Polo-like kinase-1 (PLK1) is an essential mitotic kinase regulating multiple aspects of the cell division process. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 kinase domain, but the kinase responsible for this has not yet been affirmatively identified. Here we show that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. We find that aurora A can directly phosphorylate PLK1 on Thr 210, and that activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A (ref. 7). We show that Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A.

Successful cell division requires that chromosomes attach to opposite poles of the mitotic spindle (bi-orientation). Aurora B kinase regulates chromosome-spindle attachments by phosphorylating kinetochore substrates that bind microtubules. Centromere tension stabilizes bi-oriented attachments, but how physical forces are translated into signaling at individual centromeres is unknown. Using FRET-based biosensors to measure localized phosphorylation dynamics in living cells, we found that phosphorylation of an Aurora B substrate at the kinetochore depended on its distance from the kinase at the inner centromere. Furthermore, repositioning Aurora B closer to the kinetochore prevented stabilization of bi-oriented attachments and activated the spindle checkpoint. Thus, centromere tension can be sensed by increased spatial separation of Aurora B from kinetochore substrates, which reduces phosphorylation and stabilizes kinetochore microtubules.Accurate chromosome segregation during cell division is essential to maintain genome integrity. Prior to segregation, kinetochores of sister chromatids attach to microtubules from opposite spindle poles (bi-orientation). This configuration is achieved through a trial-and-error process in which correct attachments exert tension across the centromere, which stabilizes kinetochore-microtubule interactions. Incorrect attachments, for example if both sister chromatids attach to a single spindle pole, exert less tension and are destabilized, providing a new opportunity to bi-orient (1,2). How tension is coupled to kinetochore-microtubule stability is not known.The mitotic kinase Aurora B (Ipl1 in budding yeast) localizes to the inner centromere, between sister kinetochores, and destabilizes microtubule attachments by phosphorylating kinetochore substrates, including Dam1 and the Ndc80 complex (3-10). An appealing model is that Aurora B substrates are selectively phosphorylated at incorrect attachments. To test this model we first examined phosphorylation of CENP-A Ser-7, a known kinetochore substrate (11). We used an assay in which Aurora B inhibition leads to a high frequency of syntelic attachment errors, with sister chromatids connected to a single spindle pole (6) (Fig. S1A). We compared phospho-CENP-A staining at correct and incorrect attachments 10 min after removing the reversible Aurora B kinase inhibitor ZM447439 (12), which re-activates Aurora B. Phospho-** Publisher's Disclaimer: This manuscript has been accepted for publication in Science. This version has not undergone final editing.Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the

Summary Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here, we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine tuning of kinetochore-microtubule interactions.

KNL targets PP1 to kinetochores, where it antagonizes Aurora B activity.

Many biologically important macromolecules are internalized into cells by clathrin-coated pit endocytosis. The mechanism of clathrin-coated pit budding has been investigated intensively, and considerable progress has been made in characterizing the proteins involved in internalization. Membrane lipid composition and the lateral organization of lipids and proteins within membranes are believed to play an important role in the regulation of membrane-trafficking processes. Here we report that membrane cholesterol plays a critical role in clathrin-coated pit internalization. We show that acute cholesterol depletion, using ␤-methyl-cyclodextrin, specifically reduces the rate of internalization of transferrin receptor by more than 85%, without affecting intracellular receptor trafficking back to the cell surface. The effect on endocytosis is attributable to a failure of coated pits to detach from the plasma membrane, as visualized by using a green f luorescent protein-clathrin conjugate in living cells. Ultrastructural studies indicate that acute cholesterol depletion causes accumulation of f lat-coated membranes and a corresponding decrease in deep-coated pits, consistent with the possibility that f lat clathrin lattices are direct precursors of indented pits and endocytic vesicles in intact cells. We conclude that clathrin is unable to induce curvature in the membrane depleted of cholesterol.

Summary Aneuploidy arising early in development is the leading genetic cause of birth defects and developmental disabilities in humans. Most errors in chromosome number originate from the egg, and maternal age is well established as the key risk factor. Although the importance of this problem for reproductive health is widely recognized, the underlying molecular basis for age-related aneuploidy in female meiosis is unknown. Here we show that weakened chromosome cohesion is a leading cause of aneuploidy in oocytes in a natural aging mouse model. We find that sister kinetochores are farther apart at both Metaphase I and II, indicating reduced centromere cohesion. Moreover, levels of the meiotic cohesin protein REC8 are severely reduced on chromosomes in oocytes from old mice. To test whether cohesion defects lead to the observed aneuploidies, we monitored chromosome segregation dynamics at Anaphase I in live oocytes and counted chromosomes in the resulting Metaphase II eggs. About 90% of age-related aneuploidies are best explained by weakened centromere cohesion. Together, these results demonstrate that the maternal age-associated increase in aneuploidy is often due to a failure to effectively replace cohesin proteins that are lost from chromosomes during aging.

The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle ("bioriented"). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end-directed microtubule motor CENP-E (kinesin-7).Successful cell division requires proper "biorientation" of chromosomes, whereby microtubule bundles (K fibers) connect sister kinetochores of each chromosome to opposite spindle poles (1). Biorientation errors are linked to chromosome loss and cancers (2). Formation of sister K fibers occurs asynchronously (3), and once a kinetochore captures microtubules growing from a spindle pole, the chromosome is transported toward this pole and becomes "monooriented" (4). Monooriented chromosomes remain near the spindle pole for variable times (3, 4) until they suddenly "congress" to the spindle equator. Current models of mitotic spindle formation (5, 6) postulate that chromosome congression occurs as the result of biorientation (7).We followed movements of individual chromosomes in mammalian cells by differential interference contrast (DIC) time-lapse microscopy (8 Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript oscillations that occur toward and away from spindle poles, we frequently observed monooriented chromosomes making direct movements to the metaphase plate as if they were attempting to congress ( fig. S1). Centromeres on these congressing chromosomes were frequently stretched, which indicated force generation by the leading kinetochore (Movie S1). However, these movements did not always result in a stable alignment on the metaphase plate, because chromosomes often returned to the spindle pole after a 3-to 4-μm excursion. This chromosome behavior was observed in essentially every cell we imaged and has also been previously reported (9-12). To determine whether these chromosomes were bioriented, we followed mitotic cells by DIC microscopy until one of the chromosomes exhibited an extended linear movement toward the metaphase plate, and we fixed the cell when the chromosome had almost reached the metaphase plate (~5 to 7 μm from the proximal spindle pole) (Fig.

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.