Role of microtubule organization in centrosome migration and mitotic spindle formation in PtK1 cells

Summary Quinacrine, an acridine derivative, has previously been shown to disrupt lateral associations between non-kinetochore microtubules (nkMTs) of opposite polarity in PtK₁ metaphase spindles such that the balance of spindle forces is significantly altered. We extended the analysis of the spatial relationship of spindle microtubules (MTs) in this study by using quinacrine to compare ATP-dependent requirements for early prometaphase centrosome separation and spindle formation. The route used for centrosome migration can take a variety of pathways in PtK₁ cells, depending on the location of the centrosomes at the time of nuclear envelope breakdown. Following quinacrine treatment centrosome separation decresased by 1.9 to 14.0 m depending on the pathway utilized. However, birefringence of the centrosomal region increased approximately 50% after quinacrine treatment. Quinacrine-treated mid-prometaphase cells, where chromosome attachment to MTs had occurred, showed a decrease in spindle length of approximately 6.0 m with only a slight increase in astral birefringence. Computer-generated reconstructions of quinacrine-treated prometaphase cells were used to confirm changes in MT reorganization. Early-prometaphase cells showed more astral MTs (aMTs) of varied length while mid-prometaphase cells showed only a few short aMTs. Late prometaphase cells again showed a large number of aMTs. Our results suggest that: (1) quinacrine treatment affects centrosome separation, (2) recruitment of nkMTs by kinetochores is quinacrine-sensitive, and (3) development of the prometaphase spindle is dependent on quinacrine-sensitive lateral interactions between nkMTs of opposite polarity. These data also suggest that lateral interactions between MTs formed during prometaphase are necessary for centrosome separation and normal spindle formation but not necessarily chromosome motion.Abbreviations aMT(s) astral microtubule(s) - DIC differential interference contrast - MT(s) microtubule(s) - kMT(s) kinetochore microtubule(s) - NEB nuclear envelope breakdown - nkMT(s) non-kinetochore microtubule(s)     

Dioptrics of the facet lenses in the dorsal rim area of the cricket Gryllus bimaculatus

1. The optics of the corneal facet lenses from the dorsal rim area (DRA) and from the dorso-lateral areas (DA) of the compound eye of the cricket Gryllus bimaculatus were studied.
2. The DRA of the cricket eye contains quite normally shaped facet lenses. The diameter of the facet lens in the DA is 2-fold larger compared to that in the DRA. The radius of curvature of the front surface is distinctly less in the DA facet lenses, as the surface of the facet lenses in the DRA are virtually flat.
3. The averaged axial refractive index of the facet lenses of Gryllus bimaculatus, measured by interference microscopy, was 1.496 ± 0.008 (n = 42) in the DRA and 1.469 ± 0.004 (n = 39) in the DA. The geometrical thickness of the lenses was calculated to be 77 ± 3 μm (n = 42) in the DRA and 56 ± 1 μm (n = 39) in the DA.
4. Analysis of the diffraction pattern obtained with a point light source revealed distinct focusing properties of both the DRA and the DA facet lenses; striking Airy-like diffraction patterns were obtained in both cases.
5. Focal distances measured directly at the backfocal plane were 40 ± 8 μm (n = 84) in the DRA of all the animals studied, and 60–90 μm (n = 62) in DA depending on the animal. Analysis of the diffraction of the point light source yielded very similar focal distances: 40 ± 5 μm (n = 10) in DRA and 81 ± 8 μm (n = 11) in DA. In the DRA, focal distance of the facet lenses was smaller than the cone length, 58 ± 3 μm (n = 9) while in the DA the focal distance matched the effective cone length, 71 ± 5 μm (n = 16).

     

The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly

Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.     

Microtubule disorientation in anaphase half-spindles during autosome segregation in crane fly spermatocytes

The region between the kinetochores of syntelically oriented autosomes and the pole in meta- and anaphase of Pales ferruginea spermatocytes was studied by means of serial sections. Microtubule (MT) were counted and measured, and the spindle region was reconstructed by superimposition of successive micrographs. Kinetochoric (kMTs) and non-kinetochoric microtubules (nkMTs) interdigitate with one another forming a bundle which is often arrow-shaped due to an inclination of nkMTs (skew nkMTs) with respect to the kinetochore-pole axis. The average length of MT in the bundle decreases towards anaphase while the average number increases. The extent of MT disorder in anaphase half-spindles is higher than in metaphase. The number of kMTs inserted in the kinetochore was found to remain unchanged from meta- to early anaphase. Some of the kMTs become divergent in anaphase. The relative proportion of skew nkMTs within the kMT/nkMT bundle is higher in anaphase. It is proposed that the morphological changes observed to occur from meta- to anaphase are due to fragmentation of kMTs followed by disorientation of the MTs pieces. Some aspects of the physical properties of the half-spindles are discussed.     

Dynamic changes in autosomal spindle fibers during prometaphase in crane fly spermatocytes

Different prometaphase stages of Pales ferruginea spermatocytes were serially sectioned and the regions between kinetochores and poles analysed by counting and measuring spindle microtubules. These regions are characterized by an intermingling of kinetochoric (kMTs) and non-kinetochoric microtubules (nkMTs). A considerable proportion of nkMTs is skewed with respect to kMTs, thus being responsible for microtubule disorder in these spindle areas. The degree of disorder expressed by the percentage of skew microtubules was found to decrease from early prometaphase to metaphase, parallel with an increase in kMT number. A possible causal relation between pulling forces and morphological changes in the spindle is discussed.     

Selective reduction of anaphase B in quinacrine-treated PtK1 cells

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has previously been shown to cause the reorganization of metaphase spindle microtubules (MTs) due to changes in interactions of non-kinetochore microtubules (nkMTs) of opposite polarity (Armstrong and Snyder: Cell Motil. Cytoskeleton 7:10-19, 1987). In the study presented here, mitotic PtK1 cells were treated in early anaphase with concentrations of quinacrine ranging from 2 to 12 microM to determine energy requirements for chromosome motion. The rate and extent of chromosome-to-pole movements (anaphase A) were not affected by these quinacrine treatments. The extent of anaphase B (kinetochore-kinetochore separation) was reduced with increasing concentrations of quinacrine. Five micromolar quinacrine reduced the extent of kinetochore-kinetochore separation by 20%, and addition of 12 microM quinacrine reduced the kinetochore-kinetochore separation by 40%. To determine the role of nkMTs in anaphase spindle elongation, quinacrine-treated metaphase cells were treated with hyperosmotic sucrose concentrations, and spindle elongation was measured (Snyder et al.: Eur J. Cell Biol. 39:373-379, 1985). Metaphase cells treated with 2-10 microM concentrations of quinacrine for 2-5 min reduced spindle lengths by 10-50% prior to 0.5 M sucrose treatment for 5 min. This treatment showed a significant reduction in the ability of sucrose to induce spindle elongation in cells pretreated with quinacrine. As spindle length and birefringence was reduced by quinacrine treatment, sucrose-induced elongation was concomitantly diminished. These data suggest that quinacrine-sensitive linkages are necessary for anaphase B motions. Reduction in these linkages and/or MT length in the nkMT continuum may reduce the ability of the nkMTs to hold compression at metaphase. This form of energy is thought to drive a significant proportion of normal anaphase B in PtK1 cells and sucrose-induced metaphase spindle elongation.     

Precocious cleavage furrows simultaneously move and ingress when kinetochore microtubules are depolymerized in <Emphasis Type="Italic">Mesostoma ehrenbergii</Emphasis> spermatocytes

A “precocious” cleavage furrow develops and ingresses during early prometaphase in Mesostoma ehrenbergii spermatocytes (Forer and Pickett-Heaps Eur J Cell Biol 89:607-618, 2010). In response to chromosome movements which regularly occur during prometaphase and that alter the balance of chromosomes in the two half-spindles, the precocious furrow shifts its position along the cell, moving 2–3 μm towards the half cell with fewer chromosomes (Ferraro-Gideon et al. Cell Biol Int 37:892-898, 2013). This process continues until proper segregation is achieved and the cell enters anaphase with the cleavage furrow again in the middle of the cell. At anaphase, the furrow recommences ingression. Spindle microtubules (MTs) are implicated in various furrow positioning models, and our experiments studied the responses of the precocious furrows to the absence of spindle MTs. We depolymerized spindle MTs during prometaphase using various concentrations of nocodazole (NOC) and colcemid. The expected result is that the furrow should regress and chromosomes remain in the midzone of the cell (Cassimeris et al. J Cell Sci 96:9-15, 1990). Instead, the furrows commenced ingression and all three bivalent chromosomes moved to one pole while the univalent chromosomes, that usually reside at the two poles, either remained at their poles or moved to the opposite pole along with the bivalents, as described elsewhere (Fegaras and Forer 2018). The microtubules were completely depolymerized by the drugs, as indicated by immunofluorescence staining of treated cells (Fegaras and Forer 2018), and in the absence of microtubules, the furrows often ingressed (in 33/61 cells) at a rate similar to normal anaphase ingression (~?1 μm/min), while often simultaneously moving toward one pole. Thus, these results indicate that in the absence of anaphase and of spindle microtubules, cleavage furrows resume ingression.     

Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase

Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase.     

The mitotic apparatus in the dinoflagellateAmphidinium carterae

Summary Aspects of mitosis in the dinoflagellateAmphidinium carterae have been examined using TEM, SEM and fluorescence immunochemistry. The extranuclear spindle is composed of 2–4 bundles of microtubules arranged into two interdigitated half-spindles. Unlike previous reports of dinomitosis, the spindle bundles converge at the poles. These bundles of microtubules are inserted into a multilobed, vesiculate body containing electron opaque, amorphous material. This spindle pole body has ribosomes associated with it and is continuous with the endoplasmic reticulum. Chromosomes are attached to the nuclear envelope, which is persistent throughout mitosis. Kinetochore microtubules attach to the nuclear envelope via elongate electron dense kinetochores (one microtubule per daughter kinetochore). Several microtubules pass alongside the kinetochore, forming a halo of 3–4 spindle microtubules. Electron dense connections can be seen between some of these microtubules and the kinetochore. Chromosome segregation appears to be a function of spindle elongation (anaphase B), since chromosome-to-pole distance (anaphase A) remains relatively unchanged throughout mitosis.Abbreviations DABCO 1,4 diazabicyclo(2,2,2)octane - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,NN-tetraacetic acid - PIPES piperazine-N,N-bis(2-thanesulfonic acid) Supported by a Charles and Johanna Bush Predoctoral Fellowship to S. B. B.     

The Mechanics of Anaphase B in a Basidiomycete as Revealed by Laser Microbeam Microsurgery

Bayles, C. J., Aist, J. R., and Berns, M. W. 1993. The mechanics of anaphase B in a basidiomycete as revealed by laser microbeam microsurgery. Experimental Mycology 17, 191-199. Cytoplasmic forces were found to be actively pulling on the spindle pole bodies during anaphase B in the dikaryotic, basidiomycete fungus, Helicobasidium mompa. When the spindle of one nucleus was severed with a laser microbeam at mid anaphase B, its two spindle pole bodies separated at a much faster rate than did those of the intact spindle in the other nucleus of the same cell. Since astral microtubule populations apparently reach their maximum during anaphase B in this fungus, we suggest that these microtubules may be involved in the cytoplasmic pulling forces. The spindle appears to act primarily as a governor, regulating the rate at which the spindle pole bodies are separated.     

Rapid backward movement of anaphase chromosome whose kinetochore fibers were cut by ultraviolet microbeam irradiation

Summary— kinetochore spindle fibers in meiosis I and II grasshopper spermatocytes were cut with a heterochromatic ultraviolet (UV) microbeam converging on the specimen to form a slit-shaped microspot 1.5 × 8 μm or 3 × 8 μm. A total exposure of 3 × 10^?8 joules per μm² was administered within 0.8–2.4 s, which was sufficient for severing. The cells were observed with a high extinction polarizing microscope or phase contrast optics and a record made by time-lapse video microscopy, continuously before, during and after the irradiation. When kinetochore fibers were irradiated i anaphase with UV, an area of reduced birefringence (ARB) was produced at the exposed site. The newly created + ends of the microtubules rapidly disassembled poleward, at a constant speed of 17 μm/min. The — ends at the edge of ARB also depolymerized at a slower rate. When a kinetochore fiber was cut with UV in early anaphase at which time its associated chromosome had not disjoined from the partner chromosome, the chromosome of the irradiated kinetochore fiber moved rapidly back to its partner. The speed during this movement was faster than the normal poleward chromosome movement in anaphase by an order to magnitude or more. When a kinetochore and its associated kinetochore fiber were included in the irradiation are, the effects were more pronounced than the effects of irradiation on a kinetochore fiber alone; the direction of the line connecting the irradiated half-bivalent with the partner half-bivalent deviated so much from the longitudinal axis of the original spindle with time that the division assumed a tripolar figure.     

<Emphasis Type="Italic">Myxobolus imparfinis</Emphasis> n. sp. (Myxozoa: Myxosporea), a new gill parasite of <Emphasis Type="Italic">Imparfinis mirini</Emphasis> Haseman (Siluriformes: Heptapteridae) in Brazil

A new species of myxozoan, Myxobolus imparfinis n. sp. is described based on material from the gills of Imparfinis mirini (Haseman) (Heptapteridae). Mature myxospores are round, measuring 7.1–8.4 (7.9 ± 0.3) μm in length, 4.5–6.2 (5.5 ± 0.5) μm in width and 3.1–4.2 (3.7 ± 0.3) μm in thickness. The polar capsules are of unequal size, the larger polar capsule measuring 3.4–4.5 (3.9 ± 0.3) μm in length and 1.4–2.0 (1.7 ± 0.1) μm in width and the smaller capsule measuring 3.1–3.8 (3.4 ± 0.2) μm in length and 1.2–1.8 (1.5 ± 0.2) μm in width. The polar filament presents 6–7 coils. Spores had a prevalence of infection of 75% (6/8). In histological analyses we detected the development site of spores in primary filaments, in afferent branchial artery, thus classifying the type of infection to the filamental type and vascular subtype. The phylogenetic analyses of a dataset including species Myxobolus Bütschli, 1882 and Henneguya Thélohan, 1892 from South America recovered M. imparfinis n. sp. as a sister species of Myxobolus flavus Carriero, Adriano, Silva, Ceccarelli & Maia, 2013. To our knowledge, this is the first record of a myxozoan species parasitising I. mirini.     

A regulatory model for spindle function during mitosis

Structural information on the mitotic spindle of Saccharomyces cerevisiae obtained from isolated whole mount preparations has shown that the spindle undergoes a two-fold increase in length whilst comprising only a single microtubule continuous between the two spindle pole bodies. Further data from immunofluorescence microscopy on the timing of anaphase B has suggested that microtubules do not directly produce the required force, but instead have a more passive role. Here a regulatory function for spindle microtubules during mitosis is explored and the existence of a non-microtubule force-generating system is postulated. Thus it is suggested that the continuous microtubules govern the velocity of anaphase B by providing a resistive force that is itself regulated by the number of microtubules and their rate of polymerization. On this basis a model for the forces acting on a spindle pole body during anaphase is proposed.     

Kinetochore microtubules in crane-fly spermatocytes: untreated, 2 degrees C-treated, and 6 degrees C-grown spindles

We investigated the involvement of kinetochore microtubules (kMTs) in mediating chromosome-to-pole connections in crane-fly (Nephrotoma suturalis and Nephrotoma ferruginea) spermatocytes. Two experimental treatments were used to yield spindles with reduced numbers of nonkinetochore microtubules (nkMTs). Short-term (10-15 min) exposure of spermatocytes to 2 degrees C caused depolymerization of the majority of nkMTs, resulting in a kMT:(kMT + nkMT) ratio of 0.76. Long-term (24h) exposure to 2 degrees C followed by recovery at 6 degrees C resulted in a kMT:(kMT + nkMT) ratio of 0.55, the spindle having more nkMTs than a 2 degrees C-treated spindle but fewer than an untreated spindle, in which the kMT:(kMT + nkMT) ratio was 0.27. The numbers and lengths of kMTs in 6 degrees C-grown spindles were similar to those in untreated cells, suggesting that the overall inhibition of MT assembly at 6 degrees C apparently did not affect the mechanism by which kMTs are formed. We observed most kMTs of early anaphase spindles to be long (greater than 3 microns), and many extended to the polar regions of the spindle. Thus, the crane-fly spindle appears not to be as atypical as it was previously suggested to be.     

Chromosome micromanipulation

The relationship of kinetochore orientation and reorientation to orderly chromosome distribution in anaphase has been studied experimentally by micromanipulation of living grasshopper spermatocytes. Bivalents or the X chromosome at prometaphase or metaphase I can be detached from the spindle with a microneedle and moved to any desired location within the cell. Following a pause of variable duration the detached chromosome invariably moved, kinetochores foremost, back to the spindle, reassumed its characteristic metaphase position, and, with one exception, segregated normally at anaphase I. Detachment from the spindle is demonstrated unequivocally (1) by manipulation evidence for the absence of the firm spindle connections seen both before detachment and after reattachment and (2) by a functional criterion: a given kinetochore, oriented to one pole before detachment, often orients to the opposite pole after detachment. The segregation in anaphase was always as expected from the final, post-operation, orientation. Reorientation and prometaphase and anaphase movement after detachment cannot be distinguished from their counterparts in control cells. Kinetochore position after detachment is the primary determinant of the pole to which that kinetochore will orient. Therefore, since the experimenter determines kinetochore position, he can cause any given half-bivalent to segregate to a predetermined pole at anaphase I. Similarly, orientation of both half-bivalents to the same pole can be induced. These mal-oriented bivalents invariably reorient and normal anaphase segregation ensues. Non-disjunction can, however, be produced directly in late anaphase. These experiments are based upon current views of orderly chromosome distribution; their success confirms our understanding of the fundamental orientation process.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Efficient Chromosome Biorientation and the Tension Checkpoint in Saccharomyces cerevisiae both Require Bir1

Accurate chromosome segregation requires the capture of sister kinetochores by microtubules from opposite spindle poles prior to the initiation of anaphase, a state termed chromosome biorientation. In the budding yeast Saccharomyces cerevisiae, the conserved protein kinase Ipl1 (Aurora B in metazoans) is critical for ensuring correct chromosomal alignment. Ipl1 associates with its activators Sli15 (INCENP), Nbl1 (Borealin), and Bir1 (Survivin), but while Sli15 clearly functions with Ipl1 to promote chromosome biorientation, the role of Bir1 has been uncertain. Using a temperature-sensitive bir1 mutant (bir1-17), we show that Bir1 is needed to permit efficient chromosome biorientation. However, once established, chromosome biorientation is maintained in bir1-17 cells at the restrictive temperature. Ipl1 is partially delocalized in bir1-17 cells, and its protein kinase activity is markedly reduced under nonpermissive conditions. bir1-17 cells arrest normally in response to microtubule depolymerization but fail to delay anaphase when sister kinetochore tension is reduced. Thus, Bir1 is required for the tension checkpoint. Despite their robust mitotic arrest in response to nocodazole, bir1-17 cells are hypersensitive to microtubule-depolymerizing drugs and show a more severe biorientation defect on recovery from nocodazole treatment. The role of Bir1 therefore may become more critical when spindle formation is delayed.Accurate chromosome segregation during anaphase is vital for ensuring the maintenance of genome integrity during cell division and, in turn, depends critically on the correct attachment of sister chromatids to kinetochore microtubules. For high-fidelity chromosome segregation, kinetochores must capture spindle microtubules such that sister chromatids are connected to opposite spindle poles (termed amphitelic attachment or chromosome biorientation), ensuring that they are pulled in opposite directions during the subsequent anaphase.In the budding yeast Saccharomyces cerevisiae, the majority of sister chromatids remain attached to microtubules from a single pole (mono-oriented) without the intervention of a correction mechanism to promote amphitelic attachment (36), a key element of which is the Ipl1 protein kinase. Ipl1 has been proposed to promote the detachment of incorrect microtubule-kinetochore connections so that correct attachments subsequently can form (35). In the absence of Ipl1 function, at the point of anaphase onset around two-thirds of sister chromatids remain mono-oriented, attached to microtubules originating from a single pole to which they then cosegregate (35). Kinetochore proteins such as Dam1 and Ndc80 have been proposed as key Ipl1 substrates for their role in promoting chromosome biorientation (6, 41). Ipl1 kinase also is required for cells to activate the spindle checkpoint in the absence of tension on kinetochore-microtubule attachments, and hence ipl1 mutant cells fail to delay anaphase despite their many mono-oriented chromosomes (2). Depending on the circumstances, the checkpoint role of Ipl1 involves either the generation of unattached kinetochores (26) or the phosphorylation of the checkpoint protein Mad3 (19). Ipl1 also is required in the absence of the BimC family kinesin Cin8p, probably reflecting a role in spindle assembly (9, 21), and is involved in regulating spindle disassembly following anaphase (5).Ipl1 kinase is highly conserved, and its metazoan ortholog (Aurora B) is involved in both chromosome biorientation and the spindle assembly checkpoint, forming part of the chromosomal passenger complex that also contains INCENP, Survivin, and Borealin (27, 40). The chromosomal passenger complex is so called because although these proteins colocalize throughout the cell cycle, their location changes dynamically from the chromosome arms in G₁ to centromeres in prometaphase and finally to the central spindle in anaphase. Such coordinated behavior is consistent with the recent crystal structure of the complex between INCENP, Survivin, and Borealin, in which they interact via tightly entwined helical domains (16).In budding yeast, Ipl1 interacts with Sli15, Bir1, and Nbl1, which have been proposed to be orthologs of INCENP, Survivin, and Borealin, respectively (6, 18). All three proteins are the products of essential genes. Like INCENP, Sli15 has a conserved C-terminal domain (the IN-box) that is required for Ipl1 kinase activation, and sli15 mutants have a phenotype that is very similar to that of ipl1 mutants (17, 18). Although yeast cells with reduced Bir1 function show chromosome instability, the first-described bir1 mutants failed to reveal a chromosome biorientation defect but instead conferred defects in septin dynamics during anaphase (38). Bir1 interacts with Ndc10 and is responsible for taking Ndc10 to the anaphase spindle (38, 42, 43), a role that may be linked to this septin defect (4). Yeast Bir1 is much larger than its metazoan counterpart (Survivin) and shows little sequence conservation outside the conserved BIR domain, yet this region is nonessential in yeast (42) and therefore unlikely to be involved in chromosome biorientation. Conversely, metazoan Borealin proteins are much larger than yeast Nbl1, which consists of little more than the helical region proposed to form the tight interaction with INCENP/Sli15 and Survivin/Bir1 complexes. Furthermore, a significant fraction of both Sli15 and Bir1 are present in a complex that lacks Ipl1 (29, 38) and that recent work has shown to contain Nbl1 (25), bringing into question the importance of Bir1 for chromosome biorientation. The extent to which Bir1 and Survivin function in conserved or analogous ways within the chromosomal passenger complexes of yeast and metazoans therefore was unclear at the start of our work.The Sli15-Bir1 complex has been proposed to interact both with microtubules (via the central domain of Sli15) and with kinetochores (through the Bir1-Ndc10 interaction) and through these interactions to function as a tension sensor, relaying information concerning the state of microtubule-kinetochore connections to Ipl1 kinase. Thus, when chromosomes are mono-oriented, the Bir1-Sli15-Nbl1 complex might activate Ipl1 in the absence of tension so as to promote chromosome biorientation by detaching incorrect microtubule attachments (29). This model predicts an essential role for Bir1 in promoting chromosome biorientation, but such evidence has been lacking. By generating a temperature-sensitive bir1 allele (bir1-17) and showing that it confers a profound defect in chromosome biorientation, we demonstrate that Bir1 does play a key role in the correction process needed to ensure that all yeast chromosomes become correctly aligned on the mitotic spindle. Furthermore, since the bir1-17 mutant fails to activate the spindle assembly checkpoint properly in response to reduced sister kinetochore tension, like Ipl1 it forms part of the tension checkpoint mechanism. Our data therefore are consistent with a role for Bir1 in conferring tension responsiveness on Ipl1 function.