Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.     

Centrosome-kinetochore interaction in multinucleate cells

The interaction between centrosomes and kinetochores was studied in multinucleate cells induced by Colcemid treatment or by random cell fusion. Except for prematurely condensed chromosomes (PCC) of the G₂-phase, PCCs do not develop their own spindle area. Perhaps the maturation promoting factor (MPF) fails to activate these centrosomes. In such PCCs, the kinetochore-centrosome interaction was found to be non-specific: sometimes only a few chromosomes of a group could establish connections with centrosomes, sometimes chromosomes from the same PCC group developed microtubule (MT) attachment with different centrosomes (not the pair), and sometimes kinetochores of PCC groups failed to interact with MTs. These findings explain the abnormal mitotic behaviour of PCCs as seen in the light microscope. These PCCs develop micronuclei or normal nuclei by nuclear re-formation in telophase. All the different PCC groups revealed kinetochores with kinetochore plates. It was shown that transformation of presumptive kinetochores to a trilaminar kinetochore does not depend on nuclear envelope breakdown or on the degree of chromosome condensation. This may be induced by the MPF which may initiate different events like chromosome condensation, nuclear envelope breakdown and kinetochore transformation by secondary factors. Other observations like establishment of connections by different chromosome groups to a common centrosome, kinetochore attachment of PCCs to different centrosomes, interaction of one kinetochore with two centrosomes, kinetochores being stretched and bent to receive microtubules and finally the failure of some kinetochores to develop MT attachment, all strongly suggest that the kinetochores serve as the point of termination rather than the nucleation sites of kinetochore MTs.     

Cell division in two large pennate diatoms Hantzschia and Nitzschia III. A new proposal for kinetochore function during prometaphase

Prometaphase in two large species of diatoms is examined, using the following techniques: (a) time-lapse cinematography of chromosome movements in vivo; (b) electron microscopy of corresponding stages: (c) reconstruction of the microtubules (MTs) in the kinetochore fiber of chromosomes attached to the spindle. In vivo, the chromosomes independently commence oscillations back and forth to one pole. The kinetochore is usually at the leading edge of such chromosome movements; a variable time later both kinetochores undergo such oscillations but toward opposite poles and soon stretch poleward to establish stable bipolar attachment. Electron microscopy of early prometaphase shows that the kinetochores usually laterally associate with MTs that have one end attached to the spindle pole. At late prometaphase, most chromosomes are fully attached to the spindle, but the kinetochores on unattached chromosomes are bare of MTs. Reconstruction of the kinetochore fiber demonstrates that most of its MTs (96%) extend past the kinetochore and are thus apparently not nucleated there. At least one MT terminates at each kinetochore analyzed. Our interpretation is that the conventional view of kinetochore function cannot apply to diatoms. The kinetochore fiber in diatoms appears to be primarily composed of MTs from the poles, in contrast to the conventional view that many MTs of the kinetochore fiber are nucleated by the kinetochore. Similarly, chromosomes appear to initially orient their kinetochores to opposite poles by moving along MTs attached to the poles, instead of orientation effected by kinetochore MTs laterally associating with other MTs in the spindle. The function of the kinetochore in diatoms and other cell types is discussed.     

Kinetochore-generated pushing forces separate centrosomes during bipolar spindle assembly

In animal somatic cells, bipolar spindle formation requires separation of the centrosome-based spindle poles. Centrosome separation relies on multiple pathways, including cortical forces and antiparallel microtubule (MT) sliding, which are two activities controlled by the protein kinase aurora A. We previously found that depletion of the human kinetochore protein Mcm21R^CENP-O results in monopolar spindles, raising the question as to whether kinetochores contribute to centrosome separation. In this study, we demonstrate that kinetochores promote centrosome separation after nuclear envelope breakdown by exerting a pushing force on the kinetochore fibers (k-fibers), which are bundles of MTs that connect kinetochores to centrosomes. This force is based on poleward MT flux, which incorporates new tubulin subunits at the plus ends of k-fibers and requires stable k-fibers to drive centrosomes apart. This kinetochore-dependent force becomes essential for centrosome separation if aurora A is inhibited. We conclude that two mechanisms control centrosome separation during prometaphase: an aurora A–dependent pathway and a kinetochore-dependent pathway that relies on k-fiber–generated pushing forces.     

The impact of chromosomes and centrosomes on spindle assembly as observed in living cells

We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video- enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.     

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.     

Dynamics of spindle microtubule organization: kinetochore fiber microtubules of plant endosperm

Organization of kinetochore fiber microtubules (MTs) throughout mitosis in the endosperm of Haemanthus katherinae Bak. has been analysed using serial section reconstruction from electron micrographs. Accurate and complete studies have required careful analysis of individual MTs in precisely oriented serial sections through many (45) preselected cells. Kinetochore MTs (kMTs) and non-kinetochore MTs (nkMTs) intermingle within the fiber throughout division, undergoing characteristic, time- dependent, organizational changes. The number of kMTs increases progressively throughout the kinetochore during prometaphase-metaphase. Prometaphase chromosomes which were probably moving toward the pole at the time of fixation have unequally developed kinetochores associated with many nkMTs. The greatest numbers of kMTs (74-109/kinetochore), kinetochore cross-sectional area, and kMT central density all occur at metaphase. Throughout anaphase and telophase there is a decrease in the number of kMTs and, in the kinetochore cross-sectional area, an increased obliquity of kMTs and increased numbers of short MTs near the kinetochore. Delayed kinetochores possess more kMTs than do kinetochores near the poles, but fewer kMTs than chromosomes which have moved equivalent distances in other cells. The frequency of C-shaped proximal MT terminations within kinetochores is highest at early prometaphase and midtelophase, falling to zero at midanaphase. Therefore, in Haemanthus, MTs are probably lost from the periphery of the kinetochore during anaphase in a manner which is related to both time and position of the chromosome along the spindle axis. The complex, time-dependent organization of MTs in the kinetochore region strongly suggests that chromosome movement is accompanied by continual MT rearrangement and/or assembly/disassembly.     

Mechanisms of spindle-pole organization are influenced by kinetochore activity in mammalian cells

The spindle is a fusiform bipolar-microtubule array that is responsible for chromosome segregation during mitosis. Focused poles are an essential feature of spindles in vertebrate somatic cells, and pole focusing has been shown to occur through a centrosome-independent self-organization mechanism where microtubule motors cross-link and focus microtubule minus ends. Most of our understanding of this mechanism for pole focusing derives from studies performed in cell-free extracts devoid of centrosomes and kinetochores. Here, we examine how sustained force from kinetochores influences the mechanism of pole focusing in cultured cells. We show that the motor-driven self-organization activities associated with NuMA (i.e., cytoplasmic dynein) and HSET are not necessary for pole focusing if sustained force from kinetochores is inhibited in Nuf2- or Mis12-deficient cells. Instead, pole organization relies on TPX2 as it cross-links spindle microtubules to centrosome-associated mitotic asters. Thus, both motor-driven and static-cross-linking mechanisms contribute to spindle-pole organization, and kinetochore activity influences the mechanism of spindle-pole organization. The motor-driven self-organization of microtubule minus ends at spindle poles is needed to organize spindle poles in vertebrate somatic cells when kinetochores actively exert force on spindle microtubules.     

Density and distribution of α-bungarotoxin-binding sites in postsynaptic structures of regenerated rat skeletal muscle

The polarity of kinetochore microtubules (MTs) has been studied in lysed PtK1 cells by polymerizing hook-shaped sheets of neurotubulin onto walls of preexisting cellular MTs in a fashion that reveals their structural polarity. Three different approaches are presented here: (a) we have screened the polarity of all MTs in a given spindle cross section taken from the region between the kinetochores and the poles, (b) we have determined the polarity of kinetochore MTs are more stable to cold-treated spindles; this approach takes advantage of the fact that kinetochore MTs are more stable to cold treatment than other spindle MTs; and (c) we have tracked bundles of kinetochore MTs from the vicinity of the pole to the outer layer of the kinetochore in cold- treated cells. In an anaphase cell, 90-95% of all MTs in an area between the kinetochores and the poles are of uniform polarity with their plus ends (i.e., fast growing ends) distal to the pole. In cold- treated cells, all bundles of kinetochore MTs show the same polarity; the plus ends of the MTs are located at the kinetochores. We therefore conclude that kinetochore MTs in both metaphase and anaphase cells have the same polarity as the aster MTs in each half-spindle. These results can be interpreted in two ways: (a) virtually all MTs are initiated at the spindle poles and some of the are "captured" by matured kinetochores using an as yet unknown mechanism to bind the plus ends of existing MTs; (b) the growth of kinetochore MTs is initiated at the kinetochore in such a way that the fast growing MT end is proximal to the kinetochore. Our data are inconsistent with previous kinetochore MT polarity determinations based on growth rate measurements in vitro. These studies used drug-treated cells from which chromosomes were isolated to serve as seeds for initiation of neurotubule polymerization. It is possible that under these conditions kinetochores will initiate MTs with a polarity opposite to the one described here.     

Microtubule depolymerization can drive poleward chromosome motion in fission yeast

Prometaphase kinetochores interact with spindle microtubules (MTs) to establish chromosome bi-orientation. Before becoming bi-oriented, chromosomes frequently exhibit poleward movements (P-movements), which are commonly attributed to minus end-directed, MT-dependent motors. In fission yeast there are three such motors: dynein and two kinesin-14s, Pkl1p and Klp2p. None of these enzymes is essential for viability, and even the triple deletion grows well. This might be due to the fact that yeasts kinetochores are normally juxtapolar at mitosis onset, removing the need for poleward chromosome movement during prometaphase. Anaphase P-movement might also be dispensable in a spindle that elongates significantly. To test this supposition, we have analyzed kinetochore dynamics in cells whose kinetochore-pole connections have been dispersed. In cells recovering from this condition, the maximum rate of poleward kinetochore movement was unaffected by the deletion of any or all of these motors, strongly suggesting that other factors, like MT depolymerization, can cause such movements in vivo. However, Klp2p, which localizes to kinetochores, contributed to the effectiveness of P-movement by promoting the shortening of kinetochore fibers.     

Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.     

Requirements for NuMA in maintenance and establishment of mammalian spindle poles

Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.     

Localization of kinetochore fragments isolated from single chromatids in mitotic CHO cells

Summary Chinese hamster ovary (CHO) cells are treated with hydroxurea followed by a caffeine treatment to form detached kinetochore fragments in the absence of sister chromatids. Detached kinetochores in mitotic CHO cells display a functional association with MTs initiated from one or both centrosomes such that these association(s) have a significant influence on the location and orientation of detached kinetochores and/or their fragments. Kinetochore fragments which are amphitelically oriented are positioned approximately midway between the two centrosomes. Thus, a kinetochore isolated from a single chromatid can capture MTs from both poles. Monotelic orientation of these fragments is more frequently observed with kinetochore fragments located an average distance of 2.5 m from the nearest centrosome, compared to an average distance of 4.4 m in amphitelically oriented fragments. In cells treated with the potent MT poison, nocodazole, kinetochore isolation also occurs and therefore is not dependent on the presence of MTs. CHO cells treated to produce isolated kinetochores or kinetochore fragments then subsequently hyperosmotically shocked show no MTs directly inserted into kinetochore lamina, similar to the response of sucrose-treated metapbase PtK₁ cells. This treatment shows circular kinetochores tangentially associated with bundles of MTs that are located an average of 1.5 m from the centrosome. Our results suggest that a single kinetochore fragment can attach to MTs initiated from one or both centrosomes and that their specific association to MT fibers defines orientation of detached kinetochores within the spindle domain.     

Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.     

Light and electron microscopy of rat kangaroo cells in mitosis

Kinetochores in rat kangaroo (PtK₂) cells in prophase of mitosis are finely fibrillar, globular bodies, 5000–8000 Å in diameter. Sister kinetochores are attached to opposite lateral faces in the primary constriction of chromosomes. No microtubules (MTs) occur in prophase nuclei. During prometaphase the ball-shaped kinetochores differentiate into trilaminar plaques. An outer kinetochore layer, less electron dense than chromatin, appears first in the fibrillar matrix. The inner layer, continuous with, but more electron dense than the chromosome, is formed later. Kinetochore-spindle MT interaction is evident at the very beginning of prometaphase. As a result, kinetochore shape is very variable, but three types of kinetochores can be distinguished by fine structure analysis. A comparison of kinetochore structure and chromosome position in the mitotic spindle yielded clues regarding initial orientation and congression. At the time the nuclear envelope (NE) breaks down chromosomes near asters orient first. Chromosomes approximately equidistant from the two spindle poles amphi-orient immediately. Chromosomes closer to one pole probably achieve mono-orientation first, then amphi-orient and congress. In normal metaphase all the chromosomes lie at or near the spindle equator and kinetochores are structurally uniform. Paraxial and para-equatorial sections revealed that they are trilaminar, roughly circular plaques of 4000–6000 Å diameter. Inner and outer layers are 400 Å, and the electron translucent middle layer which separates them is 270 Å thick. From 16 to 40 MTs are anchored in the outer layer. In cold-treated cells the kinetochores are trilaminar, but in colcemid-treated cells the inner layer is lacking. Both kinetochores and their MTs are disorganized beginning in late anaphase. In telophase the inner layer persists for some time as an electron dense patch apposed to the NE, while the outer layer disintegrates.     

Anaphase spindle mechanics prevent mis-segregation of merotelically oriented chromosomes

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.     

CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.     

Chromosome mal-orientation and reorientation during mitosis.

We argue that mal-orientation of mitotic chromosomes is not as rare as once believed. However, unlike bivalents during meiosis I, the reorientation of a mal-oriented mitotic chromosome has yet to be observed. This appears to be due, in part, to the difficulty in differentiating mal-oriented chromosomes from mono-oriented ones which are common during spindle formation in living mitotic cells. We assume that mitotic cells possess mechanisms for correcting chromosome mal-orientations that are similar to those operating during meiosis. However, unlike meiosis, where reorientation appears to be triggered when tension on a K-fiber is relieved or reduced, other factors related to the close proximity of sister kinetochores may also induce reorientation in mal-oriented mitotic chromosomes. We favor a model in which the reorientation of a mitotic kinetochore depends on, and is initiated by, the kinetochore capturing MTs from the pole to which it is reorienting.     

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.     

Kinetochore microtubules in PTK cells.

We have analyzed the fine structure of 10 chromosomal fibers from mitotic spindles of PtK1 cells in metaphase and anaphase, using electron microscopy of serial thin sections and computer image processing to follow the trajectories of the component microtubules (MTs) in three dimensions. Most of the kinetochore MTs ran from their kinetochore to the vicinity of the pole, retaining a clustered arrangement over their entire length. This MT bundle was invaded by large numbers of other MTs that were not associated with kinetochores. The invading MTs frequently came close to the kinetochore MTs, but a two-dimensional analysis of neighbor density failed to identify any characteristic spacing between the two MT classes. Unlike the results from neighbor density analyses of interzone MTs, the distributions of spacings between kinetochore MTs and other spindle MTs revealed no evidence for strong MT-MT interactions. A three-dimensional analysis of distances of closest approach between kinetochore MTs and other spindle MTs has, however, shown that the most common distances of closest approach were 30-50 nm, suggesting a weak interaction between kinetochore MTs and their neighbors. The data support the ideas that kinetochore MTs form a mechanical connection between the kinetochore and the pericentriolar material that defines the pole, but that the mechanical interactions between kinetochore MTs and other spindle MTs are weak.