EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B

The Aurora B kinase coordinates kinetochore–microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. We find that EB1, a microtubule plus end–tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). Our data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase.     

Kinetochore dynein generates a poleward pulling force to facilitate congression and full chromosome alignment

For proper chromosome segregation, all kinetochores must achieve bipolar microtubule （MT） attachment and subsequently align at the spindle equator before anaphase onset. The MT minus end-directed motor dynein/dynactin binds kinetoehores in prometaphase and has long been implicated in chromosome congression. Unfortunately, inactivation of dynein usually disturbs spindle organization, thus hampering evaluation of its kinetochore roles. Here we specifically eliminated kinetochore dynein/dynactin by RNAi-mediated depletion of ZW10, a protein essential for kinetochore localization of the motor. Time-lapse microscopy indicated markedly-reduced congression efficiency, though congressing chromosomes displayed similar velocities as in control cells. Moreover, cells frequently failed to achieve full chromosome alignment, despite their normal spindles. Confocal microcopy revealed that the misaligned kinetochores were monooriented or unattached and mostly lying outside the spindle, suggesting a difficulty to capture MTs from the opposite pole. Kinetoehores on monoastral spindles were dispersed farther away from the pole and exhibited only mild oscillation. Furthermore, inactivating dynein by other means generated similar phenotypes. Therefore, kinetochore dynein produces on monooriented kinetochores a poleward pulling force, which may contribute to efficient bipolar attachment by facilitating their proper microtubule captures to promote congression as well as full chromosome alignment.     

Aurora B kinase controls the targeting of the Astrin-SKAP complex to bioriented kinetochores

During mitosis, kinetochores play multiple roles to generate interactions with microtubules, and direct chromosome congression, biorientation, error correction, and anaphase segregation. However, it is unclear what changes at the kinetochore facilitate these distinct activities. Here, we describe a complex of the spindle- and kinetochore-associated protein Astrin, the small kinetochore-associated protein (SKAP), and the dynein light chain LC8. Although most dynein-associated proteins localize to unaligned kinetochores in an Aurora B-dependent manner, Astrin, SKAP, and LC8 localization is antagonized by Aurora B such that they target exclusively to bioriented kinetochores. Astrin-SKAP-depleted cells fail to maintain proper chromosome alignment, resulting in a spindle assembly checkpoint-dependent mitotic delay. Consistent with a role in stabilizing bioriented attachments, Astrin and SKAP bind directly to microtubules and are required for CLASP localization to kinetochores. In total, our results suggest that tension-dependent Aurora B phosphorylation can act to control outer kinetochore composition to provide distinct activities to prometaphase and metaphase kinetochores.     

EB1 targets to kinetochores with attached,polymerizing microtubules

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end-kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.     

Distinct sequence elements of cyclin B1 promote localization to chromatin, centrosomes, and kinetochores during mitosis

The mitotic cyclins promote cell division by binding and activating cyclin-dependent kinases (CDKs). Each cyclin has a unique pattern of subcellular localization that plays a vital role in regulating cell division. During mitosis, cyclin B1 is known to localize to centrosomes, microtubules, and chromatin. To determine the mechanisms of cyclin B1 localization in M phase, we imaged full-length and mutant versions of human cyclin B1-enhanced green fluorescent protein in live cells by using spinning disk confocal microscopy. In addition to centrosome, microtubule, and chromatin localization, we found that cyclin B1 also localizes to unattached kinetochores after nuclear envelope breakdown. Kinetochore recruitment of cyclin B1 required the kinetochore proteins Hec1 and Mad2, and it was stimulated by microtubule destabilization. Mutagenesis studies revealed that cyclin B1 is recruited to kinetochores through both CDK1-dependent and -independent mechanisms. In contrast, localization of cyclin B1 to chromatin and centrosomes is independent of CDK1 binding. The N-terminal domain of cyclin B1 is necessary and sufficient for chromatin association, whereas centrosome recruitment relies on sequences within the cyclin box. Our data support a role for cyclin B1 function at unattached kinetochores, and they demonstrate that separable and distinct sequence elements target cyclin B1 to kinetochores, chromatin, and centrosomes during mitosis.     

CAMP (C13orf8, ZNF828) is a novel regulator of kinetochore-microtubule attachment

Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore-microtubule attachment, chromosome alignment-maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc-finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1-dependent phosphorylation at multiple sites during mitosis. CAMP-depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K-fibres to the tension exerted upon establishment of sister kinetochore bi-orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C-terminal region containing the zinc-finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP-E and CENP-F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP-E and CENP-F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore-microtubule attachment during bi-orientation.     

NudC is required for Plk1 targeting to the kinetochore and chromosome congression

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.     

Merotelic kinetochores in mammalian tissue cells

Merotelic kinetochore attachment is a major source of aneuploidy in mammalian tissue cells in culture. Mammalian kinetochores typically have binding sites for about 20-25 kinetochore microtubules. In prometaphase, kinetochores become merotelic if they attach to microtubules from opposite poles rather than to just one pole as normally occurs. Merotelic attachments support chromosome bi-orientation and alignment near the metaphase plate and they are not detected by the mitotic spindle checkpoint. At anaphase onset, sister chromatids separate, but a chromatid with a merotelic kinetochore may not be segregated correctly, and may lag near the spindle equator because of pulling forces toward opposite poles, or move in the direction of the wrong pole. Correction mechanisms are important for preventing segregation errors. There are probably more than 100 times as many PtK1 tissue cells with merotelic kinetochores in early mitosis, and about 16 times as many entering anaphase as the 1% of cells with lagging chromosomes seen in late anaphase. The role of spindle mechanics and potential functions of the Ndc80/Nuf2 protein complex at the kinetochore/microtubule interface is discussed for two correction mechanisms: one that functions before anaphase to reduce the number of kinetochore microtubules to the wrong pole, and one that functions after anaphase onset to move merotelic kinetochores based on the ratio of kinetochore microtubules to the correct versus incorrect pole.     

Dynactin helps target Polo‐like kinase 1 to kinetochores via its left‐handed beta‐helical p27 subunit

Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)‐based motor. Dynactin binds both dynein and MTs via its p150^Glued subunit, but little is known about the ‘pointed‐end complex’ that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin‐dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo‐like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore–MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left‐handed β‐helix domain, with the phosphorylation site located within a disordered, C‐terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.     

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.     

Cyclin B destruction triggers changes in kinetochore behavior essential for successful anaphase

Successful mitosis requires that anaphase chromosomes sustain a commitment to move to their assigned spindle poles. This requires stable spindle attachment of anaphase kinetochores. Prior to anaphase, stable spindle attachment depends on tension created by opposing forces on sister kinetochores [1]. Because tension is lost when kinetochores disjoin, stable attachment in anaphase must have a different basis. After expression of nondegradable cyclin B (CYC-B(S)) in Drosophila embryos, sister chromosomes disjoined normally but their anaphase behavior was abnormal [2]. Chromosomes exhibited cycles of reorientation from one pole to the other. Additionally, the unpaired kinetochores accumulated attachments to both poles (merotelic attachments), congressed (again) to a pseudometaphase plate, and reacquired associations with checkpoint proteins more characteristic of prometaphase kinetochores. Unpaired prometaphase kinetochores, which occurred in a mutant entering mitosis with unreplicated (unpaired) chromosomes, behaved just like the anaphase kinetochores at the CYC-B(S) arrest. Finally, the normal anaphase release of AuroraB/INCENP from kinetochores was blocked by CYC-B(S) expression and, reciprocally, was advanced in a CycB mutant. Given its established role in destabilizing kinetochore-microtubule interactions [3], Aurora B dissociation is likely to be key to the change in kinetochore behavior. These findings show that, in addition to loss of sister chromosome cohesion, successful anaphase requires a kinetochore behavioral transition triggered by CYC-B destruction.     

Dynein is a transient kinetochore component whose binding is regulated by microtubule attachment, not tension

Cytoplasmic dynein is the only known kinetochore protein capable of driving chromosome movement toward spindle poles. In grasshopper spermatocytes, dynein immunofluorescence staining is bright at prometaphase kinetochores and dimmer at metaphase kinetochores. We have determined that these differences in staining intensity reflect differences in amounts of dynein associated with the kinetochore. Metaphase kinetochores regain bright dynein staining if they are detached from spindle microtubules by micromanipulation and kept detached for 10 min. We show that this increase in dynein staining is not caused by the retraction or unmasking of dynein upon detachment. Thus, dynein genuinely is a transient component of spermatocyte kinetochores.We further show that microtubule attachment, not tension, regulates dynein localization at kinetochores. Dynein binding is extremely sensitive to the presence of microtubules: fewer than half the normal number of kinetochore microtubules leads to the loss of most kinetochoric dynein. As a result, the bulk of the dynein leaves the kinetochore very early in mitosis, soon after the kinetochores begin to attach to microtubules. The possible functions of this dynein fraction are therefore limited to the initial attachment and movement of chromosomes and/or to a role in the mitotic checkpoint.     

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.     

Chmp4c is required for stable kinetochore-microtubule attachments

Formation of stable kinetochore-microtubule attachments is essential for accurate chromosome segregation in human cells and depends on the NDC80 complex. We recently showed that Chmp4c, an endosomal sorting complex required for transport protein involved in membrane remodelling, localises to prometaphase kinetochores and promotes cold-stable kinetochore microtubules, faithful chromosome alignment and segregation. In the present study, we show that Chmp4c associates with the NDC80 components Hec1 and Nuf2 and is required for optimal NDC80 stability and Hec1-Nuf2 localisation to kinetochores in prometaphase. However, Chmp4c-depletion does not cause a gross disassembly of outer or inner kinetochore complexes. Conversely, Nuf2 is required for Chmp4c kinetochore targeting. Constitutive Chmp4c kinetochore tethering partially rescues cold-stable microtubule polymers in cells depleted of the endogenous Nuf2, showing that Chmp4c also contributes to kinetochore-microtubule stability independently of regulating Hec1 and Nuf2 localisation. Chmp4c interacts with tubulin in cell extracts, and binds and bundles microtubules in vitro through its highly basic N-terminal region (amino acids 1–77). Furthermore, the N-terminal region of Chmp4c is required for cold-stable kinetochore microtubules and efficient chromosome alignment. We propose that Chmp4c promotes stable kinetochore-microtubule attachments by regulating Hec1–Nuf2 localisation to kinetochores in prometaphase and by binding to spindle microtubules. These results identify Chmp4c as a novel protein that regulates kinetochore-microtubule interactions to promote accurate chromosome segregation in human cells.     

Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells

During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule-nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the corona but not the plate), distributed along the surface of the kinetochore microtubules, or both.     

The microtubule catastrophe promoter Sentin delays stable kinetochore–microtubule attachment in oocytes

The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore–microtubule attachment in oocytes.     

Regulated targeting of protein phosphatase 1 to the outer kinetochore by KNL1 opposes Aurora B kinase

Regulated interactions between kinetochores and spindle microtubules are essential to maintain genomic stability during chromosome segregation. The Aurora B kinase phosphorylates kinetochore substrates to destabilize kinetochore–microtubule interactions and eliminate incorrect attachments. These substrates must be dephosphorylated to stabilize correct attachments, but how opposing kinase and phosphatase activities are coordinated at the kinetochore is unknown. Here, we demonstrate that a conserved motif in the kinetochore protein KNL1 directly interacts with and targets protein phosphatase 1 (PP1) to the outer kinetochore. PP1 recruitment by KNL1 is required to dephosphorylate Aurora B substrates at kinetochores and stabilize microtubule attachments. PP1 levels at kinetochores are regulated and inversely proportional to local Aurora B activity. Indeed, we demonstrate that phosphorylation of KNL1 by Aurora B disrupts the KNL1–PP1 interaction. In total, our results support a positive feedback mechanism by which Aurora B activity at kinetochores not only targets substrates directly, but also prevents localization of the opposing phosphatase.     

Spindle-kinetochore attachment requires the combined action of Kin I-like Klp5/6 and Alp14/Dis1-MAPs in fission yeast

Fission yeast Klp5 and Klp6 belong to the microtubule-destabilizing Kin I family. In klp5 mutants, spindle checkpoint proteins Mad2 and Bub1 are recruited to mitotic kinetochores for a prolonged duration, indicating that these kinetochores are unattached. Further analysis shows that there are kinetochores to which only Bub1, but not Mad2, localizes. These kinetochores are likely to have been captured, yet lack tension. Thus Klp5 and Klp6 play a role in a spindle- kinetochore interaction at dual steps, capture and generation of tension. The TOG/XMAP215 family, Alp14 and Dis1 are known to stabilize microtubules and be required for the bivalent attachment of the kinetochore to the spindle. Despite apparent opposing activities towards microtubule stability, Klp5/Klp6 and Alp14/Dis1 share an essential function, as either dis1klp or alp14klp mutants are synthetically lethal, like alp14dis1. Defective phenotypes are similar to each other, characteristic of attachment defects and chromosome mis-segregation. Furthermore Alp14 is of significance for kinetochore localization of Klp5. We propose that Klp5/Klp6 and Alp14/Dis1 play a collaborative role in bipolar spindle formation during prometaphase through producing spindle dynamism.     

RED, a spindle pole-associated protein, is required for kinetochore localization of MAD1, mitotic progression, and activation of the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) is essential for ensuring the proper attachment of kinetochores to the spindle and, thus, the precise separation of paired sister chromatids during mitosis. The SAC proteins are recruited to the unattached kinetochores for activation of the SAC in prometaphase. However, it has been less studied whether activation of the SAC also requires the proteins that do not localize to the kinetochores. Here, we show that the nuclear protein RED, also called IK, a down-regulator of human leukocyte antigen (HLA) II, interacts with the human SAC protein MAD1. Two RED-interacting regions identified in MAD1 are from amino acid residues 301-340 and 439-480, designated as MAD1(301-340) and MAD1(439-480), respectively. Our observations reveal that RED is a spindle pole-associated protein that colocalizes with MAD1 at the spindle poles in metaphase and anaphase. Depletion of RED can cause a shorter mitotic timing, a failure in the kinetochore localization of MAD1 in prometaphase, and a defect in the SAC. Furthermore, the RED-interacting peptides MAD1(301-340) and MAD1(439-480), fused to enhanced green fluorescence protein, can colocalize with RED at the spindle poles in prometaphase, and their expression can abrogate the SAC. Taken together, we conclude that RED is required for kinetochore localization of MAD1, mitotic progression, and activation of the SAC.     

Plk1 phosphorylates sgt1 at the kinetochores to promote timely kinetochore-microtubule attachment

Accurate chromosome segregation during cell division maintains genomic integrity and requires the proper establishment of kinetochore-microtubule attachment in mitosis. As a key regulator of mitosis, Polo-like kinase 1 (Plk1) is essential for this attachment process, but the molecular mechanism remains elusive. Here we identify Sgt1, a cochaperone for Hsp90, as a novel Plk1 substrate during mitosis. We show that Sgt1 dynamically localizes at the kinetochores, which lack microtubule attachments during prometaphase. Plk1 is required for the kinetochore localization of Sgt1 and phosphorylates serine 331 of Sgt1 at the kinetochores. This phosphorylation event enhances the association of the Hsp90-Sgt1 chaperone with the MIS12 complex to stabilize this complex at the kinetochores and thus coordinates the recruitment of the NDC80 complex to form efficient microtubule-binding sites. Disruption of Sgt1 phosphorylation reduces the MIS12 and NDC80 complexes at the kinetochores, impairs stable microtubule attachment, and eventually results in chromosome misalignment to delay the anaphase onset. Our results demonstrate a mechanism for Plk1 in promoting kinetochore-microtubule attachment to ensure chromosome stability.