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.     

Biotin-tubulin incorporates into kinetochore fiber microtubules during early but not late anaphase

The dynamic behavior of kinetochore fiber microtubules has been examined in PtK1 cells during anaphase of mitosis. Cells in anaphase were injected with biotin-tubulin and, at various intervals after injection, fixed for light or electron microscopic immunolocalization of biotin-tubulin-containing microtubules. When cells in early to mid anaphase were injected with biotin-tubulin and fixed 1-2 min later, fluorescence was observed throughout the spindle, including the region of the kinetochore fibers. Electron microscopy of early to mid anaphase cells, after processing with immunogold methods, revealed both labeled and unlabeled microtubules in the kinetochore fibers; some labeled microtubules contacted the kinetochores. When late anaphase cells were injected with biotin-tubulin, and fixed a few minutes later, little fluorescence was observed in the kinetochore fibers. Electron microscopy confirmed that kinetochore fibers in late anaphase cells were refractory to tubulin incorporation. The results of these experiments demonstrate that the kinetochore fiber incorporates new microtubules during early anaphase but that this incorporation ceases in mid to late anaphase. Thus, microtubule turnover within the kinetochore fiber does not abruptly cease at the onset of anaphase and anaphase kinetochore fiber microtubules are more dynamic than previously suspected.     

Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components, including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly.     

Sensing centromere tension: Aurora B and the regulation of kinetochore function

Maintaining genome integrity during cell division requires regulated interactions between chromosomes and spindle microtubules. To ensure that daughter cells inherit the correct chromosomes, the sister kinetochores must attach to opposite spindle poles. Tension across the centromere stabilizes correct attachments, whereas phosphorylation of kinetochore substrates by the conserved Ipl1/Aurora B kinase selectively eliminates incorrect attachments. Here, we review our current understanding of how mechanical forces acting on the kinetochore are linked to biochemical changes to control chromosome segregation. We discuss models for tension sensing and regulation of kinetochore function downstream of Aurora B, and mechanisms that specify Aurora B localization to the inner centromere and determine its interactions with substrates at distinct locations.     

Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.     

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule- and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20- to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three- to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.     

Function and Assembly of DNA Looping,Clustering, and Microtubule Attachment Complexes within a Eukaryotic Kinetochore

The kinetochore is a complex protein–DNA assembly that provides the mechanical linkage between microtubules and the centromere DNA of each chromosome. Centromere DNA in all eukaryotes is wrapped around a unique nucleosome that contains the histone H3 variant CENP-A (Cse4p in Saccharomyces cerevisiae). Here, we report that the inner kinetochore complex (CBF3) is required for pericentric DNA looping at the Cse4p-containing nucleosome. DNA within the pericentric loop occupies a spatially confined area that is radially displaced from the interpolar central spindle. Microtubule-binding kinetochore complexes are not involved in pericentric DNA looping but are required for the geometric organization of DNA loops around the spindle microtubules in metaphase. Thus, the mitotic segregation apparatus is a composite structure composed of kinetochore and interpolar microtubules, the kinetochore, and organized pericentric DNA loops. The linkage of microtubule-binding to centromere DNA-looping complexes positions the pericentric chromatin loops and stabilizes the dynamic properties of individual kinetochore complexes in mitosis.     

Visualizing kinetochore architecture

Kinetochores are large macromolecular assemblies that link chromosomes to spindle microtubules (MTs) during mitosis. Here we review recent advances in the study of core MT-binding kinetochore complexes using electron microcopy methods in vitro and nanometer-accuracy fluorescence microscopy in vivo. We synthesize these findings in novel three-dimensional models of both the budding yeast and vertebrate kinetochore in different stages of mitosis. There is a growing consensus that kinetochores are highly dynamic, supra-molecular machines that undergo dramatic structural rearrangements in response to MT capture and spindle forces during mitosis.     

Where is the right path heading from the centromere to spindle microtubules?

The kinetochore is a large protein complex that ensures accurate chromosome segregation during mitosis by connecting the centromere and spindle microtubules. One of the kinetochore sub-complexes, the constitutive centromere-associated network (CCAN), associates with the centromere and recruits another sub-complex, the KMN (KNL1, Mis12, and Ndc80 complexes) network (KMN), which binds to spindle microtubules. The CCAN-KMN interaction is mediated by two parallel pathways (CENP-C- and CENP-T-pathways) in the kinetochore, which bridge the centromere and microtubules. Here, we discuss dynamic protein-interaction changes in the two pathways that couple the centromere with spindle microtubules during mitotic progression.     

Spindle microtubules and their mechanical associations after micromanipulation in anaphase

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.     

Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K–dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.     

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.     

Structure of kinetochore fibres in crane-fly spermatocytes after irradiation with an ultraviolet microbeam: neither microtubules nor actin filaments remain in the irradiated region

We studied chromosome movement after kinetochore microtubules were severed. Severing a kinetochore fibre in living crane-fly spermatocytes with an ultraviolet microbeam creates a kinetochore stub, a birefringent remnant of the spindle fibre connected to the kinetochore and extending only to the edge of the irradiated region. After the irradiation, anaphase chromosomes either move poleward led by their stubs or temporarily stop moving. We examined actin and/or microtubules in irradiated cells by means of confocal fluorescence microscopy or serial-section reconstructions from electron microscopy. For each cell thus examined, chromosome movement had been recorded continuously until the moment of fixation. Kinetochore microtubules were completely severed by the ultraviolet microbeam in cells in which chromosomes continued to move poleward after the irradiation: none were seen in the irradiated regions. Similarly, actin filaments normally present in kinetochore fibres were severed by the ultraviolet microbeam irradiations: the irradiated regions contained no actin filaments and only local spots of non-filamentous actin. There was no difference in irradiated regions when the associated chromosomes continued to move versus when they stopped moving. Thus, one cannot explain motion with severed kinetochore microtubules in terms of either microtubules or actin-filaments bridging the irradiated region. The data seem to negate current models for anaphase chromosome movement and support a model in which poleward chromosome movement results from forces generated within the spindle matrix that propel kinetochore fibres or kinetochore stubs poleward.     

Kinetochore function is controlled by a phospho-dependent coexpansion of inner and outer components

It is widely accepted that the kinetochore is built on CENP-A–marked centromeric chromatin in a hierarchical order from inner to outer kinetochore. Recruitment of many kinetochore proteins depends on microtubule attachment status, but it remains unclear how their assembly/disassembly is orchestrated. Applying 3D structured illumination microscopy to Xenopus laevis egg extracts, here we reveal that in the absence of microtubule attachment, proteins responsible for lateral attachment and spindle checkpoint signaling expand to form micrometer-scale fibrous structures over CENP-A–free chromatin, whereas a core module responsible for end-on attachment (CENP-A, CENP-T, and Ndc80) does not. Both outer kinetochore proteins (Bub1, BubR1, Mad1, and CENP-E) and the inner kinetochore component CENP-C are integral components of the expandable module, whose assembly depends on multiple mitotic kinases (Aurora B, Mps1, and Plx1) and is suppressed by protein phosphatase 1. We propose that phospho-dependent coexpansion of CENP-C and outer kinetochore proteins promotes checkpoint signal amplification and lateral attachment, whereas their selective disassembly enables the transition to end-on attachment.     

The ABCs of CENPs

Equal distribution of DNA in mitosis requires the assembly of a large proteinaceous ensemble onto the centromeric DNA, called the kinetochore. With few exceptions, kinetochore specification is independent of the DNA sequence and is determined epigenetically by deposition at the centromeric chromatin of special nucleosomes containing an H3-related histone, CENP-A. Onto centromeric CENP-A chromatin is assembled the so-called constitutive centromere-associated network (CCAN) of 16 proteins distributed in several functional groups as follows: CENP-C, CENP-H/CENP-I/CENP-K/, CENP-L/CENP-M/CENP-N, CENP-O/CENP-P/CENP-Q/CENP-R/CENP-U(50), CENP-T/CENP-W, and CENP-S/CENP-X. One role of the CCAN is to recruit outer kinetochore components further, such as KNL1, the Mis12 complex, and the Ndc80 complex (KMN network) to which attach the spindle microtubules with their structural and regulatory proteins. Among the CENPs in CCAN, CENP-C and CENP-T are required in parallel for operational kinetochore specification and spindle attachment. This review presents discussion of the latest structural and functional data on CENP-A and CENPs from the CCAN as well as their interaction with the KMN network.     

CENP-E kinesin interacts with SKAP protein to orchestrate accurate chromosome segregation in mitosis

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochore. Although previous studies show that the mitotic kinesin CENP-E forms a link between attachment of the spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying dynamic kinetochore-microtubule interactions in mammalian cells remains elusive. Here, we identify a novel interaction between CENP-E and SKAP that functions synergistically in governing dynamic kinetochore-microtubule interactions. SKAP binds to the C-terminal tail of CENP-E in vitro and is essential for an accurate kinetochore-microtubule attachment in vivo. Immunoelectron microscopic analysis indicates that SKAP is a constituent of the kinetochore corona fibers of mammalian centromeres. Depletion of SKAP or CENP-E by RNA interference results in a dramatic reduction of inter-kinetochore tension, which causes chromosome mis-segregation with a prolonged delay in achieving metaphase alignment. Importantly, SKAP binds to microtubules in vitro, and this interaction is synergized by CENP-E. Based on these findings, we propose that SKAP cooperates with CENP-E to orchestrate dynamic kinetochore-microtubule interaction for faithful chromosome segregation.     

Micromanipulation of Chromosomes in Mitotic Vertebrate Tissue Cells: Tension Controls the State of Kinetochore Movement

In mitotic vertebrate tissue cells, chromosome congression to the spindle equator in prometaphase and segregation to the poles in anaphase depend on the movements of kinetochores at their kinetochore microtubule attachment sites. To test if kinetochores sense tension to control their states of movement poleward (P) and away from the pole (AP), we applied an external force to the spindle in preanaphase newt epithelial cells by stretching chromosome arms with microneedles. For monooriented chromosomes (only one kinetochore fiber), an abrupt stretch of an arm away from the attached pole induced the single attached kinetochore to persist in AP movement at about 2 μm/min velocity, resulting in chromosome movement away from the pole. When the stretch was reduced or the needle removed, the kinetochore switched to P movement at about 2 μm/min and pulled the chromosome back to near the premanipulation position within the spindle. For bioriented chromosomes (sister kinetochores attached to opposite poles) near the spindle equator, stretching one arm toward a pole placed the kinetochore facing away from the direction of stretch under tension and the sister facing toward the stretch under reduced tension or compression. Kinetochores under increased tension exhibited prolonged AP movement while kinetochores under reduced tension or compression exhibited prolonged P movement, moving the centromeres at about 2 μm/min velocities off the metaphase plate in the direction of stretch. Removing the needle resulted in centromere movement back to near the spindle equator at similar velocities. These results show that tension controls the direction of kinetochore movement and associated kinetochore microtubule assembly/disassembly to position centromeres within the spindle of vertebrate tissue cells. High tension induces persistent AP movement while low tension induces persistent P movement. The velocity of P and AP movement appears to be load independent and governed by the molecular mechanisms which attach kinetochores to the dynamic ends of kinetochore microtubules.     

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.     

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.