© 2014. Published by The Company of Biologists Ltd.
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James Bancroft1,2,4, Philip Auckland1,4, Catarina P. Samora1,3 and Andrew D. McAinsh1
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Mechanochemical Cell Biology Building, Division of Biomedical Cell Biology, Warwick Medical School, University of
Warwick, Coventry CV4 7AL, UK a.d.mcainsh@warwick.ac.uk
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Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RF, UK
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Present address: CRUK London Research Institute, Lincolns Inn Fields, London WC2A 3LY, UK.
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These authors contributed equally to this work.
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A key step of mitosis is the congression of chromosomes to the spindle equator. Congression
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is driven by at least two distinct mechanisms: (1) kinetochores slide along the microtubule
Accepted manuscript
Summary
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lattice using the plus-end directed CENP-E motor, and (2) kinetochores biorientating near the
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pole move to the equator through microtubule depolymerisation-coupled pulling. Here, we
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show that CENP-Q - a subunit of the CENP-O/P/Q/U complex that targets polo-like kinase
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(Plk1) to kinetochores - is also required for the recruitment of CENP-E to kinetochores. We
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further reveal a CENP-E recruitment-independent role for CENP-Q in depolymerisation-
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coupled pulling. Both these functions are abolished by a single point mutation in CENP-Q
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(S50A) – a residue that is phosphorylated in vivo. Importantly, the S50A mutant does not
Journal of Cell Science
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Chromosome congression is promoted by CENP-Q- and
CENP-E-dependent pathways
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affect Plk1 loading onto kinetochores and leaves the CENP-O complex intact. Thus, the
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function of CENP-Q in CENP-E loading and depolymerisation-coupled pulling are
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independent from its role in Plk1 recruitment and CENP-O/P/Q/U complex stabilization.
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Together, our data provide evidence that phospho-regulation of CENP-Q plays a central
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function in coordinating chromosome congression mechanisms.
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Introduction
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Chromosome congression is the process by which chromosomes align at the spindle equator,
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a position equidistant from both spindle poles (Kops et al., 2010; Walczak et al., 2010). The
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timescale of this process in human cells is ~15-20 minutes and results in the formation of the
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metaphase plate. Chromosomes remain in metaphase for a similar period of time before sister
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chromatids are segregated to opposite spindle poles in anaphase. Congression is coincident
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with the process of chromosome biorientation; that is the establishment of amphitelic (or
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bipolar) attachments in which sister kinetochores attach to microtubules emanating from
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opposite spindle poles. Such amphitelic attachments are compatible with oscillatory motion
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because sisters switch between poleward (P) and away-from-the-pole (AP) movement states
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(Skibbens et al., 1993). These chromosomes are able to congress because they undergo
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JCS Advance Online Article. Posted on 13 November 2014
Accepted manuscript
Journal of Cell Science
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sustained movements towards the spindle equator. The force for these movements comes
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from the P moving sister kinetochore remaining attached to depolymerising kinetochore-
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microtubule (K-MT) plus-ends (from here on called depolymerisation-coupled pulling) (Coue
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et al., 1991; Koshland et al., 1988; Mitchison and Salmon, 1992). Congression can also take
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place before biorientation: mono-orientated kinetochores have been shown to engage the
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lattice of a pre-existing kinetochore-microtubule fibres (K-fibres) and other stabilized
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microtubule bundles through the second (leading) sister and slide towards the spindle equator
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(Kapoor et al., 2006). Once at the metaphase plate these kinetochores then biorientate.
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Furthermore, work in human cells and mouse oocytes reveals that chromosomes are arranged
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in a ring-like conformation early in prometaphase that favors the “instantaneous”
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biorientation of sister kinetochores at the spindle equator (Kitajima et al., 2011; Magidson et
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al., 2011). Thus, cells have multiple mechanisms to position chromosomes on the metaphase
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plate.
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The key challenge is to identify the molecules that are involved in these congression
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mechanisms. The sliding of chromosomes by laterally-attached kinetochores is mediated by
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the minus-end directed motor dynein (Yang et al., 2007), or the plus-end directed kinesin
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CENP-E (Kapoor et al., 2006; Kim et al., 2010). Following depletion or inhibition of CENP-E
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the majority of chromosomes congress forming a metaphase plate, however, a subset remain
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trapped around the spindle poles; these are termed polar chromosomes (Putkey et al., 2002).
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In contrast to these lateral-sliding mechanisms, the molecules required for depolymerisation-
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coupled pulling are largely unknown. Such a mechanism must involve proteins that (1)
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mediate end-on microtubule-kinetochore attachments, (2) maintain attachment to
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depolymerising microtubules and (3) ensure the K-fibre remains in a net depolymerising state
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through control of K-MT dynamics. The Ndc80 and Ska complexes can directly bind
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microtubules in vitro, with the Ska complex (but not Ndc80) able to autonomously track
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depolymerising microtubule plus-ends (Schmidt et al., 2012). Extensive depletion of either
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complex causes a severe failure in chromosome congression and the inability to form a
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metaphase plate (Daum et al., 2009; Gaitanos et al., 2009; Welburn et al., 2010). Thus, the
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Ndc80 and Ska complexes are proposed to form the end-on microtubule attachment sites
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within the kinetochore.
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Whether the process of end-on attachment can be separated from the mechanism of
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depolymerisation-coupled pulling is unclear. In this regard, our previous work showed that
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the constitutive centromere associated network (CCAN) is required for normal chromosome
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congression, but is not required for formation of end-on attachments (Amaro et al., 2010;
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Accepted manuscript
Journal of Cell Science
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McClelland et al., 2007; Toso et al., 2009). The CCAN is a 17-subunit complex that contains
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a set of core factors that directly bind CENP-A (CENP-C, -N) and H3 nucleosomes (CENP-
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T/W/S/X) (for reviews see (McAinsh and Meraldi, 2011; Westhorpe and Straight, 2013). The
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core CCAN recruits the extended CCAN, which includes the CENP-H/I/K/M complex
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(Basilico et al., 2014) and the CENP-O/P/Q/U complex (Hori et al., 2008). However, the
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function of this extended CCAN is poorly understood. Phenotypic analysis shows that
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depletion of such CCAN subunits can interfere with timely chromosome congression as well
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as altering K-MT plus-end turnover, K-fibre stability and poleward microtubule flux (Amaro
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et al., 2010; Foltz et al., 2006; Hori et al., 2008; McClelland et al., 2007; McHedlishvili et al.,
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2012). The CENP-O complex also plays a role in recruitment of other key kinetochore
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components, including Plk1 via interaction with phospho-T78 of CENP-U (Kang et al., 2011).
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Moreover, the CENP-Q and CENP-U subunits bind directly to taxol-stabilised microtubules
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in vitro (Amaro et al., 2010; Hua et al., 2011), suggesting that they may have direct roles in
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regulating the kinetochore-microtubule, and potentially depolymerisation-coupled pulling.
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Results
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Depletion of CENP-Q causes accumulation of polar chromosomes.
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To investigate the function of human CENP-Q we depleted the protein in HeLa cells using
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small interfering RNA (siRNA) oligonucleotides. Immunoblotting with anti-CENP-Q
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antibodies demonstrated that the total protein levels were reduced to non-detectable levels
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(Fig. 1A). Moreover, quantitative immunofluorescence confirmed that the kinetochore-bound
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population of the CENP-Q protein is reduced by 86% (± 6.2) (relative to CENP-A) following
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siRNA treatment (Fig. 1B). Previous work (Hori et al., 2008; Kang et al., 2006) demonstrated
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that depletion of CENP-Q destabilized the binding of other CENP-O complex subunits to
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kinetochores. We were able to confirm these observations as depletion of CENP-Q resulted in
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the loss of CENP-O from kinetochores (Fig. S1A). Moreover, depletion of CENP-Q reduced
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the levels of Plk1 from kinetochores by 84% (Fig. S1B,C; n=3, 300 kinetochores, 30 cells).
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Consistent with a kinetochore specific effect, CENP-Q depletion did not affect polar
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localization of Plk1 (Fig. S1B,C; n=3, 120 poles, 60 cells). To rule out off-target effects, we
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carried out a rescue experiment where control or CENP-Q siRNA treated cells were
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transfected with either an siRNA-protected CENP-Q-eGFP transgene or an empty vector. The
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transgene partially rescued kinetochore bound levels of Plk1 to 50% of those observed in
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control siRNA treated cells transfected with the empty vector (Fig. S1B, C; n=3, 600
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kinetochores, 60 cells). Immunoblotting confirmed that the CENP-Q siRNA depleted the
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endogenous CENP-Q protein without affecting the expression of CENP-Q-eGFP transgene
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used for these rescue experiments (Fig. 1A). These data confirm that the CENP-O complex is
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a key platform for recruitment of Plk1 to kinetochores.
Journal of Cell Science
Accepted manuscript
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Depletion of CENP-Q did not, however, affect the kinetochore binding of Ndc80,
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Kif18A, Ska3 or MCAK (Fig. S1A), indicating that CENP-Q does not play a major role in
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global kinetochore architecture. This conclusion is consistent with the ability of CENP-Q-
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depleted cells to form a normal metaphase plate (Fig. 1C,D). We did, however, note that the
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loss of CENP-Q from kinetochores had a striking effect on the position of a subset of
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chromosomes within the spindle; a number of kinetochores were trapped around the spindle
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poles (from here-on termed polar chromosomes; Fig. 1C). This phenotype can also be
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observed in images of CENP-Q knock-out DT40 cells (Hori et al., 2008) or CENP-U depleted
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human cells (Kang et al., 2006). To further assess this phenotype, we collected time-lapse
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movies in living HeLa cells expressing Histone2B-eGFP/mRFP-alpha-Tubulin (Fig. 1D;
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Movies S1,2). Taking T=0 to be the point of nuclear envelope breakdown, we determined the
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timing of chromosome congression. Only 10.3% of CENP-Q-depleted cells had formed a
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metaphase plate and aligned all their chromosomes by T=24 min compared to >95% in
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control cells (Fig. 1D, E). Consistent with our fixed cell experiments, a number of
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chromosomes remained trapped around the spindle pole in live cell movies (yellow arrows in
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Fig. 1D). These chromosome congression defects were associated with a prolonged mitotic
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delay, with only 26% of CENP-Q-depleted cells undergoing anaphase within 96 min,
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compared to 99% of control cells (Fig. 1E). Of the cells that underwent anaphase within 114
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min, 19% had failed to congress all chromosomes. This is consistent with previous work
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showing that the knock-out of CENP-Q in chicken DT40 cells causes a mitotic delay and
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congression defects (Hori et al., 2008).
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To rule out off-target effects we depleted cells of CENP-Q using siRNA and
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transfected them with either an siRNA-protected CENP-Q-eGFP transgene or an empty
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vector. We next treated the cells with MG132 for 90 min (to prevent anaphase onset and
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therefore rule out differences associated with variable cell cycle stage) (Amaro et al., 2010)
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prior to fixation and staining with antibodies against CENP-A (to mark kinetochores) and
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DAPI (to give chromosome position). This allowed the proportion of cells with polar
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chromosomes to be calculated. Depletion of CENP-Q and transfection with empty vector
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resulted in 84% (±3.5%) of metaphase cells having unaligned kinetochores situated at the pole.
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In contrast, transfection with a siRNA protected CENP-Q transgene greatly reduced the
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proportion of cells with polar chromosomes to 48% (±8%) (Fig. 1F,G). This reduction in
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polar chromosomes following transfection with the CENP-Q transgene also resulted in an
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increased rate of chromosome congression (compared to vector alone), with approximately
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twice the number of cells entering anaphase when assessed by live-cell imaging of Histone-
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2B-GFP expressing cells (Fig. 1E). Finally, a second siRNA that targets CENP-Q gave the
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same polar chromosome and congression phenotype (Fig. S2A-C), as did depletion of the
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CCAN subunit CENP-P, which has been shown to recruit CENP-Q to kinetochores (Fig.
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S2A-C) (Foltz et al., 2006).
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Journal of Cell Science
Accepted manuscript
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Taken together, these data show that kinetochores lacking CENP-Q (and thus the
CENP-O complex) are defective in the timely congression of most chromosomes, whilst also
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being unable to transport a subset of chromosomes positioned near the spindle pole, to the
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metaphase plate.
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CENP-Q is required to load CENP-E to kinetochores.
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The polar chromosome phenotype in CENP-Q depleted cells is very reminiscent of that
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reported for the depletion of the kinesin-7 CENP-E in human cells (Putkey et al., 2002;
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Weaver et al., 2003). We therefore investigated whether CENP-Q was required for the
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loading of CENP-E to kinetochores. We confirmed by immunofluorescence that treatment of
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cells with siRNA targeting CENP-E reduced the levels of the motor protein on kinetochores
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by 95.6 % (Fig. 2A, C; 150 kinetochores). Depletion of CENP-Q also reduced the levels of
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CENP-E on kinetochores by 80% (Fig. 2A,C; number kinetochores = 100). Thus, phenotypes
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associated with CENP-Q depletion must be considered as a composite of loosing both CENP-
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Q and CENP-E. We next carried out the reciprocal experiment and found that depletion of
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CENP-E did not affect kinetochore binding by CENP-Q (Fig. 2B,C). To confirm these results
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we transfected Control or CENP-Q siRNA treated cells with either the siRNA-protected
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CENP-Q-eGFP transgene or an empty vector and quantified the levels of CENP-E on
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kinetochores. Transfection with the transgene partially rescued kinetochore-binding of CENP-
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E to ~50% of the intensity measured in control siRNA treated cells that had been transfected
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with the empty vector (Fig. 2D,E). These data indicate that CENP-Q is required to load
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CENP-E to kinetochores.
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Fate of unaligned kinetochore pairs in CENP-Q and CENP-E depleted cells
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The key question is whether the polar chromosome phenotype in CENP-Q-depleted cells
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simply reflects the unbinding of CENP-E motor proteins from kinetochores, or whether
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CENP-Q makes additional CENP-E recruitment-independent contributions to chromosome
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congression. Because of the dependencies reported above we are in a position to investigate: a
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Accepted manuscript
Journal of Cell Science
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CENP-Q depletion results in kinetochores lacking CENP-Q/CENP-E, while a CENP-E-
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depletion leaves CENP-Q kinetochore-bound. We therefore determined how these differing
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kinetochore states affected the congression of chromosomes by tracking the fates of
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uncongressed kinetochore pairs by live-cell microscopy. For this we used HeLa K cells
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expressing eGFP-CENP-A (kinetochore marker) and eGFP-Centrin1 (spindle pole marker)
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and collected time-lapse movies over the course of 5 min in both CENP-Q depleted cells and
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CENP-E-depleted cells. Sister pairs were assigned as non-biorientated (as judged in in the
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first frame of the movie; supplementary movies S3-S6) if the sister-sister axis was rotated by
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around 90º relative to the pole-to-pole axis (see schematic in Fig. 3A). Biorientation would be
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improbable with this geometry because kinetochores could not make end-on attachments with
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microtubules coming from opposite spindle poles. Similarly, kinetochore pairs positioned
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behind the spindle pole were classed as non-biorientated as in this state biorientation would be
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geometrically impossible. In contrast, kinetochore pairs with a sister-sister axis of ~45º or less
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relative to the pole-pole axis were classified as orientated. We cannot be sure that these sisters
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are biorientated because it is not possible to distinguish this state from sister pairs in which
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one sister is mono-orientated and the second laterally attached ((Kapoor et al., 2006) and
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schematic in Fig. 3A). The relative proportion of orientated and non-biorientated
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uncongressed kinetochore pairs was very similar in CENP-Q and CENP-E-depleted cells,
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with around 80% occupying a non-biorientated state (Fig. 3B). The fates of these non-
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biorientated unaligned kinetochore pairs were also very similar with the majority (>90%)
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remaining stalled and unable to progress towards the spindle equator (Fig. 3C,E).
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In CENP-Q depleted cells (CENP-E also absent from kinetochores) the orientated
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kinetochore pairs were also unable to congress and remained stalled (Fig. 3D,E; also see
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movie S4 for further example). Surprisingly, in CENP-E depleted cells (where CENP-Q
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remains kinetochore-bound) 80% of orientated sister pairs were able to congress to the
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metaphase plate (Fig. 3D,E; also see movie S5 for further example). Because CENP-E is
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essential for lateral sliding, the congression events observed in CENP-E depleted cells are
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almost certainly due to biorientated kinetochores utilising depolymerisation-coupled pulling.
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We note that the level of kinetochore-bound CENP-E is significantly less following depletion
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of CENP-E compared to depletion of CENP-Q (t-test p=0.0148; Fig. 2A,B,C). Thus, the
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stronger phenotype in CENP-Q-depleted cells cannot be attributed to more efficient loss of
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CENP-E from kinetochores. Moreover, inhibition of CENP-E with 300 nM GSK925293,
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which renders the motor in a rigor state (Wood et al., 2010), gave a similar result, with almost
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100% of non-biorientated kinetochores remaining stalled (Fig. S2D) and over 50% of
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orientated kinetochores able to congress (Fig. S2E). One prediction from these data is that
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CENP-E depleted cells should have a higher proportion of their unaligned kinetochores
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trapped behind spindle poles. Indeed, 69.7% of unaligned kinetochores are behind the pole in
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CENP-E-depleted cells (numbered 1, 2 and 3in Fig. 3A), compared to only 50.4% in CENP-
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Q-depleted cells. Overall, these data suggest that a CENP-Q-dependent (CENP-E-
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independent) mechanism is required for generating sustained plateward movement by
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depolymerisation-coupled pulling to move kinetochore pairs that reside between the pole and
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equator to the metaphase plate.
Journal of Cell Science
Accepted manuscript
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CENP-Q and CENP-E generate counter-forces on polar chromosomes.
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Behind the spindle pole the geometry of microtubules is reversed, i.e. the plus ends of astral
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microtubules are proximal to the cell cortex, whereas K-fibres have plus-ends proximal to the
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metaphase plate. If CENP-Q directs depolymerisation-coupled pulling, it would move
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chromosomes poleward, while the CENP-E driven lateral sliding would move them towards
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the plus-end (anti-poleward). Thus, we would predict that depletion of each protein would
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alter the positions of kinetochores located behind the pole relative to the spindle pole. To test
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this hypothesis we used fixed cell high-resolution wide-field fluorescence imaging to measure
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the position of polar kinetochore pairs relative to the spindle pole and astral microtubules (Fig.
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4A,B). Kinetochores were on average 1.29 μm (±0.43 μm) from the pole in CENP-E-depleted
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cells (Fig. 4B,C; n=2 159 kinetochores). In contrast, kinetochore pairs in CENP-Q depleted
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cells were further from the pole, occupying an average distance of 2.62 μm (±0.99 μm) (Fig.
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B,C; n=2 107 kinetochores). Thus supporting our prediction that depletion of CENP-Q and
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CENP-E differentially affects the position of polar chromosomes relative to the pole.
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To further validate this we measured the distance of individual kinetochores to the
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spindle pole in monopolar spindles following depletion of CENP-E or CENP-Q. To generate
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monopolar spindles, cells were treated with an Eg5 inhibitor (monastrol) for 90 min, which
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prevents centrosome separation in prophase (Mayer et al., 1999). This induces a high
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frequency of syntelic attachments (Lampson et al., 2004), enabling the assessment of
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positional changes in end-on attached kinetochores. Cells were then fixed and stained with
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antibodies against CENP-A (kinetochore marker) and gamma-tubulin (spindle pole marker).
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The fixed cells were imaged and the center of mass for the monopole was identified based on
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the gamma-tubulin signal (see methods for details). We then automatically found the
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kinetochore positions based on the CENP-A staining and calculated the Euclidian distance to
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the monopole (Fig. 4D). The kinetochore-to-pole distances for each experiment (n ≥ 4, ≥ 8
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Accepted manuscript
Journal of Cell Science
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cells per experiment per condition) were then used to calculate cumulative distribution
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frequency of kinetochore to pole distances, the average of these cumulative distribution
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frequencies was then plotted (Fig. 4E,F). These plots show that depletion of CENP-E resulted
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in the distribution of kinetochore-to-pole distances moving closer to the monopole compared
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to control cells (Fig. 4E). We confirmed this result by treating cells with 300 nM
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GSK925293 to inhibit CENP-E motor activity (Fig. 4E). Conversely, kinetochores in CENP-
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Q-depleted cells (which also lack the anti-poleward CENP-E force due to its unbinding from
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kinetochores) occupied a similar mean distance from the monopole to kinetochores in control-
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depleted cells, albeit with a much narrower standard deviation (Fig. 4F). Taken together, these
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data support our model that CENP-E slides kinetochores towards microtubule plus-ends while
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CENP-Q-dependent mechanisms (that are CENP-E recruitment-independent) drive
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depolymerisation-coupled pulling that moves chromosomes towards or away from the pole
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depending on the microtubule geometry.
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CENP-Q roles in chromosome congression are independent of Plk1 and CENP-O
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complex subunit recruitment.
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Previous reports have demonstrated that CENP-Q is phosphorylated by CENP-U-bound Plk1
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in vitro (Kang et al., 2011), and that Plk1 is required for localization of CENP-E to the
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kinetochore (Ahonen et al., 2005; Nishino et al., 2006). Moreover, serine-50 in CENP-Q is
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phosphorylated in vivo (Rigbolt et al., 2011) suggesting that this may represent a key
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regulatory event. We therefore mutated serine-50 to alanine (CENP-QS50A-eGFP) or to a
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phospho-mimicking aspartic acid residue (CENP-QS50D-eGFP). We first tested whether the
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S50A mutant affected the recruitment of Plk1 to kinetochores. Consistent with our previous
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result (Fig. S2), CENP-Q depleted cells transfected with an empty vector demonstrated an
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84% reduction in Plk1 at kinetochores (±3.7%; Fig. 5A, B n=3 300 kinetochores, 30 cells). In
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contrast, transfection with CENP-QS50A-eGFP rescued kinetochore Plk1 levels to 62% (±12%;
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Fig. 5A,B; n=3 600 kinetochores, 60 cells). Previous work has shown that recruitment of Plk1
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to kinetochores is dependent upon CENP-U (Kang et al., 2006). The presence of Plk1 and
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CENP-Q therefore indicates that CENP-U remains kinetochore bound, inferring that the
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CENP-O complex has successfully assembled at the kinetochore. Moreover, Serine 50 is not
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reported as a Plk1 phosphorylation site (Santamaria et al., 2011), therefore CENP-QS50A-
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eGFP enables the separation of CENP-O complex assembly and Plk1 recruitment and
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regulation from the role of CENP-Q in chromosome congression.
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Journal of Cell Science
Accepted manuscript
1
Next we investigated whether CENP-QS50A-eGFP could rescue the CENP-Q depletion
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phenotype. Transfection with CENP-QS50A-eGFP failed to rescue CENP-E recruitment when
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compared to control cells transfected with an empty vector (Fig. 6A,B; n=3 600 kinetochores,
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60 cells). These cells still had an alignment defect, with ~6 kinetochore pairs trapped at each
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spindle pole (Fig. 6A,B; n=3 120 poles, 60 cells). To further validate the potential importance
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of phosphorylation at Serine-50, we transfected CENP-Q depleted cells with the phospho-
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mimmetic CENP-QS50D-eGFP transgene. This partially rescued CENP-E recruitment to 43%
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(±10%), comparable with CENP-QWT-eGFP (Fig. 6A,B; n=3 600 kinetochores, 60 cells).
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These cells also displayed a less severe congression phenotype, having on average 2.4 (±0.8)
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kinetochore pairs per pole (Fig. 6A,B; n=3 120 poles, 60 cells).
Importantly, both CENP-QS50A-eGFP and CENP-QS50D-eGFP proteins are recruited to
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kinetochores (Fig. 6A). However, due to the transient transfection, some cells had a low or
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non-detectable eGFP signal on kinetochores. We thus carried out additional experiments and
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restricted our analysis to cells where a CENP-Q-eGFP, CENP-QS50A-eGFP or CENP-QS50D -
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eGFP signal could be detected on kinetochores. We first quantified this signal and confirmed
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that the levels were equivalent for wild type and mutant CENP-Q transgenes (Fig. 6C,
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n=≥150 kinetochores per condition). Next we counted the number of kinetochore pairs per
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pole in these cells and found that the CENP-Q-eGFP and CENP-QS50D-eGFP transgenes, but
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not the CENP-QS50A-eGFP transgene, almost completely rescued the phenotype (Median
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number of kinetochore pairs per pole is zero for WT/S50D, compared to 6.5 for S50A; Fig.
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6D; n=≥150 kinetochores per condition). Likewise, we found that the CENP-Q-eGFP and
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CENP-QS50D-eGFP transgenes restored CENP-E kinetochore-binding to ~75% of the levels
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measured in control cells (Fig. 6D; n=≥150 kinetochores per condition).. Thus, the partial
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(~50%) rescue in our population-based rescue experiments (see Fig. 6A,B) are simply a
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consequence of some cells not expressing the transgene. From these data, we also plotted the
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level of the transgene and that of CENP-E for individual kinetochores. This revealed a
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positive correlation for CENP-Q-eGFP and CENP-QS50D-eGFP, indicating that recruitment of
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CENP-E molecules is dependent upon the number of CENP-Q molecules at each kinetochore.
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As expected, this relationship is lost in CENP-QS50A-eGFP, with no CENP-E recruitment
30
observed at high transgene levels (Fig. 6E, n=≥150 kinetochores per condition). Taken
31
together, these data support a Plk1 and CENP-O complex independent role for CENP-Q in
32
loading CENP-E to kinetochores and for CENP-Q mediated (CENP-E independent)
33
depolymerisation-coupled pulling.
34
9
Accepted manuscript
1
Discussion
2
Recent studies in human cells indicate that the majority of chromosomes rapidly convert from
3
lateral to amphitelic attachment (biorientation) at the spindle equator as a result of
4
kinetochores being organised into a ring-like conformation following nuclear envelope
5
breakdown (Magidson et al., 2011). However, cells must have mechanisms that ensure
6
chromosomes that are close to the pole early in mitosis can congress and thereby allow
7
accurate disjunction of sister chromatids in anaphase. Our data support a model in which there
8
are at least two distinct mechanisms that drive the congression of such polar chromosomes
9
(Fig. 7A): (1) the well documented lateral sliding of (non-biorientated) kinetochores by
10
kinesin-7 CENP-E (Kapoor et al., 2006) (Fig. 7a Chr. 1), and (2) a CENP-E recruitment-
11
independent role for CENP-Q in the movement of biorientated sisters to the metaphase plate
12
(this study; Fig. 7A Chr. 2). This conclusion is based on our observation that biorientated
13
kinetochores are still able to congress in the absence of CENP-E, but not CENP-Q (where
14
CENP-E is also unbound). Importantly, it is the absence of CENP-E from kinetochores in
15
CENP-Q-depleted cells that allows us to rule out lateral sliding as the mechanism of
16
congression, and reveals a role for CENP-Q in movement of biorientated sisters.
Journal of Cell Science
17
These CENP-Q-dependent movements are analogous to those described by Skibbens
18
and colleagues 20 years ago, in which biorientated kinetochores can undergo long duration
19
motion to the spindle equator (Skibbens et al., 1993). This motion is a consequence of one
20
sister (facing the metaphase plate) remaining in a poleward (P) moving state, while the other
21
sister maintains an away-from-the-pole (AP) state. Sister kinetochores lacking CENP-Q
22
appear unable to sustain their P or AP state resulting in stalling and failure to congress. An
23
important question will be to understand how CENP-Q contributes to this process (See below).
24
How do cells initially move chromosomes to spindle poles? Existing models (see
25
(Kim et al., 2008; Skibbens et al., 1993) state that movement to the pole is necessary to
26
increase the probability of biorientation due to the higher density of microtubules. However,
27
our data show that CENP-E-dependent sliding of laterally attached kinetochores will move
28
chromosomes which are behind the pole further away from the pole, and towards the plus-end
29
of astral-microtubules. At the same time, chromosome arms are pushed away from the pole by
30
the polar ejection force (Stumpff et al., 2012). To counter these anti-poleward forces, dynein
31
motors can slide laterally attached kinetochores towards the pole (Yang et al, 2007). We
32
propose that there is a second CENP-Q-dependent process moving chromosomes poleward.
33
We suggest that this process is likely to reflect the effect of CENP-Q on mono-orientated
34
kinetochores. If direct, this would mean a requirement for CENP-Q in depolymerisation10
Accepted manuscript
Journal of Cell Science
1
coupled pulling and P-kinetochore motility. It follows that the failure of a biorientated sister
2
pair to congress is therefore most likely also due to a defective P-kinetochore movement
3
(depolymerisation-coupled pulling), rather than AP-kinetochore (pushing) movement. It will
4
be important to determine how kinetochores are converted between lateral, monoorientated
5
and biorientated states (Chrs. 1-4 in Fig. 7A, Chrs. 1 and 2 in Fig. 7B). In this regard, recent
6
work has revealed that the transition from CENP-E-dependent lateral attachment to an end-on
7
configuration requires the microtubule depolymerase MCAK (Shrestha and Draviam, 2013).
8
MCAK is proposed to depolymerise the astral-microtubule back to the kinetochore, thus
9
promoting the monoorientation of the kinetochore. Overall, current data suggests that
10
kinetochores utilise poleward and anti-poleward mechanisms to position themselves at the
11
optimum distance from the spindle pole to promote biorientation.
12
How could CENP-Q contribute to depolymerisation-coupled pulling? One possibility
13
is that CENP-Q indirectly affects depolymerisation–coupled pulling by affecting the binding
14
of other factors to kinetochores (see schematic in Fig. 7C). However, we can rule out the loss
15
of the CENP-O complex or Plk1, which both remain kinetochore-bound in the S50A mutant
16
(which phenocopies a CENP-Q depletion). In addition, our data show that CENP-E is not
17
required for depolymerisation-coupled pulling (see above). Although it remains possible that
18
an unknown protein is recruited to kinetochores in a serine-50-dependent manner, we favor
19
the alternative idea that CENP-Q mediates a direct effect on kinetochore-microtubule
20
dynamics. Support for this idea comes from in vitro biochemistry, which demonstrates that
21
the purified CENP-Q protein can directly bind taxol-stabilised microtubules in vitro (Amaro
22
et al., 2010). We do find that depletion of CENP-Q reduces the turnover of kinetochore-
23
microtubules in vivo (Fig. S3). However, CENP-E depletion is reported to have the same
24
effect (Maffini et al., 2009), while still allowing congression of biorientated kinetochores
25
through depolymerisation-coupled pulling (this study). Therefore, we cannot yet attribute
26
these changes in kinetochore-microtubule dynamics to the observed defects in chromosome
27
movement in CENP-Q-depleted cells.
28
Our data suggest that phosphoregulation of CENP-Q through serine-50 may be an
29
important regulatory step in controlling chromosome congression. An attractive idea is that
30
this phosphorylation event allows direct binding of CENP-E to CENP-Q. However, CENP-E
31
remains bound to kinetochores in CENP-H- or CENP-L-depleted cells - CCAN proteins that
32
are required for CENP-Q binding to kinetochores (Amaro et al., 2010; McClelland et al.,
33
2007; McHedlishvili et al., 2012). Moreover, binding of CENP-E to kinetochores in CENP-Q
34
depleted cells is partially rescued following depolymerisation of microtubules with
11
Accepted manuscript
Journal of Cell Science
1
nocodazole (Fig. S4). Thus, a direct mechanism seems unlikely. An alternative possibility is
2
that CENP-Q modulates kinetochore-microtubule dynamics such that CENP-E can be
3
recruited. As discussed above, these same changes in microtubule dynamics could also
4
explain the defects in depolymerization-coupled pulling. Finally, the most likely kinase
5
responsible for phosphorylation of Serine-50 would be the CENP-U-bound pool of Plk1
6
(Kang et al., 2006). CENP-Q has been shown to be a substrate for Plk1 in vitro (Kang et al.,
7
2011). However, SILAC experiments show that phosphorylation of serine-50 is not sensitive
8
to the depletion or inhibition of Plk1 in human cells (Santamaria et al., 2011). Future work
9
will clearly be required to identify the kinase responsible for regulation of CENP-Q function.
10
11
Materials and Methods
12
13
Cell culture, siRNA transfection and drug treatments
14
HeLa-E1 and HeLa-K cells were grown in Dulbecco’s modified medium (DMEM) containing
15
10% FCS, 100 U/ml penicillin and 100 μg ml-1 streptomycin at 37°C with 5% CO2 in a
16
humidified incubator. The Histone2B-mRFP cell line was maintained in 500 μg ml-1 G418
17
and the eGFP-CENP-A eGFP-Centrin1 cell line was maintained in 500 μg ml-1 G418 and 0.3
18
μg ml-1 puromycin. All other cell lines were maintained in non-selective medium. Small
19
inhibitory (si) RNA oligonucleotides (53 nM) were transfected using oligofectamine
20
(Invitrogen) for 48 hr (24 hr in MEM, medium then changed to DMEM for a further 24 hr)
21
acording to manufactures instructions. siRNA oligonucleotide sequences used: Control
22
(Samora et al., 2011) , CENP-Q (5’-GGUCUGGCAUUACUACAGGAAGAAA-3’ Stealth,
23
Invitrogen), CENP-Q-2 (5’-CAGAGUUAAUGACUGGGAAUAUUCA-3’ Stealth,
24
Invitrogen), CENP-P (Amaro et al., 2010), CENP-E (5’-
25
ACUCUUACUGCUCUCCAGUdTdT-3’, Ambion). Drug treatments were carried out at the
26
following concentrations and time periods; nocodazole (Tocris) 14 hr at 1 μg ml-1,
27
GSK923295 (CENP-E inhibitor; Haoyuan Chemexpress) 14 hr at 300nM, taxol (Tocris) at
28
10μM for 60 min, monastrol (Tocris) at 1μM for 90 min and MG132 at 1 μM for 90 min.
29
30
Molecular biology and siRNA rescue experiments
31
To generate a human CENP-Q-eGFP expression vector the CENP-Q coding sequence was
32
amplified by PCR (using primers MC246 and MC248) and ligated into pEGFP-N1 (empty
33
vector; Clontech) using BamHI and EcoRI (pMC276). pMC276 was then mutated using a
12
Accepted manuscript
Journal of Cell Science
1
quick change site-directed mutagenesis kit (Stratagene), and primers MC297 (5’-
2
gacaaagctaatgaagaaggcctagcgttgctccaagaggaaatagataaaatggtagag-3’) and MC298 (5’-
3
ctctaccattttatctatttcctcttggagc aacgctaggccttcttcattagctttgtc-3’), to render the transgene
4
resistant to the CENP-Q siRNA oligonucleotide (pMC308). This siRNA resistant CENP-Q
5
construct was then used as a template for generation of CENP-Q phospho-mutants using a
6
quick change site-directed mutagenesis kit (Stratagene). With the following primers: CENP-
7
QS50A-eGFP (5’-ataaaaatcatctgaaagatctggcttctgaaggacaaacaaagcac-3’) and (5’-
8
gtgctttgtttgtccttcagaagccagatctttcagatgatttttat-3’), CENP-QS50D-eGFP (5’-
9
aaaaaataaaaatcatctgaaagatctggattctgaaggacaaacaaagcacactaac-3’) and (5’-
10
gttagtgtgctttgtttgtccttcagaatccagatctttcagatgatttttatttttt-3’). For siRNA rescue experiments cells
11
were transfected with CENP-Q-1 or Control siRNAs and grown for 14 hr in MEM. Medium
12
was then changed to DMEM and cells transfected with 1 μg of pEGFP-N1 (empty vector;
13
Clonetech), or a CENP-Q transgene using FuGene6 transfection reagent (Roche), and
14
incubated for a further 48 hr before analysing.
15
16
Immunofluorescence microscopy
17
Cells were fixed at room temperature for 10 min in 20 mM PIPES pH 6.8 containing 10 mM
18
EGTA, 1 mM MgCl2, 0.2% Triton X-100 and 4% formaldehyde. Cells were then were then
19
washed with PBS every 10 min three times before blocking with PBS + 3% BSA for 30 min.
20
After blocking the fixed cells were incubated for 1 hr with primary antibodies: rabbit anti-
21
CENP-O (1:500; McAinsh et al, 2006), mouse anti-CENP-A (1:500; Abcam), mouse anti-α-
22
tubulin (1:1000; Sigma- Aldrich),CREST antisera (1:250; Antibodies Incorporated), rabbit
23
anti-CENP-E (1:1,500; (Meraldi et al., 2004), rabbit anti-Ska3 (1:250) (kind gift from Anna
24
Santamaria), rabbit, anti-CENP-Q (1:250; Rockland), rabbit anti-Plk1 (1:250; Santa Cruz),
25
rabbit anti-γ-tubulin (1:200; Abcam), mouse monoclonal anti Ndc80/Hec1 (1:1000; Abcam).
26
Cells were then washed PBS every 10 min three times and incubated for 30 min with
27
alexafluor conjugated highly cross-adsorbed secondary antibodies raised in goat (Invitrogen).
28
Three-dimensional image stacks of mitotic cells were acquired in 0.2 μm steps using a 100×
29
oil-immersion 1.4 numerical aperture objective lens on an Olympus DeltaVision Elite
30
microscope (Applied Precision, LLC) equipped with a DAPI, fluorescein isothiocyanate
31
(FITC), rhodamine/Texas Red, CY5 filter set (Chroma), solid state light source and a
32
CoolSNAP HQ camera (Roper Scientific). Image stacks were deconvolved using SoftWorx
33
(Applied Precision, LLC) and figures were generated with Photoshop and Illustrator (Adobe).
13
1
Fluorescence intensity measurements were made manually using SoftWorx subtracting
2
background values and normalising to intensity of CENP-A or CREST on the same
3
kinetochore.
Journal of Cell Science
Accepted manuscript
4
5
Live-cell imaging
6
Fluorescence time-lapse imaging of H2B-GFP/mRFP-α-tubulin cells was carried out on a
7
Personal Deltavision microscope (Applied Precision, LLC), using a 40 x NA 1.3 objective,
8
with GFP (excitation 475/28, emission 525/50) and mCherry (excitation 575/25, emission
9
632/60) filter set, Quad-mCherry dichroic mirror (reflection bands 381-401, 464-492, 561-
10
590, 625-644, transmission bands 409-456, 500-553, 598-617, 652-700) (Chroma), Xenon
11
light source and a CoolSNAP HQ2 camera (Roper Scientific). Image stacks (7 x 2 μm z-
12
sections) were collected every 3 min for a total time of 10 hr.
13
14
Immunoblotting
15
Whole cell protein extracts were prepared using a liquid nitrogen grinding technique as
16
previously described (McClelland et al., 2007). Immunoblotting was carried out as described
17
in McAinsh et al, 2006. Primary mouse anti-α-tubulin (1:10,000; Sigma-Aldrich) or rabbit
18
anti-CENP-Q (1:500; Rockland), and secondary anti-mouse or anti-rabbit horseradish
19
peroxidase-conjugated secondary antibodies (1:10,000; Amersham) were diluted in PBS 0.1%
20
Tween20 + 3% milk powder (Marvel). Primary antibodies were incubated for 14 hr overnight
21
at 4ºC and secondary antibodies for 1 hr at room temperature.
22
23
24
Acknowledgements
25
Thank you to A. Santamaria for the Ska3 antibody, B. Fitton for help with data analysis and C.
26
Smith for help with image processing. We also thank J. Meadows, P. Meraldi, S. Royle and C.
27
Smith for comments on the manuscript. J. Bancroft is funded by studentships from the
28
Medical Research Council (MRC), and C. Samora by a fellowship from the Fundação para a
29
Ciência e Tecnologia. P. Auckland is funded by the MRC Doctoral Training Partnership
30
(MR/J003964/1). This work was supported by BBSRC grant BB/I021353/1 to A.D. McAinsh.
31
32
Author Contributions
14
1
Project was initiated and supervised by A.D.M. Experiments in figures 1-4 were carried out
2
by J.M.B., except photoactivation experiments, live-cell rescue experiments and initial
3
observations of polar chromosomes in CENP-P siRNA (C.P.S.). Experiments in figures 5 and
4
6 were carried out by P.A. The manuscript was written by A.D.M with contributions from
5
P.A., J.M.B. and C.P.S.
6
7
8
Journal of Cell Science
Accepted manuscript
9
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Toso, A., Winter, J. R., Garrod, A. J., Amaro, A. C., Meraldi, P. and McAinsh, A.
25
D. (2009). Kinetochore-generated pushing forces separate centrosomes during bipolar spindle
26
assembly. In J Cell Biol, vol. 184, pp. 365-72.
27
28
Walczak, C. E., Cai, S. and Khodjakov, A. (2010). Mechanisms of chromosome
behaviour during mitosis. Nat Rev Mol Cell Biol 11, 91-102.
29
Weaver, B. A., Bonday, Z. Q., Putkey, F. R., Kops, G. J., Silk, A. D. and
30
Cleveland, D. W. (2003). Centromere-associated protein-E is essential for the mammalian
31
mitotic checkpoint to prevent aneuploidy due to single chromosome loss. J Cell Biol 162,
32
551-63.
18
1
2
T. and Cheeseman, I. M. (2010). Aurora B phosphorylates spatially distinct targets to
3
differentially regulate the kinetochore-microtubule interface. In Mol Cell, vol. 38, pp. 383-92.
4
5
6
Accepted manuscript
Westhorpe, F. G. and Straight, A. F. (2013). Functions of the centromere and
kinetochore in chromosome segregation. Curr Opin Cell Biol 25, 334-40.
Wood, K. W., Lad, L., Luo, L., Qian, X., Knight, S. D., Nevins, N., Brejc, K.,
7
Sutton, D., Gilmartin, A. G., Chua, P. R. et al. (2010). Antitumor activity of an allosteric
8
inhibitor of centromere-associated protein-E. Proc Natl Acad Sci U S A 107, 5839-44.
9
Journal of Cell Science
Welburn, J. P. I., Vleugel, M., Liu, D., Yates, J. R., Lampson, M. A., Fukagawa,
Yang, Z., Tulu, U. S., Wadsworth, P. and Rieder, C. L. (2007). Kinetochore dynein
10
is required for chromosome motion and congression independent of the spindle checkpoint.
11
Curr Biol 17, 973-80.
12
13
Figure Legends
14
15
Figure 1 - Depletion of outer-plate protein CENP-Q causes accumulation of polar
16
chromosomes.
17
(a) Immunoblots of whole-cell HeLa E1 lysates transfected with control or CENP-Q siRNA
18
for 12 hr and then transfected with either empty vector or an siRNA resistant vector
19
expressing CENP-Q tagged with eGFP for 48 hr. The blot was probed with antibodies against
20
CENP-Q and α-tubulin. Endogenous CENP-Q and the corresponding α-tubulin control are
21
shown in the top two panels. The pellet fraction is shown as nonspecific staining obscuring
22
the protein in the supernatant. Transgene and corresponding α-tubulin loading control are
23
shown in the lower two panels.
24
(b) Immunofluorescence microscopy images of metaphase cells and magnified kinetochore
25
pairs in control siRNA and CENP-Q siRNA depleted cells stained with CREST (red), CENP-
26
Q (green) and α-tubulin (blue) antibodies. The images of metaphase cells correspond to z-
27
projections (10 focal planes at 0.2 μm spacing) and the zoom images of the single
28
kinetochores are from a single focal plane of the stack. Values at the base of the bottom two
29
panels correspond to the relative values of CENP-Q in control and CENP-Q-depleted cells
30
(n=3, 150 kinetochores, 30 cells). Scale bar indicates 3 μm.
31
(c) Immunofluorescence microscopy images of a CENP-Q depleted metaphase HeLa E1 cell
32
stained with CREST (green) and α-tubulin (red) antibodies. The image is a z-projection (10
33
focal planes at 0.2 μm spacing). Zoom boxes 1 and 2 are centered on the spindle poles and
19
Accepted manuscript
Journal of Cell Science
1
yellow arrows point to unaligned kinetochores at around the spindle poles. Scale bar indicates
2
3 μm.
3
(d) Frames from live-cell movies of HeLa E1 cells expressing H2B–eGFP/mRFP–α-tubulin in
4
control (top row) and CENP-Q siRNA (second row). Yellow arrows point to unaligned
5
kinetochores. Scale bar indicates 10μm. Videos of control and CENP-Q depleted cells
6
available in in supplementary movies S1 and S2 respectively.
7
(e) Quantification of nuclear envelope breakdown (NEB) to the time when the last
8
chromosome congressed to the metaphase plate, and NEB to the time of anaphase onset. Blue
9
and orange lines indicate timings of HeLa E1 cells expressing H2B–eGFP/mRFP–α-tubulin in
10
control and CENP-Q siRNA treatments respectively. Black green and red lines represent
11
timing data from cells expressing H2B–mRFP treated with control siRNA + siRNA resistant
12
CENP-Q-eGFP vector (black), CENP-Q siRNA + siRNA resistant CENP-Q-eGFP vector
13
(green) and CENP-Q siRNA + eGFP vector (red).
14
(f) Immunofluorescence microscopy images (z-projections, 10 focal planes at 0.2 μm
15
spacing) of a CENP-Q siRNA rescue experiment in HeLa E1 cells. Cells were treated with
16
CENP-Q siRNA or control siRNA for 14 hr and then transfected with an siRNA resistant
17
CENP-Q-eGFP or a control eGFP expression plasmid for a further 48 hr. To reduce the effect
18
of mitotic stage on alignment, cells were treated with 1μM MG132 for 90 min prior to
19
fixation. Cells were stained with DAPI and probed with an antibody against CENP-A. Scale
20
bar indicates 3μm.
21
(g) Quantification of cells with polar chromosomes in the rescue experiment described in (f).
22
n=2, ≥100 poles, 50.
23
24
Figure 2 - CENP-Q is required to load the kinesin-7 CENP-E to kinetochores
25
(a) Immunofluorescence microscopy images of a metaphase HeLa E1 cells treated with
26
control, CENP-E or CENP-Q siRNA for 48 hr and stained with CENP-E (green), CREST
27
(red) and α-tubulin (blue) antibodies. The image is a z-projection (10 focal planes at 0.2μm
28
spacing). Yellow arrows point to unaligned kinetochores. Scale bar indicates 3μm.
29
(b) Immunofluorescence microscopy images of a metaphase HeLa E1 cells treated with
30
control, CENP-E or CENP-Q siRNA for 48 hr and stained with CENP-Q (green), CREST
31
(red) and α-tubulin (blue) antibodies. The image is a z-projection (10 focal planes at 0.2 μm
32
spacing). Yellow arrows point to unaligned kinetochores, scale bar indicates 3μm.
33
(c) Left panel: Quantification of CENP-E immunofluorescence levels in HeLa E1 cells treated
34
with control siRNA, CENP-E siRNA or CENP-Q siRNA for 48 hr. Intensities are determined
20
Accepted manuscript
Journal of Cell Science
1
at each kinetochore relative to CREST and with background subtraction, n≥150 kinetochores
2
per condition from 3 independent experiments. Dashed line indicates CENP-E levels in
3
control siRNA cells. Error bar indicate SD. Right panel: Quantification of CENP-Q
4
immunofluorescence levels in HeLa E1 cells treated with control siRNA, CENP-E siRNA or
5
CENP-Q siRNA for 48 hr. Intensities are determined at each kinetochore relative to CREST
6
and with background subtraction, n≥100 kinetochores per condition from 2 independent
7
experiments. Dashed line indicates CENP-Q levels in control cells. Error bar indicate SD.
8
(d) Immunofluorescence microscopy images (z-projections, 10 focal planes at 0.2μm spacing)
9
of CENP-Q siRNA rescue experiment in HeLa E1 cells. Cells were treated with CENP-Q
10
siRNA or Control siRNA for 12 hr and then transfected with an siRNA resistant plasmid
11
expressing CENP-Q-eGFP or a control eGFP expression plasmid for a further 48 hr. To
12
reduce the effect of mitotic stage on alignment, cells were arrested in metaphase with MG132
13
at a 1μM final concentration for 90 min followed by fixation. Cells were stained with
14
antibodies against CENP-A (blue) and CENP-E (red). Scale bar indicates 3μm.
15
(e) Quantification of CENP-E immunofluorescence levels the CENP-Q siRNA rescue
16
experiment detailed in (d). Intensities were determined at each kinetochore relative to CENP-
17
A and with background subtraction, n≥150 kinetochores per condition from 3 independent
18
experiments. Dashed line indicates CENP-Q levels in control siRNA treated cells. Error bar
19
indicate SD.
20
21
Figure 3 - Fates of unaligned kinetochore pairs
22
(a) Schematic representing the orientation of unaligned kinetochore pairs within the mitotic
23
spindle. Black dotted line represents pole-to-pole axis, dotted green lines on chromosomes 4
24
and 5 represent kinetochore sister-sister axis, the ~90° angle between the sister-sister axis of
25
chromosome 4 and the spindle pole-to-pole axis indicates non-biorientation. The reduced
26
angle (≤45°) of the sister-sister axis relative to spindle axis of chromosome 5 indicates
27
biorientation. Chromosomes 1, 2 and 3 are behind the pole and therefore non-biorientated.
28
Chromosome 6 is mono-orientated by the pole proximal kinetochore and laterally attached to
29
an adjacent k-fibre by the pole distal kinetochore. Discriminating this chromosome from
30
biorientated chromosomes (e.g. number 5) is not possible in our assay.
31
(b) Orientation state of unaligned kinetochore pairs in CENP-Q (n=217 kinetochores) and
32
CENP-E siRNA (n=211 kinetochores) treated HeLa K cells stably expressing eGFP-CENP-A
33
and eGFP-Centrin1. Pink represents non-biorientated kinetochores (classes 1, 2, 3 and 4), and
34
orange orientated kinetochores (classes 5 and 6). n≥3 independent experiments.
21
Accepted manuscript
Journal of Cell Science
1
(c) The fates of non-biorientated kinetochore pairs over 5 min in live HeLa K cells (stably
2
expressing eGFP-CENP-A and eGFP-Centrin1) after 48 hr treatment with CENP-Q or CENP-
3
E siRNA.
4
(d) The fates of orientated kinetochore pairs over 5 min in live HeLa K cells (stably
5
expressing eGFP-CENP-A and eGFP-Centrin1) after 48 hr treatment with CENP-Q or CENP-
6
E siRNA.
7
(e) Example frames from movies of kinetochore fates in HeLa K cells (stably expressing
8
eGFP-CENP-A and eGFP-Centrin1) after 48 hr treatment with CENP-Q or CENP-E siRNA.
9
Orange chevrons indicate pairs that are orientated with the spindle axis, blue chevrons
10
indicate pairs that are non-biorientated and white chevrons indicate where designation of state
11
is unclear. Yellow stars indicate spindle pole when visible. First column shows an example of
12
a biorientated kinetochore pair in a CENP-Q depleted cell, the pair does not congress, but
13
switches to a non-biorientated state (supplementary movie S3). Second column shows
14
example of an orientated kinetochore pair that fails to congress in CENP-Q depleted cells
15
(supplementary movie S4). In contrast, third column shows an example of an orientated sister
16
pair in CENP-E-depleted cells that are still able to congress to the metaphase plate
17
(supplementary movie S5). The forth column shows an aligned non-biorientated sister pair
18
that cannot move to the metaphase plate in CENP-E depleted cell (supplementary movie S6).
19
20
Figure 4 - CENP-Q and CENP-E-depleted kinetochores affect distinct steps of
21
congression.
22
(a) Schematic representing the measurement of pole to kinetochore distances during CENP-Q
23
and CENP-E depletion.
24
(b) Immunofluorescence microscopy images of HeLa K cells treated for 48 hr with CENP-Q
25
or CENP-E siRNA and stained with Crest (green), α-tubulin (blue) and pericentrin (red)
26
antibodies. The image is a single z-slice. Scale bar 5μm.
27
(c) Quantification of pole to kinetochore distances in CENP-Q siRNA (blue n=2 159
28
kinetochores) and CENP-E siRNA (green n=2 107 kinetochores). Error bars represent SD.
29
(d) Immunofluorescence microscopy image of a HeLa E1 cell stained with CENP-A (green)
30
and γ-tubulin (red) antibodies. Cells were treated for 48 hr with control, CENP-Q or CENP-E
31
siRNA followed by 90 min treatment with 1μM monastrol to induce monopolarity. The image
32
is a z-projection (all focal planes, 15μm at 0.2μm spacing). The successive analysis overlays
22
1
show the automated process of pole identification and kinetochore identification followed by
2
distance measurement from each kinetochore to the pole.
3
(e) Cumulative distribution of kinetochore distances from the monopole center in cells treated
4
with control siRNA (red lines, n=5 experiments with ≥ 8 cells), CENP-E siRNA (red lines,
5
n=4, with ≥ 8 cells) and GSK923295 (purple line, n=1, 10 cells). Shaded areas represent SD.
6
(f) Cumulative distribution of kinetochore distances from the monopole center in cells treated
7
with control siRNA (green lines, n=5, with ≥ 8 cells) and CENP-Q siRNA (red lines, n=3,
8
with ≥ 8 cells). Shaded areas represent SD.
Journal of Cell Science
Accepted manuscript
9
10
Figure 5 – CENP-QS50A-eGFP rescues kinetochore recruitment of Plk1
11
(a) Immunofluorescence microscopy images (z-slice) of CENP-QS50A-eGFP siRNA Plk1
12
rescue experiment in HeLa K cells. Cells were treated with CENP-Q siRNA or control siRNA
13
for 12hr and then transfected with an siRNA resistant plasmid expressing CENP-QS50A-eGFP
14
or a control eGFP expression plasmid for a further 48 hr. To reduce the effect of mitotic stage
15
on alignment, cells were arrested in metaphase with 1μM MG132 for 90 min prior to fixation.
16
Cells were stained with antibodies against Crest (blue) and Plk1 (red). Scale bar indicates
17
5μm.
18
(b) Quantification of Plk1 levels in the CENP-QS50A-eGFP siRNA rescue experiment.
19
Intensities were determined at each kinetochore relative to Crest and with background
20
subtraction, n=3, ≥300 kinetochores, 30 cells. Dashed line indicates CENP-Q levels in control
21
siRNA treated cells. Error bar indicate SD.
22
23
Figure 6 – CENP-Q serine 50 is required for CENP-E recruitment and orderly
24
congression
25
(a) Immunofluorescence microscopy images (z-slice) of CENP-QS50A-eGFP and CENP-
26
QS50D-eGFP siRNA rescue experiment in HeLa K cells. Cells were treated with CENP-Q
27
siRNA or control siRNA for 12hr and then transfected with an siRNA resistant plasmid
28
expressing CENP-Q-eGFP, CENP-QS50A-eGFP, CENP-QS50D-eGFP or control eGFP
29
expression plasmid for a further 48 hr. To reduce the effect of mitotic stage on alignment,
30
cells were arrested in metaphase with 1μM MG132 for 90 min prior to fixation. Cells were
31
stained with antibodies against CENP-A (blue) and CENP-E (red). Scale bar indicates 5μm.
32
(b) Left panel: Quantification of CENP-E levels in the CENP-QS50A-eGFP and CENP-QS50D-
33
eGFP siRNA rescue experiments. Intensities were determined at each kinetochore relative to
34
CENP-A and with background subtraction, n=3, ≥300 kinetochores, ≥30 cells. Dashed line
23
Accepted manuscript
Journal of Cell Science
1
indicates CENP-Q levels in control siRNA treated cells. Error bar indicate SD. Right panel:
2
Quantification of the average number of kinetochore pairs per pole in CENP-QS50A-eGFP and
3
CENP-QS50D-eGFP from 3 independent rescue experiments (120 poles per condition, 60 cells).
4
(c) Box plot showing quantification of kinetochore transgene levels for each CENP-Q variant,
5
n=3, ≥150 kinetochores.
6
(d) Analysis of cells with transgene positive kinetochores. Box plots showing quantification
7
of the average number of kinetochore pairs per pole, n=3, ≥60 poles (left panel) and
8
quantification of kinetochore CENP-E levels, n=3, ≥150 (right panel). Dotted lines indicate
9
levels in control cells.
10
(e) Plot demonstrating the relationship between kinetochore eGFP signal in cells rescued with
11
CENP-Q-eGFP, CENP-Q-eGFPS50A or CENP-Q-eGFPS50D and kinetochore CENP-E signal,
12
n=3, ≥150 kinetochores.
13
14
Figure 7 – A model for chromosome congression through the combined action of CENP-
15
Q and CENP-E.
16
(a) Schematic showing a proposed model of chromosome congression for unaligned
17
chromosomes positioned between the plate and the pole through the combined action of
18
CENP-Q and CENP-E dependent mechanisms. Chromosome 1 is mono-orientated and
19
laterally attached at the black sister kinetochore by CENP-E, this chromosome is able to
20
congress to the plate by lateral sliding driven by the kinesin-7 CENP-E, where it then able to
21
biorientate (as reported in Kapoor et al, 2006). Chromosome 2 is biorientated and is able to
22
congress to the metaphase plate by making persistent plateward movements driven by CENP-
23
Q dependent microtubule depolymerisation-coupled pulling at the P kinetochore.
24
Chromosome 3 is mono-orientated and will likely engage its free kinetochore with either the
25
microtubule lattice, or the plus-end of microtubules coming from opposite pole in order to
26
congress via CENP-E/CENP-Q-dependent pathways. Chromosome 4 is biorientated and
27
aligned at the metaphase plate.
28
(b) Schematic showing the mechanisms acting on chromosomes positioned behind the spindle
29
poles. Arrows indicate the direction of the generated forces. CENP-E lateral sliding and polar
30
ejection forces (PEFs) move chromosomes anti-poleward, while CENP-Q dependent
31
depolymerisation-coupled pulling and dynein-driven lateral sliding move chromosomes
32
poleward. Force balance amongst these mechanisms dictates a chromosomes distance from
33
the spindle pole, and kinetochores are likely able to cycle between these attachment states
34
(grey arrow).
24
(c) Schematic showing kinetochore loading and phosphorylation dependencies. Solid black
2
lines represent direct loading dependencies (percentage values indicate the level of
3
dependency). Black dashed lines represent loading dependencies, which are indirect (or have
4
not been shown to be direct). Blue lines represent phosphorylation events and red arrows
5
represent proteins that have been shown to make direct contact with the microtubule.
6
Dependencies from: [1] (Maia et al., 2012), [2], (Maffini et al., 2009), [3] (Kang et al., 2006),
7
[4] this study [5] (Kang et al., 2011). Dependencies within CCAN are from (Foltz et al., 2006;
8
McClelland et al., 2007; Okada et al., 2006). The separation of the CCAN into core and
9
extended components taken from Westhorpe and Straight, 2013.
Journal of Cell Science
Accepted manuscript
1
25
Figure 1
A
+
-
+
-
+
+
Control siRNA
CENP-Q siRNA
+
-
+
+
-
+
eGFP
CENP-Q-eGFP
1
2
3
4
37
Control siRNA
B
CENP-Q siRNA
zoom
single pairs
10 z projected
α-tubulin CENP-Q
CREST
10 z projected
α-tubulin CENP-Q
CREST
zoom
single pairs
C
α-tubulin γ-tubulin CREST
1
1
CENP-Q
(endogenous)
25
Anti-CENP-Q (Pellet)
CREST
CREST
α-tubulin
50
Anti-α-tubulin (Pellet)
2
75
50
CENP-Q-eGFP
(exogenous)
CENP-Q
Anti-CENP-Q (supernatant)
1
CENP-Q
α-tubulin
50
2
Anti-α-tubulin (supernatant)
CENP-Q levels 14%
SD +/- 6.2
CENP-Q siRNA
Control siRNA
D
40
20
0
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72
96
+ eGFP
CENP-Q siRNA
+ eGFP
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+ CENP-Q-eGFP
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Control siRNA+eGFP
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DAPI eGFP CENP-A
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siRNA
CENP-Q-eGFP
60
Control siRNA
eGFP
80
F
NEB anaphase onset
CENP-Q-eGFP
Cumulative frequency (%)
100
eGFP
Congression time
100
cells with polar
chromosomes (%)
E
Cumulative frequency (%)
Journal of Cell Science
Accepted manuscript
CENP-Q levels 100%
CENP-Q
siRNA
eGFP
CENP-Q siRNA
CENP-Q-eGFP
E
C
1.2
1.0
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anti-CENP-Q intensity [a.u.]
α-tubulin
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α-tubulin
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+ eGFP
Control siRNA
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siRNA
Control siRNA
+ CENP-Q-eGFP
eGFP
eGFP
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siRNA
anti-CENP-E intensity [a.u.]
CENP-E
D
Control
siRNA
Control siRNA
+ eGFP
CENP-A
Accepted manuscript
A
anti-CENP-E intensity [a.u.]
CENP-A
eGFP CENP-E
Journal of Cell Science
Figure 2
CENP-E
siRNA
0
CENP-Q
siRNA
1.2
1.0
0.8
0.6
0.4
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Control CENP-E CENP-Q
siRNA
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Figure 3
E
A
Orientated
congressing
non-Biorientated
stalled
*
t=15s
t=30s
t=30s
t=45s
t=45s
t=60s
t=60s
t=75s
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t=90s
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*
move to pole
Stalled
Switch to
orientated
Congress to
equator
t=165s
eGFP-CENP-A eGFP-Centrin1
t=180s
move to pole
t=150s
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t=135s
t=270s
Switch to
non-biorientated
t=120s
*
*
Congress to
equator
t=105s
Kinetochore pairs (%)
t=90s
t=247s
Kinetochore pairs (%)
t=75s
Accepted manuscript
t=60s
*
*
Journal of Cell Science
t=45s
t=225s
20
0
t=30s
*
*
40
*
t=202s
60
*
*
t=180s
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80
*
Orientated kinetochores
100
*
0
t=157s
20
*
t=135s*
40
*
*
CENP-Q siRNA
CENP-E siRNA
60
D
t=112s
Non-biorientated kinetochores
*
C
t=90s
non-biorientated orientated
*
*
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80
t=67s
80
60
40
20
0
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*
*
Unaligned kinetochores (%)
100
B
t=15s
*
t=45s
1
*
6
t=22s
3
7
t=0s
t=0s
t=15s
*
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t=0s
2
5
4
CENP-E siRNA
Orientated
stalled
*
nonnonbiorientated aligned orientated biorientated
CENP-Q siRNA
Orientated
switch to non-biorientated
Figure 4
A
B
CENP-Q siRNA
CENP-E siRNA
d
3
2
Crest alpha-tubulin Pericentrin
1
0
CENP-Q siRNA CENP-E siRNA
CENP-A gamma-tubulin
1. centre of mass identified
for gamma tubulin
E
1
0.9
Cumulative frequency
Distance from
spindle pole (µm)
D
4
0.8
0.7
0.6
0.5
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Control siRNA
CENP-E siRNA
GSK923295
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0.1
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F
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4
6
8
10
12
Distance from monopole centre (µm)
1
0.9
2. CENP-A kinetochore marker
identified by spot detection
Cumulative frequency
Journal of Cell Science
Accepted manuscript
C
0.8
0.7
0.6
0.5
0.4
0.3
Control siRNA
CENP-Q siRNA
0.2
0.1
3. Distance from each kinetochore
to centre of gamma tubulin
measured
0
0
2
4
6
8
10
12
Distance from monopole centre (µm)
Figure 5
Control siRNA
Journal of Cell Science
Crest
eGFP Plk1
Accepted manuscript
Crest
Plk1
eGFP
eGFP
CENP-QS50AeGFP
CENP-Q siRNA
eGFP
B
CENP-QS50AeGFP
1.2
Relative kinetochore Plk1 intensity (a.u)
A
1
0.8
0.6
0.4
0.2
0
eGFP
CENP-QS50AeGFP
Control siRNA
eGFP
CENP-QS50AeGFP
CENP-Q siRNA
Figure 6
A
Control siRNA
CENP-Q siRNA
CENP-Q-eGFP
eGFP
CENP-Q-eGFP
S50D
eGFP
S50A
WT
S50D
S50A
1.2
1
0.8
0.6
0.4
0.2
0
eGFP
WT
S50D
S50A
eGFP
CENP-Q-eGFP
WT
S50D
S50A
CENP-Q-eGFP
Control siRNA
C
8
4
7
6
5
4
3
2
1
0
eGFP
WT
S50D
S50A
eGFP
CENP-Q siRNA
WT
S50D
S50A
CENP-Q-eGFP
CENP-Q-eGFP
Control siRNA
Relative eGFP Intensity (a.u)
Relative CENP-E intensity
at kinetochores (a.u)
1.4
Number of kinetochore pairs per pole
CENP-A
eGFP CENP-E
B
3
2
1
CENP-Q siRNA
0
12
eGFP positive kinetochores
5
eGFP positive kinetochores
Relative CENP-E intensity
at kinetochores (a.u)
10
8
6
4
4
3
2
1
2
0
0
WT
S50D
S50A
CENP-Q siRNA + CENP-Q-eGFP
WT
S50D
S50A
CENP-Q siRNA + CENP-Q-eGFP
E
Relative kinetochore CENP-E Intensity (a.u)
D
Number of kinetochore pairs per pole
Journal of Cell Science
Accepted manuscript
CENP-A
CENP-E
eGFP
WT
WT
S50D
S50A
CENP-Q siRNA + CENP-Q-eGFP
6
CENP-Q-eGFP CENP-QS50D-eGFP CENP-QS50A-eGFP
5
4
R2 0.2
3
R2 0.27
2
1
0
R2 0.0023
0
0.5
1
1.5
2
2.5
Relative KT eGFP Intensity (a.u)
3
3.5
Figure 7
A
B
PEF
1
CENP-E
sliding
+
AP
P
+
+
L
+
3
-
1
?
4
L
L
CENP-E
sliding
dynein
sliding
P
2
PEF
(Kid)
CENP-Q dependent
end-on pulling
2
spindle
equator
P
C
[1] 50%
Clasp2
Clasp1
[2] 90%
CENP-E
Journal of Cell Science
Kif18A
Ska
Ndc80
Plk1
[5]
U
R
S50
Q
M
O/P
T78
[4]
[3]
extended
CCAN
core CCAN
KH I
Accepted manuscript
AP
N
MCAK
C
T/W
S/X
+
CENP-Q
dependent
end-on
pulling