Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins
2020; Springer Nature; Volume: 39; Issue: 23 Linguagem: Inglês
10.15252/embj.2020105432
ISSN1460-2075
AutoresYulia Steblyanko, Girish Rajendraprasad, Mariana Osswald, Susana Eibes, Ariana Jacome, Stephan Geley, António J. Pereira, Hélder Maiato, Marin Barišić,
Tópico(s)Protist diversity and phylogeny
ResumoArticle19 October 2020free access Transparent process Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins Yulia Steblyanko Yulia Steblyanko Danish Cancer Society Research Center (DCRC), Copenhagen, Denmark Search for more papers by this author Girish Rajendraprasad Girish Rajendraprasad Danish Cancer Society Research Center (DCRC), Copenhagen, Denmark Search for more papers by this author Mariana Osswald Mariana Osswald i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Search for more papers by this author Susana Eibes Susana Eibes Danish Cancer Society Research Center (DCRC), Copenhagen, Denmark Search for more papers by this author Ariana Jacome Ariana Jacome i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Search for more papers by this author Stephan Geley Stephan Geley Institute of Pathophysiology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author António J Pereira António J Pereira i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Search for more papers by this author Helder Maiato Helder Maiato i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Experimental Biology Unit, Department of Biomedicine, Faculdade de Medicina, Universidade do Porto, Porto, Portugal Search for more papers by this author Marin Barisic Corresponding Author Marin Barisic [email protected] orcid.org/0000-0001-7587-3867 Danish Cancer Society Research Center (DCRC), Copenhagen, Denmark Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Yulia Steblyanko Yulia Steblyanko Danish Cancer Society Research Center (DCRC), Copenhagen, Denmark Search for more papers by this author Girish Rajendraprasad Girish Rajendraprasad Danish Cancer Society Research Center (DCRC), Copenhagen, Denmark Search for more papers by this author Mariana Osswald Mariana Osswald i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Search for more papers by this author Susana Eibes Susana Eibes Danish Cancer Society Research Center (DCRC), Copenhagen, Denmark Search for more papers by this author Ariana Jacome Ariana Jacome i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Search for more papers by this author Stephan Geley Stephan Geley Institute of Pathophysiology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author António J Pereira António J Pereira i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Search for more papers by this author Helder Maiato Helder Maiato i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Experimental Biology Unit, Department of Biomedicine, Faculdade de Medicina, Universidade do Porto, Porto, Portugal Search for more papers by this author Marin Barisic Corresponding Author Marin Barisic [email protected] orcid.org/0000-0001-7587-3867 Danish Cancer Society Research Center (DCRC), Copenhagen, Denmark Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Yulia Steblyanko1, Girish Rajendraprasad1, Mariana Osswald2,3, Susana Eibes1, Ariana Jacome2,3, Stephan Geley4, António J Pereira2,3, Helder Maiato2,3,5 and Marin Barisic *,1,6 1Danish Cancer Society Research Center (DCRC), Copenhagen, Denmark 2i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal 3IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal 4Institute of Pathophysiology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria 5Experimental Biology Unit, Department of Biomedicine, Faculdade de Medicina, Universidade do Porto, Porto, Portugal 6Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark *Corresponding author. Tel. +45 35257323; E-mail: [email protected] The EMBO Journal (2020)39:e105432https://doi.org/10.15252/embj.2020105432 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 Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT-dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin-5/EG5 and kinesin-12/KIF15, and our data suggest that the MT-flux driving force is transmitted from non-KT-MTs to KT-MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT-flux rate correlates with spindle length, and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we find that MT-flux is required to regulate spindle length by counteracting kinesin 13/MCAK-dependent MT-depolymerization. Thus, our study unveils the long-sought mechanism of MT-flux in human cells as relying on the coordinated action of four kinesins to compensate for MT-depolymerization and regulate spindle length. Synopsis The phenomenon of continuous poleward flux of mitotic spindle microtubules has remained mysterious. This study establishes the long-sought molecular mechanisms underlying microtubule flux, and explains its role in regulating spindle length upon establishment of stable end-on kinetochore-microtubule attachments Mitotic microtubule flux in human cells is sequentially driven by the coordinated action of four kinesins. Microtubule-sliding motors EG5 and KIF15 collaboratively act on interpolar microtubules, assisted by CENPE at kinetochores in prometaphase and KIF4A on chromosome arms in metaphase. Microtubule-crosslinking proteins HSET and NuMA facilitate distribution of microtubule flux-associated spindle forces on metaphase chromosomes, enabling kinetochore microtubule flux due to coupling with non-kinetochore microtubules. Microtubule poleward flux regulates spindle length in response to MCAK-mediated depolymerization of kinetochore microtubules. Introduction Microtubule (MT) poleward flux is an evolutionarily conserved process in metazoan spindles and is defined as a continuous poleward motion of MTs, typically coordinated with addition of new tubulin subunits at the MT plus-ends and their removal at the MT minus-ends at spindle poles (Forer, 1965; Bajer & Molè-Bajer, 1972; Hiramoto & Izutsu, 1977; Hamaguchi et al, 1987; Mitchison, 1989). Although its functions remain unclear, MT-flux was proposed to play a role in various aspects of mitosis. For instance, MT-flux has been implicated in the regulation of spindle length (Gaetz & Kapoor, 2004; Rogers et al, 2004; Fu et al, 2015; Renda et al, 2017). However, this role remains controversial, as attenuation of MT-flux led to spindle elongation in Drosophila embryos (Rogers et al, 2004) and Xenopus egg extracts (Gaetz & Kapoor, 2004), while its reduction in human cells either had no effect on spindle length (Ganem et al, 2005; Jiang et al, 2017), or resulted in shorter spindles (Maffini et al, 2009; Fu et al, 2015). Additionally, MT-flux was proposed to regulate kinetochore (KT) activity (Maddox et al, 2003) and chromosome movements (Rogers et al, 2004; Ganem et al, 2005), mediate the correction of erroneous KT-MT attachments (Ganem et al, 2005), and equalize spindle forces at metaphase KTs prior to their segregation (Matos et al, 2009). In addition to its unclear function, the molecular mechanism underlying MT-flux also remains controversial. Two main models were proposed to drive spindle MT-flux. The first model envisions MT-flux to be driven by kinesin-13-induced depolymerization of MT minus-ends at the spindle poles (Rogers et al, 2004; Ganem et al, 2005). However, several lines of evidence have challenged this model. In particular, fluorescence speckle microscopy in newt lung cells showed no flux in astral MTs (Waterman-Storer et al, 1998), which originate at the poles and extend toward the cell cortex. Moreover, laser microsurgery experiments on KT-MTs (also called k-fibers) revealed normal MT-flux despite stable MT minus-ends detached from the spindle poles (Maiato et al, 2004; Matos et al, 2009). Finally, MTs continued to flux at unchanged rates even when MT-depolymerization at spindle poles was inhibited by controlled mechanical compression applied to metaphase mitotic spindles (Dumont & Mitchison, 2009). Together, these experiments demonstrate that MT-flux can be uncoupled from MT minus-end depolymerization. The second model proposes that MT-depolymerization (and polymerization) are a response to kinesin-5-mediated sliding of antiparallel interpolar MTs (Brust-Mascher & Scholey, 2002; Miyamoto et al, 2004; Matos et al, 2009; Pereira & Maiato, 2012), which could then be translated from interpolar- to KT-MTs via MT crosslinking molecules (Shimamoto et al, 2011; Vladimirou et al, 2013). However, this mechanism was challenged by experiments showing that inhibition of kinesin-5 (EG5 in humans) only slightly reduced MT-flux rates both in bipolar and monopolar spindles (Cameron et al, 2006). Moreover, the ability of monopolar spindles to flux (Cameron et al, 2006) further suggests that antiparallel interpolar MTs do not play an essential role in this process in mammalian cells. Thus, although MT-flux has been studied over three decades, the molecular mechanisms underlying spindle MT-flux, as well as its cellular function(s), remain to be elucidated. In this study, we demonstrate that MT-flux is sequentially driven from prometaphase to metaphase by CENP-E/kinesin-7 at KTs and KIF4A/kinesin-4 on chromosome arms, respectively, and these kinesins work in close cooperation with the MT-crosslinking motors EG5 and KIF15. We further propose that these activities are coordinated with the action of the MT-crosslinking molecules HSET and NuMA, which couple non-KT-MTs to KT-MTs, thereby ensuring a uniform distribution of spindle forces on metaphase KTs. Lastly, we shed new light on the cellular function of MT-flux in human cells and propose a role in the regulation of mitotic spindle length by counteracting MCAK-mediated MT-depolymerizing activity on KT-MTs. Results KIF4A mediates MT-flux In order to gain insight into the molecular mechanisms underlying MT-flux in human cells, we combined RNA interference (RNAi; Fig EV1) and chemical inhibitors with photoactivation-based spinning disk confocal live-cell imaging of late prometaphase/metaphase bipolar spindles and S-trityl-l-cysteine (STLC)-treated monopolar spindles in U2OS cells stably expressing photoactivatable (PA)-GFP tubulin (Fig 1A–C, Movies EV1 and EV2). We tested established contributors to MT-flux (kinesin-13/KIF2A, CLASP1 + 2 and kinesin-5/EG5), as well as several additional potential candidates (kinesin-12/KIF15, kinesin-7/CENP-E, kinesin-10/hKID, and kinesin-4/KIF4A). KIF2A was investigated because of its ability to depolymerize MT minus-ends at spindle poles and because of its previous implication in MT-flux (Ems-McClung & Walczak, 2010), whereas CLASPs contribute to MT polymerization at the KT (Maiato et al, 2005; Maffini et al, 2009). hKID and KIF4A are chromokinesins, MT plus-end directed motor proteins localized on chromosome arms that generate chromosomal polar ejection forces (PEFs), promoting proper positioning of chromosome arms and accurate chromosome alignment and segregation (Rieder et al, 1986; Rieder & Salmon, 1994; Vernos et al, 1995; Antonio et al, 2000; Funabiki & Murray, 2000; Levesque & Compton, 2001; Mazumdar et al, 2004; Brouhard & Hunt, 2005; Wandke et al, 2012; Barisic et al, 2014; Tipton et al, 2017; Dong et al, 2018). KIF4A also interacts with PRC1 at interpolar MTs, facilitating accurate cytokinesis (Zhu & Jiang, 2005). CENP-E is a KT- and MT-localized plus-end directed motor protein required for congression of peripheral chromosomes and proper chromosome segregation (Yen et al, 1991; Schaar et al, 1997; Wood et al, 1997; Kapoor et al, 2006; Barisic et al, 2014). KIF15 is another plus-end directed motor with the ability to slide antiparallel MTs in a way similar to EG5 (Tanenbaum et al, 2009; Drechsler et al, 2014). Analysis of sum-projected kymographs (Fig 1D) excluded EG5, KIF15, and hKID as strong individual drivers of MT-flux (Fig 1E and F). However, depletion of KIF4A revealed a strong contribution to MT-flux, not only in bipolar spindles (0.32 ± 0.14 μm/min, compared with 0.61 ± 0.22 μm/min in controls), as previously reported (Wandke et al, 2012), but also in monopolar spindles (0.33 ± 0.11 μm/min, compared with 0.5 ± 0.14 μm/min in controls). Intriguingly, while inhibition of CENP-E did not significantly affect MT-flux in bipolar spindles (0.52 ± 0.21 μm/min; see also (Logarinho et al, 2012)), we detected a significant reduction of MT-flux in monopolar spindles (0.34 ± 0.07 μm/min; Fig 1E and F). As expected, depletion of KIF2A and CLASPs led to a strong reduction in flux rates in bipolar (0.17 ± 0.12 μm/min and 0.27 ± 0.18 μm/min, respectively (see also (Maffini et al, 2009)) and monopolar spindles (0.17 ± 0.09 μm/min and 0.28 ± 0.07 μm/min, respectively; Fig 1E and F), consistent with their respective roles in MT minus-end depolymerization and plus-end polymerization. These data are consistent with either of the proposed models of flux; however, in one model, KIF2A and CLASPs are interpreted as flux drivers, whereas in the other these are interpreted as part of cell's response to flux to regulate spindle length. Click here to expand this figure. Figure EV1. Efficient RNAi-mediated knockdown of selected MT-flux driving candidatesImmunoblot analysis of lysates from U2OS cells stably expressing PA-GFP/mCherry-α-tubulin treated with control or indicated siRNAs. Antibodies against respective proteins were used to validate siRNA mediated knockdown, with GAPDH, α-tubulin and vinculin serving as the loading controls. Download figure Download PowerPoint Figure 1. KIF4A mediates MT-flux A. Representative spinning disk confocal live-cell imaging time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), treated with control and KIF4A siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 μm. Time, min:s. B. Representative spinning disk confocal live-cell imaging time series images of S-trityl-l-cysteine (STLC)-treated U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red). Note that MT-flux is abrogated in the presence of taxol (lower panel). Scale bars, 10 μm. Time, min:s. C. Illustration of 405 nm laser-photoactivated regions in monopolar spindles (blue circles, left). The effect of photoactivation and region selected for kymograph generation (dashed white rectangle, right). Scale bar, 10 μm. D. Corresponding kymograph profiles of the photoactivated regions in bipolar and monopolar spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 30 s. E, F. Quantification of MT-flux in bipolar (E) and monopolar (F) spindles subjected to indicated treatments. Graphs represent MT-flux of individual cells with mean ± SD. N (number of cells, number of independent experiments): – bipolar spindles: siControl (49, 5), STLC (31, 3), siKIF15 (39, 3), siKID (32, 3), siKIF4A (28, 3), siKIF2A (36, 3), siCLASPs (21, 2), GSK923295 (44, 3); – monopolar spindles: siControl (35, 3), siKIF15 (44, 3), siKID (38, 3), siKIF4A (44, 3), siKIF2A (38, 3), siCLASPs (12, 2), GSK923295 (33, 3). P-values were calculated against control using one-way ANOVA and Kruskal–Wallis H-test. n.s.—not significant, ****P ≤ 0.0001. Download figure Download PowerPoint KIF4A drives MT-flux via its chromosome arm-based motor activity Because of its strong contribution, we sought to characterize the molecular mechanisms by which the chromokinesin KIF4A drives MT-flux. First, we examined the effect of KIF4A overexpression on MT-flux rates. To do so, we designed a dose-response system by cloning KIF4A into an adenoviral vector to infect target cells with increasing virus titers (multiplicity of infection ratios [MOI]; Fig EV2A). KIF4A overexpression resulted in increased MT-flux rates (Fig EV2B), supporting the hypothesis that KIF4A drives MT-flux. Although RNAi and overexpression experiments clearly showed the importance of KIF4A for MT-flux, it remained unclear how KIF4A contributed to this process. To address this question, we conditionally reconstituted KIF4A-depleted cells expressing near physiological levels of RNAi-resistant versions of wild-type KIF4A, the ATPase-dead K94A motor mutant, and a chromatin non-binding KIF4A mutant (∆Zip1) that was still able to bind to central spindle interpolar MTs (Wu & Chen, 2008; Sigl et al, 2014) (Figs 2A and B, and EV2C and Movie EV3). While wild-type KIF4A successfully rescued the reduced MT-flux rates in KIF4A RNAi cells (0.53 ± 0.15 μm/min, compared with 0.57 ± 0.1 μm/min in uninduced cells and to 0.29 ± 0.13 μm/min in KIF4A shRNA), neither the motor mutant (K94A; 0.32 ± 0.14 μm/min, compared with 0.61 ± 0.15 μm/min in uninduced cells), nor the chromatin-binding mutant (ΔZip1; 0.3 ± 0.1 μm/min, compared with 0.63 ± 0.13 μm/min in uninduced cells) were able to recover MT-flux rates (Fig 2A and C). These results indicate that both the motor activity and localization of KIF4A on chromosome arms are essential for MT-flux (Fig 2D). Click here to expand this figure. Figure EV2. Evaluation of localization of KIF4A expression variants and the effect of KIF4A overexpression on MT-flux Immunoblot analysis of cell lysates obtained from U2OS cells stably expressing PA-GFP-α-tubulin infected with different multiplicity of infection (MOI) ratios of mCherry-KIF4A expressing adenovirus. Expression levels of KIF4A upon adenoviral titration were detected using anti-KIF4A antibody, with GAPDH as loading control. Quantification of the MT-flux rates upon KIF4A overexpression. The individual values and their mean ± SD are plotted. N (number of cells, number of independent experiments): Control (untreated) (23, 3); 1 MOI (33, 3); 2 MOI (26, 3), 4 MOI (9, 1). P-values were calculated using Mann–Whitney U-test. **P ≤ 0.01, ****P ≤ 0.0001. Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α-tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-KIF4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. Scale bars, 10 μm. Time, h:min. Schematic model illustrating KIF4A-mediated forces in MT-flux driving. Forces exerted by KIF4A motors promote motion of chromosomes relative to MTs. Once a bi-oriented chromosome aligns at the metaphase plate, KIF4A forces can no longer do work on it. Rather, the reactive force on MTs may promote non-KT-MTs to flux toward the poles. Download figure Download PowerPoint Figure 2. KIF4A drives MT-flux via its chromosome arm-based motor activity Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α-tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 μm. Time, min:s. Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi-resistant mCherry-KIF4A variants. The anti-KIF4A antibody was used, together with anti-vinculin antibody as a loading control. Quantification of MT-flux upon shRNA-mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi-resistant mCherry-KIF4A variants. The bars in graph represent mean ± SD. N (number of cells, number of independent experiments): uninduced shKIF4A (48, 4), shKIF4A (60, 4), uninduced KIF4A WT (36, 5), shKIF4A + KIF4A WT (43, 5), uninduced KIF4A K94A (42, 7), shKIF4A + KIF4A K94A (38, 6), uninduced KIF4A ΔZip1 (32, 4), and shKIF4A + ΔZip1 (30, 4). P-values were calculated using Student's t-test and Mann–Whitney U-test. n.s.—not significant, ****P ≤ 0.0001. Model illustrating chromosome arms-localized KIF4A driving MT-flux. Download figure Download PowerPoint Based on these data, we speculate that after helping chromosomes to congress to the metaphase plate during early mitosis (Wandke et al, 2012), KIF4A cannot push the chromosomes any further due to equivalent forces applied from opposite spindle sides. Nevertheless, processive KIF4A on interacting MTs would provide the reactive force necessary to drive poleward flux (Fig EV2D). To further elucidate the involvement of chromatin in MT-flux, we established U2OS cells to undergo mitosis with unreplicated genomes (MUGs) (Brinkley et al, 1988; O'Connell et al, 2008), in which chromatin is physically detached from spindle MTs (Figs 3A and C, and EV4B, and Movie EV4). In agreement with the data obtained using KIF4A mutants, MT-flux in bipolar MUG spindles was strongly reduced (0.31 ± 0.13 μm/min, compared with 0.5 ± 0.13 μm/min in control cells treated with caffeine alone; Fig 3B), consistent with the importance of chromatin as the locus of KIF4A activity relevant for MT-flux. However, other features that are normally present in MUGs, such as merotelic KT-MT attachments, cannot be excluded to contribute to the observed attenuation of MT-flux. Figure 3. Bipolar spindles with expelled chromatin in MUGs exhibit reduced MT-flux Representative spinning disk confocal time series of MT-flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes (MUGs). U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), labeled for chromosomes with SiR-DNA (gray) are shown, with 5 mM caffeine used as a control. Scale bar, 10 μm. Time, min:s. Quantification of the MT-flux rates from indicated conditions. MT-flux values with mean ± SD are plotted. N (number of cells, number of independent experiments): Caffeine-only control (30, 3) and MUGs (34, 3). P-values from Mann–Whitney U-test. ****P ≤ 0.0001. Representative point-scanning confocal maximum-intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions, immunostained with antibodies against KIF4A and α-tubulin. DNA was counterstained with DAPI. KIF4A in cyan and α-tubulin in red in merged image. Scale bar, 10 μm. Download figure Download PowerPoint The MT-crosslinking proteins HSET and NuMA facilitate the distribution of MT-flux associated spindle forces on metaphase chromosomes If our model based on KIF4A driving MT-flux from chromosome arms was correct, one would predict that the action of MT crosslinking molecules is required to transmit spindle forces from fluxing non-KT-MTs to KT-MTs, as proposed by the “coupled spindle” model (Matos et al, 2009). In order to test this hypothesis, we investigated the contribution of three well-established MT-crosslinking proteins to MT-flux, namely NuMA, kinesin-14/HSET, and PRC1. NuMA localizes to the spindle poles together with the MT minus-end-directed motor Dynein to crosslink and focus MT minus-ends (Merdes et al, 1996; Radulescu & Cleveland, 2010; Hueschen et al, 2017). HSET is a MT minus-end-directed kinesin motor with the ability to crosslink and slide antiparallel MTs, thereby generating an inward force within the spindle, similar to that proposed for Dynein (Mountain et al, 1999). PRC1 is involved in the assembly of the central spindle during late mitosis (Mollinari et al, 2002) and acts as a crosslinker between KT-MTs and bridging interpolar MTs throughout mitosis (Kajtez et al, 2016; Polak et al, 2017; Vukusic et al, 2017; Suresh et al, 2020). We found that, while depletion of NuMA and/or HSET significantly reduced MT-flux rates (0.48 ± 0.13, 0.36 ± 0.13, and 0.35 ± 0.16 μm/min, respectively; Figs 4A and B, and EV3A, and Movie EV5), depletion of PRC1 had no such effect (0.61 ± 0.22 μm/min, compared with 0.61 ± 0.22 μm/min in control cells; Figs 4B and EV3A). Thus, KIF4A's requirement for MT-flux is independent of its PRC1-dependent localization at interpolar MTs (Kurasawa et al, 2004; Nguyen et al, 2018), further strengthening the argument of its association with chromatin in driving MT-flux. Figure 4. The MT-crosslinking proteins HSET and NuMA facilitate the distribution of MT-flux associated spindle forces on metaphase chromosomes Representative spinning disk confocal live-cell image series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 μm. Time, min:s. Quantification of the impact of the MT-crosslinking proteins on MT-flux in U2OS PA-GFP/mCherry-α-tubulin cells transfected with respective siRNAs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siPRC1 (45, 3), siNuMA (40, 3), siHSET (37, 3), and siHSET + siNuMA (39, 3). P-values were calculated against control using one-way ANOVA. n.s., not significant, **P ≤ 0.01, ****P ≤ 0.0001. Quantification of the impact of the motor activity of HSET on MT-flux in U2OS cells stably expressing mEOS-α-tubulin treated with control or HSET 3′UTR siRNAs in presence or absence of the respective RNAi-resistant GFP-HSET constructs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (23, 3), siHSET 3′UTR (33, 3), siHSET 3′UTR + HSET WT (29, 5), and siHSET 3′UTR + HSET N593K (30, 4). P-values were calculated against control using one-way ANOVA. n.s., not significant, ****P ≤ 0.0001. Representative kymographs of the photoactivated spindles from (B) displaying asynchronous flux motion (split of the two red dashed lines toward individual poles) upon RNAi-mediated depletion of MT-crosslinkers. Scale bars, 30 s. Percentage of cells with asynchronous flux movements calculated from (B). Model illustrating the role of MT-crosslinking activities of NuMA and HSET in uniform distribution of poleward forces across the mitotic spindle. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Analysis of the roles of MT-crosslinking proteins NuMA, HSET and PRC1 in MT-flux Immunoblot analysis of the respective knockdown efficiencies in U2OS cells stably expressing PA-GFP/mCherry-α-tubulin treated with control or indicated siRNAs. GAPDH, α-tubulin and vinculin were used as a loading control. Spinning disk confocal time series images of photoconverted mEOS-α-tubulin (red) in U2OS cells stably expressing mEOS-α-tubulin (cyan). White arrowheads highlight the photoconverted regions fluxing toward the poles. Scale bar, 10 μm. Time, min:s. Immunoblot analysis of U2OS cells stably expressing mEOS-α-tubulin transfected with control or HSET 3′UTR-targeting siRNAs and co-transfected with the RNAi-resistant GFP-HSET constructs. Expression levels of HSET was observed using anti-HSET antibody with α-tubulin serving as the loading control. Download figure Download PowerPoint In order to dissect whether the contribution of HSET for MT-flux depends on its MT-crosslinking ability, or on its motor activity, we designed a rescue experiment in which we individually expressed WT HSET and a non-processive motor mutant that is still able to crosslink MTs (N593K) (Cai et al, 2009b). Because our HSET constructs were GFP-tagged and could thus interfere with the PA-GFP tubulin signal, we used a U2OS cell line stably expressing green-red photoconvertible mEOS-tubulin (Wandke et al, 2012) (Fig EV3B). By monitoring MT-flux rates after depletion of endogenous HSET by 3′UTR-targeting siRNAs (0.37 ± 0.13 μm/min, compared with 0.61 ± 0.13 μm/min in control cells), we observed that both WT HSET and the motor mutant successfully ensured normal MT-flux (0.59 ± 0.1 μm/min and 0.55 ± 0.11 μm/min, respectively; Figs 4C and EV3C). This suggests that MT-flux relies on HSET MT-crosslinking capacity, rather than its motor activity (Fig 4F). Interestingly, spindles deplete
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