylation (Fig 1A and B), we find that Polo is more active at KTs during prometaphase and its activity markedly decreases at metaphase, when KT-MT attachments are more stable. Maintaining Plk1 constitutively active in different human cell lines produced conflicting results regarding its effect on KT-MT attachments and chromosome congression (Liu et al, 2012; Paschal et al, 2012). Thus, we decided to assess the impact of constitutively active Polo on mitotic progression in vivo. The expression of either UASPoloWT (wild-type) or UASPoloT182D was induced in neuroblasts with the inscuteable-Gal4 driver (Betschinger et al, 2006) and mitotic progression analysed (Fig 1C; Movie EV1). In contrast to PoloWT, the expression of PoloT182D caused a significant extension of the time from nuclear envelope breakdown to anaphase onset (NEBD-AO) (Fig 1D). This mitotic delay results mainly from chromosome congression defects, as the time measured from NEBD to alignment of the last chromosome was twice as long in PoloT182D-expressing neuroblasts (Fig 1E). In vivo measurement of inter-kinetochore distances revealed that the increased time in prometaphase duration was accompanied by reduced centromeric tension (Fig 1F), indicating that PoloT182D delays the establishment of KT-MT end-on attachments. This observation suggests that KT-MT interactions are more labile in neuroblasts expressing PoloT182D. To confirm this, we monitored the localization of Mad2-GFP, a SAC protein that decorates unattached KTs. In PoloWT-expressing neuroblasts, Mad2-GFP accumulates at KTs during early prometaphase and the signal rapidly fades as stable KT-MT attachments are established, allowing chromosomes to align at the metaphase plate within a few minutes (Fig 1G and H; Movie EV2). In contrast, Mad2-GFP persisted for longer periods at KTs of neuroblasts expressing PoloT182D, indicating a reduced MT occupancy on unaligned KTs (Fig 1G and H; Movie EV3). This conclusion is further supported by immunofluorescence analysis showing increased levels of Mad1 at KTs of neuroblasts that express the constitutively active kinase (Appendix Fig S1A and B). Figure 1. Expression of constitutively active Polo kinase destabilizes kinetochore–microtubule (KT-MT) interactions Representative immunofluorescence images of Polo-phospho(ph)-T182 levels at KTs of Drosophila neuroblasts throughout mitotic progression. Insets show magnifications of the outlined regions showing kinetochores (KTs) in early prometaphase (EP), late prometaphase (LP) KTs and aligned KTs in metaphase (M). Plotted profiles of signal intensities of phospho(ph)-T182 and the KT protein Spc105 are shown for the highlighted KTs. Graph represents the levels of Polo phT182 at KTs for neuroblasts shown in (A). phT182 signal was determined relative to Spc105 and all values normalized to the mean value determined for EP, which was set to 100% (n ≥ 67 KTs from at least 19 neuroblasts for each condition, n = 4 independent experiments). Selected stills from live imaging analysis of mitotic progression in neuroblasts expressing UASPoloWT or UASPoloT182D under the control of inscuteable-Gal4 driver. Neuroblasts without Polo overexpression were used as control. Mitotic progression was followed in vivo by direct visualization of tubulin-RFP and the centromere marker CID-GFP. Time 0 refers to nuclear envelope breakdown (NEBD). Quantification of the mitotic time (from NEBD to anaphase onset) for neuroblasts shown in (C). NEBD was identified as the time tubulin entered the nuclear space and anaphase onset as the time sister KTs separated (n ≥ 12 neuroblasts for each condition, n ≥ 3 independent experiments). Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for neuroblasts shown in (C) (n ≥ 11 neuroblasts for each condition, n ≥ 3 independent experiments). Measurement of inter-kinetochore distance during mitotic progression for neuroblasts shown in (C). Only KT pairs within the maximum of two consecutive z-planes were considered eligible for quantification. Images were acquired every 20 s. The graph shows the mean distance between two CID centroids in a KT pair over time (t = 0 is NEBD). A linear regression was applied to the data set. Vertical dashed lines highlight the time at which cells overexpressing PoloWT (grey) or PoloT182D (red) reach the average inter-kinetochore distance measured in metaphase cells without Polo overexpression (black) (n ≥ 7 neuroblasts for each condition). Selected stills from live imaging of neuroblasts expressing Mad2-GFP to follow KT-MT attachment status upon expression of PoloWT or PoloT182D. CID-RFP was used as KT reference. Insets show magnifications of the outlined regions showing single KT pairs that take longer to align at the metaphase plate. Asterisks indicate direction of chromosome segregation (putative spindle pole positions). Graph represents the mean fluorescence intensity (MFI) for Mad2-GFP at KTs measured from NEBD to anaphase onset for neuroblasts shown in (G). Each line represents the average of all KTs measured from a single neuroblast at each time point. Mad2-GFP MFI was determined relative to CID-RFP MFI (n ≥ 16 neuroblasts for each condition, n = 5 independent experiments). Data information: Statistical analysis was calculated using a Kruskal–Wallis test for multiple comparisons. P values: ns, not significant; *< 0.05; **< 0.01; ****< 0.0001. Data are shown as mean ± SD. Scale bar: 5 μm. Download figure Download PowerPoint To assess in greater detail the configuration of KT-MT attachments, we turned to cultured S2 cells which also showed increased frequency of unaligned chromosomes when Polo is constitutively active (Fig 2A and B). Detailed analysis of KT-MT interactions shows that these cells have aligned KTs attached to the side of a MT end, which occurs to a lesser extent in PoloWT-EGFP expressing cells (Fig 2C and D). Importantly, analysis of neuroblasts expressing PoloT182D allowed us to determine the dynamics and outcome of mitotic progression in a more reproducible manner than cultured cells (Fig 2E–G). Time course analysis shows that these cells undergo a highly asynchronous chromatid migration during anaphase as opposed to the synchrony observed in PoloWT neuroblasts (Fig 2E and G). Moreover, whereas virtually all PoloWT neuroblasts displayed a normal karyotype, we frequently detected aneuploidy in neuroblasts expressing PoloT182D (Fig 2F and G). Collectively, these results demonstrate that constitutive activation of Polo renders KT-MT attachments unstable and compromises the fidelity of chromosome segregation. These errors are unlikely to result from hyperactivation of Aurora B, as the expression of PoloT182D failed to cause a significant increase in the phosphorylation of Spc105 Ser35 (Appendix Fig S1C and D), a well-described Aurora B substrate (Welburn et al, 2010; Bajaj et al, 2018). Figure 2. Expression of PoloT182D compromises mitotic fidelity Representative immunofluorescence images of KT alignment efficiency in mitotic S2 cells expressing PoloWT-EGFP, PoloT182D-EGFP or lacking expression of any Polo transgene. Cells were treated with MG132 prior to fixation to prevent cells from exiting mitosis and increase the number of pre-anaphase figures. Insets display magnifications of the outlined regions, which highlight both low-tension misaligned (insets 1 and 2) and high-tension aligned KTs (inset 3). Graph represents the percentage of cells in each indicated mitotic state, as shown in (A) (n ≥ 532 cells for each condition, n = 2 independent experiments). Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Asterisk highlights either an aligned KT pair attached to MTs in an end-on fashion (PoloWT-EGFP) or an aligned KT pair in which a sister KT is laterally attached to the end of a MT fibre (PoloT182D-EGFP). Plotted profiles show the overlap between Polo-EGFP and tubulin signals for the highlighted KT. Graph represents the percentage of metaphase cells showing at least 1 KT with a lateral interaction, as shown in (C) (asterisk) (n ≥ 58 cells for each condition, n = 2 independent experiments). Selected frames from live image analysis of chromosome segregation fidelity in neuroblasts expressing either PoloWT or PoloT182D. Neuroblasts without Polo overexpression were used as control. Jupiter-GFP was imaged for direct visualization of the mitotic spindle. Time 0 refers to anaphase onset. Representative immunofluorescence images of mitotic neuroblasts from squashed brains showing distinct chromosome content. Spc105 was used as reference for KTs. Chromosome content is shown for each neuroblast. Table shows the quantification of the percentage of asynchronous migration (n ≥ 23 neuroblasts for each condition, n ≥ 3 independent experiments) and of aneuploid cells shown in (E and F), respectively. For the quantification of aneuploidy, the whole brain was analysed and prometaphase and metaphase neuroblasts were scored for chromosome number. An aneuploid cell was considered when > 8 chromosomes (chr) were visualized (n ≥ 360 neuroblasts from at least seven larvae brains for each condition, n ≥ 3 independent experiments). ND, not determined. Data information: Data are shown as mean ± SD. Scale bar: 5 μm. Download figure Download PowerPoint RNAi-based screen identifies rough deal as a suppressor of PoloT182D expression To uncover how Polo destabilizes KT-MT attachments, we designed a small-scale RNAi screen to identify molecular players mis-regulated by constitutively active Polo kinase (Fig 3A). We selected 222 candidates (Appendix Tables S1 and S2) biased towards putative Polo interactors annotated on DroID database (www.droidb.org) and induced RNAi-mediated depletion of individual genes in the eye imaginal disc in a PoloT182D background. The expression of PoloT182D with the eye-specific driver eyeless Gal4 caused abnormal eye development, which ultimately led to a dramatic reduction in the adult structure and a significant reduction in male survival at 25°C (Fig 3B and C). Thus, simply by scoring for the increase in male viability, we identified 24 suppressors of the PoloT182D phenotype (Table EV1). Among these, we identified rough deal, the gene encoding for Rod, a subunit of the RZZ complex (Karess & Glover, 1989). Previous studies in Caenorhabditis elegans showed that Rod interacts with Ndc80 preventing the formation of stable end-on attachments (Cheerambathur et al, 2013). Thus, we hypothesized that constitutively active Polo might promote KT-MT instability through inappropriate Rod function. Figure 3. RNAi-based screen identifies Rod as a suppressor of the PoloT182D phenotype Schematics of the RNAi screen strategy. Females expressing UASPoloT182D under the control of eyless-Gal4 promoter were crossed with males carrying a specific UAS-RNAi transgene. Crosses were left either at 18°C or at 25°C, and adult offspring viability was assessed. Representative images of the eye phenotype observed in adult flies that were expressing UASPoloT182D in the eye imaginal disc during development at indicated temperatures. An example of an eye from an adult fly co-expressing UASPoloT182D with UASRodRNAi is also shown. The expression of UASlacZ transgene in the eye imaginal disc was used as control for overexpression and UAS dilution effect. Graph represents the mean percentage of males born at 25°C either expressing eyGal4-driven UASPoloT182D together with UASlacZ or in combination with UASRodRNAi (n ≥ 3 independent crosses for each condition). The number of males with the genotype of interest born in each cross was normalized to the mean total number of males that are born in a control cross (eyGal4 > UASlacZ, n = 9 independent crosses). Selected stills from live-cell imaging of neuroblasts expressing either in UASPoloT182D alone or in Rod-depleted background (UASPoloT182D + UASRodRNAi). Neuroblasts without transgene expression were used as control, and neuroblasts expressing UASPoloT182D together with UASlacZ were used as control for UAS dilution effect. Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for neuroblasts shown in (D) (n ≥ 17 neuroblasts for each condition, n ≥ 4 independent experiments). Statistical analysis was calculated using an one-way ANOVA test for multiple comparisons. P values: ns, not significant; **< 0.01. Quantification of the percentage of prometaphase/metaphase mitotic figures per brain showing abnormal chromosome number (> 8 chromosomes (chr) as shown in Fig 2F and G) (n ≥ 360 neuroblasts from at least seven larvae brains for each condition, n ≥ 3 independent experiments). Statistical analysis was calculated using a Kruskal–Wallis test for multiple comparisons. P values: ns, not significant; **< 0.01. Data information: Data are shown as mean ± SD. Scale bar: 5 μm. Download figure Download PowerPoint To evaluate the suppressor effect of Rod depletion in PoloT182D-expressing neuroblasts, we monitored mitotic progression by live-cell imaging (Fig 3D and E; Movie EV4). While control neuroblasts efficiently congressed all chromosomes to the metaphase plate within 3.5 min, the expression of PoloT182D significantly compromised this process, extending prometaphase duration up to 13 min. Notably, depletion of Rod partially rescued this delay, with neuroblasts achieving full KT alignment in 8 min (Fig 3E; Movie EV4). Analysis of data dispersion indicates that the absence of Rod particularly improves chromosome congression in PoloT182D neuroblasts (Fig 3E). Importantly, Rod depletion by itself did not significantly affect prometaphase duration, suggesting that SAC mis-regulation is not responsible for the observed rescue (Fig EV1A–C; Movie EV5). Moreover, depletion of Rod in neuroblasts expressing PoloT182D also rescued the fidelity of chromosome segregation as indicated by the significant decrease in the frequency of aneuploidy (Fig 3F). Rod RNAi led to an equivalent reduction in the accumulation of KT-associated ZW10 in colchicine-treated neuroblasts regardless of PoloT182D expression, suggesting that the same level of depletion occurred in both conditions (Fig EV1D and E). Moreover, total levels of PoloT182D were unaffected by Rod RNAi (Fig EV1F and G) and Gal4-driven expression of UASlacZ failed to suppress the prometaphase delay and aneuploidy caused by PoloT182D (Fig 3D–F). These results exclude titration of the Gal4 transcription factor as the underlying cause for the observed rescue in mitotic fidelity. Taken together, these data strongly suggest that chromosome congression defects and segregation errors observed in PoloT182D neuroblasts are linked to Rod mis-regulation. Click here to expand this figure. Figure EV1. Absence of the RZZ complex does not impact on the time required for chromosome congression in Drosophila neuroblasts Selected stills from live imaging analysis of mitotic progression in neuroblasts depleted of Rod (UASRodRNAi). Neuroblasts without transgene expression were used as control. Quantification of the mitotic time (time from NEBD to anaphase onset) for neuroblasts shown in (A) (n ≥ 9 neuroblasts for each condition, n = 2 independent experiments). Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for neuroblasts shown in (A) (n ≥ 9 neuroblasts for each condition, n = 2 independent experiments). Representative immunofluorescence images of ZW10 levels in colchicine-treated neuroblasts expressing UASPoloWT, UASPoloT182D, UASRodRNAi or UASPoloT182D in a Rod-depleted background (UASPoloT182D + UASRodRNAi). Insets show magnifications of the outlined regions. Neuroblasts expressing UASPoloWT were used as control. Graph represents ZW10 levels in neuroblasts for the conditions shown in (D). ZW10 levels were determined relative to Spc105, and all values were normalized to the control mean fluorescence intensity, which are set to 100% (n ≥ 1116 KTs for each condition, n ≥ 2 independent experiments). Western blot analysis of UAS dilution effect on Polo overexpression levels in brain extracts from 3rd-instar larvae expressing UASPoloWT, UASPoloT182D, UASPoloT182D together with UASlacZ or in a Rod-depleted background (UASPoloT182D + UASRodRNAi). Tubulin was used as a loading control. Graph represents the fold increase in Polo protein levels relative to the absence of transgene overexpression. Expected Polo protein levels were determined after normalization to tubulin levels, and fold increase was calculated comparing to the actual Polo levels. Data information: Statistical analysis was calculated using an unpaired t-test (Mann–Whitney). P values: **< 0.01. Data are shown as mean ± SD. Scale bar: 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Depletion of ZW10 rescues chromosome congression in PoloT182D expressing cells independently of SAC impairment Selected stills from live imaging analysis of mitotic progression in Drosophila S2 cells expressing PoloWT-EGFP, PoloT182D-EGFP and PoloT182D-EGFP in a ZW10- or Mad2-depleted background. Time 0 refers to nuclear envelope breakdown (NEBD). Black box highlights full KT alignment. Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for cells shown in A (n ≥ 8 cells for each condition, n ≥ 3 independent experiments). Note that the Mad2-depleted cell does not achieve full KT alignment. Graph represents the percentage of cells that enter anaphase with different levels of KT alignment (n = 15 cells, n ≥ 3 independent experiments). Data information: Statistical analysis was calculated using a Kruskal–Wallis test for multiple comparisons. P values: *< 0.05; **< 0.01. Data are shown as mean ± SD. Scale bar: 5 μm. Download figure Download PowerPoint Constitutively active Polo impairs RZZ stripping from late congressing KTs To determine how Polo activity regulates Rod, we imaged neuroblasts expressing Rod-GFP under the regulation of its endogenous promoter in the presence of either PoloWT or PoloT182D. In accordance with previous studies (Basto et al, 2004; Siller et al, 2005; Défachelles et al, 2015), Rod-GFP localized prominently to KTs during prometaphase and was significantly reduced during metaphase in PoloWT neurob
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