Effect of superexpression of DNA-binding protein heterochromatin Abp1p on frequency of loss of minichromosomes and growth of Schizosaccharomyces pombe with mutations of the gene coding cofactor D

Mitotic chromosome segregation is partly determined by interaction between microtubules (MTs) and the kinetochores of sister chromatids. The precise mechanism of the interaction between kinetochores and MTs remains unclear. This process has been studied in fission yeast Schizosaccharomyces pombe by analyzing interaction between genes encoding kinetochore components, such as DNA-binding protein Abp1p, and genes whose protein products affect the dynamics of MTs, such as cofactor D of tubulin dimer assembly. Analysis of cell growth and minichromosome loss frequency has demonstrated that mutations in the gene of cofactor D, especially mutation tsm1-512, increase the rate of minichromosome loss and the sensitivity to changes in Abp1p concentration in cells compared to wild-type cells Probably, mutations alp1-1315 and tsm1-512 of the cofactor D gene cause defects in the kinetochore-MT interaction.     

Phallacidin stains the kinetochore region in the mitotic spindle of the green algaeOedogonium spp.

Summary We found previously that in living cells ofOedogonium cardiacum andO. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (<2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore ofOedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.Abbreviations CD cytochalasin D - EM electron microscopy - MBS m-maleimidobenzoyl N-hydroxysuccinimide ester - MTs 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.     

Fine structural studies of apolar mitosis

A fine structural analysis of apolar mitosis induced by chloral hydrate was made on Haemanthus katherinae Bak. endosperm. Under the influence of chloral hydrate MTs disappear initially and then are formed de novo. Kinetochore fibers grow away from kinetochores and their formation is asynchronous for all chromosomes in the set and also for sister kinetochores. Bundles of MTs forming kinetochore fibers converge toward one of the poorly defined polar regions during formation of kinetochore fibers (metaphase) and in motionless kinetochores. Such MTs increasingly diverge when kinetochores move during anaphase. The relation of ER to formation of MTs is evident and briefly discussed. A continuous transition exists between NE and ER during formation and disintegration of the NE. Some theoretical aspects of these problems were also discussed.     

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 biorientation-deficient 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.     

Functional redundancies, distinct localizations and interactions among three fission yeast homologs of centromere protein-B

Several members of protein families that are conserved in higher eukaryotes are known to play a role in centromere function in the fission yeast Schizosaccharomyces pombe, including two homologs of the mammalian centromere protein CENP-B, Abp1p and Cbh1p. Here we characterize a third S. pombe CENP-B homolog, Cbh2p (CENP-B homolog 2). cbh2Delta strains exhibited a modest elevation in minichromosome loss, similar to cbh1Delta or abp1Delta strains. cbh2Delta cbh1Delta strains showed little difference in growth or minichromosome loss rate when compared to single deletion strains. In contrast, cbh2Delta abp1Delta strains displayed dramatic morphological and chromosome segregation defects, as well as enhancement of the slow-growth phenotype of abp1Delta strains, indicating partial functional redundancy between these proteins. Both cbh2Delta abp1Delta and cbh1Delta abp1Delta strains also showed strongly enhanced sensitivity to a microtubule-destabilizing drug, consistent with a mitotic function for these proteins. Cbh2p was localized to the central core and core-associated repeat regions of centromeric heterochromatin, but not at several other centromeric and arm locations tested. Thus, like its mammalian counterpart, Cbh2p appeared to be localized exclusively to a portion of centromeric heterochromatin. In contrast, Abp1p was detected in both centromeric heterochromatin and in chromatin at two of three replication origins tested. Cbh2p and Abp1p homodimerized in the budding yeast two-hybrid assay, but did not interact with each other. These results suggest that indirect cooperation between different CENP-B-like DNA binding proteins with partially overlapping chromatin distributions helps to establish a functional centromere.     

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.     

Plc1p is required for proper chromatin structure and activity of the kinetochore in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by facilitating recruitment of the RSC complex

High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p’s involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells

Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40–50 nm thick. On average, each S2 kinetochore binds 11±2 MTs, in contrast to the 4–6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly, nearly all of the kinetochore MT plus ends terminate in the peripheral surface layer, which we argue is analogous to the outer plate in vertebrate kinetochores. Our structural observations provide important data for assessing the results of RNAi studies of mitosis, as well as for the development of mathematical modelling and computer simulation studies in Drosophila and related organisms.Electronic supplementary material Supplementary material is available for this article at and is accessible to authorized users.     

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.     

The attachment of kinetochores to the pro-metaphase spindle in PtK1 cells

When late prophase PtK₁ cells are chilled to 6 ° C the nuclear envelope (NE) breaks down as in normal cells but the spindle is inhibited from forming. When these cells are subsequently warmed to 18 ° C the spindle slowly forms and pro-metaphase congression ensues. Using this approach we have been able to experimentally eliminate the influence of asynchronous NE breakdown on the formation and development of the spindle, and also to slow down (and thus increase the temporal separation of) the subsequent events which occur during the initial stages of spindle formation. Correlative light and high voltage electron microscopic studies on these cells, fixed after various times of recovery, reveal the following results: 1) the centrosomes generate microtubules (MTs) well before MTs are seen to be associated with the kinetochores; 2) as in untreated PtK₁ cells (Roos, 1973a, 1976) the order in which chromosomes attach to the forming spindle is influenced by their proximity to a centrosome-kinetochores closest to a centrosome appear stretched towards the centrosome at a time during recovery when other kinetochores, more distal to the centrosome appear unstretched and unoriented; 3) as in untreated cells (Heneen, 1970; Roos, 1976) the predominant behavior during recovery is for a chromosome to initially mono-orient and associate with the near centrosome and only later to develop a bipolar association; and 4) MTs associated with early pro-metaphase kinetochores are almost always oriented towards a centrosome. — From our results we conclude that the proximity effect and the tendency of pro-metaphase chromosomes in PtK₁ to initially mono-orient and associate with the near centrosome cannot be ascribed, as suggested by Roos (1976), to influences arising during the asynchronous breakdown of the NE. Rather, our data clearly demonstrate that a kinetochore-centrosome interaction occurs during spindle formation which cannot be attributed to transient influences. The proximity effect and the predominant tendency of PtK₁ pro-metaphase chromosomes to mono-orient to the near pole are taken to signify the existance of a centrosomal influence on the attachment and orientation of chromosomes. Two possible mechanisms for this influence, both involving a structural interaction between the centrosome and the kinetochore, are outlined.     

Yeast UBL-UBA proteins have partially redundant functions in cell cycle control

Abp1, and the closely related Cbh1 and Cbh2 are homologous to the human centromere-binding protein CENP-B that has been implicated in the assembly of centromeric heterochromatin. Fission yeast cells lacking Abp1 show an increase in mini-chromosome instability suggesting that Abp1 is important for chromosome segregation and/or DNA synthesis. Here we show that Abp1 interacts with the DNA replication protein Cdc23 (MCM10) in a two-hybrid assay, and that the Δabp1 mutant displays a synthetic phenotype with a cdc23 temperature-sensitive mutant. Moreover, genetic interactions were also observed between abp1 ⁺ and four additional DNA replication initiation genes cdc18 ⁺, cdc21 ⁺, orc1 ⁺, and orc2 ⁺. Interestingly, we find that S phase is delayed in cells deleted for abp1 ⁺ when released from a G1 block. However, no delay is observed when cells are released from an early S phase arrest induced by hydroxyurea suggesting that Abp1 functions prior to, or coincident with, the initiation of DNA replication.     

Captivating capture: how microtubules attach to kinetochores

Accurate chromosome segregation is essential to ensure genomic stability because the aneuploidy that results from segregation errors leads to birth defects and contributes to the development of cancer. Chromosome segregation is directed by the kinetochore, the chromosomal site of attachment to dynamic polymers called microtubules (MTs). Although the fidelity of chromosome segregation depends on precise interactions between kinetochores and MTs, it is still unclear how this interaction is mediated and regulated. Here we discuss current progress in determining how kinetochores assemble and attach to MTs during mitosis as well as how they correct errors.     

Centromere protein F includes two sites that couple efficiently to depolymerizing microtubules

Firm attachments between kinetochores and dynamic spindle microtubules (MTs) are important for accurate chromosome segregation. Centromere protein F (CENP-F) has been shown to include two MT-binding domains, so it may participate in this key mitotic process. Here, we show that the N-terminal MT-binding domain of CENP-F prefers curled oligomers of tubulin relative to MT walls by approximately fivefold, suggesting that it may contribute to the firm bonds between kinetochores and the flared plus ends of dynamic MTs. A polypeptide from CENP-F’s C terminus also bound MTs, and either protein fragment diffused on a stable MT wall. They also followed the ends of dynamic MTs as they shortened. When either fragment was coupled to a microbead, the force it could transduce from a shortening MT averaged 3–5 pN but could exceed 10 pN, identifying CENP-F as a highly effective coupler to shortening MTs.     

Kinetochore fiber formation in animal somatic cells: dueling mechanisms come to a draw

The attachment to and movement of a chromosome on the mitotic spindle are mediated by the formation of a bundle of microtubules (MTs) that tethers the kinetochore on the chromosome to a spindle pole. The origin of these “kinetochore fibers” (K fibers) has been investigated for over 125 years. As noted in 1944 by Schrader [Mitosis, Columbia University Press, New York, 110 pp.], there are three possible ways to form a K fiber: (a) it grows from the pole until it contacts the kinetochore, (b) it grows directly from the kinetochore, or (c) it forms as a result of an interaction between the pole and the chromosome. Since Schrader's time, it has been firmly established that K fibers in centrosome-containing animal somatic cells form as kinetochores capture MTs growing from the spindle pole (route a). It is now similarly clear that in cells lacking centrosomes, including higher plants and many animal oocytes, K fibers “self-assemble” from MTs generated by the chromosomes (route b). Can animal somatic cells form K fibers in the absence of centrosomes by the “self-assembly” pathway? In 2000, the answer to this question was shown to be a resounding “yes.” With this result, the next question became whether the presence of a centrosome normally suppresses K fiber self-assembly or if this route works concurrently with centrosome-mediated K-fiber formation. This question, too, has recently been answered: observations on untreated live animal cells expressing green fluorescent protein-tagged tubulin clearly show that kinetochores can nucleate the formation of their associated MTs in a unique manner in the presence of functional centrosomes. The concurrent operation of these two “dueling” routes for forming K fibers in animal cells helps explain why the attachment of kinetochores and the maturation of K fibers occur as quickly as they do on all chromosomes within a cell.     

Changes in prometaphase chromosome motion are dependent on experimentally induced changes in kinetochore structure.

Prometaphase PtK₁ cells are treated with low concentrations of sucrose in order to analyze its effects on kinetochore structure, microtubule (MT) associations with the developing kinetochore and chromosome congression. Prometaphase cells treated with 0.15M sucrose slows chromosome congression, yet chromosomes form a metaphase configuration. However, 0.2M sucrose treatment prevents chromosome congression and affects some of the kinetochore MT linkages with the kinetochore, resulting in loss of chromosome congression. We use time lapse video microscopy and ultrastructural analysis to correlate changes in the linkages in the kinetochore MTs and the kinetochore to explain these findings. It appears hyperosmotic shock treatment can produce non-functional linkages between kinetochore MTs and kinetochores such that chromosome congression is affected. When non-functional linkages are formed, the presence of both a corona and matrix-like material is also present, proximal to the kinetochore. The role of this material and its organization at the klnetochore is discussed in its relation to generating mitotic forces.     

On the interaction of ribosomal protein RPL22e with microtubules

Microtubule (MT) protein preparations often contain components of the translation machinery, including ribosome proteins. To understand the biological meaning of it we studied the interaction of ribosomal protein RPL22e with the MT. We found that bacteria expressed purified RPL22e‐GFP‐6His did co‐sediment with brain tubulin MTs with 1.3 µM dissociation coefficient. Such a K_D is comparable to some specific MT‐associated proteins. Distinct in vitro interaction of RPL22e‐GFP with MTs was also observed by TIRF microscopy. In real‐time assay, RPL22e‐GFP molecules stayed bound to MTs for several seconds, and 15% of them demonstrated random‐walk along MTs with diffusion coefficient 0.03 µ²/s. Deletion of basic areas of RPL22e did not have an impact on K_D, and deletion of acidic tail slightly increased association with MTs. Interestingly, the deletion of acidic tail increased diffusion coefficient as well. The interaction of RPL22e with MTs is hardly noticeable in vivo in cultured cells, probably since a significant part of the protein is incorporated into the ribosomes. The mobility of ribosomal protein on the MTs probably prevents its interfering with MT‐dependent transport and could ameliorate its transport to the nucleus.     

Minichromosomes inDrosophila melanogaster derived from the transposing elementTE1

A minichromosome has originated from the transposing elementTE1. This autonomously replicating chromosome contains the structural genes white and roughest, from theDrosophila X chromosome. It arose within a stock carryingTE1 at 45F on chromosome2. In addition to thew andrst genes, the minichromosome may carry section 45C–45F from chromosome2. It is inherited by 33%–47% of the offspring. By this criterion it carries a centromere, although the origin of the centromere is unknown. From this minichromosome a still smaller one has originated, probably through the loss of all material from chromosome2 together with some heterochromatin. At the same time a duplication of white and roughest could have taken place. This chromosome has a strange morphology and is more frequently lost in meiosis than the larger one, but is still transmitted to about 29%–37% of the progeny of one parent heterozygous for the minichromosome. In both cases the flies have variegated eyes, probably as a result of position-effect variegation. The variegation pattern is influenced by factors in theX chromosome. The size of the smaller minichromosome is little more than 1 Mb as determined by pulsed field gel electrophoresis.     

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.     

Slk19 enhances cross-linking of microtubules by Ase1 and Stu1

The Saccharomyces cerevisiae protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important role for spindle formation and stabilization in prometaphase and metaphase, albeit this role is unresolved. Here we show that Slk19’s localization to metaphase spindles in vivo and to microtubules (MTs) in vitro depends on the MT cross-linking protein Ase1 and the MT cross-linking and stabilizing protein Stu1. By analyzing a slk19 mutant that specifically fails to localize to spindles and MTs, we surprisingly found that the presence of Slk19 amplified the amount of Ase1 strongly and that of Stu1 moderately at the metaphase spindle in vivo and at MTs in vitro. Furthermore, Slk19 markedly enhanced the cross-linking of MTs in vitro when added together with Ase1 or Stu1. We therefore suggest that Slk19 recruits additional Ase1 and Stu1 to the interpolar MTs (ipMTs) of metaphase spindles and thus increases their cross-linking and stabilization. This is in agreement with our observation that cells with defective Slk19 localization exhibit shorter metaphase spindles, an increased number of unaligned nuclear MTs, and most likely reduced ipMT overlaps.