Laser microsurgery reveals conserved viscoelastic behavior of the kinetochore

Accurate chromosome segregation depends on proper kinetochore–microtubule attachment. Upon microtubule interaction, kinetochores are subjected to forces generated by the microtubules. In this work, we used laser ablation to sever microtubules attached to a merotelic kinetochore, which is laterally stretched by opposing pulling forces exerted by microtubules, and inferred the mechanical response of the kinetochore from its length change. In both mammalian PtK1 cells and in the fission yeast Schizosaccharomyces pombe, kinetochores shortened after microtubule severing. Interestingly, the inner kinetochore–centromere relaxed faster than the outer kinetochore. Whereas in fission yeast all kinetochores relaxed to a similar length, in PtK1 cells the more stretched kinetochores remained more stretched. Simple models suggest that these differences arise because the mechanical structure of the mammalian kinetochore is more complex. Our study establishes merotelic kinetochores as an experimental model for studying the mechanical response of the kinetochore in live cells and reveals a viscoelastic behavior of the kinetochore that is conserved in yeast and mammalian cells.     

MITOSIS IN THE FUNGUS THRAUSTOTHECA CLAVATA

The ultrastructure of mitosis is described in Thraustotheca clavata, an oömycete fungus. An intranuclear spindle develops between differentiated regions of the nuclear envelope which move apart, each associated with 180° oriented centriole pairs. The spindle contains low numbers of continuous and interdigitating microtubules in addition to chromosomal microtubules. Each kinetochore is attached to only one microtubule. Serial section analysis shows that at meiosis there are probably 12 chromosomes in the diploid nucleus, yet at mitosis the methods utilized in the present study suggest that there may be less than 12 kinetochores connected to each pole. At mitosis many of the kinetochores within a given spindle are not arranged in opposite pairs. The behavior of the spindle microtubules during mitosis is comparable to that of higher organisms but the rarity of short intertubular distances appears to preclude significant force generation by means of intertubular bridge mechanisms. Evidence is presented for a nuclear envelope-microtubule interaction which is capable of generating shear forces during both mitosis and interphase nuclear movements.     

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.     

Bi-orienting chromosomes: acrobatics on the mitotic spindle

To maintain their genetic integrity, eukaryotic cells must segregate their chromosomes properly to opposite poles during mitosis. This process mainly depends on the forces generated by microtubules that attach to kinetochores. During prometaphase, kinetochores initially interact with a single microtubule that extends from a spindle pole and then move towards a spindle pole. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles (sister kinetochore bi-orientation). If sister kinetochores interact with microtubules in wrong orientation, this must be corrected before the onset of anaphase. Here, I discuss the processes leading to bi-orientation and the mechanisms ensuring this pivotal state that is required for proper chromosome segregation.     

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.     

Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint

Kinetochore attachment to the ends of dynamic microtubules is a conserved feature of mitotic spindle organization that is thought to be critical for proper chromosome segregation. Although kinetochores have been described to transition from lateral to end-on attachments, the phase of lateral attachment has been difficult to study in yeast due to its transient nature. We have previously described a kinetochore mutant, DAM1-765, which exhibits lateral attachments and misregulation of microtubule length. Here we show that the misregulation of microtubule length in DAM1-765 cells occurs despite localization of microtubule associated proteins Bik1, Stu2, Cin8 and Kip3 to microtubules. DAM1-765 kinetochores recruit the spindle checkpoint protein Bub1, however Bub1 localization to DAM1-765 kinetochores is not sufficient to cause a cell cycle arrest. Interestingly, the DAM1-765 mutation rescues the temperature sensitivity of a biorientationdeficient ipl1-321 mutant, and DAM1-765 chromosome loss rates are similar to wild-type cells. the spindle checkpoint in DAM1-765 cells responds properly to unattached kinetochores created by nocodazole treatment and loss of tension caused by a cohesin mutant. progression of DAM1-765 cells through mitosis therefore suggests that satisfaction of the checkpoint depends more highly on biorientation of sister kinetochores than on achievement of a specific interaction between kinetochores and microtubule plus ends.Key words: spindle assembly checkpoint, kinetochore-microtubule attachments, biorientation, DAM1-765     

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.     

Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle

We argue that hypotheses for how chromosomes achieve a metaphase alignment, that are based solely on a tug-of-war between poleward pulling forces produced along the length of opposing kinetochore fibers, are no longer tenable for vertebrates. Instead, kinetochores move themselves and their attached chromosomes, poleward and away from the pole, on the ends of relatively stationary but shortening/elongating kinetochore fiber microtubules. Kinetochores are also "smart" in that they switch between persistent constant-velocity phases of poleward and away from the pole motion, both autonomously and in response to information within the spindle. Several molecular mechanisms may contribute to this directional instability including kinetochore-associated microtubule motors and kinetochore microtubule dynamic instability. The control of kinetochore directional instability, to allow for congression and anaphase, is likely mediated by a vectorial mechanism whose magnitude and orientation depend on the density and orientation or growth of polar microtubules. Polar microtubule arrays have been shown to resist chromosome poleward motion and to push chromosomes away from the pole. These "polar ejection forces" appear to play a key role in regulating kinetochore directional instability, and hence, positions achieved by chromosomes on the spindle.     

What generates flux of tubulin in kinetochore microtubules?

Summary. We discuss models for production of tubulin flux in kinetochore microtubules. Current models concentrate solely on microtubules and their associated motors and enzymes. For example, in some models the driving force for flux is enzymes at the poles and the kinetochores; in others the driving force is motor molecules that are associated with a stationary spindle matrix. We present a different viewpoint, that microtubules are propelled poleward by forces arising from the spindle matrix, that the forces on the microtubules “activate” polymerising and depolymerising enzymes at kinetochores and poles, that matrix forces utilise actin, myosin, and microtubule motors, and that the matrix itself may not necessarily be static. Correspondence: A. Forer, Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

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.     

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.     

Immunofluorescence analysis of sucrose-induced changes in spindle morphology

Metaphase and anaphase PtK1 cells show spindle elongation without concomitant chromosome motion when treated with culture medium containing 0.5 M sucrose. Electron microscopy has shown sucrose-induced changes in microtubule (MT) organization, changes in trilaminar kinetochore structure, and specific kinetochore-MT associations which may account for these results. In this paper we employ double-label immunofluorescence techniques using antibodies against tubulin and the kinetochore to analyze changes in spindle microtubule and kinetochore distribution produced by sucrose treatment. Cells treated from prometaphase through anaphase with 0.5 M sucrose from 10 min to 2 h showed spindle elongation and a distinct rearrangement of spindle microtubules into bundles, with a pronounced increase in length of interpolar microtubule bundles. In sucrose-treated mitotic cells kinetochores remained as antigenically distinct structures, similar to those found in untreated interphase cells. Kinetochore determinants remained positioned within a diffuse chromatin mass, but the orientation of sister kinetochores to opposite spindle poles was lost. Instead, kinetochore pairs were found in lateral association with microtubule bundles, with several pairs of determinants associated with a single bundle in many instances. Cells released from 0.5 M sucrose treatment showed a return of the spindle to a pretreatment arrangement for both the microtubules and kinetochore determinants.     

Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells

Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.     

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.     

Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension

A single unattached kinetochore can delay anaphase onset in mitotic tissue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994. J. Cell Biol. 127:1301–1310). Kinetochores in vertebrate cells contain multiple binding sites, and tension is generated at kinetochores after attachment to the plus ends of spindle microtubules. Checkpoint component Mad2 localizes selectively to unattached kinetochores (Chen, R.-H., J.C. Waters, E.D. Salmon, and A.W. Murray. 1996. Science. 274:242–246; Li, Y., and R. Benezra. Science. 274: 246–248) and disappears from kinetochores by late metaphase, when chromosomes are properly attached to the spindle. Here we show that Mad2 is lost from PtK₁ cell kinetochores as they accumulate microtubules and re-binds previously attached kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells are stabilized with taxol, tension at kinetochores is lost. The phosphoepitope 3f3/2, which has been shown to become dephosphorylated in response to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929–939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol. In contrast, Mad2 only localized to an average of 2.6 out of the 22 kinetochores in taxol-treated PtK₁ cells. Therefore, loss of tension at kinetochores occupied by microtubules is insufficient to induce Mad2 to accumulate on kinetochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells arrested with taxol to exit mitosis after ~12 min, while uninjected cells remained in mitosis for at least 6 h, demonstrating that Mad2 is necessary for maintenance of the taxol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 interactions at kinetochores which are important for inhibiting anaphase onset.     

Kinetochore microtubules and chromosome movement during prometaphase in Drosophila melanogaster spermatocytes studied in life and with the electron microscope

Prometaphase I chromosome behavior was examined in wild-type Drosophila melanogaster primary spermatocytes. Cine analysis of live cells reveals that bivalents exhibit complex motions that include (1) transient bipolar orientations, (2) simultaneous reorientation of homologous kinetochores, (3) movements not parallel to the spindle axis, and (4) movement along the nuclear membrane. — Kinetochores and kinetochore microtubule have been analyzed for bivalents previously studied in life. The results suggest that most chromosome motions (complex though they may be) can be explained by poleward forces acting on or through kinetochore microtubules that span the distance between the kinetochore and the vicinity of a pole. The results also suggest that the majority of short kinetochore microtubules may be remnants of previous microtubule-mediated associations between a kinetochore and a pole.     

Tension,microtubule rearrangements,and the proper distribution of chromosomes in mitosis

The basis for stable versus unstable kinetochore orientation was investigated by a correlated living-cell/ultrastructural study of grasshopper spermatocytes. Mal-oriented bivalents having both kinetochores oriented to one spindle pole were induced by micromanipulation. Such malorientations are stable while the bivalent is subject to tension applied by micromanipulation but unstable after tension is released. Unstable bivalents always reorient with movement of one kinetochore toward the opposite pole. Microtubules associated with stably oriented bivalents, whether they are mal-oriented or in normal bipolar orientation, are arranged in orderly parallel bundles running from each kinetochore toward the pole. Similar orderly kinetochore microtubule arrangements characterize mal-oriented bivalents fixed just after release of tension. A significantly different microtubule arrangement is found only some time after tension release, when kinetochore movement is evident. The microtubules of a reorienting kinetochore always include a small number of microtubules running toward the pole toward which the kinetochore was moving at the time of fixation. All other microtubules associated with such a moving kinetochore appear to have lost their anchorage to the original pole and to be dragged passively as the kinetochore proceeds to the other pole. Thus, the stable anchorage of kinetochore microtubules to the spindle is associated with tension force and unstable anchorage with the absence of tension. The effect of tension is readily explained if force production and anchorage are both produced by mitotic motors, which link microtubules to the spindle as they generate tension forces.     

Reconstituting the kinetochore–microtubule interface: what, why, and how

The kinetochore is the proteinaceous complex that governs the movement of duplicated chromosomes by interacting with spindle microtubules during mitosis and meiosis. Faithful chromosome segregation requires that kinetochores form robust load-bearing attachments to the tips of dynamic spindle microtubules, correct microtubule attachment errors, and delay the onset of anaphase until all chromosomes have made proper attachments. To understand how this macromolecular machine operates to segregate duplicated chromosomes with exquisite accuracy, it is critical to reconstitute and study kinetochore–microtubule interactions in vitro using defined components. Here, we review the current status of reconstitution as well as recent progress in understanding the microtubule-binding functions of kinetochores in vivo.     

Mps1 promotes poleward chromosome movements in meiotic prometaphase

In prophase of meiosis I, homologous chromosomes pair and become connected by cross-overs. Chiasmata, the connections formed by cross-overs, enable the chromosome pair, called a bivalent, to attach as a single unit to the spindle. When the meiotic spindle forms in prometaphase, most bivalents are associated with one spindle pole and then go through a series of oscillations on the spindle, attaching to and detaching from microtubules until the partners of the bivalent become bioriented—attached to microtubules from opposite sides of the spindle. The conserved kinase, Mps1, is essential for the bivalents to be pulled by microtubules across the spindle in prometaphase. Here we show that MPS1 is needed for efficient triggering of the migration of microtubule-attached kinetochores toward the poles and promotes microtubule depolymerization. Our data support the model Mps1 acts at the kinetochore to coordinate the successful attachment of a microtubule and the triggering of microtubule depolymerization to then move the chromosome.