A Biosensor for the Mitotic Kinase MPS1 Reveals Spatiotemporal Activity Dynamics and Regulation

•Development of a FRET-based biosensor of MPS1 kinase activity•Active MPS1 detected at centromeres and chromatin is derived from kinetochores•MPS1 activity is initiated ∼12 min before NEB in a PP2A-B56-dependent manner•Colon cancer cell lines and organoids have lower MPS1 activity than healthy lines Accurate chromosome segregation during cell division critically depends on error correction of chromosome-spindle interactions and the spindle assembly checkpoint (SAC) [1Tanaka T.U. Bi-orienting chromosomes: acrobatics on the mitotic spindle.Chromosoma. 2008; 117: 521-533Crossref PubMed Scopus (30) Google Scholar, 2Sacristan C. Kops G.J. Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling.Trends Cell Biol. 2015; 25: 21-28Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 3London N. Biggins S. Signalling dynamics in the spindle checkpoint response.Nat. Rev. Mol. Cell Biol. 2014; 15: 736-747Crossref PubMed Scopus (215) Google Scholar]. The kinase MPS1 is an essential regulator of both processes, ensuring full chromosome biorientation before anaphase onset [3London N. Biggins S. Signalling dynamics in the spindle checkpoint response.Nat. Rev. Mol. Cell Biol. 2014; 15: 736-747Crossref PubMed Scopus (215) Google Scholar, 4Pachis S.T. Kops G.J.P.L. Leader of the SAC: molecular mechanisms of Mps1/TTK regulation in mitosis.Open Biol. 2018; 8: 180109Crossref PubMed Scopus (52) Google Scholar]. To understand when and where MPS1 activation occurs and how MPS1 signaling is modulated during mitosis, we developed MPS1sen, a sensitive and specific FRET-based biosensor for MPS1 activity. By placing MPS1sen at different subcellular locations, we show that MPS1 activity initiates in the nucleus ∼9–12 min prior to nuclear envelope breakdown (NEB) in a kinetochore-dependent manner and reaches the cytoplasm at the start of NEB. Soon after initiation, MPS1 activity increases with switch-like kinetics, peaking at completion of NEB. We further show that timing and extent of pre-NEB MPS1 activity is regulated by Aurora B and PP2A-B56. MPS1sen phosphorylation declines in prometaphase as a result of formation of kinetochore-microtubule attachments, reaching low but still detectable levels at metaphase. Finally, leveraging the sensitivity and dynamic range of MPS1sen, we show deregulated MPS1 signaling dynamics in colorectal cancer cell lines and tumor organoids with diverse genomic instability phenotypes. Accurate chromosome segregation during cell division critically depends on error correction of chromosome-spindle interactions and the spindle assembly checkpoint (SAC) [1Tanaka T.U. Bi-orienting chromosomes: acrobatics on the mitotic spindle.Chromosoma. 2008; 117: 521-533Crossref PubMed Scopus (30) Google Scholar, 2Sacristan C. Kops G.J. Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling.Trends Cell Biol. 2015; 25: 21-28Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 3London N. Biggins S. Signalling dynamics in the spindle checkpoint response.Nat. Rev. Mol. Cell Biol. 2014; 15: 736-747Crossref PubMed Scopus (215) Google Scholar]. The kinase MPS1 is an essential regulator of both processes, ensuring full chromosome biorientation before anaphase onset [3London N. Biggins S. Signalling dynamics in the spindle checkpoint response.Nat. Rev. Mol. Cell Biol. 2014; 15: 736-747Crossref PubMed Scopus (215) Google Scholar, 4Pachis S.T. Kops G.J.P.L. Leader of the SAC: molecular mechanisms of Mps1/TTK regulation in mitosis.Open Biol. 2018; 8: 180109Crossref PubMed Scopus (52) Google Scholar]. To understand when and where MPS1 activation occurs and how MPS1 signaling is modulated during mitosis, we developed MPS1sen, a sensitive and specific FRET-based biosensor for MPS1 activity. By placing MPS1sen at different subcellular locations, we show that MPS1 activity initiates in the nucleus ∼9–12 min prior to nuclear envelope breakdown (NEB) in a kinetochore-dependent manner and reaches the cytoplasm at the start of NEB. Soon after initiation, MPS1 activity increases with switch-like kinetics, peaking at completion of NEB. We further show that timing and extent of pre-NEB MPS1 activity is regulated by Aurora B and PP2A-B56. MPS1sen phosphorylation declines in prometaphase as a result of formation of kinetochore-microtubule attachments, reaching low but still detectable levels at metaphase. Finally, leveraging the sensitivity and dynamic range of MPS1sen, we show deregulated MPS1 signaling dynamics in colorectal cancer cell lines and tumor organoids with diverse genomic instability phenotypes. Accurate chromosome segregation requires biorientation of all chromosomes before the onset of anaphase. Central to guarding chromosome segregation fidelity is the mitotic kinase MPS1 [4Pachis S.T. Kops G.J.P.L. Leader of the SAC: molecular mechanisms of Mps1/TTK regulation in mitosis.Open Biol. 2018; 8: 180109Crossref PubMed Scopus (52) Google Scholar]. Its activation causes phosphorylation of various substrates that promote efficient correction of erroneous kinetochore-microtubule attachments and a robust response of the SAC to lack of such attachments [1Tanaka T.U. Bi-orienting chromosomes: acrobatics on the mitotic spindle.Chromosoma. 2008; 117: 521-533Crossref PubMed Scopus (30) Google Scholar, 2Sacristan C. Kops G.J. Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling.Trends Cell Biol. 2015; 25: 21-28Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar]. Conversely, inhibition of MPS1 upon biorientation is necessary for timely cell-cycle progression [5Jelluma N. Dansen T.B. Sliedrecht T. Kwiatkowski N.P. Kops G.J. Release of Mps1 from kinetochores is crucial for timely anaphase onset.J. Cell Biol. 2010; 191: 281-290Crossref PubMed Scopus (87) Google Scholar, 6Hayward D. Bancroft J. Mangat D. Alfonso-Pérez T. Dugdale S. McCarthy J. Barr F.A. Gruneberg U. Checkpoint signaling and error correction require regulation of the MPS1 T-loop by PP2A-B56.J. Cell Biol. 2019; 218: 3188-3199Crossref PubMed Google Scholar]. Much can be learned concerning maintenance of chromosomal stability from studying mechanisms and dynamics of MPS1 activation prior to and during mitosis and of its subsequent inactivation. FRET-based biosensors have uncovered localized regulation of mitotic kinase activity and greatly increased our understanding of their signaling pathways in single cells [7Liu D. Vader G. Vromans M.J. Lampson M.A. Lens S.M. Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates.Science. 2009; 323: 1350-1353Crossref PubMed Scopus (416) Google Scholar, 8Zaytsev A.V. Segura-Peña D. Godzi M. Calderon A. Ballister E.R. Stamatov R. Mayo A.M. Peterson L. Black B.E. Ataullakhanov F.I. et al.Bistability of a coupled Aurora B kinase-phosphatase system in cell division.eLife. 2016; 5: e10644Crossref PubMed Scopus (35) Google Scholar, 9Liu D. Davydenko O. Lampson M.A. Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing.J. Cell Biol. 2012; 198: 491-499Crossref PubMed Scopus (118) Google Scholar]. Inspired by these studies, we set out to develop a FRET-based biosensor specific for MPS1 kinase in order to study its (de)activation dynamics during mitosis. To study spatiotemporal dynamics of MPS1 kinase signaling, we modified an existing PLK1 biosensor with the substrate sequence LLLDS(pThr)LSINW, previously shown to be also targeted by MPS1 during mitosis [10Macůrek L. Lindqvist A. Lim D. Lampson M.A. Klompmaker R. Freire R. Clouin C. Taylor S.S. Yaffe M.B. Medema R.H. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.Nature. 2008; 455: 119-123Crossref PubMed Scopus (524) Google Scholar, 11Bruinsma W. Macurek L. Freire R. Lindqvist A. Medema R.H. Bora and Aurora-A continue to activate Plk1 in mitosis.J. Cell Sci. 2014; 127: 801-811Crossref PubMed Scopus (77) Google Scholar]. Substrate consensus motifs for MPS1 and PLK1 kinases are similar in some positions (e.g., negative charges at −2 or −3 relative to phosphorylated S/T) but also have distinct features [12Alexander J. Lim D. Joughin B.A. Hegemann B. Hutchins J.R. Ehrenberger T. Ivins F. Sessa F. Hudecz O. Nigg E.A. et al.Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling.Sci. Signal. 2011; 4: ra42Crossref PubMed Scopus (0) Google Scholar, 13Dou Z. von Schubert C. Körner R. Santamaria A. Elowe S. Nigg E.A. Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1.PLoS ONE. 2011; 6: e18793Crossref PubMed Scopus (56) Google Scholar]. We utilized these distinctions to increase specificity of the sensor for MPS1 kinase. We introduced a glycine at position −1, an additional negative charge at −3, and an alanine at +2, creating the substrate sequence LLEDG(pThr)LIANW. We furthermore optimized the dynamic range of the sensor by inserting higher quantum yield fluorophores (Figure 1A). A prominent pool of active MPS1 is located at the outer kinetochore where it binds to the HEC1/NUF2 components of the NDC80 complex [14Kemmler S. Stach M. Knapp M. Ortiz J. Pfannstiel J. Ruppert T. Lechner J. Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling.EMBO J. 2009; 28: 1099-1110Crossref PubMed Scopus (119) Google Scholar, 15Hiruma Y. Sacristan C. Pachis S.T. Adamopoulos A. Kuijt T. Ubbink M. von Castelmur E. Perrakis A. Kops G.J.P.L. CELL DIVISION CYCLE. Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling.Science. 2015; 348: 1264-1267Crossref PubMed Scopus (151) Google Scholar, 16Ji Z. Gao H. Yu H. CELL DIVISION CYCLE. Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80C.Science. 2015; 348: 1260-1264Crossref PubMed Scopus (138) Google Scholar]. To verify specificity of the biosensor (hereafter named MPS1sen), we placed it close to the MPS1 activation site by fusion to the N terminus of SPC24 (hereafter named MPS1sen-KT). We then monitored FRET (mTurquoise2/YPet ratio after mTurquoise excitation) in mitotic HeLa cells treated with the spindle poison nocodazole. MPS1sen-KT showed a robust FRET signal, which was abolished upon inhibition of MPS1 by the small-molecule inhibitor Cpd-5 [17Koch A. Maia A. Janssen A. Medema R.H. Molecular basis underlying resistance to Mps1/TTK inhibitors.Oncogene. 2016; 35: 2518-2528Crossref PubMed Scopus (34) Google Scholar] (Figures 1B and 1C) or upon depletion of MPS1 by RNAi (Figures S1A and S1B). Importantly, despite similarity in consensus substrate sequences and overlapping localization [12Alexander J. Lim D. Joughin B.A. Hegemann B. Hutchins J.R. Ehrenberger T. Ivins F. Sessa F. Hudecz O. Nigg E.A. et al.Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling.Sci. Signal. 2011; 4: ra42Crossref PubMed Scopus (0) Google Scholar, 13Dou Z. von Schubert C. Körner R. Santamaria A. Elowe S. Nigg E.A. Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1.PLoS ONE. 2011; 6: e18793Crossref PubMed Scopus (56) Google Scholar, 18Hennrich M.L. Marino F. Groenewold V. Kops G.J. Mohammed S. Heck A.J. Universal quantitative kinase assay based on diagonal SCX chromatography and stable isotope dimethyl labeling provides high-definition kinase consensus motifs for PKA and human Mps1.J. Proteome Res. 2013; 12: 2214-2224Crossref PubMed Scopus (35) Google Scholar], inhibition of PLK1 with BI-2536 [19Lénárt P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar] did not reduce FRET, further validating specificity of MPS1sen (Figures 1B and 1C). Aurora B prefers basic amino acids at position −2 and −3 instead of acidic ones [12Alexander J. Lim D. Joughin B.A. Hegemann B. Hutchins J.R. Ehrenberger T. Ivins F. Sessa F. Hudecz O. Nigg E.A. et al.Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling.Sci. Signal. 2011; 4: ra42Crossref PubMed Scopus (0) Google Scholar], and indeed the Aurora B inhibitor ZM447439 [20Ditchfield C. Johnson V.L. Tighe A. Ellston R. Haworth C. Johnson T. Mortlock A. Keen N. Taylor S.S. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.J. Cell Biol. 2003; 161: 267-280Crossref PubMed Scopus (1045) Google Scholar] did not prevent MPS1sen phosphorylation. In line with the known role of Aurora B in promoting MPS1 activity during mitosis [21Saurin A.T. van der Waal M.S. Medema R.H. Lens S.M. Kops G.J. Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis.Nat. Commun. 2011; 2: 316Crossref PubMed Scopus (165) Google Scholar, 22Nijenhuis W. von Castelmur E. Littler D. De Marco V. Tromer E. Vleugel M. van Osch M.H. Snel B. Perrakis A. Kops G.J. A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.J. Cell Biol. 2013; 201: 217-231Crossref PubMed Scopus (98) Google Scholar, 23Santaguida S. Vernieri C. Villa F. Ciliberto A. Musacchio A. Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction.EMBO J. 2011; 30: 1508-1519Crossref PubMed Scopus (147) Google Scholar], we observed a slight but significant reduction of FRET, illustrating the ability of MPS1sen to report on minor changes to MPS1 activity states. MPS1sen-KT showed similar dynamic range and responded similarly to inhibition of Aurora B or PLK1 in non-transformed RPE-1 cells (Figures S1C and S1D). Taken together, we conclude that MPS1sen is a specific reporter for MPS1 with a dynamic range that enables it to detect relatively subtle changes to MPS1 activity. To examine whether MPS1 activity can be detected beyond the kinetochore, we placed MPS1sen at centromeres or chromatin by fusing it to CENP-B (MPS1sen-cent) or histone H2B (MPS1sen-chr), respectively. In agreement with reported MPS1 substrates at these locations [24Jelluma N. Brenkman A.B. van den Broek N.J. Cruijsen C.W. van Osch M.H. Lens S.M. Medema R.H. Kops G.J. Mps1 phosphorylates Borealin to control Aurora B activity and chromosome alignment.Cell. 2008; 132: 233-246Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 25Kagami Y. Nihira K. Wada S. Ono M. Honda M. Yoshida K. Mps1 phosphorylation of condensin II controls chromosome condensation at the onset of mitosis.J. Cell Biol. 2014; 205: 781-790Crossref PubMed Scopus (18) Google Scholar], both MPS1sen-cent and MPS1sen-chr revealed substantial MPS1 activity, which was sensitive to cpd-5 (Figures 1D and 1E). The MPS1 activity reported by these probes originated from kinetochores, as evidenced by the following observations: first, FRET signals in mitotic cells were strongly reduced following HEC1 depletion by RNAi or Aurora B inhibition by ZM447439 (Figures 1F, S1E, and S1F). Second, conditional knockout of CENP-C [26McKinley K.L. Sekulic N. Guo L.Y. Tsinman T. Black B.E. Cheeseman I.M. The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface.Mol. Cell. 2015; 60: 886-898Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar] in roughly half the cell population (Figure S1G) abolished or strongly reduced FRET signals in a similar fraction of cells (Figures 1G, S1H, and S1I). Assembly of the outer kinetochore is thus required for generating most if not all cellular MPS1 activity. Third, if MPS1 reaches distant sites by spreading from kinetochores, we reasoned that this should result in local differences in the concentration of active molecules. In support of this, full inhibition of MPS1 at kinetochores required 500 nM of Cpd-5 (IC50 18 nM) while at more kinetochore-distal locations 100 nM Cpd-5 sufficed (IC50 2–3 nM) (Figures 1D and 1E). Although differences in phosphatase activities at these various sites may exist, our data are consistent with a model in which MPS1 is activated at the outer kinetochore by binding the NDC80 complex and subsequently travels to distant sites. In prophase, for example, if sufficient MPS1 can be activated at kinetochores (see below), this would then allow phosphorylation of condensin II [25Kagami Y. Nihira K. Wada S. Ono M. Honda M. Yoshida K. Mps1 phosphorylation of condensin II controls chromosome condensation at the onset of mitosis.J. Cell Biol. 2014; 205: 781-790Crossref PubMed Scopus (18) Google Scholar] and nuclear pore- MAD1 complexes [27Rodriguez-Bravo V. Maciejowski J. Corona J. Buch H.K. Collin P. Kanemaki M.T. Shah J.V. Jallepalli P.V. Nuclear pores protect genome integrity by assembling a premitotic and Mad1-dependent anaphase inhibitor.Cell. 2014; 156: 1017-1031Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 28Ji Z. Gao H. Jia L. Li B. Yu H. A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling.eLife. 2017; 6: e22513Crossref PubMed Scopus (91) Google Scholar, 29Cunha-Silva S. Osswald M. Goemann J. Barbosa J. Santos L.M. Resende P. Bange T. Ferrás C. Sunkel C.E. Conde C. Mps1-mediated release of Mad1 from nuclear pores ensures the fidelity of chromosome segregation.J. Cell Biol. 2020; 2: e201906039Crossref Scopus (9) Google Scholar]. We and others have shown that formation of end-on kinetochore-microtubule attachments disrupt the MPS1-HEC1/NUF2 interaction and can silence the SAC in the absence of tension [15Hiruma Y. Sacristan C. Pachis S.T. Adamopoulos A. Kuijt T. Ubbink M. von Castelmur E. Perrakis A. Kops G.J.P.L. CELL DIVISION CYCLE. Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling.Science. 2015; 348: 1264-1267Crossref PubMed Scopus (151) Google Scholar, 16Ji Z. Gao H. Yu H. CELL DIVISION CYCLE. Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80C.Science. 2015; 348: 1260-1264Crossref PubMed Scopus (138) Google Scholar, 30Etemad B. Kuijt T.E.F. Kops G.J.P.L. Kinetochore-microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint.Nat. Commun. 2015; 6: 8987Crossref PubMed Scopus (56) Google Scholar, 31Tauchman E.C. Boehm F.J. DeLuca J.G. Stable kinetochore-microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells.Nat. Commun. 2015; 6: 10036Crossref PubMed Scopus (65) Google Scholar, 32Dou Z. Liu X. Wang W. Zhu T. Wang X. Xu L. Abrieu A. Fu C. Hill D.L. Yao X. Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment.Proc. Natl. Acad. Sci. USA. 2015; 112: E4546-E4555Crossref PubMed Scopus (42) Google Scholar]. Our MPS1 biosensor allowed us to examine how MPS1 substrate phosphorylation is affected by various kinetochore-microtubule attachment configurations. To this end, we modulated kinetochore-microtubule attachments using various perturbations in cells expressing MPS1sen-KT. In these cells, mCherry-BUB1 [33Etemad B. Vertesy A. Kuijt T.E.F. Sacristan C. van Oudenaarden A. Kops G.J.P.L. Spindle checkpoint silencing at kinetochores with submaximal microtubule occupancy.J. Cell Sci. 2019; 132: jcs231589Crossref PubMed Scopus (14) Google Scholar] was expressed from the endogenous locus to monitor whether and how BUB1 kinetochore localization correlates with MPS1 activity. While unattached kinetochores in nocodazole-treated cells displayed high MPS1sen-KT phosphorylation, stably bioriented kinetochores at metaphase in MG132-treated cells showed low phosphorylation that was further reduced by addition of Cpd-5 (Figures 2A, 2B, and S2A). MPS1 thus displays detectable activity in metaphase. This is in line with residual levels of BUB1 (Figures 2B and S2F; [33Etemad B. Vertesy A. Kuijt T.E.F. Sacristan C. van Oudenaarden A. Kops G.J.P.L. Spindle checkpoint silencing at kinetochores with submaximal microtubule occupancy.J. Cell Sci. 2019; 132: jcs231589Crossref PubMed Scopus (14) Google Scholar]) and KNL1 phosphorylation [15Hiruma Y. Sacristan C. Pachis S.T. Adamopoulos A. Kuijt T. Ubbink M. von Castelmur E. Perrakis A. Kops G.J.P.L. CELL DIVISION CYCLE. Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling.Science. 2015; 348: 1264-1267Crossref PubMed Scopus (151) Google Scholar, 33Etemad B. Vertesy A. Kuijt T.E.F. Sacristan C. van Oudenaarden A. Kops G.J.P.L. Spindle checkpoint silencing at kinetochores with submaximal microtubule occupancy.J. Cell Sci. 2019; 132: jcs231589Crossref PubMed Scopus (14) Google Scholar, 34von Schubert C. Cubizolles F. Bracher J.M. Sliedrecht T. Kops G.J.P.L. Nigg E.A. Plk1 and Mps1 Cooperatively Regulate the Spindle Assembly Checkpoint in Human Cells.Cell Rep. 2015; 12: 66-78Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar] on metaphase kinetochores, and with observations that targeting of MAD1 to metaphase kinetochores re-elicits an MPS1-dependent SAC response [35Ballister E.R. Riegman M. Lampson M.A. Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint.J. Cell Biol. 2014; 204: 901-908Crossref PubMed Scopus (43) Google Scholar, 36Kuijt T.E.F. Omerzu M. Saurin A.T. Kops G.J.P.L. Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase.Chromosoma. 2014; 123: 471-480Crossref PubMed Scopus (32) Google Scholar, 37Maldonado M. Kapoor T.M. Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation.Nat. Cell Biol. 2011; 13: 475-482Crossref PubMed Scopus (133) Google Scholar]. The reason for residual MPS1 activity in metaphase is unclear. Since we observed occasional loss of metaphase plate integrity upon MPS1 inhibition (Figures S2B and S2D), perhaps it functions to maintain correct microtubule dynamics or retain some BUB1-SGO1 (Figure S2F) to promote centromeric CPC localization and thus preserve sister chromatid cohesion [38Williams S.J. Abrieu A. Losada A. Bub1 targeting to centromeres is sufficient for Sgo1 recruitment in the absence of kinetochores.Chromosoma. 2017; 126: 279-286Crossref PubMed Scopus (10) Google Scholar, 39El Yakoubi W. Buffin E. Cladière D. Gryaznova Y. Berenguer I. Touati S.A. Gómez R. Suja J.A. van Deursen J.M. Wassmann K. Mps1 kinase-dependent Sgo2 centromere localisation mediates cohesin protection in mouse oocyte meiosis I.Nat. Commun. 2017; 8: 694Crossref PubMed Scopus (25) Google Scholar]. Low metaphase MPS1 activity could additionally aid in fast SAC reactivation upon loss of kinetochore-microtubule attachments. To directly compare various attachment states in a single cell, we inhibited the kinesin CENP-E. Cells with inactive CENP-E have many bioriented chromosomes at the metaphase plate and a few unaligned (unattached or transiently attached) ones near centrosomes [40Wood K.W. Lad L. Luo L. Qian X. Knight S.D. Nevins N. Brejc K. Sutton D. Gilmartin A.G. Chua P.R. et al.Antitumor activity of an allosteric inhibitor of centromere-associated protein-E.Proc. Natl. Acad. Sci. USA. 2010; 107: 5839-5844Crossref PubMed Scopus (163) Google Scholar, 41Barisic M. Aguiar P. Geley S. Maiato H. Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces.Nat. Cell Biol. 2014; 16: 1249-1256Crossref PubMed Scopus (95) Google Scholar]. MPS1sen-KT phosphorylation on kinetochores of those bioriented chromosomes was as low as on those of metaphase cells (Figures 2A, 2B, S2A, and S2B). On kinetochores of unaligned chromosomes, however, MSP1sen-KT was highly phosphorylated. Similar results were obtained in RPE-1 cells (Figure S2G). Interestingly, BUB1 levels at kinetochores did not fully correlate with MPS1sen-KT phosphorylation under these conditions (Figure S2F), suggesting there may be MPS1-independent regulation of BUB1 at kinetochores [34von Schubert C. Cubizolles F. Bracher J.M. Sliedrecht T. Kops G.J.P.L. Nigg E.A. Plk1 and Mps1 Cooperatively Regulate the Spindle Assembly Checkpoint in Human Cells.Cell Rep. 2015; 12: 66-78Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 42Ikeda M. Tanaka K. Plk1 bound to Bub1 contributes to spindle assembly checkpoint activity during mitosis.Sci. Rep. 2017; 7: 8794Crossref PubMed Scopus (25) Google Scholar]. MPS1sen modulations by attachment states were generally recapitulated by placing it on centromeres or chromatin. Interestingly, however, on unaligned chromosomes in CENP-E-inhibited cells, MPS1sen phosphorylation on centromeres was substantially lower than on kinetochores (Figures 2C, 2D, and S2C–S2H). The reason for this is unclear. Perhaps it reflects differences in local phosphatase activities or in the ability of MPS1 to reach centromeric chromatin regions in these cells. Taken together, the data show that kinetochore-microtubule interactions and chromosome biorientation modulate MPS1 kinase signaling throughout the cell. Kinase biosensors revealed that nuclear translocation of cyclin B/CDK1 in prophase does not restrict CDK1 activity solely to the nucleus [43Gavet O. Pines J. Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis.J. Cell Biol. 2010; 189: 247-259Crossref PubMed Scopus (216) Google Scholar] and that PLK1 activity is restricted to the nucleus in G2 phase [10Macůrek L. Lindqvist A. Lim D. Lampson M.A. Klompmaker R. Freire R. Clouin C. Taylor S.S. Yaffe M.B. Medema R.H. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.Nature. 2008; 455: 119-123Crossref PubMed Scopus (524) Google Scholar]. MPS1 localizes to the cytoplasm in interphase and translocates to the nucleus upon mitotic entry [44Mills G.B. Schmandt R. McGill M. Amendola A. Hill M. Jacobs K. May C. Rodricks A.M. Campbell S. Hogg D. Expression of TTK, a novel human protein kinase, is associated with cell proliferation.J. Biol. Chem. 1992; 267: 16000-16006Abstract Full Text PDF PubMed Google Scholar, 45Alfonso-Pérez T. Hayward D. Holder J. Gruneberg U. Barr F.A. MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling.J. Cell Biol. 2019; 218: 1108-1117Crossref PubMed Scopus (39) Google Scholar]. To visualize the spatial dynamics of MPS1 activation, we expressed MPS1sen without specifying its subcellular localization, resulting in nuclear and cytoplasmic pools. We co-expressed mScarlet-NLS to accurately monitor nuclear envelope breakdown (NEB). Phosphorylation of nuclear MPS1sen became apparent roughly 10 min before completion of NEB, while phosphorylation of cytoplasmic MPS1sen was delayed by several minutes (Figures 3A and 3B ; Video S1). Phosphorylation of this pool correlated with leakage of mScarlet-NLS (cytoplasmic/nuclear ratio) from the nucleus as NEB commenced. These data agree with a recent observation that MPS1 kinetochore localization correlates with nuclear import of cyclin B [45Alfonso-Pérez T. Hayward D. Holder J. Gruneberg U. Barr F.A. MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling.J. Cell Biol. 2019; 218: 1108-1117Crossref PubMed Scopus (39) Google Scholar] and suggest MPS1 is activated in the nucleus. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJkNDZiYzg5MzU3M2I3MWU5ZWM2NDdkZjhjODViMWJkNyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MzI2OTg2fQ.pwriQWbXuR27bndhAdq7jVqgAjzJDKy7w8O9DrsXeYWSqvseKnygZeXqwsiRD5U2MZmk9k1HOmqlBNNcU_NMSV_bIXNGvtURh0uAAHvUJlkz8Qe7NDHvtbOYkMNEsYxoTAl6eaKvSuVBYD-zgegkpthxKgDeyViS80CylVFnLHJlQhgBd0udGQYBw7kDaLz1Zsfni7tnPLSOARWGsx-pGtIghHMQShwD7P5Igsu7jOQhqL9sCdCWRlU7c5nMRUVByKsWixBIlqtR16h91IbapiTtd0eqFABMq0cP8aMwpR5JYCDmWWsSxlC8ibf3HL9h-w6yPegWtVRKM4h-yoV3tA Download .mp4 (1.97 MB) Help with .mp4 files Video S1. MPS1 Activation Is Restricted to the Nucleus during Prophase, Related to Figure 3HeLa cells expressing MPS1sen and IRES-PURO-P2A-mScarlet-NLS, released from RO-3306 block and imaged every ± 20 s. Fluorescent channels are mTurq2 (top left), mScarlet (top, right), FRET (YPet, bottom left), smoothed mTurq2/FRET pseudo colored ratio (bottom, right). Scale bar, 10 μm. To more accurately monitor pre-mitotic activation of MPS1, we next wished to immobilize MPS1sen in the nucleus. Unfortunately, MPS1sen-KT was hardly detectable at early prophase kinetochores, likely due to immature outer-kinetochore assembly [46Cheeseman I.M. Desai A. Molecular architecture of the kinetochore-microtubule interface.Nat. Rev. Mol. Cell Biol. 2008; 9: 33-46Crossref PubMed Scopus (689) Google Scholar]. We thus opted to use MPS1sen-chr, as it is stably bound to chromatin throughout the cell cycle and correctly reports on kinetochore-derived MPS1 activity (see Figures 1D–1F and also an additional mutation of the substrate sequence in Figure 3C). In further support of this, MPS1sen-chr did not show any phosphorylation during mitotic progression of MPS1-inhibited or -depleted cells (Figures 3C and S3A). This was not an indirect effect of SAC inactivation, because MAD2 depletion hardly impacted MPS1sen-chr phosphorylation (Figure S3A). Notably, during a normal, unperturbed mitosis, MPS1sen-chr phosphorylation was detectable 9–12 min before full NEB (monitored by mScarlet-NLS [Video S2]), rapidly increased thereafter, and peak