Cell, Vol. 70, 451-459,
August
7, 1992, Copyright
0 1992 by Cell Press
Kinesin-Related Proteins Required for Structural Integrity of the Mitotic Spindle William S. Saunders and M. Andrew Department of Biology The Johns Hopkins University Baltimore, Maryland 21218
Hoyt
Summary For S. cerevisiae cells, the assembly of a bipolar mitotic spindle requires the action of either Cin8p or Kipl p, gene products related to the mechanochemical enzyme kinesln. In this paper we demonstrate that the activity of either one of these proteins is also required following spindle assembly. When their function was eliminated, preanaphase bipolar spindles rapidly collapsed, with previously separated poles being drawn together. In contrast, anaphase spindles were apparently resistant to collapse. Deletion of kinesin-related KM3 partially suppressed the phenotypes associated with loss of Cin8plKiplp function. Our findings sug gest that the structure of the preanaphase bipolar spindle is maintained by counteracting forces produced by kinesin-related proteins. Introduction The mitotic spindle is unique among subcellular structures in the extent of its morphological dynamics. In every mitotic cycle, the spindle undergoes a well-defined program of complex structural changes, many of which produce motile force (reviewed in Hyams and Brinkley, 1989; Inoue, 1981; McIntosh and Pfarr, 1991; Nicklas, 1988). In anaphase, spindle structural changes cause the physical segregation of replicated chromosomes. Prior to anaphase, the cell assembles a bipolar spindle competent to perform segregation. Our view of the preanaphase bipolar spindle is that of a structure subjected to self-generated mechanical tension. Various treatments that physically perturb the structure of these spindles cause immediate motile responses (see Discussion for examples). The close relationship between force-generating mechanisms and spindle structure suggested by these observations was examined in the experiments reported in this paper. The assembly of the bipolar spindle from two halfspindles produces motility and therefore presumably requires a force-generating mechanism. During assembly, the microtubules from duplicated spindle poles interact in a manner such that the poles are separated from each other and an antiparallel microtubule array is established between them. It is to this array that the kinetochores of duplicated chromosomes make bipolar connections prior to segregation in anaphase. Recently, we and others demonstrated that the assembly of the Saccharomyces cerevisiae spindle requires the actions of the C/N8 and K/P 7 gene products (Hoyt et al., 1992; Roof et al., 1992). Cin8p and Kiplp both contain a region of high primary sequence similarity to the force-
producing (or “mote?‘) domain of the microtubule-based mechanochemical enzyme kinesin. Similar kinesin-related gene products, bimC and cut7, are required for spindle assembly in Aspergillus nidulans and Schizosaccharomyces pombe, respectively (Enos and Morris, 1990; Hagan and Yanagida, 1990). From our studies, two observations suggested that Cin8p and Kiplp redundantly perform an essential function. First, although double deletion mutants were inviable, each single deletion mutant strain grew well at most temperatures. Deletion of the K/P7 gene (/@l-A) caused no detectable phenotype. Deletion and missense alleles of C/N8 caused a block in mitosis, but only at 37X, near the upper limit for S. cerevisiae growth. Second, the temperature sensitivity of cin8-A mutants could be suppressed by additional copies of K/P 7. When shifted to the nonpermissive temperature early in the cell cycle, cin8 single mutants and viable cind temperaturesensitive kipl-A double mutants arrested growth with duplicated, but unseparated spindle poles. Cin8p and Kipl p were localized to the spindle microtubules that lie between the poles, suggesting that these proteins produce a poleseparating force from within the spindle (Hoyt et al., 1992; Roof et al., 1992). Another S. cerevisiae kinesin-related gene, KAR3, has been linked to mitosis by mutant phenotype (Meluh and Rose, 1990). Originally identified by its requirement for karyogamy (nuclear fusion during mating), KAR3 also has a nonessential but important mitotic role. kar3-A mutants are viable but appear to have difficulty traversing M-phase. The findings reported in this paper reveal that CinEpl Kipl p function is required not only for spindle assembly, but also at a subsequent stage in mitosis. The action of one or the other of these gene products is required to maintain the integrity of the assembled bipolar spindle. Following spindle assembly, CinEp and Kipl p apparently act to oppose a force that draws separated poles back together. We present evidence that Kar3p contributes to this inwardly directed force. These findings suggest that the preanaphase bipolar spindle is stabilized by counteracting mechanical forces generated by kinesin-related proteins. Results Requirement for either Cin8p or Kiplp Following Spindle Assembly We previously demonstrated that the function of either Cin8p or Kiplp is required for the assembly of a bipolar spindle from duplicated poles (Hoyt et al., 1992; Roof et al., 1992). We next examined the effect of loss of Cin8p and Kipl p function on preanaphase spindles assembled under permissive conditions. Unlike many eukaryotic cells, S. cerevisiae will assemble a bipolar spindle even if DNA replication is blocked (Pringle and Hartwell, 1981). In contrast, entry into anaphase, characterized by elongation of the spindle, requires the completion of DNA synthesis. Cells arrested with the DNA synthesis inhibitor hydroxy-
Cell
452
0
20
40
60
60
100
Minutes Figure 1. Nuclear Division missive temperature
after Release
from Hydroxyurea
lo Nonper-
Ceils of the genotypes indicated below were synchronized with hydroxyurea at 26% and released into fresh medium at 37OC. The percentage of total cells with a large-budded mononucleate morphology was determined as a function of time at 37%. Monitoring the frequency of large-budded binucleate cells in the cultures yielded essentially reciprocal curves (data not shown). Identical results were obtained in several experimental trials. Genotypes and strains utilized: wild type (MAY1094) (triangles); kipl-A (MAY2077) (open squares); cin8-3 (MAY1562) (open circles); cinbA (MAY2061) (closed circles); cin8-3 /@i-A (MAY2075) (closed squares).
urea are large budded and mononucleate and possess a short bipolar spindle that spans the nucleus (see Figures 2A and 3A). Cells of various genotypes were synchronized with hydroxyurea at 26% and then released into fresh media. Their ability to pass through mitosis was examined by following the loss of large-budded mononucleate cells from each culture and their replacement with large-budded binucleate cells. Cells of all genotypes were able to proceed efficiently through mitosis after release into media at 26% (data not shown) as were wild-type and kipl-A cells when released at 37% (Figure 1). Previously we observed that cin8 mutants were unable to perform spindle assembly at 37% (Hoyt et al., 1992). In contrast, when released from the hydroxyurea block, both cin8-3 (a temperaturesensitive allele) and cinb-A mutant strains could complete mitosis at 37X, but at a reduced rate relative to wild type (Figure 1). Although Cin8p is required to assemble a short spindle at 37%, its action is apparently no longer necessary once the preanaphase spindle is formed. In contrast, cin8-3 kipl-A double mutants were completely blocked for mitosis following the shift from hydroxyurea into 37%. Therefore, the action of either Cin8p or Kiplp is required for cells to transit from the hydroxyurea arrest point through mitosis. To determine the nature of the mitotic defect in the cin8-3 kipl-A double mutants, we examined the cells by electron microscopy (Figure 2). Double mutant cells arrested with hydroxyurea at 26% possessed short bipolar spindles that appeared normal. These extended between spindle pole bodies (SPBs) separated to opposite sides of the nucleus (Figure 2A). After a shift to 37% for 30 min, bipolar spindles were rarely observed (only one out of ten cells with two poles visible). SPBs were no longer separated to
Figure
2. Electron
Microscopic
Analysis
of Mutant
Cells
cin8-3 !iipl-A cells (MAY2169) were treated as described below, fixed, and examined by thin-section electron microscopy. (A) shows cells arrested with hydroxyurea at 26%; in (6) and (C), cells received the same treatment as in (A), but then were incubated at 37% for 30 min. Arrowheads point to each SPB. Bar, 0.5 pg.
opposite sides of the nucleus, but had now assumed a side-by-side position (Figures 28 and 2C). Long microtubules extending from the poles into the nucleus were usually visible. These observations suggested that previously separated spindle poles had become drawn back together following the shift to 37% When separated SPBs are brought back together by nocodazole-induced microtubule depolymerization, they assume a nuclear face-to-face orientation (Jacobs et al., 1988). In the cinb3 kipl-A cells,
Kinesin-Related
Proteins
and Spindle
Structure
453
Figure 3. lmmunofluorescence Mutant Cells
Microscopy
of
Cells were treated as described below, fixed, and examined by immunofluorescence microscopy. (A) and(C) show antitubulin staining, and (B) and (D) show the same cells stained with the DNA-specific dye DAPI. (A)and (B)show acirt&3kipl-A cell (MAY2169) arrested with hydroxyurea at 26%; (C)and (D), same as (A), but after subsequent incubation at 37% for 10 min. Five mononucleate cells are depicted. Bar in (A) applies to (A)-(D) and equals 5 pm. (E)-(H) show cells stained with antibodies directed against the 90 kd SPB component. (E) shows CM-3 kipl-A cells (MAY2169) arrested with hydroxyurea at 26W; (F), same as(E), but after subsequent incubation at 37% for 15 min; (G), cin8-3 &ipl-A kar3-A cells (MAY2375) arrested with hydroxyureaat 26%; (H), same as(G), but after subsequent incubation at 37’C for 15 min. Note the presence of two cells in (H) with a double dot staining pattern indicating that their spindles have not collapsed. Bar in (E) applies to (E)(H) and equals 10 urn.
the SPBs assumed a side-by-side rather than a face-toface orientation, possibly owing to interference from the microtubules extending out from the SPB nuclear face. The electron microscopic observations suggested that bipolar spindles formed in the presence of hydroxyurea were collapsing inwardly upon a shift to 37%. To investigate this possibility, immunofluorescence microscopy was performed on populations of cells, allowing quantitative analysis of spindle morphologies. For this study, we used an antibody preparation directed against a 90 kd protein component of SPBs (Rout and Kilmartin, 1990). In cells possessing short preanaphase spindles (i.e., hydroxyurea-arrested cells), staining with the anti-90 kd antibody revealed two dots on opposite sides of the nucleus (Figures 3E and 3G). Cells with a single SPB or side-by-side SPBs showed only a single dot (Figure 3F and Experimen-
tal Procedures). After hydroxyurea synchronization, but prior to a temperature shift, 70% of the cin8-3 kipl-A double mutant cells contained two clearly distinguishable dots of anti-90 kd-stained material. After 10 min at 37% only 5% of the cells had two dots. (See Table 1 for a similar experiment.) Therefore, we can conclude that a large fraction of separated SPBs were brought back together after the temperature shift. Similarly treated wild-type cells did not show a significant drop in double dot cells (Table 1). Similar results were obtained when spindle morphologies were scored using antitubulin staining. Prior to a temperature shift, the double mutant cells possessed mostly short spindles, consisting of a brightly stained bar of nuclear microtubules, with fainter staining cytoplasmic microtubules connected to each end (Figure 3A). After as little as 5 min at 37% the observed frequency of bipolar
Cell 454
Table 1. Separation Hydroxyurea
of Spindle
Pole Bodies
after
Release
from
Percent Cells with Separated Poles Strain MAY591 MAY2169 MAY2375 MAY2035
Genotype Wild type
26OC
33%
37v
50 22 36 45
57 4.5 25 50
cinll-3 kipl-A
KAR3
60 56
cin&3
kar3-A
46
kar3-A
kipl-A
54
Cells of the indicated genotype were synchronized with hydroxyurea at 26V and released at 33%’ or 37%. Samples were taken from before and 15 min after the temperature shift were fixed. These were stained with the antibody directed against the 90 kd SPB component and examined by immunofluorescence microscopy. The percentage of cells with two clearly separated spindle poles was determined. Between 300 and 800 cells were counted for each sample.
cells dropped at least 1O-fold (Figure 3C). A similar phenotype was observed for five additional cin8 temperaturesensitive alleles when combined with kipl-A (N. Cole, W. S. S., and M. A. H., unpublished observations). This indicated that the spindle collapse phenotype is not the property of an unusual allelic form of cin8. Spindles in hydroxyurea-synchronized cin8-3 kipl-A cells were observed to collapse rapidly upon a shift to 37OC independently of whether or not hydroxyurea was removed. This indicated that cell cycle progression beyond the hydroxyurea arrest point was not required in order for spindle collapse to occur. In addition, this change in spindle structure was not a consequence of hydroxyurea
pretreatment; collapse could be observed in untreated culmicrotures as well. Antitubulin immunofluorescence scopic examination of a 26OC asynchronous cin8-3 kipl-A culture revealed that 16% of the cells possessed spindles of approximately the same size (1.5 pm between poles) and morphology as hydroxyurea-arrested cells. Following a shift to 37V for 10 min, less than 1% of the cells had identifiable short spindles, and in 94% of the cells, the spindles appeared monopolar. Therefore, nearly all of the short spindles in the culture had lost their structure during the temperature shift. Anaphase Spindles Are Resistant to Collapse culture described In the asynchronous cin&3 kipl-A above, the fraction of cells with bipolar spindles longer than 1.5 pm remained about constant before and after a 10 min shift to 37OC (3.5% before and 5% after). This suggests that spindles that had begun anaphase were resistant to collapse triggered by loss of CirBp/Kipl p function. In support of this possibility, we have found that spindles in cells arrested at late anaphase are resistant to collapse. Itel-A mutant strains arrest growth at 11 “C (Wickner et al., 1987) with long spindles (approximately 8-10 pm) connecting two segregated chromosomal masses (E. Woodhouse and M. A. H., unpublished observations). Itel-A cin8-3 kipl-A triple mutants express both cold-sensitive and temperature-sensitive arrest phenotypes. Triple mutants were shifted to 11 OC for 24 hr, resulting in a population of cells containing long spindles (approximately 80%; Figures 4A and 46). The cells were then shifted from 1 l°C directly to 37OC for various
Figure 4. Antitubulin lmmunofluorescence croscopy of ffel cin8 kipl Cells
Mi-
Strain MAY2282 (Itel-A cin8-3 kipl-A) was grown at 11% for 24 hr (A and B) and then shifted directly to 37% for 15 min (C and D). Cells are stained with antitubulin (A and C) and for DNA with DAPI (B and D). Bar, 10 pm.
Kinesin-Related 455
Proteins
and Spindle
26’
Structure
30’
33’
35’
37’
ing to the collapsing force. The inviability tant strain at 37% (Figure 5) is probably in bipolar spindle assembly. Triple mutant this temperature prior to spindle assembly with unseparated poles (data not shown).
of the triple mudue to a defect cells shifted to arrested growth
Discussion
Figure
5. Temperature
Resistance
of Mutant
Strains
Cells of the indicated genotypes were suspended in water and spotted onto solid rich medium at the indicated temperatures. Strains: wildtype (MAY591); cin&3 kipl-A (MAY2082); kar3-A cin8-3 kipbA (MAY2324).
amounts of time and examined by immunofluorescence microscopy. Following the shift to 37%, the spindles in triple mutant cells did not collapse (Figures 4C and 40). Instead, the cells completed mitosis as efficiently as did similarly treated kel-A single mutants; after 3 hr at 37%, approximately half of the cells of each genotype had recovered from the cold-induced arrest and completed mitosis. While it is conceivable that the Ire7 mutation caused changes to the spindles rendering them resistant to collapse, the reversibility of the ltel phenotype and the asynchronous culture experiment described above argue against this possibility. Therefore, we conclude that elongated bipolar spindles are stabilized against collapse and CinBplKipl p function is no longer required for completion of mitosis following the ltel arrest point.
kar3-A Suppresses
cin8 kipl In an attempt to identify gene products that interact with Cin8p and Kiplp, we have selected mutations that supphenotype. press the cin8-3 kipl-A temperature-sensitive Seven extragenic suppressors analyzed were found to be mutant alleles of another S. cerevisiae kinesin-related gene, KAR3. The identification and characterization of these kar3 alleles will be published elsewhere. This finding prompted us to test the effect of a kar3 loss-of-function allele. Deleting KAR3 in a cin&3 kipl-A background clearly had a beneficial effect on temperature resistance (Figure 5). Although not as resistant as wild type, the constructed triple mutant strains grew well at 33% a temperature that inhibited cin8-3 kipl-A cells. kar3-A was able to increase the temperature resistance of cin8-7 kipl-A cells as well (data not shown). The observed suppression of cin8-3 kipl-A temperature sensitivity suggested that the deletion of KAR3 might prevent spindle collapse. Cells of various genotypes were synchronized with hydroxyurea at 26% and transferred to 33OC or 37% for 15 min. The number of cells with separated spindle poles was determined before and after the shift by using the anti-90 kd antibody (Table 1). The frequency of collapsed spindles was greater in the cin8-3 kipl-A KAR3 strain than in the cin8-3 kipl-A kar3-A strain. At 37%, less than 10% of the KAR3 spindles remained intact, compared with greater than 50% of the kar3-A spindles. This finding suggests that Kar3p is directly contribut-
The Role of Cin8p/Kiplp in the Preanaphase Spindle Cin8p and Kipl p redundantly perform a role essential for mitotic spindle function. In their absence, the phenotypes observed are most simply interpreted as resulting from the loss of a force-producing mechanism. The action of at least one of these proteins is required for pole separation during assembly of the bipolar spindle. After the spindle is assembled, this activity is also required to resist an inwardly directed force acting upon the separated poles. From these findings and the relationships of Cin8p and Kiplp to the mechanochemical enzyme kinesin, it is reasonable to conclude that these proteins produce a pole-separating force. Cin8pand Kipl p have both been localized to the spindle fibers that lie between the poles, suggesting that they produce force from within the spindle (Hoyt et al., 1992; Roof et al., 1992). Observations of spindles in numerous eukaryotic cell types revealed that they possess a conserved architecture (reviewed in McDonald, 1989). Each halfspindle consists of a single pole with attached microtubules of the same polarity (plus ends out). These overlap in the midzone with microtubules of opposite polarity originating from the other pole. Experimental observations have suggested that forces that push spindle poles away from each other are produced by interactions between the antiparallel midzone microtubules (reviewed in Hogan and Cande, 1990). In the simplest formulation for CinbplKipl p function, they act by cross-linking the overlapping antiparallel microtubules and sliding them past one another to generate an outward force (Figure 6). This force is first utilized to separate duplicated poles and then is required to oppose the inwardly directed force acting upon the poles prior to anaphase. We propose that Cin8p and Kipl p constitute or contribute to bridges between the two half-spindles. Bridges bein electween midzone microtubules have been ObSeNed tron micrographs of diatom spindles (McDonald et al., 1979). As represented in Figure 6, Cin8p and Kipl p may each possess two microtubule-binding domains. Alternatively, these polypeptides may participate in higher order complexes that bridge the half-spindles. These proteins could operate on overlapping nonkinetochore microtubules (nkMTs) as depicted (Figure 6) or on kinetochore microtubules (kMTs). The only requirement for outward force generation is that the interacting microtubules be in an antiparallel configuration. We have not yet determined the direction of force generation by Cin8p and Kipl p with respect to microtubule polarity. While it is hypothetically possible to drive poles apart with either a plus end- or a minus end-directed activity, a plus end activity, like that of kinesin, is strongly favored by our understanding of spindle dynamics (Figure 6). In
Cell 456
SPB Figure
6. Model for CinEplKipl
p Function
in the Preanaphase
Spindle
Cin8p and Kiplp, represented by the “lollypop” structures, are proposed to function by cross-linking antiparallel microtubules and sliding them past each other (see Discussion). The thin lines represent kinetochore, nonkinetochore, and cytoplasmic microtubules (kMT, nkMT, and cMT. respectively). The motor domains (round part of the motor proteins) are proposed to move along the microtubules toward the plus ends (in the direction of the closed arrows). The stalk represents a second domain involved in a static interaction with an antiparallel microtubule. This produces an outwardly directed force that separates the duplicated spindle pole bodies (SPB) and opposes an inwardly directed force (open arrows). A component of the inwardly directed force appears to be produced by Kar3p. The kMTs may also contribute to the inwardly directed force (see Discussion).
order for a minusend-directed activity to push poles apart, it must act by extruding a microtubule from the pole in toward the center of the spindle. As poles separate, tubulin subunits would have to be added at the poleward minus ends, a process that has not been observed. Numerous studies have demonstrated that tubulin subunits are added primarily at the plus end8 of spindle fibers in vivo and in vitro (reviewed in Hogan and Cande, 1990; Salmon, 1989). The Inwardly Directed Force Our findings demonstrate that the action8 of Cin8p and Kipl p are opposing a force that draws separated spindle poles back together. A number of other experimental observations support our finding of a compressive force acting upon the poles. Severing of diatom, newt, and mammalian metaphase spindles with ultraviolet microbeam irradiation was observed to cause spindle buckling or collapse (Leslie and Pickett-Heaps, 1983; Spurck et al., 1990). Treatment of mammalian cells in culture with nocodazole at low concentration preferentially depolymerized nkMTsand caused metaphase spindle shortening (Snyder et al., 1985). Nocodazole treatment has also been observed to cause S. cerevisiae spindle poles to collapse together (Jacobs et al., 1988). It is probable that the inward force evident upon inactivation of Cin8p and Kiplp is of the same origin as these other compressive forces.
There are two pole-to-pole microtubule connections, the kMTs and the nkMTs, through which the inwardly directed force may be applied. The nkMTS connect the poles through antiparallel interactions in the spindle midzone. Experimental observations have suggested that interactions between the antiparallel nkMT microtubules produce the forces that push spindle poles outwardly (reviewed in Hogan and Cande, 1990) although a role in inward force generation cannot be ruled out. An attractive hypothesis is that the inwardly directed force is mediated through the kMTs. The kMTs connect the two poles prior to anaphase through the paired kinetochores of sister chromatids. When chromosomes establish a bipolar connection, they are pulled simultaneously toward each pole; irradiation of one of the paired kinetochores in a metaphase chromosome caused the chromatids to move to the pole opposite to the irradiated kinetochore (McNeil and Berns, 1981). These balanced poleward forces should therefore be acting on the poles to pull them in toward each other. Indeed, spindle shortening has been observed at metaphase when chromosomes establish bipolar connections (LaFountain, 1972; Taylor, 1980). Therefore, force8 that drive kinetochores toward poles may be contributing to spindle collapse when the outwardly directed Cin8pl Kipl p function is lost. As structural detail for the S. cerevisiae spindle is lacking, however, we do not know whether the chromosomes in our hydroxyurea-inhibited cells had assumed a bipolar connection. If spindle collapse is caused by forces acting on the kinetochores, then this force should abate upon the dissolution of the sister kinetochore connections at anaphase. Our observations suggest that upon entering anaphase, spindles become resistant to collapse despite the loss of Cin8plKipl p function. Similarly, when anaphase spindles were cut by microbeam irradiation, the compressive force observed acting upon metaphase poles was no longer present (Spurcket al., 1990). Alternatively, if the collapsing force is mediated through nkMTs, it may be eliminated in anaphase simply by attenuating the activity of an inward motor function. In anaphase B, poles are separated further, resulting in an often extreme elongation of the spindle. We have demonstrated a requirement for Cin8plKipl p function for pole separation prior to anaphase. This finding suggests the possibility that these proteins also contribute to pole separation during anaphase. We observed that the rate of progression from the hydroxyurea arrest point through mitosis was reduced for the cin8 single mutant strains at 37% (Figure l), perhaps indicating an anaphase role for Cin8p. Kar3p Acts Antagonistically to CinlplKipl p Loss of KAR3 function was found to partially suppress the temperature sensitivity and spindle collapse associated with the cin8-3 kip7-A genotype. The simplest interpretation of this finding is that the kinesin-related Kar3p directly contributes to the inwardly directed force. Since our knowledge of the mitotic role and localization of Kar3p is limited, we can only speculate as to how it may be producing this force. One possibility is that Kar3p is positioned at kineto-
Kinesin-Related 457
Table 2. Yeast
Proteins
and Spindle
Structure
Strains
Strain
Relevant
MAY591 MAY 1094 MAY 1397 MAY 1562 MAY2035 MAY2061 MAY2075 MAY2077 MAY2082 MAY2169 MAY2282 MAY2324 MAY2375
CIN8 K/PI KAR3 LTEl C/N8 K/PI KAR3 LTE7 /fel::URA3 cin8-3 kaA-A 102::LEU2 cin8::LJRAS cinB3 kipl:tH/SS kip I::H/.S3 cin8-3 kipl::H/SS cin8-3 kipl::H/S3 cin8-3 kipl::H/S3 /tel::URAS cinB-3 kipl::H/SS kar3-A 702::LEU2 cin&3 kip l::H/S3 kar3-A 102::LEU2
Genotype
chores and exerts a poleward (or minus end-directed) force. Alternatively, it could be cross-linking antiparallel midzone microtubules, as has been proposed for Cin8pl Kipl p, but exerting force in the direction opposite to Cin8pl Kiplp. In consideration of either of these possibilities, the similarity of Kar3p to the minus end-directed ncd motor protein from Drosophila should be noted (Rose, 1992; Endow et al., 1990; McDonald and Goldstein, 1990; McDonald et al., 1990; Walker et al., 1990). We conclude that Cin8p and Kipl p, components of the mitotic spindle required for a motility event, are apparently essential structural elements as well. These findings contribute to the view that spindle structure is generated and maintained by opposing mechanical forces. Of particular novelty is our conclusion that two antagonistic forces acting upon the spindle poles are produced by kinesin-related proteins. Experimental
Procedures
Yeast Strains and Media The yeast strains used in these experiments are derivatives of S288C and are listed in Table 2. Different strains with the same CIN8, K/PI, KAR3, and LTE7 genotypes behaved identically in assays for temperature resistance and spindle collapse. The kar3 deletion allele, kar3A102:tLEU2, removes almost the entire reading frame (Meluh and Rose, 1990) and was supplied by M. Rose and P. Meluh. kar3-A102:: LEU2 was introduced into cin8-3 kipl-A strains by standard genetic crosses. Suppression of the cin8-3 kipl-A temperature-sensitive phenotype was found to be 100% linked to the LEU2 marker. In various crosses in which both genotypes segregated, 39 cin8-3 kipl-A double mutants and 36 cin&3 kipl-A kar3-A702::LEU2 triple mutants were obtained. All triple mutants were more temperature resistant than the double mutants. The Itel::URAS allele was supplied by R. Wickner and is described in Wickner (1987). Rich medium (YPD) was as described (Sherman et al., 1983). Hy droxyurea (Sigma) was added to log-phase cultures in YPD (pli 5.8) to a final concentration of 0.1 M. The cultures were then incubated at 26°C until more than 70% of the cells had assumed a large-budded morphology (4-5 hr). Cells were released from arrest by centrifugation and resuspension in the same medium minus the inhibitor. Microscopic Analysis of Cells To determine the distribution of cell morphologies, culture samples were briefly fixed in 70% ethanol and stained for DNA with 0.5 mg/ml4, Wiamidino-2-phenylindole (DAPI) plus 1 mg/ml p-phenylenediamine (used as an anti-fade agent). Using differential interference contrast optics, cells were scored as unbudded, small budded (bud size roughly
50% size of mother). The number of nuclei per cell wasdetermined by epifluorescent illumination. Microtubule structures were observed following formaldehyde fixation using the anti-tubulin monoclonal antibody YOL1134 (Kilmartin et al., 1982; Eioproducts for Science, Inc.) and a fluorochrome-conjugated secondary antibody as previously described (Stearns et al., 1990). Spindle length was measured using a calibrated eyepiece reticule. The SPB-associated 90 kd antigen (Rout and Kilmartin, 1990) was detected by using an undiluted pooled sample of monoclonal antibodies directed against this polypeptide (gift from J. Kilmartin). We obtained best results by performing the primary antibody incubation at 37OC for 30 min. The secondary antibody was a 1:50 dilution of FITCconjugated goat anti-mouse immunoglobulin G (Cappel). Because 90 kd antigenicity was sensitive to formaldehyde treatment, short fixation times of 5-10 min were used (in 3.7% formaldehyde, 50 mM KPO. (pH 6.8) 150 mM NaCI). Control experiments indicated that cells with a single SPB (i.e, a-factor arrested cells) or duplicated but unseparated SPBs (i.e., early cell cycle-shifted cin8-3 kipl-A cells) yielded a single dot of stained material. Cells with separated SPBs showed double dots (also see Rout and Kilmartin, 1990). Cells were prepared for thin-section electron microscopy as previously described (Hoyt et al., 1992). Acknowledgments We gratefully thank John Kilmartin for the gift of antibodies, Mark Rose and Pam Meluh for the kar3 mutation, Mike Sepanski for assistance with the electron microscopy, and Don Cleveland, Doug Koshland, and Tibor Roberts for comments on the manuscript. This work was supported by National Institutes of Health grant GM40714 awarded to M. A. H. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received
February
12, 1992;
revised
June
1, 1992
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Leslie, R. J., and Picket&Heaps, J. D. (1983). Ultraviolet microbeam irradiations of mitotic diatoms: investigations of spindle elongation. J. Cell Biol. 96, 548-581.
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McDonald, H. B., and Goldstein, L. S. B. (1990). Identification and characterization Of a gene encoding a kinesin-like protein in Drosoph. ila. Cell 67, 991-1000. McDonald, H. B., Stewart, Ft. J., and Goldstein, L. S. B. (1990). The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor. Cell 63, 1159-l 165. McDonald, K. (1989). Mitoticspindle ultrastructureanddesign. In Mitosis: Molecules and Mechanisms, J. S. Hyams and B. R. Brinkley, eds. (San Diego: Academic Press), pp. l-313. McDonald, K. L., Edwards, M. K., and McIntosh, J. R. (1979). Crosssectional structure of the central mitotic spindle of Diatoma vulgare. J. Cell Biol. 83. 443-461. McIntosh, J. Ft., and Pfarr, 115. 577-505.
C. M. (1991).
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McNeil, P. A., and Bern& M. W. (1981). Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtKP cells. J. Cell Biol. 88, 543-553. Meluh, P. B., and Rose, M. D. (1990). KAR3, a kinesin-related required for yeast nuclear fusion. Cell 60, 1029-1041. Nicklas, R. B. (1988). The forces that move Annu. Rev. Biophys. Chem. 17, 431-449.
chromosomes
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Pringle, J. R., and Hartwell. L. H. (1981). The Saccharomyces cerevisiae cell cycle. In The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, J. N. Strathern. E. W. Jones, and J. R. Broach, eds. (Cold Spring Harbor: Cold Spring Harbor Laboratory), pp. 97-142. Roof, D. M., Meluh, P. B., and Rose, M. D. (1992). proteins required for assembly of the mitotic spindle. press.
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Rose, M. D. (1992). The kinesin-related genes. In Guidebook to Cytoskeletal Proteins, T. Kreis and R. D. Vale, eds. (Oxford: Oxford University Press), in press. Rout, M. P., and Kilmartin, J. V. (1990). Components of the yeast spindle and spindle pole body. J. Cell Biol. 117, 1913-1927. Salmon, E. D. (1989). Microtubule dynamics and chromosome movement. In Mitosis: Molecules and Mechanisms, J. S. Hyams and B R. Brinkley, eds. (San Diego: Academic Press), pp. 119-181. Sherman, F., Fink, G. R., and Hicks, J. B. (1983). Methods in Yeast Genetics (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Snyder, J. A., Golub, R. J., and Berg, S. P. (1985). Role of nonkinetochore microtubules in spindle elongation in mitotic PtKl cells. Eur. J. Cell Biol. 39, 373379. Spurck, T. P., Stonington, 0. G., Snyder, J. A., Pickett-Heaps, Bajer, A., and Mole-Bajer, J. (1990). UV microbeam irradiations mitotic spindle. Il. Spindle fiber dynamics and force production. Biol. 777. 1505-1518.
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Stearns, T., Hoyt, M. A., and Botstein, D. (1990). Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function. Genetics 124, 251-262. Taylor, E. W. (1960). Sci. 90. 430-434.
Dynamics
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Walker, R. A., Salmon, E. D., and Endow, S. A. (1990). The Drosophila claret segregation protein is a minus end-directed motor molecule. Nature 347, 780-762. Wickner, R. B.. Koh, T. J., Crowley, J. C., Cl’Neil, J., and Kaback, D. B. (1987). Molecular cloning of chromosome I DNA from Saccharoomyces cerevisiae: isolation of the MAK7 gene and analysis of an adjacent gene essential for growth at low temperatures. Yeast 3. 51-57.
August
7, 1992, Copyright
0 1992 by Cell Press
Kinesin-Related Proteins Required for Structural Integrity of the Mitotic Spindle William S. Saunders and M. Andrew Department of Biology The Johns Hopkins University Baltimore, Maryland 21218
Hoyt
Summary For S. cerevisiae cells, the assembly of a bipolar mitotic spindle requires the action of either Cin8p or Kipl p, gene products related to the mechanochemical enzyme kinesln. In this paper we demonstrate that the activity of either one of these proteins is also required following spindle assembly. When their function was eliminated, preanaphase bipolar spindles rapidly collapsed, with previously separated poles being drawn together. In contrast, anaphase spindles were apparently resistant to collapse. Deletion of kinesin-related KM3 partially suppressed the phenotypes associated with loss of Cin8plKiplp function. Our findings sug gest that the structure of the preanaphase bipolar spindle is maintained by counteracting forces produced by kinesin-related proteins. Introduction The mitotic spindle is unique among subcellular structures in the extent of its morphological dynamics. In every mitotic cycle, the spindle undergoes a well-defined program of complex structural changes, many of which produce motile force (reviewed in Hyams and Brinkley, 1989; Inoue, 1981; McIntosh and Pfarr, 1991; Nicklas, 1988). In anaphase, spindle structural changes cause the physical segregation of replicated chromosomes. Prior to anaphase, the cell assembles a bipolar spindle competent to perform segregation. Our view of the preanaphase bipolar spindle is that of a structure subjected to self-generated mechanical tension. Various treatments that physically perturb the structure of these spindles cause immediate motile responses (see Discussion for examples). The close relationship between force-generating mechanisms and spindle structure suggested by these observations was examined in the experiments reported in this paper. The assembly of the bipolar spindle from two halfspindles produces motility and therefore presumably requires a force-generating mechanism. During assembly, the microtubules from duplicated spindle poles interact in a manner such that the poles are separated from each other and an antiparallel microtubule array is established between them. It is to this array that the kinetochores of duplicated chromosomes make bipolar connections prior to segregation in anaphase. Recently, we and others demonstrated that the assembly of the Saccharomyces cerevisiae spindle requires the actions of the C/N8 and K/P 7 gene products (Hoyt et al., 1992; Roof et al., 1992). Cin8p and Kiplp both contain a region of high primary sequence similarity to the force-
producing (or “mote?‘) domain of the microtubule-based mechanochemical enzyme kinesin. Similar kinesin-related gene products, bimC and cut7, are required for spindle assembly in Aspergillus nidulans and Schizosaccharomyces pombe, respectively (Enos and Morris, 1990; Hagan and Yanagida, 1990). From our studies, two observations suggested that Cin8p and Kiplp redundantly perform an essential function. First, although double deletion mutants were inviable, each single deletion mutant strain grew well at most temperatures. Deletion of the K/P7 gene (/@l-A) caused no detectable phenotype. Deletion and missense alleles of C/N8 caused a block in mitosis, but only at 37X, near the upper limit for S. cerevisiae growth. Second, the temperature sensitivity of cin8-A mutants could be suppressed by additional copies of K/P 7. When shifted to the nonpermissive temperature early in the cell cycle, cin8 single mutants and viable cind temperaturesensitive kipl-A double mutants arrested growth with duplicated, but unseparated spindle poles. Cin8p and Kipl p were localized to the spindle microtubules that lie between the poles, suggesting that these proteins produce a poleseparating force from within the spindle (Hoyt et al., 1992; Roof et al., 1992). Another S. cerevisiae kinesin-related gene, KAR3, has been linked to mitosis by mutant phenotype (Meluh and Rose, 1990). Originally identified by its requirement for karyogamy (nuclear fusion during mating), KAR3 also has a nonessential but important mitotic role. kar3-A mutants are viable but appear to have difficulty traversing M-phase. The findings reported in this paper reveal that CinEpl Kipl p function is required not only for spindle assembly, but also at a subsequent stage in mitosis. The action of one or the other of these gene products is required to maintain the integrity of the assembled bipolar spindle. Following spindle assembly, CinEp and Kipl p apparently act to oppose a force that draws separated poles back together. We present evidence that Kar3p contributes to this inwardly directed force. These findings suggest that the preanaphase bipolar spindle is stabilized by counteracting mechanical forces generated by kinesin-related proteins. Results Requirement for either Cin8p or Kiplp Following Spindle Assembly We previously demonstrated that the function of either Cin8p or Kiplp is required for the assembly of a bipolar spindle from duplicated poles (Hoyt et al., 1992; Roof et al., 1992). We next examined the effect of loss of Cin8p and Kipl p function on preanaphase spindles assembled under permissive conditions. Unlike many eukaryotic cells, S. cerevisiae will assemble a bipolar spindle even if DNA replication is blocked (Pringle and Hartwell, 1981). In contrast, entry into anaphase, characterized by elongation of the spindle, requires the completion of DNA synthesis. Cells arrested with the DNA synthesis inhibitor hydroxy-
Cell
452
0
20
40
60
60
100
Minutes Figure 1. Nuclear Division missive temperature
after Release
from Hydroxyurea
lo Nonper-
Ceils of the genotypes indicated below were synchronized with hydroxyurea at 26% and released into fresh medium at 37OC. The percentage of total cells with a large-budded mononucleate morphology was determined as a function of time at 37%. Monitoring the frequency of large-budded binucleate cells in the cultures yielded essentially reciprocal curves (data not shown). Identical results were obtained in several experimental trials. Genotypes and strains utilized: wild type (MAY1094) (triangles); kipl-A (MAY2077) (open squares); cin8-3 (MAY1562) (open circles); cinbA (MAY2061) (closed circles); cin8-3 /@i-A (MAY2075) (closed squares).
urea are large budded and mononucleate and possess a short bipolar spindle that spans the nucleus (see Figures 2A and 3A). Cells of various genotypes were synchronized with hydroxyurea at 26% and then released into fresh media. Their ability to pass through mitosis was examined by following the loss of large-budded mononucleate cells from each culture and their replacement with large-budded binucleate cells. Cells of all genotypes were able to proceed efficiently through mitosis after release into media at 26% (data not shown) as were wild-type and kipl-A cells when released at 37% (Figure 1). Previously we observed that cin8 mutants were unable to perform spindle assembly at 37% (Hoyt et al., 1992). In contrast, when released from the hydroxyurea block, both cin8-3 (a temperaturesensitive allele) and cinb-A mutant strains could complete mitosis at 37X, but at a reduced rate relative to wild type (Figure 1). Although Cin8p is required to assemble a short spindle at 37%, its action is apparently no longer necessary once the preanaphase spindle is formed. In contrast, cin8-3 kipl-A double mutants were completely blocked for mitosis following the shift from hydroxyurea into 37%. Therefore, the action of either Cin8p or Kiplp is required for cells to transit from the hydroxyurea arrest point through mitosis. To determine the nature of the mitotic defect in the cin8-3 kipl-A double mutants, we examined the cells by electron microscopy (Figure 2). Double mutant cells arrested with hydroxyurea at 26% possessed short bipolar spindles that appeared normal. These extended between spindle pole bodies (SPBs) separated to opposite sides of the nucleus (Figure 2A). After a shift to 37% for 30 min, bipolar spindles were rarely observed (only one out of ten cells with two poles visible). SPBs were no longer separated to
Figure
2. Electron
Microscopic
Analysis
of Mutant
Cells
cin8-3 !iipl-A cells (MAY2169) were treated as described below, fixed, and examined by thin-section electron microscopy. (A) shows cells arrested with hydroxyurea at 26%; in (6) and (C), cells received the same treatment as in (A), but then were incubated at 37% for 30 min. Arrowheads point to each SPB. Bar, 0.5 pg.
opposite sides of the nucleus, but had now assumed a side-by-side position (Figures 28 and 2C). Long microtubules extending from the poles into the nucleus were usually visible. These observations suggested that previously separated spindle poles had become drawn back together following the shift to 37% When separated SPBs are brought back together by nocodazole-induced microtubule depolymerization, they assume a nuclear face-to-face orientation (Jacobs et al., 1988). In the cinb3 kipl-A cells,
Kinesin-Related
Proteins
and Spindle
Structure
453
Figure 3. lmmunofluorescence Mutant Cells
Microscopy
of
Cells were treated as described below, fixed, and examined by immunofluorescence microscopy. (A) and(C) show antitubulin staining, and (B) and (D) show the same cells stained with the DNA-specific dye DAPI. (A)and (B)show acirt&3kipl-A cell (MAY2169) arrested with hydroxyurea at 26%; (C)and (D), same as (A), but after subsequent incubation at 37% for 10 min. Five mononucleate cells are depicted. Bar in (A) applies to (A)-(D) and equals 5 pm. (E)-(H) show cells stained with antibodies directed against the 90 kd SPB component. (E) shows CM-3 kipl-A cells (MAY2169) arrested with hydroxyurea at 26W; (F), same as(E), but after subsequent incubation at 37% for 15 min; (G), cin8-3 &ipl-A kar3-A cells (MAY2375) arrested with hydroxyureaat 26%; (H), same as(G), but after subsequent incubation at 37’C for 15 min. Note the presence of two cells in (H) with a double dot staining pattern indicating that their spindles have not collapsed. Bar in (E) applies to (E)(H) and equals 10 urn.
the SPBs assumed a side-by-side rather than a face-toface orientation, possibly owing to interference from the microtubules extending out from the SPB nuclear face. The electron microscopic observations suggested that bipolar spindles formed in the presence of hydroxyurea were collapsing inwardly upon a shift to 37%. To investigate this possibility, immunofluorescence microscopy was performed on populations of cells, allowing quantitative analysis of spindle morphologies. For this study, we used an antibody preparation directed against a 90 kd protein component of SPBs (Rout and Kilmartin, 1990). In cells possessing short preanaphase spindles (i.e., hydroxyurea-arrested cells), staining with the anti-90 kd antibody revealed two dots on opposite sides of the nucleus (Figures 3E and 3G). Cells with a single SPB or side-by-side SPBs showed only a single dot (Figure 3F and Experimen-
tal Procedures). After hydroxyurea synchronization, but prior to a temperature shift, 70% of the cin8-3 kipl-A double mutant cells contained two clearly distinguishable dots of anti-90 kd-stained material. After 10 min at 37% only 5% of the cells had two dots. (See Table 1 for a similar experiment.) Therefore, we can conclude that a large fraction of separated SPBs were brought back together after the temperature shift. Similarly treated wild-type cells did not show a significant drop in double dot cells (Table 1). Similar results were obtained when spindle morphologies were scored using antitubulin staining. Prior to a temperature shift, the double mutant cells possessed mostly short spindles, consisting of a brightly stained bar of nuclear microtubules, with fainter staining cytoplasmic microtubules connected to each end (Figure 3A). After as little as 5 min at 37% the observed frequency of bipolar
Cell 454
Table 1. Separation Hydroxyurea
of Spindle
Pole Bodies
after
Release
from
Percent Cells with Separated Poles Strain MAY591 MAY2169 MAY2375 MAY2035
Genotype Wild type
26OC
33%
37v
50 22 36 45
57 4.5 25 50
cinll-3 kipl-A
KAR3
60 56
cin&3
kar3-A
46
kar3-A
kipl-A
54
Cells of the indicated genotype were synchronized with hydroxyurea at 26V and released at 33%’ or 37%. Samples were taken from before and 15 min after the temperature shift were fixed. These were stained with the antibody directed against the 90 kd SPB component and examined by immunofluorescence microscopy. The percentage of cells with two clearly separated spindle poles was determined. Between 300 and 800 cells were counted for each sample.
cells dropped at least 1O-fold (Figure 3C). A similar phenotype was observed for five additional cin8 temperaturesensitive alleles when combined with kipl-A (N. Cole, W. S. S., and M. A. H., unpublished observations). This indicated that the spindle collapse phenotype is not the property of an unusual allelic form of cin8. Spindles in hydroxyurea-synchronized cin8-3 kipl-A cells were observed to collapse rapidly upon a shift to 37OC independently of whether or not hydroxyurea was removed. This indicated that cell cycle progression beyond the hydroxyurea arrest point was not required in order for spindle collapse to occur. In addition, this change in spindle structure was not a consequence of hydroxyurea
pretreatment; collapse could be observed in untreated culmicrotures as well. Antitubulin immunofluorescence scopic examination of a 26OC asynchronous cin8-3 kipl-A culture revealed that 16% of the cells possessed spindles of approximately the same size (1.5 pm between poles) and morphology as hydroxyurea-arrested cells. Following a shift to 37V for 10 min, less than 1% of the cells had identifiable short spindles, and in 94% of the cells, the spindles appeared monopolar. Therefore, nearly all of the short spindles in the culture had lost their structure during the temperature shift. Anaphase Spindles Are Resistant to Collapse culture described In the asynchronous cin&3 kipl-A above, the fraction of cells with bipolar spindles longer than 1.5 pm remained about constant before and after a 10 min shift to 37OC (3.5% before and 5% after). This suggests that spindles that had begun anaphase were resistant to collapse triggered by loss of CirBp/Kipl p function. In support of this possibility, we have found that spindles in cells arrested at late anaphase are resistant to collapse. Itel-A mutant strains arrest growth at 11 “C (Wickner et al., 1987) with long spindles (approximately 8-10 pm) connecting two segregated chromosomal masses (E. Woodhouse and M. A. H., unpublished observations). Itel-A cin8-3 kipl-A triple mutants express both cold-sensitive and temperature-sensitive arrest phenotypes. Triple mutants were shifted to 11 OC for 24 hr, resulting in a population of cells containing long spindles (approximately 80%; Figures 4A and 46). The cells were then shifted from 1 l°C directly to 37OC for various
Figure 4. Antitubulin lmmunofluorescence croscopy of ffel cin8 kipl Cells
Mi-
Strain MAY2282 (Itel-A cin8-3 kipl-A) was grown at 11% for 24 hr (A and B) and then shifted directly to 37% for 15 min (C and D). Cells are stained with antitubulin (A and C) and for DNA with DAPI (B and D). Bar, 10 pm.
Kinesin-Related 455
Proteins
and Spindle
26’
Structure
30’
33’
35’
37’
ing to the collapsing force. The inviability tant strain at 37% (Figure 5) is probably in bipolar spindle assembly. Triple mutant this temperature prior to spindle assembly with unseparated poles (data not shown).
of the triple mudue to a defect cells shifted to arrested growth
Discussion
Figure
5. Temperature
Resistance
of Mutant
Strains
Cells of the indicated genotypes were suspended in water and spotted onto solid rich medium at the indicated temperatures. Strains: wildtype (MAY591); cin&3 kipl-A (MAY2082); kar3-A cin8-3 kipbA (MAY2324).
amounts of time and examined by immunofluorescence microscopy. Following the shift to 37%, the spindles in triple mutant cells did not collapse (Figures 4C and 40). Instead, the cells completed mitosis as efficiently as did similarly treated kel-A single mutants; after 3 hr at 37%, approximately half of the cells of each genotype had recovered from the cold-induced arrest and completed mitosis. While it is conceivable that the Ire7 mutation caused changes to the spindles rendering them resistant to collapse, the reversibility of the ltel phenotype and the asynchronous culture experiment described above argue against this possibility. Therefore, we conclude that elongated bipolar spindles are stabilized against collapse and CinBplKipl p function is no longer required for completion of mitosis following the ltel arrest point.
kar3-A Suppresses
cin8 kipl In an attempt to identify gene products that interact with Cin8p and Kiplp, we have selected mutations that supphenotype. press the cin8-3 kipl-A temperature-sensitive Seven extragenic suppressors analyzed were found to be mutant alleles of another S. cerevisiae kinesin-related gene, KAR3. The identification and characterization of these kar3 alleles will be published elsewhere. This finding prompted us to test the effect of a kar3 loss-of-function allele. Deleting KAR3 in a cin&3 kipl-A background clearly had a beneficial effect on temperature resistance (Figure 5). Although not as resistant as wild type, the constructed triple mutant strains grew well at 33% a temperature that inhibited cin8-3 kipl-A cells. kar3-A was able to increase the temperature resistance of cin8-7 kipl-A cells as well (data not shown). The observed suppression of cin8-3 kipl-A temperature sensitivity suggested that the deletion of KAR3 might prevent spindle collapse. Cells of various genotypes were synchronized with hydroxyurea at 26% and transferred to 33OC or 37% for 15 min. The number of cells with separated spindle poles was determined before and after the shift by using the anti-90 kd antibody (Table 1). The frequency of collapsed spindles was greater in the cin8-3 kipl-A KAR3 strain than in the cin8-3 kipl-A kar3-A strain. At 37%, less than 10% of the KAR3 spindles remained intact, compared with greater than 50% of the kar3-A spindles. This finding suggests that Kar3p is directly contribut-
The Role of Cin8p/Kiplp in the Preanaphase Spindle Cin8p and Kipl p redundantly perform a role essential for mitotic spindle function. In their absence, the phenotypes observed are most simply interpreted as resulting from the loss of a force-producing mechanism. The action of at least one of these proteins is required for pole separation during assembly of the bipolar spindle. After the spindle is assembled, this activity is also required to resist an inwardly directed force acting upon the separated poles. From these findings and the relationships of Cin8p and Kiplp to the mechanochemical enzyme kinesin, it is reasonable to conclude that these proteins produce a pole-separating force. Cin8pand Kipl p have both been localized to the spindle fibers that lie between the poles, suggesting that they produce force from within the spindle (Hoyt et al., 1992; Roof et al., 1992). Observations of spindles in numerous eukaryotic cell types revealed that they possess a conserved architecture (reviewed in McDonald, 1989). Each halfspindle consists of a single pole with attached microtubules of the same polarity (plus ends out). These overlap in the midzone with microtubules of opposite polarity originating from the other pole. Experimental observations have suggested that forces that push spindle poles away from each other are produced by interactions between the antiparallel midzone microtubules (reviewed in Hogan and Cande, 1990). In the simplest formulation for CinbplKipl p function, they act by cross-linking the overlapping antiparallel microtubules and sliding them past one another to generate an outward force (Figure 6). This force is first utilized to separate duplicated poles and then is required to oppose the inwardly directed force acting upon the poles prior to anaphase. We propose that Cin8p and Kipl p constitute or contribute to bridges between the two half-spindles. Bridges bein electween midzone microtubules have been ObSeNed tron micrographs of diatom spindles (McDonald et al., 1979). As represented in Figure 6, Cin8p and Kipl p may each possess two microtubule-binding domains. Alternatively, these polypeptides may participate in higher order complexes that bridge the half-spindles. These proteins could operate on overlapping nonkinetochore microtubules (nkMTs) as depicted (Figure 6) or on kinetochore microtubules (kMTs). The only requirement for outward force generation is that the interacting microtubules be in an antiparallel configuration. We have not yet determined the direction of force generation by Cin8p and Kipl p with respect to microtubule polarity. While it is hypothetically possible to drive poles apart with either a plus end- or a minus end-directed activity, a plus end activity, like that of kinesin, is strongly favored by our understanding of spindle dynamics (Figure 6). In
Cell 456
SPB Figure
6. Model for CinEplKipl
p Function
in the Preanaphase
Spindle
Cin8p and Kiplp, represented by the “lollypop” structures, are proposed to function by cross-linking antiparallel microtubules and sliding them past each other (see Discussion). The thin lines represent kinetochore, nonkinetochore, and cytoplasmic microtubules (kMT, nkMT, and cMT. respectively). The motor domains (round part of the motor proteins) are proposed to move along the microtubules toward the plus ends (in the direction of the closed arrows). The stalk represents a second domain involved in a static interaction with an antiparallel microtubule. This produces an outwardly directed force that separates the duplicated spindle pole bodies (SPB) and opposes an inwardly directed force (open arrows). A component of the inwardly directed force appears to be produced by Kar3p. The kMTs may also contribute to the inwardly directed force (see Discussion).
order for a minusend-directed activity to push poles apart, it must act by extruding a microtubule from the pole in toward the center of the spindle. As poles separate, tubulin subunits would have to be added at the poleward minus ends, a process that has not been observed. Numerous studies have demonstrated that tubulin subunits are added primarily at the plus end8 of spindle fibers in vivo and in vitro (reviewed in Hogan and Cande, 1990; Salmon, 1989). The Inwardly Directed Force Our findings demonstrate that the action8 of Cin8p and Kipl p are opposing a force that draws separated spindle poles back together. A number of other experimental observations support our finding of a compressive force acting upon the poles. Severing of diatom, newt, and mammalian metaphase spindles with ultraviolet microbeam irradiation was observed to cause spindle buckling or collapse (Leslie and Pickett-Heaps, 1983; Spurck et al., 1990). Treatment of mammalian cells in culture with nocodazole at low concentration preferentially depolymerized nkMTsand caused metaphase spindle shortening (Snyder et al., 1985). Nocodazole treatment has also been observed to cause S. cerevisiae spindle poles to collapse together (Jacobs et al., 1988). It is probable that the inward force evident upon inactivation of Cin8p and Kiplp is of the same origin as these other compressive forces.
There are two pole-to-pole microtubule connections, the kMTs and the nkMTs, through which the inwardly directed force may be applied. The nkMTS connect the poles through antiparallel interactions in the spindle midzone. Experimental observations have suggested that interactions between the antiparallel nkMT microtubules produce the forces that push spindle poles outwardly (reviewed in Hogan and Cande, 1990) although a role in inward force generation cannot be ruled out. An attractive hypothesis is that the inwardly directed force is mediated through the kMTs. The kMTs connect the two poles prior to anaphase through the paired kinetochores of sister chromatids. When chromosomes establish a bipolar connection, they are pulled simultaneously toward each pole; irradiation of one of the paired kinetochores in a metaphase chromosome caused the chromatids to move to the pole opposite to the irradiated kinetochore (McNeil and Berns, 1981). These balanced poleward forces should therefore be acting on the poles to pull them in toward each other. Indeed, spindle shortening has been observed at metaphase when chromosomes establish bipolar connections (LaFountain, 1972; Taylor, 1980). Therefore, force8 that drive kinetochores toward poles may be contributing to spindle collapse when the outwardly directed Cin8pl Kipl p function is lost. As structural detail for the S. cerevisiae spindle is lacking, however, we do not know whether the chromosomes in our hydroxyurea-inhibited cells had assumed a bipolar connection. If spindle collapse is caused by forces acting on the kinetochores, then this force should abate upon the dissolution of the sister kinetochore connections at anaphase. Our observations suggest that upon entering anaphase, spindles become resistant to collapse despite the loss of Cin8plKipl p function. Similarly, when anaphase spindles were cut by microbeam irradiation, the compressive force observed acting upon metaphase poles was no longer present (Spurcket al., 1990). Alternatively, if the collapsing force is mediated through nkMTs, it may be eliminated in anaphase simply by attenuating the activity of an inward motor function. In anaphase B, poles are separated further, resulting in an often extreme elongation of the spindle. We have demonstrated a requirement for Cin8plKipl p function for pole separation prior to anaphase. This finding suggests the possibility that these proteins also contribute to pole separation during anaphase. We observed that the rate of progression from the hydroxyurea arrest point through mitosis was reduced for the cin8 single mutant strains at 37% (Figure l), perhaps indicating an anaphase role for Cin8p. Kar3p Acts Antagonistically to CinlplKipl p Loss of KAR3 function was found to partially suppress the temperature sensitivity and spindle collapse associated with the cin8-3 kip7-A genotype. The simplest interpretation of this finding is that the kinesin-related Kar3p directly contributes to the inwardly directed force. Since our knowledge of the mitotic role and localization of Kar3p is limited, we can only speculate as to how it may be producing this force. One possibility is that Kar3p is positioned at kineto-
Kinesin-Related 457
Table 2. Yeast
Proteins
and Spindle
Structure
Strains
Strain
Relevant
MAY591 MAY 1094 MAY 1397 MAY 1562 MAY2035 MAY2061 MAY2075 MAY2077 MAY2082 MAY2169 MAY2282 MAY2324 MAY2375
CIN8 K/PI KAR3 LTEl C/N8 K/PI KAR3 LTE7 /fel::URA3 cin8-3 kaA-A 102::LEU2 cin8::LJRAS cinB3 kipl:tH/SS kip I::H/.S3 cin8-3 kipl::H/SS cin8-3 kipl::H/S3 cin8-3 kipl::H/S3 /tel::URAS cinB-3 kipl::H/SS kar3-A 702::LEU2 cin&3 kip l::H/S3 kar3-A 102::LEU2
Genotype
chores and exerts a poleward (or minus end-directed) force. Alternatively, it could be cross-linking antiparallel midzone microtubules, as has been proposed for Cin8pl Kipl p, but exerting force in the direction opposite to Cin8pl Kiplp. In consideration of either of these possibilities, the similarity of Kar3p to the minus end-directed ncd motor protein from Drosophila should be noted (Rose, 1992; Endow et al., 1990; McDonald and Goldstein, 1990; McDonald et al., 1990; Walker et al., 1990). We conclude that Cin8p and Kipl p, components of the mitotic spindle required for a motility event, are apparently essential structural elements as well. These findings contribute to the view that spindle structure is generated and maintained by opposing mechanical forces. Of particular novelty is our conclusion that two antagonistic forces acting upon the spindle poles are produced by kinesin-related proteins. Experimental
Procedures
Yeast Strains and Media The yeast strains used in these experiments are derivatives of S288C and are listed in Table 2. Different strains with the same CIN8, K/PI, KAR3, and LTE7 genotypes behaved identically in assays for temperature resistance and spindle collapse. The kar3 deletion allele, kar3A102:tLEU2, removes almost the entire reading frame (Meluh and Rose, 1990) and was supplied by M. Rose and P. Meluh. kar3-A102:: LEU2 was introduced into cin8-3 kipl-A strains by standard genetic crosses. Suppression of the cin8-3 kipl-A temperature-sensitive phenotype was found to be 100% linked to the LEU2 marker. In various crosses in which both genotypes segregated, 39 cin8-3 kipl-A double mutants and 36 cin&3 kipl-A kar3-A702::LEU2 triple mutants were obtained. All triple mutants were more temperature resistant than the double mutants. The Itel::URAS allele was supplied by R. Wickner and is described in Wickner (1987). Rich medium (YPD) was as described (Sherman et al., 1983). Hy droxyurea (Sigma) was added to log-phase cultures in YPD (pli 5.8) to a final concentration of 0.1 M. The cultures were then incubated at 26°C until more than 70% of the cells had assumed a large-budded morphology (4-5 hr). Cells were released from arrest by centrifugation and resuspension in the same medium minus the inhibitor. Microscopic Analysis of Cells To determine the distribution of cell morphologies, culture samples were briefly fixed in 70% ethanol and stained for DNA with 0.5 mg/ml4, Wiamidino-2-phenylindole (DAPI) plus 1 mg/ml p-phenylenediamine (used as an anti-fade agent). Using differential interference contrast optics, cells were scored as unbudded, small budded (bud size roughly
50% size of mother). The number of nuclei per cell wasdetermined by epifluorescent illumination. Microtubule structures were observed following formaldehyde fixation using the anti-tubulin monoclonal antibody YOL1134 (Kilmartin et al., 1982; Eioproducts for Science, Inc.) and a fluorochrome-conjugated secondary antibody as previously described (Stearns et al., 1990). Spindle length was measured using a calibrated eyepiece reticule. The SPB-associated 90 kd antigen (Rout and Kilmartin, 1990) was detected by using an undiluted pooled sample of monoclonal antibodies directed against this polypeptide (gift from J. Kilmartin). We obtained best results by performing the primary antibody incubation at 37OC for 30 min. The secondary antibody was a 1:50 dilution of FITCconjugated goat anti-mouse immunoglobulin G (Cappel). Because 90 kd antigenicity was sensitive to formaldehyde treatment, short fixation times of 5-10 min were used (in 3.7% formaldehyde, 50 mM KPO. (pH 6.8) 150 mM NaCI). Control experiments indicated that cells with a single SPB (i.e, a-factor arrested cells) or duplicated but unseparated SPBs (i.e., early cell cycle-shifted cin8-3 kipl-A cells) yielded a single dot of stained material. Cells with separated SPBs showed double dots (also see Rout and Kilmartin, 1990). Cells were prepared for thin-section electron microscopy as previously described (Hoyt et al., 1992). Acknowledgments We gratefully thank John Kilmartin for the gift of antibodies, Mark Rose and Pam Meluh for the kar3 mutation, Mike Sepanski for assistance with the electron microscopy, and Don Cleveland, Doug Koshland, and Tibor Roberts for comments on the manuscript. This work was supported by National Institutes of Health grant GM40714 awarded to M. A. H. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received
February
12, 1992;
revised
June
1, 1992
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