Chromosome alignment relies on spindle-localized control of Cdk1 activity

Chromosome alignment relies on spindle-localized control of Cdk1 activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Chromosome alignment relies on spindle-localized control of Cdk1 activity Angela Flavia Serpico, Caterina Pisauro, Asia Trano, Domenico Grieco This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4594196/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract During mitosis, chromosome alignment at the mitotic spindle equator grants correct chromosome segregation and proper nuclei formation in daughter cells. A central role for chromosome alignment is exerted by the kinesin 8 family member Kif18A that localizes at the kinetochore-microtubule (K-MT) plus ends where it dampens MT dynamics stabilizing K-MT attachments. Kif18A action is directly antagonized by the master mitotic kinase cyclin B-dependent kinase 1 (Cdk1) and promoted by protein phosphatase 1 (PP1). Since chromosome alignment precedes Cdk1 inactivation by cyclin B proteolysis it is unclear how Kif18A evicts Cdk1 inhibition. We show here that chromosome alignment in human cells relies on a recently identified fraction of Cdk1 that is inhibited by phosphorylation in mitosis (i-Cdk1, for inhibited/inactive-Cdk1), localized at spindle structures and required for proper spindle assembly. Indeed, lowering i-Cdk1 induced several spindle defects including spindles with misaligned, bipolarly attached, chromosomes that showed poor Kif18A localization at K-MT plus ends. Both alignment defects and Kif18A localization were reversed by restoring i-Cdk1. In i-Cdk1-lowered cells, alignment defects were also significantly rescued by expressing a phosphorylation-resistant Kif18A version at Cdk1-dependent sites. Mechanistically, our evidence indicates that i-Cdk1 and active PP1 promoted spindle-localized Kif18A dephosphorylation. Given the relevance of Kif18A for survival of aneuploid cancer cells, these observations may also have relevance for cancer therapy. Chromosome alignment i-Cdk1 micronuclei aneuploidy Wee1 spindle assembly cancer vulnerability. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction During mitosis, proper chromosome alignment at the equator of the mitotic spindle at metaphase grants synchronous chromosome segregation in anaphase, helping to prevent mis-segregation and formation of micronuclei into daughter cells [ 1 , 2 , 3 ]. During spindle assembly, chromosomes congress towards the spindle equator in an oscillating fashion, promoted by the action of kinesins and the control of MT dynamics. To reach the metaphase configuration, chromosomes are pushed away from centrosomes by polar ejection forces, mainly acting on chromosome arms, and pulled towards centrosomes by kinetochore-dependent forces, until the final alignment at the center of the spindle [ 1 ]. This oscillatory behavior is also supposed to facilitate correct chromosome attachments, while the final alignment at the spindle equator is reached by the progressive decline of these oscillatory movements. A central action to dampen chromosome oscillations and promote alignment is exerted by localization of the plus end-directed kinesin 8 family member Kif18A at the plus ends of K-MTs where it suppresses MT dynamics [ 1 , 2 , 3 , 4 , 5 , 6 ]. Kif18A concentration at K-MT plus ends has also been involved in the tension-dependent mechanisms that silence the Spindle Assembly Checkpoint (SAC), the safeguard mechanism that prevents anaphase onset until correct spindle assembly completion [ 7 ]. Although loss of Kif18A function causes micronuclei formation, it appears to be rather tolerated by euploid cells and inactivating mutations in Kif18A have been shown to produce micronuclei in mice without leading to cellular transformation or cancer [ 2 , 3 , 7 ]. Nevertheless, Kif18A does appear to be essential for survival of aneuploid cancer cells, perhaps by preventing unbearable chromosome mis-segregation in cells with more complicated spindle architecture, and appears a vulnerability target for certain aneuploid cancers [ 8 , 9 , 10 , 11 , 12 ]. The ability of Kif18A to reach the K-MT plus ends and dampen chromosome oscillations has been shown to be antagonized by phosphorylation operated by Cdk1 and stimulated by its reversal operated by PP1 [ 13 ]. How Kif18A evicts inhibition by Cdk1 during spindle assembly is unknown. We show here that the ability of Kif18A to reach K-MT plus ends and promote chromosome alignment depends on i-Cdk1, a spindle-localized fraction of Cdk1 that we have recently described to be inhibited by phosphorylation in mitosis and to be crucial for spindle assembly [ 14 , 15 ]. Materials and Methods Cell lines and cell culture Human Henrietta Lacks (HeLa; CEINGE Cell Culture Facility) cells were grown in Dulbecco’s Modified Eagle Medium high glucose (DMEM; Sigma-Aldrich) supplemented with 10% foetal bovine serum (FBS; Gibco), 1% GlutaMAX-supplement (Gibco), 1% penicillin/streptomycin (Euroclone). Human Telomerase Reverse Transcriptase-immortalized Retinal Pigment Epithelial (hTERT-RPE1; Dr. A. Musacchio’s gift; Max Planck Institute of Molecular Physiology, Dortmund) cells were grown in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; Gibco, Thermo Fisher Scientific) supplemented with 10% foetal bovine serum (FBS; Gibco), 1% GlutaMAX-supplement (Gibco), 1% penicillin/streptomycin (Euroclone). All cells were incubated in a humidified incubator at 37°C with 5% CO 2 . DNA and siRNA constructs Cdk1-WT and cdk1-AF mammalian expression plasmids were purchased from Addgene (Cat# 61840; Cat# 39872, respectively). 3XFlag-Wee1 expression construct was obtained as previously described [ 16 ]. Myc-Flag-tagged-human-Kif18A-WT (Kif18A-WT) plasmid was purchased from Origene (Cat# RC208235); Kif18A-AA mutant version was generated via mutagenesis: serine 674 was mutagenized into alanine (2020–2022 nt TCT◊GCT) and serine 684 into alanine (2050–2052 nt TCT◊GCT) by QuikChange II XL site-directed mutagenesis kit (Agilent Technologies) using Kif18A-WT as template. Custom siRNAs targeting the 3’-UTR of human WEE1 (#1: 5’-CUGUAAACUUGUAGCAUUAUU-3’; #2: 5’-GUACAUAGCUGUUUGAAAUUU-3’; #3: 5’-GGGCUUUAUUACAGACAUAUU-3’) were designed using siDESIGN Center tool by Horizon and purchased from Dharmacon. Transfection and RNA interference Cells were seeded at a cell density of 7000/cm 2 either into 6–10 cm dishes or onto glass coverslips for biochemical and immunofluorescence studies, respectively. Manufacturer’s protocols were followed for both procedures and cells were plated 24 hours prior to treatment. Transient expression transfections were performed using MegaTran 2.0 Transfection Reagent (OriGene; Cat# TT210003); protein downregulation via siRNAs was obtained using DharmaFECT 1 siRNA Transfection Reagent (Dharmacon; Cat# T-2001-03). Briefly, MegaTran (1 µg/µL): DNA (1 µg/µL) (3:1) or DharmaFECT 1: siRNAs duplex (25 nM) (1:1) were mixed in cell culture media (DMEM or DMEM/F12) and incubated at room temperature (rt) for 20 minutes. Then the mixtures were added to the cells maintained in antibiotic-free complete medium and incubated for 24 hours (see also cell synchronization section). For Wee1-siRNA treatment and complementation, cells were transfected with mock- or 3XFlag-Wee1 expression construct 24 hours prior to treatment with non-targeting or specific siRNAs and 6 hours after siRNA-treatment cells were ad hoc synchronized (see also cell synchronization section). Exogenous cdk1-WT or cdk1-AF overexpression was achieved by transfection of the respective plasmids (empty or cdk1-WT or cdk1-AF expression vectors) 8 hours prior to specific synchronization (see also cell synchronization section). For co-overexpression experiments, cdk1-AF + empty vector, cdk1-AF + Kif18A-WT and cdk1-AF + Kif18A-AA vectors were simultaneously transfected 8 hours prior to synchronization. Cell synchronization Prometaphase-arrested HeLa cells were obtained by a 14-hour incubation with nocodazole (1 µg/ml; Abcam; Cat# ab120630). G2-arrested cells were obtained by incubation with RO-3306 at 9 µM (Calbiochem; Cat# 217699) for 16 hours. To obtain metaphase-arrested cells, G2- or prometaphase-arrested cells were washed twice with fresh medium and twice with phosphate buffer saline 1X (PBS; Corning; Cat# 21-031-CV) solution before plating into fresh medium containing MG-132 (20 µM; Calbiochem; Cat# 474790) and cycloheximide (CHX; 60 µg/mL; Santa Cruz Biotechnology; Cat# sc-3508) for further 60 minutes of incubation. Where indicated, mild inhibition of Cdk1 activity (+ RO) was obtained by adding RO-3306 at 0.5 µM (Calbiochem). Cell fractionation Cell fractionation was performed as previously described [ 14 ]. To separate cytoplasmic and spindle-bound proteins we adjusted a previously described method [ 17 ]. Cells were collected by centrifugation and washed once with PBS containing taxol (1µM; Sigma-Aldrich). 5 x 10 6 cells were carefully resuspended in 800 µl of fractionation lysis buffer (FLB; 40 mM b-glycerophosphate, 15 mM MgCl 2 , 20 mM EGTA, 0.25% Igepal, 20 mM Hepes, 5 µM taxol, 6 µg/mL latrunculin B; Sigma-Aldrich) completed with 300 µg/mL RNase A and 120 U/mL DNase I (Roche) and then incubated in thermomixer (Eppendorf thermomixer comfort) with constant shaking at 1.200 rpm for 20 min at 34° C. Lysates were centrifuged at 6.800 rpm (Eppendorf centrifuge 5425) for 2 minutes: the supernatants were harvested into new tubes, supplemented with 250 mM NaCl and stored on ice (soluble fractions). The pellet fractions were once again resuspended in FLB + 300 µg/mL RNase A and 120 U/mL DNase I and incubated in thermomixer at 34°C for 20 minutes. Samples were centrifuged and pellet fraction were washed twice with washing buffer (WB; 5 mM Hepes, 5 µM taxol; Sigma-Aldrich). Then, pellet fractions were resuspended in 200 µL of FLB minus taxol and supplemented with 250 mM NaCl and incubated on ice for 30 min. Finally, soluble and pellet fractions were spun at 13.200 rpm at 4°C for 10 minutes (Eppendorf centrifuge 5424 R) and both final supernatants were collected and processed for Immunoblottings (Ibs) or Immunoprecipitations (Ips). The pellet fraction proteins were extracted in a 200 µL buffer volume from a total cell lysate of 800 µl volume (mainly representing the soluble fraction volume), hence pellet fraction proteins were enriched 4 folds over the soluble fraction proteins, relatively to their cellular distribution and, in the experiment shown in Fig. 3 , a double volume of the pellet relatively to the soluble fractions was loaded on gels, thus, pellet fraction proteins were enriched a total of 8 folds over the soluble fraction proteins. Immunoprecipitation For immunoprecipitations from total cell lysates 5x 10 6 cells were lysed in 180 µl lysis buffer (LB; 80 mM b-glycerophosphate, 15 mM MgCl2, 20 mM EGTA, 20 mM Hepes pH 7.4, 100 mM NaCl, 0.1% Igepal; Sigma-Aldrich) supplemented with a phosphatase inhibitor cocktail (PhosSTOP; Roche) and incubated on ice for 30 min. Then, lysates were cleared via centrifugation at 13.200 rpm at 4°C for 10 min (Eppendorf centrifuge 5424 R) and incubated with agarose bead-conjugated antibody overnight at 4°C in constant rotation (mouse anti-cyclin B1 agarose, Santa Cruz Biotechnology, Cat# sc-245AC; normal mouse IgG-AC, Santa Cruz Biotechnology, Cat# sc-2343). For immunoprecipitations from soluble and pellet fractions, lysates were diluted in LB supplemented with PhosSTOP (Roche) until a final volume of 800 µl and incubated with agarose conjugated antibodies overnight at 4°C in constant rotation. For both above-mentioned cases, beads were washed twice in LB and proteins eluted in Laemmli denaturing buffer by boiling for 5 minutes (Laemmli sample buffer; BioRad, Cat# 1610747). Finally, samples were loaded into polyacrylamide gels and analyzed by immunoblotting. Immunoblotting Immunoblotting was performed as described [ 14 ]. Briefly, samples in SDS Laemmli buffer were incubated for 10 min at 99°C and then loaded and run on SDS-PAGE gels (polyacrylamide percentage spanning from 10 to 12%). Proteins were transferred onto nitrocellulose membrane (Cytiva-Amersham; Cat# GEH10600002) using a wet-transfer system (Thermofisher) and membranes were blocked with 5% nonfat dry milk (NFDM; AppliChem; Cat# A0830) in PBS supplemented with 0.01% Tween20 (TPBS; Sigma-Aldrich; Cat# P1379) for 1 hour at rt. Then, filters were incubated with primary antibodies, diluted in TPBS, at 4°C overnight. After 2 TPBS washes, membranes were incubated with secondary peroxidase-conjugated (HRP) antibodies, also diluted in TPBS, for 1 hour at rt. Enhanced ChemiLuminescence (ECL) kit (Cytiva-Amersham; Cat# RPN2106) enabled the detection of HRP enzyme activity. Blots were acquired using Canon CanoScan LiDE 300 scanner (Canon) and scanned at 300 dpi. For western blot analysis, primary antibodies were used as follows: rabbit anti-Wee1 (1:1000; Cell Signaling Technology; Cat# 13084); rabbit anti-cyclin B1 (1:2000; Bethyl; Cat# A305-000A); mouse anti-cdc2 (1:500; BD Biosciences; Cat# 610038); rabbit anti-Kif18A (1:1000; Bethyl; Cat# A301-080A); mouse anti-g-tubulin (1:2000; Sigma-Aldrich; Clone GTU-88; Cat# T5326), rabbit anti-phospho-tyrosine-15-cdc2 (1:1000; p-Y15-cdc2; Cell Signaling Technology, Cat# 4539), rabbit anti-phospho-Threonine-320-PP1a (1:1000; p-T320-PP1a; 1:1000; Cell Signaling Technology; Cat# 2581), mouse anti-PP1a (1:1000; Santa Cruz Biotechnology, Cat# sc-7482). Sheep anti-mouse IgG HRP linked (1:2000; GE Healthcare; Cat# NA931), donkey anti-rabbit IgG HRP linked (1:2000; GE Healthcare; Cat# NA934) were used as secondary antibodies. Immunofluorescence and microscopy Cells were plated onto poly-D-lysine (0,1 mg/mL; Sigma-Aldrich; Cat# P6407) coated glass coverslips at a cell density of 7000/cm 2 . After a brief wash in PBS, cells were fixed and permeabilized with 4% paraformaldehyde + 0,5% Triton X-100 (Sigma-Aldrich; Cat# P6148; T9284 respectively) in PBS for 12 min at rt. Then, cells were washed twice with PBS and incubated with 1.5% bovine serum albumin (BSA; Sigma-Aldrich; Cat# A7030) in PBS for 1 hour at rt. After 2 PBS washes, cells were incubated with primary antibodies in 1.5% (w/v) BSA-PBS solution for 3 hours into a humidity chamber at rt. Afterwards, cells were washed 3 times with PBS and incubated with fluorescently labelled secondary antibodies solution (1.5% BSA-PBS) for 1 hour at rt. DNA was stained with Hoechst 33258 (1 µg/ml; Invitrogen; Cat# 94403) by incubation for 10 minutes. Finally, cells were washed 4 times with PBS and slides mounted with Mowiol 40–88 (Sigma Aldrich; Cat# 81381). For immunofluorescence staining, the following antibodies were used: mouse anti-a-tubulin (1:1000; Sigma-Aldrich; Clone DM1A; Cat# T9026); human anti-centromere (CREST; 1:100; Antibodies Incorporated; Cat# 15–234); rabbit anti-Kif18A (1:2000; Abcam; Cat# ab251863); donkey anti-mouse IgG Alexa Fluor 594 (1:1000; Invitrogen; Cat# A21203); goat anti-human IgG Alexa Fluor 488 (1:1000; Invitrogen; Cat# A11013); donkey anti-rabbit IgG Alexa Fluor 594 (1:1000; Invitrogen; Cat# A21207). Inverted confocal fluorescence microscope LSM 980 (Zeiss) equipped with a 63X/1.4 oil objective (Zeiss) was used to image fixed cells. Three image planes with 0.44 mm Z-stack size were acquired and projected into one plane using ZEN3.1 software (Zeiss). Results Dependence of chromosome alignment on inhibitory control of Cdk1 We have recently gathered evidence that mitotic spindle assembly in human cells relies on the inhibitory control of a small and localized fraction of Cdk1, i-Cdk1, that represents less than 10% of total Cdk1 present in mitotic cells [ 14 , 15 ]. While bulk, active, Cdk1 maintains the cytoplasm of mitotic cells substantially free of MTs, i-Cdk1 appears required at spindle structures to locally reactivate MT-stabilizing MT-Associated Proteins (MAPs) to promote spindle assembly [ 14 , 15 ]. In experiments in which i-Cdk1 was lowered in mitotic cells either by downregulation of Cdk1 inhibitory kinase Wee1, by small interfering RNAs (siRNAs), or by overexpression of an inhibitory phosphorylation-resistant cdk1 version at the inhibitory sites threonine 14 and tyrosine 15 (cdk1-AF), we observed severe alterations in spindle assembly relatively to control cells, ranging from monopolar spindles to bipolar spindles with deranged microtubular structures and chromosome alignment [ 14 ]. To further study the alignment defects caused by i-Cdk1 loss, we analyzed in more details HeLa cells that assembled bipolar spindles upon Wee1 downregulation or cdk1-AF overexpression (Fig. 1 A, B) [ 14 ]. HeLa cells treated with non-targeting (NT) siRNAs, as control, or with siRNAs targeting Wee1 were arrested at G2, by a 16-hour treatment with the selective and reversible inhibitor RO-3306 (9 µM), released into fresh medium, containing the proteasome inhibitor MG-132 and the protein synthesis inhibitor cycloheximide, to block mitosis exit and prevent excess protein accumulation (MC medium), fixed after 80 minutes of further incubation and analyzed by immunofluorescence (IF; Fig. 1 A) [ 18 ]. Part of Wee1-siRNA-treated cells were also complemented with a siRNA-resistant Wee1 expression vector (Figs. 1 A, Wee1-siRNAs comp.) [ 16 ]. As previously shown under similar experimental conditions, the majority (around 80%) of control NT-siRNA- or Wee1-complemented Wee1-siRNA-treated cells were able to build normal bipolar spindles within 60 minutes of incubation, that remained stable for further 20 min incubation, while spindle assembly was substantially impaired in the majority of Wee1-downregulated cells [ 14 ]. Nevertheless, about 40% of the Wee1-downregulated cells could mount bipolar spindles but spindles were rather elongated and showed alignment defects with bioriented chromosome pairs that failed to align at the equator of the metaphase plate in the majority of these cells (Fig. 1 A) [ 14 ]. Similar alignment defects in bipolar spindles were also observed by overexpressing cdk1-AF, but not by wild type cdk1 (cdk1-WT), in HeLa cells (Fig. 1 B) and in the non-transformed hTERT-RPE1 cells (Supplementary Fig. 1). Importantly, mild Cdk1 inhibition in both Wee1-siRNA- and cdk1-AF-treated cells, by addition of low concentrations of RO-3306 (0.5 µM) from 60 to 80 min post G2 release, substantially reversed spindle defects compacting spindles and inducing tight chromosome alignments (Fig. 1 A, B; + RO; see also Supplementary Fig. 1) [ 14 ]. I-Cdk1 drives K-MT plus end Kif18A accumulation The alignment of bipolarly attached chromosomes at the spindle equator has been shown to require the MT-plus end-directed kinesin Kif18A that accumulates at the center of the spindle where it dampens K-MT dynamics [ 1 , 4 , 5 , 6 ]. Since the ability of Kif18A to concentrate at the K-MT plus ends and inhibit chromosome oscillations has been shown to be antagonized by direct Cdk1-dependent phosphorylation [ 13 ], we asked whether i-Cdk1 was required for K-MT plus end localization of Kif18A on bipolar spindles (Fig. 2 ). Under similar experimental conditions of the experiments described in Fig. 1 , we found that in control metaphase cells Kif18A concentrated at the center of the spindle, accumulating at the plus ends of K-MT facing centromeres (Fig. 2 A, B). Conversely, in Wee1-downregulated or cdk1-AF-overepressing cells the overall amount of Kif18A on spindles was lower than control cells and in particular it failed to concentrate at the spindle center and at the plus ends of K-MT, rather remaining around the spindle pole area (Fig. 2 A, B). Mild Cdk1 inhibition, in Wee1-downregulated or cdk1-AF-overepressing cells, restored high Kif18A concentrations at the spindle center in close centromere proximity, along with tight chromosome alignments (Fig. 2 A, B; + RO). Kif18A and Cdk1 interaction Since i-Cdk1 appears required for correct spindle assembly [ 14 ], the poor localization of Kif18A at the spindle center in i-Cdk1-lowered cells may be an indirect effect due to the overall spindle assembly impairment in those cells rather than a direct requirement for i-Cdk1 in the control of Kif18A. Nevertheless, Kif18A function has been shown to be inhibited by direct phosphorylation of Kif18A operated by Cdk1 in mitosis [ 13 ]. Kif18A has been shown to resolve in fast and slow migrating forms when analyzed from metaphase-arrested cell lysates on SDS/PAGE, suggesting different phosphorylation states [ 13 ]. In addition, the slow migrating form of Kif18 was converted into the fast migrating form upon treatment of mitotic cell lysates with calf intestinal phosphatase, in vitro , or upon treatment of mitotic cells, in vivo , with a chemical Cdk1 inhibitor, indicating that the slower migrating form was phosphorylated by Cdk1 [ 13 ]. In our SDS/PAGE we were unable to clearly detect differently migrating forms of Kif18A from total mitotic cell lysates, however, in metaphase-arrested cells the band appeared broader than that from prometaphase-arrested cells, perhaps indicating that, from prometaphase to metaphase, some Kif18A had undergone dephosphorylation (Fig. 3 A; lanes 1 and 2). Nevertheless, also under our conditions Kif18A was indeed converted into a faster migrating form after treatment of metaphase-arrested cells with a chemical Cdk1 inhibitor (Fig. 3 A; lane 3). To gain further insight into the relationship between Cdk1 and Kif18A, we first asked whether Cdk1 and Kif18A physically interacted in mitotic cells by probing cyclin B1 co-immunoprecipitations from total cell extracts and found that Kif18A indeed interacted with Cdk1 at prometaphase and at metaphase and even after treatment with the Cdk1 inhibitor (Fig. 3 B). Since i-Cdk1 is tightly bound to spindle MTs, we analyzed Kif18A distribution upon fractionating mitotic cells into insoluble pellet fraction (P), containing spindles and spindle-associated proteins, and soluble fraction (S) to further dissect the relationship between Kif18A and Cdk1 activity (Fig. 3 C) [ 17 ]. Control, Wee1-downregulated and Wee1-downregulated/Wee1-complemented cells were prometaphase-arrested by a 14-hour treament with the reversible microtubule inhibitor nocodazole, added shortly after siRNA treatments, and released into MC medium for further 60 min of incubation (Fig. 3 C). To a portion of control cells, nocodazole was added back at the beginning of the 60 min incubation to block spindle assembly and keep cells arrested at prometaphase (Fig. 3 C; Noco). The majority of control and Wee1-depleted/complemented cells built spindles during the 60 min incubation after nocodazole wash out and arrested at metaphase, on the contrary spindle assembly in Wee1-depleted cells was severely impaired, as previously shown (Fig. 3 C; top images) [ 14 ]. Cells were fractionated and Kif18A analyzed from soluble and pellet fractions (Fig. 3 C; bottom blots; pellet protein samples were enriched 8 folds relatively to soluble proteins; see Material and Methods section). In control prometaphase-arrested cells Kif18A was more abundant in the soluble rather than in the pellet fraction (Fig. 3 C; bottom blots; Noco), while in control metaphase cells substantial Kif18A amounts were recovered in the pellet fraction (Fig. 3 C; bottom blots). In Wee1-downregulated cells, Kif18A distribution between soluble and pellet fractions resembled that of prometaphase cells, while in Wee1-downregulated/complemented cells, that correctly assembled spindles, distribution was similar to control metaphase cells (Fig. 3 C; Wee1-siRNAs and Wee1-siRNAs comp.; bottom blots). As control, blots were also probed for γ-tubulin since centrosomes, that are enriched in γ-tubulin, did pelleted even from prometaphase cells and the relative γ-tubulin content in pellet and soluble fractions did not vary substantially between prometaphase and metaphase cells, as previously shown (Fig. 3 C; bottom blots) [ 14 ]. Analyzing short exposures of the Kif18A blots, it became evident that Kif18A from the pellet fractions of control and Wee1-downregulated/complemented cells resolved into two differently migrating forms of which the faster form was predominant over the slower migrating form, suggesting that Kif18A was present on spindles in a dephosphorylated form (Fig. 3 C; Kif18A SE; bottom blots). Thus, we further analyzed Kif18A/Cdk1 interaction in the soluble and pellet fractions of prometaphase- and metaphase-arrested cells, respectively (Fig. 3 D). To this end, we immunoprecipitated comparable amounts of cyclin B1 from the soluble fraction of prometaphase-arrested cells (Fig. 3 D; lane 3) and from the pellet fraction of metaphase-arrested cells (Fig. 3 D; lane 4) and found that Kif18A coprecipitating with cyclin B1 from the metaphase-arrested cell pellet fraction had a faster migration on SDS/PAGE compared to Kif18A co-precipitating with cyclin B1 from the soluble fraction of prometaphase-arrested cells, indicating its dephosphorylated state (Fig. 3 D). In addition, as previously shown, cyclin B1 immunoprecipitated from the soluble fraction of prometaphase-arrested cells was in an active Cdk1 complex, since it bound cdk1 non-phosphorylated at inhibitory Y-15-cdk1 site, and bound to inactive PP1a, phosphorylated at the inhibitory T-320, conversely, cyclin B1 from the metaphase-arrested cell pellet fraction was mostly in an i-Cdk1 complex, since it bound cdk1 substantially phosphorylated at the inhibitory Y-15 site, and with a presumably active PP1a, since it was dephosphorylated at the inhibitory T-320 (Fig. 3 D) [ 14 ]. Together, these data strongly suggest that i-Cdk1 promoted localized PP1-dependent Kif18A dephosphorylation and activation at spindle MTs. Alignment defects rescue by expression of a Cdk1-phosphorylation-resistant Kif18A mutant in i-Cdk1-downregulated cells It has been shown that the serines 674 and 684 of human Kif18A are major sites of inhibitory phosphorylation by Cdk1, thus, we asked whether expressing a Kif18A mutant version in which S674 and S684 are mutated into non-phosphorylatable alanine (S674A/S684A; Kif18A-AA) could compensate alignment defects in i-Cdk1-downregulated cells. To this end, HeLa cells were co-transfected with cdk1-AF-expression vector plus either an empty vector or a wild type Kif18A-expression vector (Kif18A-WT) or a S674A/S684A-Kif18A-expression vector (Kif18A-AA), arrested at G2, released into MC medium (as described in Fig. 1 ) and fixed after 60 minutes of further incubation (Fig. 4 A, B). Scoring chromosome alignment in cells with bipolar spindles showed that although co-expressing Kif18A-WT with cdk1-AF had already some mild effect relatively to the sole cdk1-AF expression, co-expressing Kif18A-AA with cdk1-AF had a strong effect in compensating alignment defects reducing them in more than 50% of cells (Fig. 4 A). Discussion Kif18A has been shown to promote chromosome alignment at the spindle equator by accumulating at the K-MT plus ends to confine centromere movements by suppressing MT dynamics [ 1 , 4 , 5 , 6 ]. This action has been shown to be antagonized by direct phosphorylation of Kif18A operated by Cdk1 [ 13 ]. Thus, how chromosome alignment at the spindle equator proceeds in the presence of Cdk1 activity is unclear. Here we asked whether Kif18A action is licensed in mitosis by virtue of i-Cdk1, a recently identified subpopulation of spindle-localized Cdk1 that remains inhibited in mitosis by phosphorylation and is required for spindle assembly [ 14 ]. Defective bipolar spindles, in mitotic human cells in which i-Cdk1 was lowered, were often elongated with bioriented chromosome pairs that failed to align at the equator of the metaphase plate and showed poor Kif18A accumulation at their K-MT plus ends (Figs. 1 and 2 ). These effects were reversed by mild Cdk1 inhibition, that restored i-Cdk1 as previously shown (Figs. 1 and 2 ) [ 14 ]. By cell fractionation experiments, we found that at prometaphase Kif18A was predominantly in the soluble fraction rather than in the pellet fraction, conversely, at metaphase Kif18A accumulated in the microtubular pellet fraction and in a faster migrating form on SDS/PAGE, relatively to Kif18A migration from the soluble fraction, indicative of its dephosphorylation (Fig. 3 C). Accumulation of Kif18A in the pellet fraction, in its faster migrating form, was strongly reduced in i-Cdk1-lowered cells, while both distribution and migration were reversed upon i-Cdk1 restoration (Fig. 3 C). In addition, we found that in the pellet fraction, the faster migrating form of Kif18A interacted with i-Cdk1 that was in complex with the presumably active form of PP1a, dephosphorylated at the inhibitory T320 (Fig. 3 D). Moreover, expression of a cdk1-dependent phosphorylation-resistant Kif18A mutant partly compensated chromosome alignment defects in i-Cdk1-lowered cells (Fig. 4 ). Together, our data suggest a scenario in which the distribution of i-Cdk1 along spindle MTs allows Kif18A to be locally dephosphorylated by PP1 in order to regain its ability to concentrate at K-MT plus ends, dampen MT dynamics, and promote chromosome alignment. Although loss of Kif18A function appears to be tolerated by euploid cells, it does appear to be essential for survival of some aneuploid cancers, revealing a strong antitumor therapeutic potential [ 2 , 3 , 8 – 12 ]. In addition, it has been shown that the sensitivity of aneuploid cancer cells to chemical Kif18A inhibition correlates with their ability to delay mitosis exit by implementing an efficient SAC response [ 12 , 19 , 20 ]. Since Cdk1 activity is a major SAC-activating kinase and inhibiting Wee1 prolongs mitosis in a SAC-dependent manner, it is possible to hypothesize that the combination of Wee1 and Kif18A inhibition will provide a means to potentiate the therapeutic efficacy towards aneuploid cancers [ 16 , 21 ]. Declarations Funding This work was supported by AIRC, Associazione Italiana per la Ricerca sul Cancro: IG grant 2017; Id. 19851 to D.G. Competing Interests The authors declare no competing financial interests. Author Contributions A.F.S. and D.G. designed and performed experiments and analyzed data. C.P. and A.T. performed immunoblot and immunofluorescence experiments and analyzed data. D. G. wrote the manuscript. A.F.S. revised the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated are available from the corresponding author on reasonable request. Ethics approval Not applicable Consent to participate Not applicable Consent to publish Not applicable Acknowledgments The authors thank AIRC, Associazione Italiana per la Ricerca sul Cancro for support (IG grant 2017; Id. 19851 to D.G.) References Risteski P, Jagrić M, Pavin N, Tolić IM (2021) Biomechanics of chromosome alignment at the spindle midplane. Curr Biol 31(10):R574–R585. 10.1016/j.cub.2021.03.082 Gomes AM, Orr B, Novais-Cruz M, De Sousa F, Macário-Monteiro J, Lemos C, Ferrás C, Maiato H (2022) Micronuclei from misaligned chromosomes that satisfy the spindle assembly checkpoint in cancer cells. Curr Biol 32:4240–4254. 10.1016/j.cub.2022.08.026 Sepaniac LA, Martin W, Dionne LA, Stearns TM, Reinholdt LG, Stumpff J (2021) Micronuclei in Kif18a mutant mice form stable micronuclear envelopes and do not promote tumorigenesis. J Cell Biol 220:e202101165. 10.1083/jcb.202101165 Mayr MI, Hümmer S, Bormann J, Grüner T, Adio S, Woehlke G, Mayer TU (2007) The human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression. Curr Biol 17:488–498. 10.1016/j.cub.2007.02.036 Stumpff J, von Dassow G, Wagenbach M, Asbury C, Wordeman L (2008) The kinesin-8 motor Kif18A suppresses kinetochore movements to control mitotic chromosome alignment. Dev Cell 14(2):252–262. 10.1016/j.devcel.2007.11.014 Du Y, English CA, Ohi R (2010) The kinesin-8 Kif18A dampens microtubule plus-end dynamics. Curr Biol 20(4):374–380. 10.1016/j.cub.2009.12.049 Janssen LME, Averink TV, Blomen VA, Brummelkamp TR, Medema RH, Raaijmakers JA (2018) Loss of Kif18A results in spindle assembly checkpoint activation at microtubule-attached kinetochores. Curr Biol 28(17):2685–2696e4. 10.1016/j.cub.2018.06.026 Quinton RJ, DiDomizio A, Vittoria MA, Kotýnková K, Ticas CJ, Patel S, Koga Y, Vakhshoorzadeh J, Hermance N, Kuroda TS, Parulekar N, Taylor AM, Manning AL, Campbell JD, Ganem NJ (2021) Whole-genome doubling confers unique genetic vulnerabilities on tumour cells. Nature 590(7846):492–497. 10.1038/s41586-020-03133-3 Cohen-Sharir Y, McFarland JM, Abdusamad M, Marquis C, Bernhard SV, Kazachkova M, Tang H, Ippolito MR, Laue K, Zerbib J, Malaby HLH, Jones A, Stautmeister LM, Bockaj I, Wardenaar R, Lyons N, Nagaraja A, Bass AJ, Spierings DCJ, Foijer F, Beroukhim R, Santaguida S, Golub TR, Stumpff J, Storchová Z, Ben-David U (2021) Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition. Nature 590(7846):486–491. 10.1038/s41586-020-03114-6 Marquis C, Fonseca CL, Queen KA, Wood L, Vandal SE, Malaby HLH, Clayton JE, Stumpff J (2021) Chromosomally unstable tumor cells specifically require KIF18A for proliferation. Nat Commun 12(1):1213. 10.1038/s41467-021-21447-2 Payton M, Belmontes B, Hanestad K, Moriguchi J, Chen K, McCarter JD, Chung G, Ninniri MS, Sun J, Manoukian R, Chambers S, Ho SM, Kurzeja RJM, Edson KZ, Dahal UP, Wu T, Wannberg S, Beltran PJ, Canon J, Boghossian AS, Rees MG, Ronan MM, Roth JA, Minocherhomji S, Bourbeau MP, Allen JR, Coxon A, Tamayo NA, Hughes PE (2024) Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers. Nat Cancer 5(1):66–84. 10.1038/s43018-023-00699-5 Gliech CR, Yeow ZY, Tapias-Gomez D, Yang Y, Huang Z, Tijhuis AE, Spierings DC, Foijer F, Chung G, Tamayo N, Bahrami-Nejad Z, Collins P, Nguyen TT, Plata Stapper A, Hughes PE, Payton M, Holland AJ (2024) Weakened APC/C activity at mitotic exit drives cancer vulnerability to KIF18A inhibition. EMBO J 43(5):666–694. 10.1038/s44318-024-00031-6 Häfner J, Mayr MI, Möckel MM, Mayer TU (2014) Pre-anaphase chromosome oscillations are regulated by the antagonistic activities of Cdk1 and PP1 on Kif18A. Nat Commun 5:4397. 10.1038/ncomms5397 Serpico AF, Febbraro F, Pisauro C, Grieco D (2022) Compartmentalized control of Cdk1 drives mitotic spindle assembly. Cell Rep 38(4):110305. 10.1016/j.celrep.2022.110305 Serpico AF, Pisauro C, Grieco D (2023) On the assembly of the mitotic spindle, bistability and hysteresis. Cell Mol Life Sci 80(4):83. 10.1007/s00018-023-04727-6 Visconti R, Della Monica R, Palazzo L, D'Alessio F, Raia M, Improta S, Villa MR, Del Vecchio L, Grieco D (2015) The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs. Cell Death Differ 22(9):1551–1560. 10.1038/cdd.2015.13 Silljé HH, Nigg EA (2006) Purification of mitotic spindles from cultured human cells. Methods 38(1):25–28. 10.1016/j.ymeth.2005.07.006 Vassilev LT (2006) Cell cycle synchronization at the G2/M phase border by reversible inhibition of CDK1. Cell Cycle 5(22):2555–2556. 10.4161/cc.5.22.3463 Varetti G, Guida C, Santaguida S, Chiroli E, Musacchio A (2011) Homeostatic control of mitotic arrest. Mol Cell 44(5):710–720. 10.1016/j.molcel.2011.11.014 Serpico AF, Grieco D (2020) Recent advances in understanding the role of Cdk1 in the Spindle Assembly Checkpoint F1000Res 9:F1000 Faculty Rev-57. 10.12688/f1000research.21185.1 Toledo CM, Ding Y, Hoellerbauer P, Davis RJ, Basom R, Girard EJ, Lee E, Corrin P, Hart T, Bolouri H, Davison J, Zhang Q, Hardcastle J, Aronow BJ, Plaisier CL, Baliga NS, Moffat J, Lin Q, Li XN, Nam DH, Lee J, Pollard SM, Zhu J, Delrow JJ, Clurman BE, Olson JM, Paddison PJ (2015) Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells. Cell Rep 13(11):2425–2439. 10.1016/j.celrep.2015.11.021 Additional Declarations No competing interests reported. Supplementary Files Serpicoetal.SupplementaryFigures.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4594196","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":315478645,"identity":"f0eb45b2-afb8-4c83-9a8e-3d958203e86c","order_by":0,"name":"Angela Flavia Serpico","email":"","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":false,"prefix":"","firstName":"Angela","middleName":"Flavia","lastName":"Serpico","suffix":""},{"id":315478646,"identity":"d111bbfe-8d2d-43ba-8fc2-90dae86ec29f","order_by":1,"name":"Caterina Pisauro","email":"","orcid":"","institution":"CEINGE Biotecnologie Avanzate “Franco Salvatore”","correspondingAuthor":false,"prefix":"","firstName":"Caterina","middleName":"","lastName":"Pisauro","suffix":""},{"id":315478647,"identity":"1d8d7671-19cc-4306-bb2a-7b7a2db749a3","order_by":2,"name":"Asia Trano","email":"","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":false,"prefix":"","firstName":"Asia","middleName":"","lastName":"Trano","suffix":""},{"id":315478651,"identity":"46d1d7db-0ab7-4e22-b25f-0f369afec53a","order_by":3,"name":"Domenico Grieco","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDCCAyDCAMGWY2BgbADSEsRrMYZpwa3nABo/sQHKwKmF73h34uOCAjs5Bv61Dw98qLFJ33C7ue3BDwaLOlxaJM+c3Ww8wyDZmEHiucHBGcfScjfcOdhu2IPHYQY3crdJ8xgcSGyQOMZwmLfhcO6GG4ltEjz4tWz/Ddfyt+FwugFQi+QfArYwg7XwtzEcZmw4nADSIo3PFpBfgA5LNmaTYGM42HMszXAmSIuMgYRkAw4tfMd7N37m+WMnx89/jPnDjxobeb4b6c8k31TU8eOyBQ7YJBJQHExQAxDwHyBG1SgYBaNgFIxEAAA6lVdm+tcLxQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":true,"prefix":"","firstName":"Domenico","middleName":"","lastName":"Grieco","suffix":""}],"badges":[],"createdAt":"2024-06-17 12:41:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4594196/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4594196/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59549634,"identity":"e8151713-be63-4ca2-9e1b-790a60f57afb","added_by":"auto","created_at":"2024-07-03 06:05:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1957797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDependence of chromosome alignment on i-Cdk1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) HeLa cells were treated with: non-targeting siRNAs (Control), Wee1-targeting siRNAs (Wee1-siRNAs) and Wee1-targeting siRNAs in cells previously transfected with siRNA-resistant Flag-tagged-Wee1 expression vector (Wee1-siRNA comp.). Cells were arrested at G2, by a 16-hour treatment with high concentrations of the selective and reversible inhibitor RO-3306 (9 mM; added 6 hours post siRNA-treatments), released into fresh MC medium (see Material and Methods section), fixed after 80 minutes of further incubation and analyzed by immunofluorescence. To a sample of Wee1-siRNAs, low concentrations of RO-3306 (0.5 mM) were added by 60 min from G2-arrest release (Wee1-siRNAs + RO). Immunofluorescence images: cells were stained for CREST (centromere marker, green), a-tubulin (a-TUB, red) and DNA (blue). Addition of vehicle (DMSO) had no effect on spindle assembly in Wee1-siRNA-treated cells. Graph: percent of bipolar spindles with misaligned chromosomes (more than three chromosomes falling outside the two internal quarters of the interpolar distance) in Control, Wee1-siRNAs, Wee1-siRNA comp. and Wee1-siRNAs + RO cells. Around 100 cells were scored in 4 independent microscopy slide fields, error bars refer to variability within three independent experiments performed under similar experimental conditions. Scale bar: 10 mm. Blots: cell samples from Control (non-targeting; Cont.), Wee1-siRNAs, Wee1-siRNA comp. were lysed and lysates probed for the indicated antigens. (\u003cstrong\u003eB\u003c/strong\u003e) HeLa cells were transfected with an empty vector (Control), with a wild type cdk1 (Cdk1-WT) expression vector or with a mutant cdk1 expression vector in which cdk1-threonine 14 and tyrosine 15 are mutated into non-phosphorylatable alanine and phenylalanine, respectively (Cdk1-AF). 6 hours post transfection, cells were arrested at G2 by addition of RO-3306 (9 mM) for further 16 hours of incubation, released into fresh MC medium (see Material and Methods section), fixed after 80 minutes of further incubation and analyzed by immunofluorescence. Cells were stained for DNA (blue), a-tubulin (a-TUB, red) and CREST (green) at 80 min upon release from G2 arrest. A portion of cdk1-AF-transfected cells received low-RO at 60 min post release from G2-arrest (Cdk1-AF + RO); as control vehicle was added (DMSO). Graph: percent of bipolar spindles with misaligned chromosomes (more than three chromosomes falling outside the two internal quarters of the interpolar distance) in Control, Cdk1-WT, Cdk1-AF, and Cdk1-AF + RO cells. Around 100 cells were scored in 4 independent microscopy slide fields, error bars refer to variability within three independent experiments performed under similar experimental conditions. Scale bar: 10 mm. Blots: cell samples from Control, Cdk1-WT and Cdk1-AF were lysed and lysates probed for the indicated antigens.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4594196/v1/5cb8060018fc27dfaa3ea0a6.png"},{"id":59549635,"identity":"cb703160-e6cc-4820-a59f-a30ed67df928","added_by":"auto","created_at":"2024-07-03 06:05:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2675602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKif18A accumulation at K-MT plus ends and i-Cdk1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) HeLa cells were treated as described in Figure 1A and analyzed by immunofluorescence by 80 minutes of further incubation after release from G2-arrest into fresh MC medium. Kif18A (red), CREST (green), and DNA (blue). Scale bar: 10 mm. (\u003cstrong\u003eB\u003c/strong\u003e) HeLa cells were treated as described in Figure 1B and analyzed by immunofluorescence by 80 minutes of further incubation after release from G2-arrest into fresh MC medium. Kif18A (red), CREST (green), and DNA (blue). Scale bar: 10 mm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4594196/v1/18a581b82cf6457a0fd41c66.png"},{"id":59550441,"identity":"bd45f37d-ebbf-4ba9-a362-0d12936ea70a","added_by":"auto","created_at":"2024-07-03 06:13:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1041140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKif18A and Cdk1 interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells werearrested at prometaphase by 14 hour treatment with nocodazole, released into MC medium and either nocodazole, DMSO or RO3306 (9 mM) was added and incubation prolonged for further 60 min. (\u003cstrong\u003eA\u003c/strong\u003e) \u0026nbsp;Total cell lysate blots were directly probed for the indicated antigen (lane 1, nocodazole; lane 2, DMSO; lane 3, RO3306) (\u003cstrong\u003eB\u003c/strong\u003e) Blots of cyclin B1 immunoprecipitates (Ips) from cell lysates were probed for indicated antigens (lane 1: total cell lysate from nocodazole-treated sample; lane 2: mock Ip from nocodazole-treated sample, lane 3, 4 and 5: cyclin B1 Ips from nocodazole, DMSO and RO3306 treated samples, respectively). (\u003cstrong\u003eC\u003c/strong\u003e) HeLa cells were treated with non-targeting siRNAs (Control), Wee1-targeting siRNAs (Wee1-siRNAs) and Wee1-targeting siRNAs to previously transfected with siRNA-resistant Flag-tagged-Wee1 expression vector (Wee1-siRNAs comp.), arrested at prometaphase by 14 hour treatment with nocodazole and released into MC medium for 60 min. To a Control sample, nocodazole was added back to keep cells arrested at prometaphase (Noco). Immunofluorescence images: cells were fixed and stained for CREST (green), a-tubulin (a-TUB, red) and DNA (blue). Scale bar: 10 mm. Blots: Cells were fractionated into soluble (S) and pellet (P) fractions that were probed for indicated antigens (for Kif18A short, SE, and long, LE, blot exposures are shown). (\u003cstrong\u003eD\u003c/strong\u003e) Cyclin B1 Ips from S fraction of Control-Noco-cells (arrested at prometaphase) and from P fraction of Control cells (arrested at metaphase) were probed for indicated antigens (lane 1: total S fraction of Noco cells; lane 2: mock Ip from S fraction of Noco cells, lane 3, and 4: cyclin B1 Ips from S fraction of Control-Noco-cells and from P fraction of Control cells, respectively).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4594196/v1/e11e4e777db1fcd96f10e6aa.png"},{"id":59549632,"identity":"58ee6a37-055c-42fc-b2f1-344efafbc2e7","added_by":"auto","created_at":"2024-07-03 06:05:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":544679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCdk1-phosphorylation-resistant Kif18A mutant expression improves alignment in i-Cdk1-downregulated cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells were co-transfected with a cdk1-AF expression vector plus an empty vector (Cdk1-AF), with a cdk1-AF expression vector plus a wild type Kif18A-expression vector (Cdk1-AF + Kif18A-WT) or with a cdk1-AF expression vector plus a mutant S674A/S684A-Kif18A-expression vector (Cdk1-AF + Kif18A-AA), arrested at G2, released into MC medium and fixed after 60 minutes of further incubation and stained for CREST (green), a-tubulin (a-TUB, red) and DNA (blue). Graph: percent of bipolar spindles with misaligned chromosomes (more than three chromosomes falling outside the two internal quarters of the interpolar distance). Around 100 cells were scored in 4 independent microscopy slide fields, error bars refer to variability within three independent experiments performed under similar experimental conditions. Scale bar: 10 mm. Graph: percent of bipolar spindles with misaligned chromosomes. Blots: Lysates of HeLa cells co-transfected with a cdk1-AF expression vector plus an empty vector (Mock) or plus Kif18A-WT or Kif18A-AA expression vectors were probed for the indicated antigens.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4594196/v1/1bca74bfd817744297d28254.png"},{"id":63674394,"identity":"70bf979e-77ad-4c6a-913d-d0b60f6a0cfe","added_by":"auto","created_at":"2024-08-31 03:01:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7762343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594196/v1/b5ecdd53-7e59-4c5c-b9b8-df7b71ed27c2.pdf"},{"id":59549636,"identity":"4f12eba3-2931-4249-8c85-b5f0f49c1f6f","added_by":"auto","created_at":"2024-07-03 06:05:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":419032,"visible":true,"origin":"","legend":"","description":"","filename":"Serpicoetal.SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594196/v1/170243b84e85005513db024d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chromosome alignment relies on spindle-localized control of Cdk1 activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDuring mitosis, proper chromosome alignment at the equator of the mitotic spindle at metaphase grants synchronous chromosome segregation in anaphase, helping to prevent mis-segregation and formation of micronuclei into daughter cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During spindle assembly, chromosomes congress towards the spindle equator in an oscillating fashion, promoted by the action of kinesins and the control of MT dynamics. To reach the metaphase configuration, chromosomes are pushed away from centrosomes by polar ejection forces, mainly acting on chromosome arms, and pulled towards centrosomes by kinetochore-dependent forces, until the final alignment at the center of the spindle [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This oscillatory behavior is also supposed to facilitate correct chromosome attachments, while the final alignment at the spindle equator is reached by the progressive decline of these oscillatory movements. A central action to dampen chromosome oscillations and promote alignment is exerted by localization of the plus end-directed kinesin 8 family member Kif18A at the plus ends of K-MTs where it suppresses MT dynamics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Kif18A concentration at K-MT plus ends has also been involved in the tension-dependent mechanisms that silence the Spindle Assembly Checkpoint (SAC), the safeguard mechanism that prevents anaphase onset until correct spindle assembly completion [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although loss of Kif18A function causes micronuclei formation, it appears to be rather tolerated by euploid cells and inactivating mutations in Kif18A have been shown to produce micronuclei in mice without leading to cellular transformation or cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Nevertheless, Kif18A does appear to be essential for survival of aneuploid cancer cells, perhaps by preventing unbearable chromosome mis-segregation in cells with more complicated spindle architecture, and appears a vulnerability target for certain aneuploid cancers [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The ability of Kif18A to reach the K-MT plus ends and dampen chromosome oscillations has been shown to be antagonized by phosphorylation operated by Cdk1 and stimulated by its reversal operated by PP1 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. How Kif18A evicts inhibition by Cdk1 during spindle assembly is unknown. We show here that the ability of Kif18A to reach K-MT plus ends and promote chromosome alignment depends on i-Cdk1, a spindle-localized fraction of Cdk1 that we have recently described to be inhibited by phosphorylation in mitosis and to be crucial for spindle assembly [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eHuman Henrietta Lacks (HeLa; CEINGE Cell Culture Facility) cells were grown in Dulbecco\u0026rsquo;s Modified Eagle Medium high glucose (DMEM; Sigma-Aldrich) supplemented with 10% foetal bovine serum (FBS; Gibco), 1% GlutaMAX-supplement (Gibco), 1% penicillin/streptomycin (Euroclone). Human Telomerase Reverse Transcriptase-immortalized Retinal Pigment Epithelial (hTERT-RPE1; Dr. A. Musacchio\u0026rsquo;s gift; Max Planck Institute of Molecular Physiology, Dortmund) cells were grown in Dulbecco\u0026rsquo;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; Gibco, Thermo Fisher Scientific) supplemented with 10% foetal bovine serum (FBS; Gibco), 1% GlutaMAX-supplement (Gibco), 1% penicillin/streptomycin (Euroclone). All cells were incubated in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA and siRNA constructs\u003c/h2\u003e \u003cp\u003eCdk1-WT and cdk1-AF mammalian expression plasmids were purchased from Addgene (Cat# 61840; Cat# 39872, respectively). 3XFlag-Wee1 expression construct was obtained as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Myc-Flag-tagged-human-Kif18A-WT (Kif18A-WT) plasmid was purchased from Origene (Cat# RC208235); Kif18A-AA mutant version was generated via mutagenesis: serine 674 was mutagenized into alanine (2020\u0026ndash;2022 nt TCT\u0026loz;GCT) and serine 684 into alanine (2050\u0026ndash;2052 nt TCT\u0026loz;GCT) by QuikChange II XL site-directed mutagenesis kit (Agilent Technologies) using Kif18A-WT as template. Custom siRNAs targeting the 3\u0026rsquo;-UTR of human WEE1 (#1: 5\u0026rsquo;-CUGUAAACUUGUAGCAUUAUU-3\u0026rsquo;; #2: 5\u0026rsquo;-GUACAUAGCUGUUUGAAAUUU-3\u0026rsquo;; #3: 5\u0026rsquo;-GGGCUUUAUUACAGACAUAUU-3\u0026rsquo;) were designed using siDESIGN Center tool by Horizon and purchased from Dharmacon.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTransfection and RNA interference\u003c/h2\u003e \u003cp\u003eCells were seeded at a cell density of 7000/cm\u003csup\u003e2\u003c/sup\u003e either into 6\u0026ndash;10 cm dishes or onto glass coverslips for biochemical and immunofluorescence studies, respectively. Manufacturer\u0026rsquo;s protocols were followed for both procedures and cells were plated 24 hours prior to treatment. Transient expression transfections were performed using MegaTran 2.0 Transfection Reagent (OriGene; Cat# TT210003); protein downregulation via siRNAs was obtained using DharmaFECT 1 siRNA Transfection Reagent (Dharmacon; Cat# T-2001-03). Briefly, MegaTran (1 \u0026micro;g/\u0026micro;L): DNA (1 \u0026micro;g/\u0026micro;L) (3:1) or DharmaFECT 1: siRNAs duplex (25 nM) (1:1) were mixed in cell culture media (DMEM or DMEM/F12) and incubated at room temperature (rt) for 20 minutes. Then the mixtures were added to the cells maintained in antibiotic-free complete medium and incubated for 24 hours (see also \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003ecell synchronization\u003c/span\u003e section). For Wee1-siRNA treatment and complementation, cells were transfected with mock- or 3XFlag-Wee1 expression construct 24 hours prior to treatment with non-targeting or specific siRNAs and 6 hours after siRNA-treatment cells were ad hoc synchronized (see also \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003ecell synchronization\u003c/span\u003e section). Exogenous cdk1-WT or cdk1-AF overexpression was achieved by transfection of the respective plasmids (empty or cdk1-WT or cdk1-AF expression vectors) 8 hours prior to specific synchronization (see also \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003ecell synchronization\u003c/span\u003e section). For co-overexpression experiments, cdk1-AF\u0026thinsp;+\u0026thinsp;empty vector, cdk1-AF\u0026thinsp;+\u0026thinsp;Kif18A-WT and cdk1-AF\u0026thinsp;+\u0026thinsp;Kif18A-AA vectors were simultaneously transfected 8 hours prior to synchronization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell synchronization\u003c/h2\u003e \u003cp\u003ePrometaphase-arrested HeLa cells were obtained by a 14-hour incubation with nocodazole (1 \u0026micro;g/ml; Abcam; Cat# ab120630). G2-arrested cells were obtained by incubation with RO-3306 at 9 \u0026micro;M (Calbiochem; Cat# 217699) for 16 hours. To obtain metaphase-arrested cells, G2- or prometaphase-arrested cells were washed twice with fresh medium and twice with phosphate buffer saline 1X (PBS; Corning; Cat# 21-031-CV) solution before plating into fresh medium containing MG-132 (20 \u0026micro;M; Calbiochem; Cat# 474790) and cycloheximide (CHX; 60 \u0026micro;g/mL; Santa Cruz Biotechnology; Cat# sc-3508) for further 60 minutes of incubation. Where indicated, mild inhibition of Cdk1 activity (+\u0026thinsp;RO) was obtained by adding RO-3306 at 0.5 \u0026micro;M (Calbiochem).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell fractionation\u003c/h2\u003e \u003cp\u003eCell fractionation was performed as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To separate cytoplasmic and spindle-bound proteins we adjusted a previously described method [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Cells were collected by centrifugation and washed once with PBS containing taxol (1\u0026micro;M; Sigma-Aldrich). 5 x 10\u003csup\u003e6\u003c/sup\u003e cells were carefully resuspended in 800 \u0026micro;l of fractionation lysis buffer (FLB; 40 mM b-glycerophosphate, 15 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 mM EGTA, 0.25% Igepal, 20 mM Hepes, 5 \u0026micro;M taxol, 6 \u0026micro;g/mL latrunculin B; Sigma-Aldrich) completed with 300 \u0026micro;g/mL RNase A and 120 U/mL DNase I (Roche) and then incubated in thermomixer (Eppendorf thermomixer comfort) with constant shaking at 1.200 rpm for 20 min at 34\u0026deg; C. Lysates were centrifuged at 6.800 rpm (Eppendorf centrifuge 5425) for 2 minutes: the supernatants were harvested into new tubes, supplemented with 250 mM NaCl and stored on ice (soluble fractions). The pellet fractions were once again resuspended in FLB\u0026thinsp;+\u0026thinsp;300 \u0026micro;g/mL RNase A and 120 U/mL DNase I and incubated in thermomixer at 34\u0026deg;C for 20 minutes. Samples were centrifuged and pellet fraction were washed twice with washing buffer (WB; 5 mM Hepes, 5 \u0026micro;M taxol; Sigma-Aldrich). Then, pellet fractions were resuspended in 200 \u0026micro;L of FLB minus taxol and supplemented with 250 mM NaCl and incubated on ice for 30 min. Finally, soluble and pellet fractions were spun at 13.200 rpm at 4\u0026deg;C for 10 minutes (Eppendorf centrifuge 5424 R) and both final supernatants were collected and processed for Immunoblottings (Ibs) or Immunoprecipitations (Ips). The pellet fraction proteins were extracted in a 200 \u0026micro;L buffer volume from a total cell lysate of 800 \u0026micro;l volume (mainly representing the soluble fraction volume), hence pellet fraction proteins were enriched 4 folds over the soluble fraction proteins, relatively to their cellular distribution and, in the experiment shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e, a double volume of the pellet relatively to the soluble fractions was loaded on gels, thus, pellet fraction proteins were enriched a total of 8 folds over the soluble fraction proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation\u003c/h2\u003e \u003cp\u003eFor immunoprecipitations from total cell lysates 5x 10\u003csup\u003e6\u003c/sup\u003e cells were lysed in 180 \u0026micro;l lysis buffer (LB; 80 mM b-glycerophosphate, 15 mM MgCl2, 20 mM EGTA, 20 mM Hepes pH 7.4, 100 mM NaCl, 0.1% Igepal; Sigma-Aldrich) supplemented with a phosphatase inhibitor cocktail (PhosSTOP; Roche) and incubated on ice for 30 min. Then, lysates were cleared via centrifugation at 13.200 rpm at 4\u0026deg;C for 10 min (Eppendorf centrifuge 5424 R) and incubated with agarose bead-conjugated antibody overnight at 4\u0026deg;C in constant rotation (mouse anti-cyclin B1 agarose, Santa Cruz Biotechnology, Cat# sc-245AC; normal mouse IgG-AC, Santa Cruz Biotechnology, Cat# sc-2343). For immunoprecipitations from soluble and pellet fractions, lysates were diluted in LB supplemented with PhosSTOP (Roche) until a final volume of 800 \u0026micro;l and incubated with agarose conjugated antibodies overnight at 4\u0026deg;C in constant rotation. For both above-mentioned cases, beads were washed twice in LB and proteins eluted in Laemmli denaturing buffer by boiling for 5 minutes (Laemmli sample buffer; BioRad, Cat# 1610747). Finally, samples were loaded into polyacrylamide gels and analyzed by immunoblotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eImmunoblotting was performed as described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Briefly, samples in SDS Laemmli buffer were incubated for 10 min at 99\u0026deg;C and then loaded and run on SDS-PAGE gels (polyacrylamide percentage spanning from 10 to 12%). Proteins were transferred onto nitrocellulose membrane (Cytiva-Amersham; Cat# GEH10600002) using a wet-transfer system (Thermofisher) and membranes were blocked with 5% nonfat dry milk (NFDM; AppliChem; Cat# A0830) in PBS supplemented with 0.01% Tween20 (TPBS; Sigma-Aldrich; Cat# P1379) for 1 hour at rt. Then, filters were incubated with primary antibodies, diluted in TPBS, at 4\u0026deg;C overnight. After 2 TPBS washes, membranes were incubated with secondary peroxidase-conjugated (HRP) antibodies, also diluted in TPBS, for 1 hour at rt. Enhanced ChemiLuminescence (ECL) kit (Cytiva-Amersham; Cat# RPN2106) enabled the detection of HRP enzyme activity. Blots were acquired using Canon CanoScan LiDE 300 scanner (Canon) and scanned at 300 dpi. For western blot analysis, primary antibodies were used as follows: rabbit anti-Wee1 (1:1000; Cell Signaling Technology; Cat# 13084); rabbit anti-cyclin B1 (1:2000; Bethyl; Cat# A305-000A); mouse anti-cdc2 (1:500; BD Biosciences; Cat# 610038); rabbit anti-Kif18A (1:1000; Bethyl; Cat# A301-080A); mouse anti-g-tubulin (1:2000; Sigma-Aldrich; Clone GTU-88; Cat# T5326), rabbit anti-phospho-tyrosine-15-cdc2 (1:1000; p-Y15-cdc2; Cell Signaling Technology, Cat# 4539), rabbit anti-phospho-Threonine-320-PP1a (1:1000; p-T320-PP1a; 1:1000; Cell Signaling Technology; Cat# 2581), mouse anti-PP1a (1:1000; Santa Cruz Biotechnology, Cat# sc-7482). Sheep anti-mouse IgG HRP linked (1:2000; GE Healthcare; Cat# NA931), donkey anti-rabbit IgG HRP linked (1:2000; GE Healthcare; Cat# NA934) were used as secondary antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence and microscopy\u003c/h2\u003e \u003cp\u003eCells were plated onto poly-D-lysine (0,1 mg/mL; Sigma-Aldrich; Cat# P6407) coated glass coverslips at a cell density of 7000/cm\u003csup\u003e2\u003c/sup\u003e. After a brief wash in PBS, cells were fixed and permeabilized with 4% paraformaldehyde\u0026thinsp;+\u0026thinsp;0,5% Triton X-100 (Sigma-Aldrich; Cat# P6148; T9284 respectively) in PBS for 12 min at rt. Then, cells were washed twice with PBS and incubated with 1.5% bovine serum albumin (BSA; Sigma-Aldrich; Cat# A7030) in PBS for 1 hour at rt. After 2 PBS washes, cells were incubated with primary antibodies in 1.5% (w/v) BSA-PBS solution for 3 hours into a humidity chamber at rt. Afterwards, cells were washed 3 times with PBS and incubated with fluorescently labelled secondary antibodies solution (1.5% BSA-PBS) for 1 hour at rt. DNA was stained with Hoechst 33258 (1 \u0026micro;g/ml; Invitrogen; Cat# 94403) by incubation for 10 minutes. Finally, cells were washed 4 times with PBS and slides mounted with Mowiol 40\u0026ndash;88 (Sigma Aldrich; Cat# 81381). For immunofluorescence staining, the following antibodies were used: mouse anti-a-tubulin (1:1000; Sigma-Aldrich; Clone DM1A; Cat# T9026); human anti-centromere (CREST; 1:100; Antibodies Incorporated; Cat# 15\u0026ndash;234); rabbit anti-Kif18A (1:2000; Abcam; Cat# ab251863); donkey anti-mouse IgG Alexa Fluor 594 (1:1000; Invitrogen; Cat# A21203); goat anti-human IgG Alexa Fluor 488 (1:1000; Invitrogen; Cat# A11013); donkey anti-rabbit IgG Alexa Fluor 594 (1:1000; Invitrogen; Cat# A21207). Inverted confocal fluorescence microscope LSM 980 (Zeiss) equipped with a 63X/1.4 oil objective (Zeiss) was used to image fixed cells. Three image planes with 0.44 mm Z-stack size were acquired and projected into one plane using ZEN3.1 software (Zeiss).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDependence of chromosome alignment on inhibitory control of Cdk1\u003c/h2\u003e \u003cp\u003eWe have recently gathered evidence that mitotic spindle assembly in human cells relies on the inhibitory control of a small and localized fraction of Cdk1, i-Cdk1, that represents less than 10% of total Cdk1 present in mitotic cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While bulk, active, Cdk1 maintains the cytoplasm of mitotic cells substantially free of MTs, i-Cdk1 appears required at spindle structures to locally reactivate MT-stabilizing MT-Associated Proteins (MAPs) to promote spindle assembly [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In experiments in which i-Cdk1 was lowered in mitotic cells either by downregulation of Cdk1 inhibitory kinase Wee1, by small interfering RNAs (siRNAs), or by overexpression of an inhibitory phosphorylation-resistant cdk1 version at the inhibitory sites threonine 14 and tyrosine 15 (cdk1-AF), we observed severe alterations in spindle assembly relatively to control cells, ranging from monopolar spindles to bipolar spindles with deranged microtubular structures and chromosome alignment [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To further study the alignment defects caused by i-Cdk1 loss, we analyzed in more details HeLa cells that assembled bipolar spindles upon Wee1 downregulation or cdk1-AF overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. HeLa cells treated with non-targeting (NT) siRNAs, as control, or with siRNAs targeting Wee1 were arrested at G2, by a 16-hour treatment with the selective and reversible inhibitor RO-3306 (9 \u0026micro;M), released into fresh medium, containing the proteasome inhibitor MG-132 and the protein synthesis inhibitor cycloheximide, to block mitosis exit and prevent excess protein accumulation (MC medium), fixed after 80 minutes of further incubation and analyzed by immunofluorescence (IF; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Part of Wee1-siRNA-treated cells were also complemented with a siRNA-resistant Wee1 expression vector (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Wee1-siRNAs comp.) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As previously shown under similar experimental conditions, the majority (around 80%) of control NT-siRNA- or Wee1-complemented Wee1-siRNA-treated cells were able to build normal bipolar spindles within 60 minutes of incubation, that remained stable for further 20 min incubation, while spindle assembly was substantially impaired in the majority of Wee1-downregulated cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nevertheless, about 40% of the Wee1-downregulated cells could mount bipolar spindles but spindles were rather elongated and showed alignment defects with bioriented chromosome pairs that failed to align at the equator of the metaphase plate in the majority of these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similar alignment defects in bipolar spindles were also observed by overexpressing cdk1-AF, but not by wild type cdk1 (cdk1-WT), in HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and in the non-transformed hTERT-RPE1 cells (Supplementary Fig.\u0026nbsp;1). Importantly, mild Cdk1 inhibition in both Wee1-siRNA- and cdk1-AF-treated cells, by addition of low concentrations of RO-3306 (0.5 \u0026micro;M) from 60 to 80 min post G2 release, substantially reversed spindle defects compacting spindles and inducing tight chromosome alignments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B; + RO; see also Supplementary Fig.\u0026nbsp;1) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eI-Cdk1 drives K-MT plus end Kif18A accumulation\u003c/h2\u003e \u003cp\u003eThe alignment of bipolarly attached chromosomes at the spindle equator has been shown to require the MT-plus end-directed kinesin Kif18A that accumulates at the center of the spindle where it dampens K-MT dynamics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Since the ability of Kif18A to concentrate at the K-MT plus ends and inhibit chromosome oscillations has been shown to be antagonized by direct Cdk1-dependent phosphorylation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], we asked whether i-Cdk1 was required for K-MT plus end localization of Kif18A on bipolar spindles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Under similar experimental conditions of the experiments described in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e, we found that in control metaphase cells Kif18A concentrated at the center of the spindle, accumulating at the plus ends of K-MT facing centromeres (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Conversely, in Wee1-downregulated or cdk1-AF-overepressing cells the overall amount of Kif18A on spindles was lower than control cells and in particular it failed to concentrate at the spindle center and at the plus ends of K-MT, rather remaining around the spindle pole area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Mild Cdk1 inhibition, in Wee1-downregulated or cdk1-AF-overepressing cells, restored high Kif18A concentrations at the spindle center in close centromere proximity, along with tight chromosome alignments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B; + RO).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eKif18A and Cdk1 interaction\u003c/h2\u003e \u003cp\u003eSince i-Cdk1 appears required for correct spindle assembly [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the poor localization of Kif18A at the spindle center in i-Cdk1-lowered cells may be an indirect effect due to the overall spindle assembly impairment in those cells rather than a direct requirement for i-Cdk1 in the control of Kif18A. Nevertheless, Kif18A function has been shown to be inhibited by direct phosphorylation of Kif18A operated by Cdk1 in mitosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Kif18A has been shown to resolve in fast and slow migrating forms when analyzed from metaphase-arrested cell lysates on SDS/PAGE, suggesting different phosphorylation states [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, the slow migrating form of Kif18 was converted into the fast migrating form upon treatment of mitotic cell lysates with calf intestinal phosphatase, \u003cem\u003ein vitro\u003c/em\u003e, or upon treatment of mitotic cells, \u003cem\u003ein vivo\u003c/em\u003e, with a chemical Cdk1 inhibitor, indicating that the slower migrating form was phosphorylated by Cdk1 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our SDS/PAGE we were unable to clearly detect differently migrating forms of Kif18A from total mitotic cell lysates, however, in metaphase-arrested cells the band appeared broader than that from prometaphase-arrested cells, perhaps indicating that, from prometaphase to metaphase, some Kif18A had undergone dephosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; lanes 1 and 2). Nevertheless, also under our conditions Kif18A was indeed converted into a faster migrating form after treatment of metaphase-arrested cells with a chemical Cdk1 inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; lane 3). To gain further insight into the relationship between Cdk1 and Kif18A, we first asked whether Cdk1 and Kif18A physically interacted in mitotic cells by probing cyclin B1 co-immunoprecipitations from total cell extracts and found that Kif18A indeed interacted with Cdk1 at prometaphase and at metaphase and even after treatment with the Cdk1 inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Since i-Cdk1 is tightly bound to spindle MTs, we analyzed Kif18A distribution upon fractionating mitotic cells into insoluble pellet fraction (P), containing spindles and spindle-associated proteins, and soluble fraction (S) to further dissect the relationship between Kif18A and Cdk1 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Control, Wee1-downregulated and Wee1-downregulated/Wee1-complemented cells were prometaphase-arrested by a 14-hour treament with the reversible microtubule inhibitor nocodazole, added shortly after siRNA treatments, and released into MC medium for further 60 min of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To a portion of control cells, nocodazole was added back at the beginning of the 60 min incubation to block spindle assembly and keep cells arrested at prometaphase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; Noco). The majority of control and Wee1-depleted/complemented cells built spindles during the 60 min incubation after nocodazole wash out and arrested at metaphase, on the contrary spindle assembly in Wee1-depleted cells was severely impaired, as previously shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; top images) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Cells were fractionated and Kif18A analyzed from soluble and pellet fractions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; bottom blots; pellet protein samples were enriched 8 folds relatively to soluble proteins; see Material and Methods section). In control prometaphase-arrested cells Kif18A was more abundant in the soluble rather than in the pellet fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; bottom blots; Noco), while in control metaphase cells substantial Kif18A amounts were recovered in the pellet fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; bottom blots). In Wee1-downregulated cells, Kif18A distribution between soluble and pellet fractions resembled that of prometaphase cells, while in Wee1-downregulated/complemented cells, that correctly assembled spindles, distribution was similar to control metaphase cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; Wee1-siRNAs and Wee1-siRNAs comp.; bottom blots). As control, blots were also probed for γ-tubulin since centrosomes, that are enriched in γ-tubulin, did pelleted even from prometaphase cells and the relative γ-tubulin content in pellet and soluble fractions did not vary substantially between prometaphase and metaphase cells, as previously shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; bottom blots) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Analyzing short exposures of the Kif18A blots, it became evident that Kif18A from the pellet fractions of control and Wee1-downregulated/complemented cells resolved into two differently migrating forms of which the faster form was predominant over the slower migrating form, suggesting that Kif18A was present on spindles in a dephosphorylated form (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; Kif18A SE; bottom blots). Thus, we further analyzed Kif18A/Cdk1 interaction in the soluble and pellet fractions of prometaphase- and metaphase-arrested cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). To this end, we immunoprecipitated comparable amounts of cyclin B1 from the soluble fraction of prometaphase-arrested cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; lane 3) and from the pellet fraction of metaphase-arrested cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; lane 4) and found that Kif18A coprecipitating with cyclin B1 from the metaphase-arrested cell pellet fraction had a faster migration on SDS/PAGE compared to Kif18A co-precipitating with cyclin B1 from the soluble fraction of prometaphase-arrested cells, indicating its dephosphorylated state (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In addition, as previously shown, cyclin B1 immunoprecipitated from the soluble fraction of prometaphase-arrested cells was in an active Cdk1 complex, since it bound cdk1 non-phosphorylated at inhibitory Y-15-cdk1 site, and bound to inactive PP1a, phosphorylated at the inhibitory T-320, conversely, cyclin B1 from the metaphase-arrested cell pellet fraction was mostly in an i-Cdk1 complex, since it bound cdk1 substantially phosphorylated at the inhibitory Y-15 site, and with a presumably active PP1a, since it was dephosphorylated at the inhibitory T-320 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Together, these data strongly suggest that i-Cdk1 promoted localized PP1-dependent Kif18A dephosphorylation and activation at spindle MTs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAlignment defects rescue by expression of a Cdk1-phosphorylation-resistant Kif18A mutant in i-Cdk1-downregulated cells\u003c/h2\u003e \u003cp\u003eIt has been shown that the serines 674 and 684 of human Kif18A are major sites of inhibitory phosphorylation by Cdk1, thus, we asked whether expressing a Kif18A mutant version in which S674 and S684 are mutated into non-phosphorylatable alanine (S674A/S684A; Kif18A-AA) could compensate alignment defects in i-Cdk1-downregulated cells. To this end, HeLa cells were co-transfected with cdk1-AF-expression vector plus either an empty vector or a wild type Kif18A-expression vector (Kif18A-WT) or a S674A/S684A-Kif18A-expression vector (Kif18A-AA), arrested at G2, released into MC medium (as described in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and fixed after 60 minutes of further incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Scoring chromosome alignment in cells with bipolar spindles showed that although co-expressing Kif18A-WT with cdk1-AF had already some mild effect relatively to the sole cdk1-AF expression, co-expressing Kif18A-AA with cdk1-AF had a strong effect in compensating alignment defects reducing them in more than 50% of cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eKif18A has been shown to promote chromosome alignment at the spindle equator by accumulating at the K-MT plus ends to confine centromere movements by suppressing MT dynamics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This action has been shown to be antagonized by direct phosphorylation of Kif18A operated by Cdk1 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, how chromosome alignment at the spindle equator proceeds in the presence of Cdk1 activity is unclear. Here we asked whether Kif18A action is licensed in mitosis by virtue of i-Cdk1, a recently identified subpopulation of spindle-localized Cdk1 that remains inhibited in mitosis by phosphorylation and is required for spindle assembly [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Defective bipolar spindles, in mitotic human cells in which i-Cdk1 was lowered, were often elongated with bioriented chromosome pairs that failed to align at the equator of the metaphase plate and showed poor Kif18A accumulation at their K-MT plus ends (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These effects were reversed by mild Cdk1 inhibition, that restored i-Cdk1 as previously shown (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. By cell fractionation experiments, we found that at prometaphase Kif18A was predominantly in the soluble fraction rather than in the pellet fraction, conversely, at metaphase Kif18A accumulated in the microtubular pellet fraction and in a faster migrating form on SDS/PAGE, relatively to Kif18A migration from the soluble fraction, indicative of its dephosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Accumulation of Kif18A in the pellet fraction, in its faster migrating form, was strongly reduced in i-Cdk1-lowered cells, while both distribution and migration were reversed upon i-Cdk1 restoration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, we found that in the pellet fraction, the faster migrating form of Kif18A interacted with i-Cdk1 that was in complex with the presumably active form of PP1a, dephosphorylated at the inhibitory T320 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Moreover, expression of a cdk1-dependent phosphorylation-resistant Kif18A mutant partly compensated chromosome alignment defects in i-Cdk1-lowered cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Together, our data suggest a scenario in which the distribution of i-Cdk1 along spindle MTs allows Kif18A to be locally dephosphorylated by PP1 in order to regain its ability to concentrate at K-MT plus ends, dampen MT dynamics, and promote chromosome alignment. Although loss of Kif18A function appears to be tolerated by euploid cells, it does appear to be essential for survival of some aneuploid cancers, revealing a strong antitumor therapeutic potential [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, it has been shown that the sensitivity of aneuploid cancer cells to chemical Kif18A inhibition correlates with their ability to delay mitosis exit by implementing an efficient SAC response [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Since Cdk1 activity is a major SAC-activating kinase and inhibiting Wee1 prolongs mitosis in a SAC-dependent manner, it is possible to hypothesize that the combination of Wee1 and Kif18A inhibition will provide a means to potentiate the therapeutic efficacy towards aneuploid cancers [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by AIRC, Associazione Italiana per la Ricerca sul Cancro: IG grant 2017; Id. 19851 to D.G.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.F.S. and D.G. designed and performed experiments and analyzed data. C.P. and A.T. performed immunoblot and immunofluorescence experiments and analyzed data. D. G. wrote the manuscript. A.F.S. revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank AIRC, Associazione Italiana per la Ricerca sul Cancro for support (IG grant 2017; Id. 19851 to D.G.)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRisteski P, Jagrić M, Pavin N, Tolić IM (2021) Biomechanics of chromosome alignment at the spindle midplane. 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Cell Rep 13(11):2425\u0026ndash;2439. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.celrep.2015.11.021\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2015.11.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chromosome alignment, i-Cdk1, micronuclei, aneuploidy, Wee1, spindle assembly, cancer vulnerability.","lastPublishedDoi":"10.21203/rs.3.rs-4594196/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4594196/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDuring mitosis, chromosome alignment at the mitotic spindle equator grants correct chromosome segregation and proper nuclei formation in daughter cells. A central role for chromosome alignment is exerted by the kinesin 8 family member Kif18A that localizes at the kinetochore-microtubule (K-MT) plus ends where it dampens MT dynamics stabilizing K-MT attachments. Kif18A action is directly antagonized by the master mitotic kinase cyclin B-dependent kinase 1 (Cdk1) and promoted by protein phosphatase 1 (PP1). Since chromosome alignment precedes Cdk1 inactivation by cyclin B proteolysis it is unclear how Kif18A evicts Cdk1 inhibition. We show here that chromosome alignment in human cells relies on a recently identified fraction of Cdk1 that is inhibited by phosphorylation in mitosis (i-Cdk1, for inhibited/inactive-Cdk1), localized at spindle structures and required for proper spindle assembly. Indeed, lowering i-Cdk1 induced several spindle defects including spindles with misaligned, bipolarly attached, chromosomes that showed poor Kif18A localization at K-MT plus ends. Both alignment defects and Kif18A localization were reversed by restoring i-Cdk1. In i-Cdk1-lowered cells, alignment defects were also significantly rescued by expressing a phosphorylation-resistant Kif18A version at Cdk1-dependent sites. Mechanistically, our evidence indicates that i-Cdk1 and active PP1 promoted spindle-localized Kif18A dephosphorylation. Given the relevance of Kif18A for survival of aneuploid cancer cells, these observations may also have relevance for cancer therapy.\u003c/p\u003e","manuscriptTitle":"Chromosome alignment relies on spindle-localized control of Cdk1 activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 06:05:35","doi":"10.21203/rs.3.rs-4594196/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1ce81f57-7003-4604-bf29-d6d32890bf95","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-31T02:53:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 06:05:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4594196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4594196","identity":"rs-4594196","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}