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* Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085; and Departments of Pediatric Oncology, The Dana-Farber Cancer Institute, and Pediatric Hematology, The Children's Hospital,
Harvard Medical School, Boston, Massachusetts 02115
Abstract
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
Abbreviations used in this paper
References
Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus.
MICROTUBULES mediate a series of movements in
Saccharomyces cerevisiae that culminate in chromosome segregation, including migration of the
nucleus to the neck between the mother and daughter cells,
assembly of a bipolar spindle, translocation of the spindle through the neck, and elongation of the spindle during
anaphase. Movements have been associated with the action of particular microtubule-based motor proteins by examining mutants defective in motor function and identifying the perturbed movement. One complication in this approach is that a single movement may be generated by
two or more motor proteins, as occurs in yeast cells during
spindle pole separation (Hoyt et al., 1992 The position of the nucleus and mitotic spindle within
the cell is under complex control at all stages of the cell cycle. In budding cells, the preanaphase nucleus is typically
located a short distance from the bud neck, and microtubules from one spindle pole pass through the neck and
into the bud (Byers and Goetsch, 1975 Upon initiation of anaphase, the spindle is approximately aligned along the mother-daughter axis so that one
spindle pole inserts through the narrow opening of the bud
neck, allowing spindle elongation to place the divided genomes at the distal edges of the mother and daughter cells.
One motor protein that appears to exert force on the nucleus as anaphase commences is dynein. Mutants defective
in the gene for dynein heavy chain (dhc1 or dyn1 mutants)
fail to translocate the early anaphase nucleus through the
neck, which causes the spindle to elongate within the
mother cell and results in the formation of a binucleate
cell (Eshel et al., 1993 Dynein has also been implicated in generating forces for
spindle pole separation during anaphase B, along with the
kinesin-related proteins Kip1p and Cin8p (Saunders et al.,
1995 Given the cooperation between motor proteins in generating movements, a thorough understanding of the underlying mechanisms requires identification and characterization of all of the motor proteins involved. The
recently completed DNA sequence of the S. cerevisiae genome revealed the complete set of kinesin-related proteins in this eukaryote. The sixth and final S. cerevisiae protein with substantial homology to the force-generating
domain of kinesin is encoded by a previously undescribed
open reading frame. Here we report that this protein,
named Kip3p, is required for the first phase of nuclear migration: alignment of the mitotic spindle along the mother-
daughter axis and movement of the preanaphase nucleus
to the bud neck. We show that loss of dynein function has
little effect on preanaphase nuclear migration and spindle orientation and that loss of KIP3 function has little effect
on movement of the anaphase nucleus through the bud
neck. Kip3p is located on both astral and nuclear microtubules at all stages of the cell cycle. Kip3p is essential in
strains lacking kinesin-related Kar3p and in strains lacking
dynein heavy chain, indicating that Kip3p performs overlapping or dependent functions with Kar3p and dynein.
Strains, Media, and Genetic Techniques
Genotypes and sources of the strains and plasmids used in this study are
listed in Table I. Strains were constructed by standard genetic methods
(Rose et al., 1990 Table I.
Strains and Plasmids Used
; Roof et al.,
1992). Disruption of a motor for a redundantly powered
movement may fail to generate a terminal cell morphology
that reveals all roles performed by the motor. However, S. cerevisiae has a modest set of cytoskeletal movements and
motors, which has allowed significant progress to be made
in associating particular movements with the activity of
one or more motor proteins. One movement that has yet
to be associated with a force-generating protein is migration of undivided nuclei to the neck of the emerging bud,
which proceeds in all motor mutants examined to date.
). Analysis of specific 
-tubulin alleles has demonstrated that astral (or cytoplasmic) microtubules play a key role in nuclear and
spindle positioning (Palmer et al., 1992; Sullivan and Huffaker, 1992). Microtubule-independent mechanisms also
appear to affect the position of the nucleus. When cell cycle progression is arrested in metaphase after nuclear migration to the neck is complete, the nucleus is retained at
the neck upon microtubule depolymerization (Jacobs et
al., 1988). Furthermore, the actin cytoskeleton is thought
to play a role because disruption of actin in cells arrested in metaphase causes the spindle to be improperly oriented
with respect to the neck (Palmer et al., 1992).
; Li et al., 1993; Yeh et al., 1995). Dynein mutants also fail to exhibit the back and forth movement of the anaphase spindle within the neck observed in
wild-type cells, supporting a role for dynein in spindle positioning during anaphase (Yeh et al., 1995). Dynactin, a
protein complex associated with dynein, also appears to be
required for nuclear translocation because null alleles of
the putative S. cerevisiae dynactin components JNM1 and
ACT5 have an identical phenotype to dynein disruption mutants (McMillan and Tatchell, 1994; Muhua et al.,
1994). Although dynein has a role in positioning the nucleus after the onset of anaphase, analysis of cells arrested
in metaphase suggests that dynein is not required to position the nucleus at the bud neck (Yeh et al., 1995).
; Yeh et al., 1995). Kinetic analysis of spindle pole
separation in live cells has shown that loss of dynein function slows the rate of spindle pole separation in late
anaphase (Yeh et al., 1995). Analysis of populations of
fixed cells has shown that loss of Kip1p and Cin8p function abolishes pole separation during spindle assembly and
causes an inward collapse of metaphase spindle poles
(Hoyt et al., 1992; Roof et al., 1992; Saunders and Hoyt,
1992). Simultaneous loss of dynein, Kip1p, and Cin8p
function abolishes anaphase spindle elongation, while individual or pair-wise loss of function of these proteins had
smaller effects (Saunders et al., 1995). Mutations in another kinesin-related motor, Kar3p, can suppress the spindle collapse caused by loss of Kip1p and Cin8p function.
This suggests that Kar3p contributes to a force that opposes an outwardly directed force on the spindle poles
generated by Kip1p and Cin8p (Hoyt et al., 1993). Non-motor microtubule-associated proteins are also required
for anaphase B (Pellman et al., 1995).
Materials and Methods
) and as described in the text. The yeast media used were
SD minimal medium and sporulation medium supplemented with adenine, uracil, and appropriate amino acids; SC complete medium containing 5-fluoroorotic acid (5-FOA)1 (1 mg/ml); and YPD rich medium (Rose
et al., 1990). The frequency of chromosome loss was assayed by scoring
kip3/kip3 and wild-type diploid strains for loss of heterozygosity at the
mating type locus of chromosome III. The diploid strains were mated to
MATa and MAT
tester strains on YPD plates and replica printed to score prototrophic triploid colonies (Spencer et al., 1990). Sensitivity to
benomyl was tested on YPD medium containing 1% DMSO and the desired concentration of benomyl (E.I. du Pont de Nemours, Wilmington, DE). Strains expressing the Nuf2p-green fluorescent protein (GFP) fusion protein for localization of spindle poles (Kahana et al., 1995) were
constructed by integrating the NUF2-GFP gene fusion at the NUF2 locus
using plasmid pJK67 (a gift of J. Kahana and P. Silver, Dana Farber Cancer Institute, Boston, MA). The NUF2-GFP gene fusion is expressed from
the NUF2 promoter, and the GFP gene contains S65T and V163A mutations.
KIP3 Deletion Construction, Gene Recovery, and Epitope Tagging
A precise deletion of the KIP3 open reading frame (kip3
) was constructed by PCR (Baudin et al., 1993). The 3
ends of primers 1 and 2 contained sequences for PCR amplification of the HIS3 or TRP1 genes
present in pRS403 or pRS404 (Sikorski and Hieter, 1989), and the 5 ends
of the primers contained sequences immediately flanking the KIP3 gene.
Primer 1 was of sequence 5-ACTTGAGTTTTCTTTCCAGCTGTATACTATTGACACTAACGAATTCGATTGTACTGAGAGTGCACC- 3 , and primer 2 was of sequence 5-GAAAGAAGTTATATTCGAT-AGTTTACGTAGGATATGTATGGAATTCCTGTGCGGTAT T TC A- CACC-3. (Sequences flanking KIP3 are underlined, and EcoRI restriction sites are in bold type.) The KIP3 locus in a diploid yeast strain was replaced by this PCR product using the one-step gene replacement procedure (Rothstein, 1983). Correct recombinants were identified by PCR amplification and restriction digest analysis of genomic DNA. The primers
used were primer 3, 5-CCGGGATCCGACTCTTCTAATTGGTCTCT-3 and primer 4, 5-GGCGTCGACGTCTCCTGAGAAACGTTTT-3.
(BamHI and SalI restriction sites are in bold type.)
To recover the KIP3 gene, we screened a library of S. cerevisiae genomic DNA fragments cloned into the vector YCp50 (URA3 CEN ARS)
by colony hybridization using a radiolabeled probe. The probe was a 556-bp sequence adjacent to the 5 end of KIP3 that was prepared from a plasmid carrying the kip31::HIS3 allele using the EcoRI and BamHI sites introduced by primers 1 and 3. We identified a single plasmid that carried
the full-length KIP3 gene on a 10-15-kb genomic DNA insert (pDR605)
out of ~4,100 colonies screened.
For epitope tagging of KIP3, the myc tag coding sequence (Evan et al.,
1985) was introduced into the KIP3 coding sequence as follows. A 3.8-kb
fragment containing the entire KIP3 open reading frame, but lacking the
termination codon, was generated by PCR using the Expand High Fidelity
PCR system (Boehringer Mannheim Corp., Indianapolis, IN). This fragment was cloned into a vector containing six tandem copies of the myc tag
(pB896 [Li, 1997]). The resulting construct contained 1,419 bases of 5
noncoding sequence, the entire KIP3 open reading frame, and the sequence encoding the myc epitopes just 5 of the termination codon. To eliminate the possibility of PCR-generated errors, a 2.25-kb fragment of
wild-type KIP3 was used to replace most of the PCR-generated clone.
The remaining PCR-generated KIP3 sequences were verified using DNA
sequencing. KIP3-6MYC (pB956) appears to be functional, as it fully
complements the lethality of kip3 kar3 and kip3 dyn1 strains using
the plasmid shuffle test described below.
Tests of Synthetic Lethality
To test whether the kip3 mutation was lethal in combination with other
mutations, we generally constructed double mutants in the presence of a
plasmid-based wild-type copy of one of the genes and then determined
whether the strain remained viable after plasmid loss. The presence of the
wild-type gene during strain construction prevented the potential isolation
of aneuploid strains because of genomic instability. Synthetic lethality of
kip3 and dyn1 mutations was tested by replacing the wild-type DYN1
gene with the dyn1::HIS3 deletion allele (from plasmid p2-1H, a gift of
E. Yeh and K. Bloom, University of North Carolina, Chapel Hill, NC) in
the kip3 strain DS667, which carries the KIP3 URA3 plasmid pDR605,
using one-step gene replacement. Correct deletion of DYN1 was confirmed by restriction analysis and Southern blotting of genomic DNA.
Five independently constructed double mutants were inviable when
plated on 5-FOA medium, which selects against cells carrying the URA3
plasmid (Boeke et al., 1987), indicating that kip3 is synthetically lethal
with dyn1.
Synthetic lethality of kip3 with kar3 was tested by crossing a kip3::
HIS3 strain with strain DS276 containing kar3102::LEU2 and a KAR3
plasmid. The resultant diploid strain was sporulated, and the tetrads were
dissected. The spores were 83% viable, and all 11 viable kip3 kar3 spores
carried the KAR3 plasmid. An additional 16 inviable spores were inferred
to carry both the kip3 and kar3 mutations based on analysis of markers in
the remaining spores of each tetrad; these spores may have been inviable
because they did not inherit the KAR3 plasmid. The viable kip3 kar3
strains were sensitive to 5-FOA at 30°C, indicating that the KAR3 plasmid was essential and that kip3 is synthetically lethal with kar3. In a second
test of synthetic lethality, a diploid strain homozygous for kip3 and heterozygous for kar3 was sporulated, and tetrads were dissected. Two spores
in each tetrad were kip3 KAR3 and viable, and two spores inferred to be
kip3 kar3 germinated and divided two to six times before ceasing growth,
confirming lethality of the double mutant.
Synthetic lethality of kip3 with kip1 and cin8 was tested by crossing a
kip3::HIS3 strain with strain DS118 containing kip11 and a KIP1 plasmid, and strain DS379 containing cin8112 and a CIN8 plasmid. The resultant diploid strains were sporulated, and 24 tetrads from each strain
were dissected. The spores were >90% viable, and double kip3 kip1 and
kip3 cin8 mutants were recovered at a frequency of ~25%. Some of the
double mutants did not contain the complementing plasmid, and those
that did were resistant to 5-FOA, indicating the plasmid was not essential.
The kip3 kip1 and kip3 cin8 double mutants were capable of vegetative
growth at 16, 23, 30, and 37°C, indicating that kip3 is not synthetically lethal with kip1 or cin8.
Synthetic lethality of kip3 with kip2 and smy1 was tested by crossing
kip3 strains with kip2 strain MS2309 or smy1 strain SLY57. The resultant diploid strains were sporulated, and 24 tetrads were dissected. The
spores were >90% viable, the kip3 kip2 and kip3 smy1 double mutant
progeny were recovered at a frequency of ~25%, and the double mutants
were capable of vegetative growth at 16, 23, 30, and 37°C, indicating that
kip3 is not synthetically lethal with kip2 or smy1.
Synthetic lethality of dyn1 with kar3 was tested by crossing a kar3
strain bearing a KAR3 plasmid (strain DS276) with the dyn1 strain
DS730. The resultant diploid was sporulated, and tetrads were dissected.
Spore viability was 65%, and only 5 out of 24 tetrads yielded 4 viable
spores that exhibited 2:2 segregation of mating type and the three auxotrophic markers present. Three dyn1 kar3 double mutants were recovered from these valid tetrads, and each double mutant carried the KAR3
plasmid. The double mutants were unable to grow on medium containing
5-FOA at 30°C, indicating that the plasmid was essential and that dyn1
and kar3 are synthetically lethal.
Construction of kip3 Temperature-sensitive Alleles
A 4,822-bp EcoRI/SpeI fragment from plasmid pDR605 was subcloned
into the EcoRI and SpeI sites of pRS314 (TRP1 CEN ARS) (Sikorski and
Hieter, 1989) to yield the plasmid pB893 and further subcloned into
pRS416 (URA3 CEN ARS) to yield plasmid pDR622. These plasmids carry the entire KIP3 open reading frame with 1,391 bp 5 and 966 bp 3
flanking DNA. Hydroxylamine mutagenesis of pB893 was performed by
the method of Rose and Fink (1987), and temperature-sensitive (ts) alleles
of KIP3 were obtained after transformation of strain DS738 or DS716 using the plasmid shuffle technique (Boeke et al., 1987). Transformation of
the kip3-20(ts) dyn1 strain DS765 with the KIP3 plasmid pDR622 complemented the temperature-sensitive growth, but transformation with the
vector (pRS416) did not. This confirmed that the mutation conferring
temperature-sensitive growth was in KIP3 and that the mutation is recessive.
Morphological Observations
Cells were fixed for immunofluorescence microscopy by adding formaldehyde to 3.7% directly to the culture medium and incubating for 2 h at
23°C, or overnight at 4°C. Cells were prepared for immunofluorescence
microscopy as described (Rose et al., 1990). For the Kip3p localization experiment, Kip3p-6myc was visualized with mAb 9E10, which recognizes
the myc epitope (Santa Cruz Biotechnology, Santa Cruz, CA), and microtubules were visualized with the rat antitubulin antibody YOL 1/34 (Accurate Chemical and Scientific Corp., Westbury, NY). Fluorochrome-conjugated secondary antibodies were from Jackson Immunoresearch Labs,
Inc. (West Grove, PA). For double labeling of Kip3p and microtubules,
controls demonstrated that cross-reactivity and light channel spill-over did
not contribute to the final images when species-specific secondary antibodies were used. For all other immunofluorescence experiments, tubulin was stained with the antitubulin monoclonal antibody BIBE2, a gift of F. Solomon (Massachusetts Institute of Technology, Cambridge, MA). The
secondary antibody was FITC-conjugated goat anti-mouse antibody (Accurate Chemical and Scientific Corp.), which was absorbed against fixed
yeast cells for 3 h at 4°C before use to minimize background signal. DNA
was stained using 4,6-diamidino-2-phenylindole (DAPI) (Boehringer
Mannheim Corp.). To observe Nuf2p-GFP localization in fixed cells, cells
in culture medium were fixed for 1 h with 3.7% formaldehyde, washed,
and applied to polylysine-treated Teflon®-masked slides for viewing.
For live cell microscopy of spindle pole body location, cells were applied to a microscope slide with a thin pad of 1% agarose containing SC complete medium, and a coverslip was applied and sealed with a thin bead of a mixture of equal parts Vaseline, lanolin, and paraffin at the edges. Cells were imaged at 23-25°C using a microscope (model DMRBE; Leica, Inc., Deerfield, IL) equipped with a 100×/1.4 NA objective, a monochrome CCD camera (Cohu Inc., San Diego, CA), a 100-W mercury vapor lamp, and a fluorescein filter set. Images were captured using LG3 frame grabber hardware (Scion Corp., Frederick, MD) and NIH Image software (written by W. Rasband and available at http://rsb.info.nih.gov/nih-image/) with a custom set of macro programs to control the camera exposure settings, fluorescence illumination shutter, and motorized microscope stage. At each time point, both differential interference contrast (DIC) and fluorescence images were photographed at three focal planes spaced at 1-µm intervals to increase the likelihood of detecting both spindle pole bodies. Illumination during fluorescence photography was controlled by a shutter and was 200 ms or less for each image.
Micrographs were spatially calibrated using NIH image software and a
stage micrometer. Distance and angle measurements were made using
Object Image software, a modified version of NIH Image available at the
above internet site. Measurements to determine nuclear migration indices
were made on digital images of cells by the method of Jacobs et al. (1988),
and the statistical significance of the difference between the variances of
nuclear migration indices was tested using the two-tailed variance ratio
test at a 5% significance level.
Predicted Structure of the Kinesin-related Gene KIP3
Upon completion of the S. cerevisiae genome sequencing
projects, we searched the public databases using the
BLAST program to identify additional kinesin-related
genes. In addition to known genes KIP1, KIP2, KAR3,
CIN8, and SMY1, this search detected an open reading
frame on chromosome VII (YGL216w) that is predicted to encode an 805-amino acid protein of molecular mass 91 kD. Residues 86-464 showed 39% identity to the motor
domain of human kinesin and include putative ATP-binding and microtubule-binding motifs (Fig. 1 A). Using the
nomenclature employed for the kinesin-related genes
KIP1 and KIP2, we have designated this gene KIP3. Subfamilies of kinesin-related proteins have been defined by
exceptionally high sequence conservation in the motor domain (Moore and Endow, 1996), but sequence comparisons with Kip3p did not reveal any obvious subfamily relationship. The nonmotor, COOH-terminal 340 amino acids
did not exhibit obvious sequence similarity to the nonmotor regions of other kinesin-related proteins, nor to any other protein in the current DNA, protein, and EST databases. The COOH-terminal domain contains several regions predicted to form -helical coiled coils that potentially mediate protein multimerization (Fig. 1 B).
)
residues 1-340 was performed
using the Gap program (Genetics Computer Group, Inc.,
Madison, WI). Vertical lines
indicate amino acid identities, and dots indicate gaps
introduced to improve alignment. Horizontal lines indicate key residues of the
ATP-binding site (GX4GKT)
and amino acids suspected
to play a role in nucleotide
phosphate sensing and microtubule binding. (B) The
probability of coiled-coil formation in the predicted Kip3p amino acid sequence
was calculated using the algorithm of Lupas et al. (1991)
with a window size of 14 amino
acids.
KIP3 Is Not Essential for Mitosis, Meiosis, or Karyogamy
No known mutations map to KIP3. Therefore, to determine the function of KIP3 a deletion mutant was made by
replacing the KIP3 coding sequence with the HIS3 gene in
a diploid strain via one-step gene replacement (see Materials and Methods). Tetrad analysis demonstrated that
KIP3 is not essential for mitotic growth. The efficiency of
karyogamy was tested by crossing a kip3 mutant to wild-type and kip3 strains in a qualitative limited mating assay
(Conde and Fink, 1976) and was found to occur at the
wild-type efficiency. Meiosis was tested by sporulating the
homozygous kip3 diploid strain and dissecting the tetrads. The efficiency of sporulation was normal and spore
viability was >90%. Chromosome instability was tested
using homozygous kip3 diploid strains in a qualitative assay for chromosome III loss and was found to occur at the
wild-type frequency.
To investigate potential functions of KIP3 that are not
essential for viability, we examined mitotic cultures of the
kip3 mutant in more detail. The growth rate of the kip3
mutant at 30 and 37°C in liquid YPD medium was the
same as an isogenic wild-type strain, and flow cytometry
showed that the mutant cultures at 30 and 37°C had the
same proportion of G1 and G2 cells as wild-type cultures.
However, a subtle cell cycle delay that was not evident from flow cytometry analysis was evident from the analysis of cell morphology shown in Table II, where there was
an increased frequency of preanaphase cells whose buds
had grown beyond the size at which nuclear division normally occurs. Cells defined as delayed in metaphase had a
single nucleus and a bud >50% the diameter of the
mother cell. In the wild-type strain, cells delayed in
metaphase comprised 3-4% of the culture, while in the
kip3 mutant these cells comprised 5-14% of the culture,
depending on incubation temperature.
|
Table II. Cell-Type Distributions of Wild-Type and Mutant Cultures |
Increased Microtubule Stability in kip3 Mutants
Mutation of genes that regulate microtubule function often results in altered sensitivity to drugs that destabilize
microtubules (Stearns et al., 1990). Sensitivity of the kip3
mutant to the antimicrotubule drug benomyl was tested by
spotting known numbers of cells on YPD plates containing
different concentrations of benomyl (Fig. 2). Both the
wild-type and mutant strains exhibited substantial growth
at 30°C on 0-20 µg/ml benomyl, while only the kip3 mutant grew at 30 µg/ml benomyl, indicating that kip3 mutants are more resistant to benomyl than wild-type. A similar degree of increased resistance to benomyl occurred in
an isogenic strain containing a deletion mutation of the kinesin-related gene KAR3. The same relative pattern of increased resistance of the kip3 mutant was observed when
the plates were incubated at 23 or 37°C. Consistent with
increased microtubule stability in the kip3 mutant, immunofluorescent localization of tubulin in the kip3 mutant showed brighter staining of astral microtubules in the
kip3 mutant than in wild-type (Fig. 3). In budded kip3
and wild-type cells, the astral microtubules extended from
the nucleus and passed through the neck into the bud.
mutant. Strains wild-type (DS140), kar3102 (DS276), kip3 (DS614), and dyn1
(DS730) were incubated at 23°C for 2 d in liquid YPD medium. Serial dilutions were prepared, and ~104, ~103, ~102, and ~10 cells
were spotted in horizontal rows on solid YPD medium containing 1% DMSO and benomyl at the indicated concentration and photographed after 3 d of growth at 30°C.
strain DS614 (A and B) and wild-type strain DS141 (C and D)
were grown in YPD medium at 30°C to mid-logarithmic phase and
prepared for immunofluorescence microscopy. (A and C) DIC images overlaid with corresponding DAPI-stained images to show
position of nuclear DNA. (B and D) Antitubulin staining to visualize microtubules. A and B show kip3 cells with nuclei located
distal to the neck and with brightly staining astral microtubules extending into the bud (arrows in B). Also present is a mother cell
with two nuclei (arrows in A) that are still connected by a mitotic
spindle. The small-budded wild-type cells in C and D contain significantly fewer prominent astral microtubules. Bar, 5 µm.
kip3 Mutants Are Defective in Migration of the Nucleus to the Bud Neck
In addition to nuclear division, meiosis, and karyogamy,
microtubule function is required for migration of undivided nuclei to a site near the neck of the newly emerging
daughter bud, and for the subsequent insertion of nuclei
into the neck as spindle elongation begins (Huffaker et al.,
1988; Jacobs et al., 1988; Snyder et al., 1991; Yeh et al.,
1995). Anaphase spindle insertion into the bud neck requires dynein function, but no microtubule-based motor
protein has yet been shown to be required for migration of
the nucleus to the neck of the emerging bud. We examined
nuclear migration in the kip3 mutant using DIC and fluorescence microscopy of fixed cells and found that the
kip3 mutant was defective in positioning nuclei within
the cell, both before and during anaphase.
To assess nuclear migration before anaphase, we measured the nucleus-to-neck distance in budded cells with
undivided nuclei. The nucleus-to-neck distances were measured on digital micrographs and were corrected for differences in the sizes of the mother cells using the nuclear migration index, which is a ratio of these two values (Jacobs
et al., 1988). Fig. 3 shows representative cells, and Fig. 4
shows histograms of the number of cells observed with a
given nuclear migration index. Wild-type cells from exponentially growing populations exhibited the expected nonrandom placement of nuclei near the bud neck; when the
culture was grown at 30°C, the mean distance between the
proximal edge of the nuclear DNA mass and the bud neck
was 1.1 ± 0.66 µm (mean ± SD) and the corresponding nuclear migration index was 0.17 ± 0.093 (Fig. 4). In contrast to wild-type, the kip3 mean distance between the
nucleus and bud was 1.8 ± 1.3 µm, and the corresponding
nuclear migration index was 0.23 ± 0.17. The larger standard deviation observed with the kip3 mutant is due to
the fact that undivided nuclei tended to be positioned
more randomly in the mother cell, with larger populations
of cells with the nucleus located both more closely and
more distantly to the neck (Fig. 4). A variance ratio test
demonstrated that the differences were significant for cultures grown at 11, 30, and 37°C.
(DS614), dyn1
(DS730), and kar3 (DS276)
strains grown to mid-logarithmic phase at the indicated temperature. (B) Histograms of kip3(ts) kar3
(DS752) and kip3(ts) dyn
(DS765) strains grown to
mid-logarithmic phase at the
permissive temperature of
30°C and shifted to the nonpermissive temperature of
37°C for 4 h. The mean nuclear migration index and
one standard deviation (mean ± SD) is inset on each
histogram. The nuclear migration index of a cell is proportional to the proximity of
its nucleus to the bud neck
and is calculated as the distance between the neck and
the nearest edge of the nucleus visualized by DAPI
staining divided by the distance between the neck and
the most distal edge of the
cell wall (Jacobs et al., 1988). The statistical significance of the difference between the variances of the nuclear migration indices was
tested with a variance ratio test. The variance ratio for the kip3 and wild-type 30°C cultures was found to be 3.4, well above the 95%
significance point of 1.5, indicating that we have significant evidence that the position of nuclei in the kip3 mutant is more random
than in wild-type. The position of nuclei in the kip3 mutant grown at 11 and 37°C was also significantly more random than in wild-type.
The variance ratio for the kip3(ts) dyn1 double mutant and wild-type cultures was 2.5, above the 95% significance point of 1.5, but
there was no significant difference between the kip3(ts) dyn1 double mutant and the kip3 single mutant. 50-100 cells were measured
for each strain and temperature.
To examine potential defects of the kip3 mutant in positioning the nuclei during anaphase spindle elongation,
we used DIC and fluorescence microscopy to score the
frequency of mother cells that contained two nuclei. In
wild-type cells that contain two nuclei, one nucleus is
found within the mother and another is found in the
daughter. In contrast, 15% of the kip3 cells grown at
11°C contained both nuclei within the mother cell (Table
II). The binucleate cells comprised 55% of the anaphase
stage cells in the asynchronously growing culture. Microtubules were observed using tubulin immunofluorescence
microscopy, and we found that all binucleate mother cells
with an empty bud contained an intact mitotic spindle connecting the two nuclear DNA masses (Fig. 3). Frequently,
the spindle was not aligned along the mother-bud axis as
in wild-type cells undergoing anaphase.
kip3 Mutants Are Defective in Preanaphase Mitotic Spindle Orientation
Because the kip3 mutant was defective in nuclear positioning, we tested whether loss of KIP3 function also
caused a defect in the potentially related process of spindle orientation. A specific orientation of the nucleus and
its associated spindle pole body can be detected in wild-type cells when the bud begins to emerge and the spindle
pole body faces the neck. Upon assembly of a bipolar mitotic spindle, one spindle pole is typically oriented toward the neck so that the spindle is approximately aligned along
the mother-daughter axis. To investigate spindle orientation, we fixed cells from exponentially growing populations and measured the orientation of preanaphase spindles with respect to the mother-daughter axis. Spindle
orientation was observed by fluorescence microscopy, using strains expressing a fusion of the spindle pole body
protein Nuf2p to green fluorescent protein developed by
Kahana et al. (1995). The Nuf2p-GFP fusion protein complements a nuf2 null mutation, and strains containing the
fusion grow normally and exhibit anaphase spindle elongation kinetics similar to spindle movements observed independently by DIC microscopy of live cells (Kahana et
al., 1995; Yeh et al., 1995). The measurements in Fig. 5
show that the majority of preanaphase spindles in the
wild-type strain were oriented within 30 degrees of the
mother-daughter axis, while the preanaphase spindles in
the kip3 mutant were nearly randomly oriented. Thus,
KIP3 function is required for normal preanaphase spindle
orientation.
(DS786), and (C) dyn1 (DS940) strains
were grown at 30°C in YPD medium and fixed for microscopy.
The location of the spindle poles and spindle orientation was determined by overlaying images of Nuf2p-GFP fusion fluorescence onto DIC images of the corresponding cells. Spindle orientation was measured in all cells in the population that had a
preanaphase spindle with both spindle pole bodies in a single focal plane and a pole-to-pole length of 0.8-1.2 µm. More than 120 preanaphase spindles from two independent cultures were measured for each strain.
Before Anaphase, kip3 Mutants Exhibit Large Oscillations in Nuclear Position
The pathway of nuclear migration begins at the end of
anaphase when the spindle pole bodies of the newly divided nuclei are positioned distal to the neck. After mitotic spindle disassembly, the nuclei migrate to a position
~1 µm from the neck of the new bud. In haploid cells, the
site of bud emergence is usually adjacent to the previous
division site (axial budding pattern), necessitating that the
nucleus traverse the diameter of the cell. The timing of this
event in the cell cycle and the path and rate of movement is not apparent by examining populations of fixed cells because there is not yet a bud to provide a temporal and spatial reference point. However, by observing movement of
the nucleus in a live cell after anaphase, the timing, path,
and rate of nuclear movement can be investigated. To observe mitotic spindle pole movements in live cells, we used
time-lapse fluorescence microscopy of strains expressing
the Nuf2p-GFP fusion (Kahana et al., 1995). We collected both fluorescence and DIC images at three focal planes at
each time point and overlaid the fluorescence images of
spindle poles on the corresponding DIC images of cells to
accurately position the spindle poles within the cell.
To examine nuclear migration in haploid G1 phase cells,
we monitored the position of the spindle pole body relative to the prebud site after anaphase. In wild-type cells after maximal anaphase spindle pole separation, the spindle
pole body typically moved 1-2 µm toward the cell center
in 4 to 8 min (Fig. 6 A). This immediate and rapid movement preceded cytokinesis and probably corresponds to a
similar movement described previously in diploid cells
(Yeh et al., 1995). After the rapid movement, the spindle
pole body in wild-type cells typically traversed an additional 2-3 µm to the site near the neck within 10 to 30 min,
and then its position became relatively stable until anaphase, exhibiting movements of 0.5 µm or less.
mutant. (A, C, and E)
A time-lapse series of DIC
micrographs of wild-type
(A), kip3 (C), and dyn (E)
cells. The elapsed time (min)
is indicated, and T = 0 is the
time of maximal anaphase
spindle elongation. The position of the spindle poles is indicated by open circle symbols, and the position of the
bud neck of the new daughter cell is indicated by the asterisk. At early times in the
series when the bud had not
yet emerged, the prebud site
was defined by comparison
to later time points. Spindle
pole position was defined by
overlaying images of Nuf2p- GFP fluorescence onto corresponding DIC images. To
obtain DIC images in which
the bud was always in focus,
images of two focal planes
spaced 1 µm apart were projected onto a single plane.
(B, D, and F) Two examples
of the distance of the spindle
pole in the mother cell from
the new bud neck, plotted as
a function of time. The
closed symbols represent
pole position in the mother cells shown in A, C, and E. The open symbols represent
pole position from an independent set of micrographs
(not shown), to convey the
range of movements observed. Note that in addition
to nuclear migration, small
changes in pole position
could also be caused by nuclear rotation. Bar, 5 µm.
In the kip3 mutant, the initial rapid phase of spindle
pole body movement after maximal anaphase spindle
elongation appeared similar to that of wild-type strains
(Fig. 6). However, subsequent to the initial movement, the
spindle poles exhibited large oscillations in position, with
the spindle pole alternately moving closer and farther
from the neck. A single spindle pole body movement toward or away from the neck was typically 1.5-2.5 µm and
occurred in 10-15 min (Fig. 6, C and D). The spindle pole
also exhibited lateral movements that sometimes occurred
without apparent large changes in the pole to neck distance (Fig. 6 C). The highly variable position of the spindle
pole relative to the neck revealed by real-time analysis is
consistent with the defective nuclear positioning shown by
analysis of nuclear position in fixed cells. Furthermore, the
variable nuclear positioning occasionally results in the
presence of the nucleus near the neck, constituting an alternate pathway for nuclear migration that could account
for the viability of kip3 mutants.
Nuclear Segregation during Anaphase Proceeds Normally in the kip3 Mutant
Microscopy of individual live cells has revealed several
discrete phases of spindle elongation (Palmer et al., 1989;
Kahana et al., 1995; Yeh et al., 1995; Yang et al., 1997).
When the initial fast phase of anaphase commences, spindle elongation moves one pole of the mitotic spindle to the
neck, and the nucleus passes through the narrow opening.
The fast phase of elongation continues until the spindle is
~4 µm long, and then elongation continues at a slower
rate, sometimes with a pause between the two phases. To
determine whether loss of KIP3 function affects the kinetics of spindle elongation, we used live cell microscopy of
the Nuf2p-GFP fusion to observe these movements in a
kip3 mutant. To focus specifically on anaphase, cells with
a short bipolar spindle located near the neck were selected
for time-lapse photography. The average rates of fast and
slow spindle elongation were similar in wild-type and kip3
strains (Fig. 7 A). Fast elongation in the kip3 mutant was
0.69 ± 0.25 µm/min (n = 10) over the first 6-8 min of elongation, and in the wild-type strain it was 0.60 ± 0.19 µm/
min (n = 6). Slow elongation in the kip3 mutant was 0.14 ± 0.06 µm/min (n = 6), and in the wild type strain it was
0.14 ± 0.08 µm/min (n = 4). Thus loss of KIP3 function
does not dramatically affect anaphase B kinetics.
mutant. (A, D,
and G) Pole-to-pole distance
as a function of time in an individual cell of the indicated
strain. Each cell exhibited the fast and slow phases of
anaphase spindle elongation,
followed by a reduction of
the pole to pole distance
upon spindle disassembly. (B, E, and H) Data from the
same cells and recordings in
A, D, and G, with the first 16 min of anaphase shown on an
expanded time axis. To illustrate movement of the spindle poles relative to the neck,
distances between the neck
and the pole in the mother cell were plotted as positive
numbers, and distances between the neck and the pole
in the bud were plotted as
negative numbers. (C, F, and
I) Data recorded from different cells. Measurement of
spindle pole position relative
to the neck was performed
on images of Nuf2p-GFP fluorescence overlaid onto corresponding DIC images. The
haploid strains used were
wild-type strain DS723 (A-C), kip3 strain DS786 (D-F),
and dyn1 strain DS940 (G-I).
During the early period of anaphase spindle elongation,
when the dividing nucleus is in the neck, the nucleus and
spindle can exhibit forward and reverse motion along the
mother-daughter axis. These movements range in distance
from 0.5-1 µm in wild-type diploid cells, and they are absent in dyn1 mutants (Yeh et al., 1995). We observed few
forward and reverse motions of the nucleus relative to the
neck in the dyn1 mutant, but we did observe such movements in wild-type and kip3 strains (Fig 7, B-I), suggesting that KIP3 is not essential for these movements.
The early anaphase spindle undergoes proportionally
greater elongation before insertion of the nucleus into the
neck in dyn1 mutants compared to wild-type (Yeh et al.,
1995). To identify possible defects in this process in the
kip3 mutant, we measured the pole-to-pole length of the
spindle at the time that the daughter-bound pole passed
through the neck (Fig. 7). Some elongation of the spindle
occurred before insertion into the neck in all strains examined. Spindle length upon neck contact in the kip3 mutant was 2.9 ± 0.8 µm (n = 10), and in the wild-type control it
was 2.3 ± 0.5 µm (n = 5). This difference in spindle
lengths was not statistically significant (unpaired t test,
P = 0.12), indicating that loss of KIP3 function has little
effect on insertion of the spindle through the neck. In contrast, the spindle length of an isogenic dyn1 mutant at the
time of insertion was 4.1 ± 0.7 µm (n = 8), which was significantly different from wild-type (P = 0.0005). Translocation was complete within 5 min in the wild-type, kip3,
and dyn1 cells observed, indicating that transient elongated nuclei at the beginning of successful anaphase
events make only a small contribution to the populations
of binucleate cells reported in Table II.
Loss of Dynein Function Has Little Effect on Nuclear Migration to the Neck or on Preanaphase Spindle Orientation
Mutants defective in the gene for the dynein heavy chain
(dyn1 or dhc1) have been reported to be defective in nuclear orientation, insertion of the dividing nucleus into the
neck, and oscillatory movement of the anaphase-stage nucleus within the neck (Eshel et al., 1993; Li et al., 1993;
Yeh et al., 1995). To compare the effect of loss of KIP3
function to loss of DYN1 function in isogenic strains, we
constructed a dyn1 mutant and scored the frequency of
binucleate cells and the position of undivided nuclei within mother cells. Consistent with the previously reported defects in anaphase-stage cells, the dyn1 mutant cultures
contained binucleate mother cells at a frequency of 11-
31%, depending on incubation temperature (Table II).
Proficiency in migration of undivided nuclei was assessed
by determining the mean nuclear migration index and
standard deviation. The histograms in Fig. 4 show that the
dyn1 mutant grown at 11, 30, and 37°C showed a distribution of nuclear migration indices that was similar to the
wild-type strain. The ratio of variances between wild-type
and dyn1 strains at each temperature was below the 95%
significance point, indicating no significant difference was
present. Furthermore, spindle pole movement observed
by microscopy of live dyn1 cells containing the Nuf2p-
GFP fusion showed that the path and rate of movement was similar in dyn1 and wild-type cells (Fig. 6, E and F), although we did observe that the time between anaphase
spindle disassembly and bud emergence was prolonged in
the dyn1 mutant. Thus, in contrast to the kip3 mutant,
the dyn1 mutant is not significantly impaired in migration of undivided nuclei to the site near the bud neck. This
is consistent with previous measurements of nuclear distribution in hydroxyurea-arrested dyn1 cells, where nuclear
translocation into the bud decreased, but accumulation of
nuclei at the bud neck was largely unimpaired (Yeh et al.,
1995).
To determine whether orientation of the preanaphase
mitotic spindle is affected by loss of dynein function, we
used the Nuf2p-GFP fusion protein employed above for
the kip3 mutant to measure spindle orientation in fixed
dyn1 mutant cells. Preanaphase spindles in the dyn1
mutant were oriented within 30 degrees of the mother- daughter axis in 68% of the cells, compared to 72% in
wild-type cells. There was a small increase in the number
of cells with a spindle misoriented by 60-90 degrees (13%,
compared to 8% in wild-type). Thus, loss of dynein heavy
chain function has little effect on preanaphase spindle orientation.
Synthetic Lethal Interactions of the kip3
Mutation with Mutations in Other Microtubule-based
Motor Genes
The phenotypes presented above of mutants singly defective in KIP3 or DYN1 indicate that KIP3 is required for nuclear migration to the bud neck and preanaphase spindle orientation, but that DYN1 is not. However, Kip3p and Dyn1p may perform overlapping or dependent functions that are not evident in the single mutants. For instance, the binucleate mother cell phenotype manifested by both kip3 and dyn1 mutants may reflect a common function in insertion of the anaphase nucleus through the bud neck. Alternatively, the cumulative effect caused by loss of the sequential migration and insertion steps of nuclear migration may be greater than that caused by loss of migration or insertion individually. Similarly, KIP3 could overlap with any of the other kinesin-related genes to perform an essential function, and this overlap would only result in a phenotype upon loss of both activities.
We tested for synthetic phenotypes by constructing double deletion mutants in the presence of a plasmid-based
copy of one wild-type gene and then determined whether
the strain remained viable after plasmid segregation (see
Materials and Methods). The kip3 dyn1, kip3 kar3, and
kar3 dyn1 strains all required a complementing plasmid
for viability (Fig. 8 A), indicating that these combinations
were synthetically lethal. The kip3 kip1, kip3 cin8, kip3
smy1, and kip3 kip2 double mutants were all viable and
formed colonies equal in size to the single mutants at 16, 23, 30, and 37°C (at 37°C the cin8 and kip3 cin8 strains
showed equally poor growth), indicating that these pairs of
genes are not solely responsible for an essential function.
and kip3(ts) kar3 strains. Strains wild-type (DS138), kip3 (DS613),
dyn (DS749), kip3-20(ts) dyn1 (DS765), kar3102 (DS750), and kip3-30(ts) kar3 (DS752) were incubated at 23°C for 2 d in liquid
YPD medium. Approximately 104 cells were spotted on solid YPD medium containing 1% DMSO and benomyl at the indicated concentration, and photographed after 3 d of growth at the indicated temperature. (D) KIP3 and KAR3 differ in their genetic interactions
with KIP1 and CIN8. Strains kip1 cin8-101 (DS49), wild-type (DS141), kar3 (DS276), kip3 (DS614), kar3 kip1 cin8-101
(DS689), and kip3 kip1 cin8-101 (DS737) were incubated for 2 d at 23°C in YPD medium. Approximately 5 × 104 cells were spotted
on YPD medium and incubated for 4 d at the indicated temperature. kar3 remediates the temperature-sensitive spindle assembly defect of the cin8-101 kip1 double mutant, whereas kip3 does not.
Simultaneous Loss of KIP3 and Dynein Function Causes Lethality but Does Not Abolish Any Specific Stage of Nuclear Migration
To distinguish whether kip3 and dyn1 mutations are synthetically lethal because of a functional overlap or due to a
cumulative effect caused by partial loss of sequential functions, we examined a conditional double mutant. Temperature-sensitive kip3(ts) dyn1 strains were constructed by
the plasmid shuffle technique (Fig. 8 B). A culture of the
kip3-20(ts) dyn1 strain grown at 23°C contained 48% inviable cells, while kip3 and dyn1 single mutant cultures
contained 95-100% viable cells. Incubation of the double mutant at 37°C for 4 h resulted in a doubling of cell number and a reduction of cell viability to 25%. Continued
incubation at 37°C resulted in a second doubling of cell
number by 16 h, at which time cell number ceased to increase and cell viability remained at 25%. This suggests
that the synthetic lethality is not solely due to induction of
a checkpoint that arrests cell cycle progression and prevents inviability.
Preanaphase nuclear migration was examined by microscopy of the double mutants after incubation at the
nonpermissive temperature. The position of undivided nuclei was significantly more random in the kip3(ts) dyn1
double mutant than in the dyn1 single mutant and wild-type strains (Fig. 4). However, the variance of the kip3(ts) dyn1 double mutant was not significantly different from
the kip3 single mutant. This indicates that combination
of dyn1 and kip3(ts) mutations causes no greater defect
in nuclear migration to the neck than that caused by the
kip3 mutation alone.
Both kip3 and dyn1 mutations individually cause a small
delay in the nuclear division cycle before anaphase (see
above and Yeh et al., 1995). The combination of kip3(ts)
dyn1 mutations also caused a delay in the nuclear division cycle, which we observed as an increased frequency of
preanaphase cells whose buds had grown beyond the size
at which nuclear division normally occurs. Double mutant
cells incubated at 37°C for 4 h showed the delay at a frequency 2.5-fold higher than the kip3 and dyn1 single mutants and 10-fold higher than the wild-type strain (Table
II). The kip3(ts) dyn1 cells incubated at the nonpermissive
temperature exhibited an abnormal morphology that was
not observed in either single mutant. Mother cells and mature buds were enlarged and elongated, but small buds had
a normal ellipsoidal shape (Fig. 9). Abnormal cell elongation can be caused by mutations affecting a variety of functions (Blacketer et al., 1995), including defects in the switch from apical to isotropic growth that occurs at the
G2/M transition (Lew and Reed, 1993).
To determine whether KIP3 and DYN1 overlap in function for insertion of the nucleus into the neck during
anaphase spindle elongation, we measured the frequency
of mother cells containing two discrete nuclear DNA
masses. The kip3(ts) dyn1 double mutant culture incubated at 37°C for 4 h contained 10% binucleate mother cells (Table II), compared to 6% binucleate cells in kip3
and dyn1 single mutants. An independent kip3(ts) allele in
a dyn1 strain had a similar cell type distribution. Since
the double mutant culture incubated for 4 h at 37°C still
contained some anaphase cells, we examined a culture incubated for 8 h at 37°C and found that the binucleate
mother cells increased to 23%. Thus, simultaneous loss of
KIP3 and DYN1 function causes defective insertion of
anaphase spindles through the bud neck, in addition to
causing defective preanaphase nuclear migration and a
metaphase delay. This suggests that the synthetic lethality
of kip3 dyn1 mutants is a cumulative effect caused by loss
of partially overlapping and sequential functions.
Since one phenotype of the kip3 single mutant and the
kip3(ts) dyn1 double mutant is increased microtubule
abundance (Figs. 3 and 9), we tested whether the microtubule-depolymerizing drug benomyl could suppress the lethality of the kip3(ts) dyn1 double mutant. The double
mutant showed little growth at 37°C in the absence of
benomyl but exhibited significant growth on media containing 5 or 10 µg/ml benomyl (Fig. 8 C). This suggests
that altered microtubule polymerization in the double mutant is a contributing factor to the synthetic lethality.
Simultaneous Loss of KIP3 and KAR3 Function Generates No Strong Synthetic Morphological Defect
The kinesin-related Kar3p is a candidate to provide some
of the same functions as Kip3p because loss of either KIP3
or KAR3 causes increased astral microtubule abundance
(above and Saunders et al., 1997), and Kar3p is known to
mediate one nuclear movement, nuclear congression, during karyogamy (Meluh and Rose, 1990). To determine the
nature of the defects caused by simultaneous loss of KIP3
and KAR3 function, the plasmid shuffle technique was
used to isolate a temperature-conditional double mutant
(Fig. 8 B). Like the kip3(ts) dyn1 double mutant, lethality
of the kip3-30(ts) kar3 double mutant was partially suppressed on medium containing a low concentration of
benomyl (Fig. 8 C). To examine morphological defects,
the kip3(ts) kar3 double mutant was incubated at the nonpermissive temperature, and the cells were compared
to kar3 and kip3 single mutants by microscopy. The
kar3 single mutant showed a high proportion of cells before anaphase (Table II), with many of the nuclei in very
close proximity to the bud neck (Fig. 4), as previously reported (Meluh and Rose, 1990). When the kip3 kar3 double mutant was incubated at 37°C for 4 h, fewer nuclei
were in very close proximity to the neck, and more were at
the distal edge of the mother cell (Fig 4). This suggests that the migration defect caused by kip3 mutation is epistatic
to the accumulation of nuclei in very close proximity to the
bud caused by kar3 mutation. No other aberrant morphology unique to the double mutant was detected (Fig. 9).
The data in Table II show that the frequency of cells in
metaphase in the kip3 kar3 double mutant culture was
similar to that in kar3 single mutant, and the frequency of
binucleate cells was similar to that in the kip3 single mutant. Thus, the combination of kip3 and kar3 mutations
caused the aberrant morphologies characteristic of the individual mutations to be superimposed and did not result
in a detectable synthetic morphological phenotype.
KIP3 and KAR3 Differ in Their Genetic Interactions with KIP1 and CIN8
Given the possibility of functional overlap suggested by
the synthetic lethality of kip3 and kar3, we examined
whether Kip3p could perform a previously described function of Kar3p in contributing to the balance of inwardly
and outwardly acting forces that maintain mitotic spindle
pole separation during metaphase. Mutations in kinesin-related genes KIP1 and CIN8 can cause collapse of the metaphase spindle, suggesting that Kip1p and Cin8p act to
maintain spindle pole separation, and mutations in KAR3
can suppress this collapse phenotype and restore growth,
suggesting that Kar3p generates an inward force on the
spindle poles (Saunders and Hoyt, 1992; Hoyt et al., 1993).
Mutations in KAR3 may suppress the kip1 cin8 collapse
phenotype by stabilizing microtubules, and a mutation in
the -tubulin gene that stabilizes microtubules has an effect similar to kar3 mutation (Saunders et al., 1997). To
determine whether Kip3p can perform a role similar to
Kar3p in countering the forces generated by Kip1p and
Cin8p, we tested whether a kip3 mutation can suppress
the kip1 cin8 growth defect. Meiotic crosses were used to
construct kip3 kip1 cin8 and kar3 kip1 cin8 triple mutants,
and the sensitivity of growth to temperature was examined (Fig. 8 D). The kar3 mutation partially suppressed the
temperature-sensitive growth of the kip1 cin8 mutant as
expected, but the kip3 mutation did not. Thus, KIP3 fails
to show genetic interactions with KIP1 and CIN8, indicating that Kip3 does not overlap with the suggested role of
Kar3p in generating an inwardly directed spindle force, and further suggests that Kip3p and Kar3p perform separate functions.
Kip3p is Present on Both Astral and Central Spindle Microtubules
The phenotypes of the kip3 mutant suggested roles for
Kip3p primarily in astral microtubule function. To determine if Kip3p is associated with microtubules in vivo, we
determined the intracellular localization of a functional
epitope-tagged protein. A single-copy plasmid expressing
myc-tagged Kip3p (KIP3-6MYC, see Materials and Methods) from its own promoter was introduced into a kip3 strain. Western blotting of an extract from this strain identified a single band at the predicted molecular mass of 100 kD (data not shown). In G1 cells, Kip3p colocalized with
the astral microtubules (Fig. 10, A-C). In late S phase or
early mitotic cells, Kip3p colocalized with short bipolar
spindles (Fig. 10, D-F). In late anaphase or telophase cells,
Kip3p colocalized with spindle microtubules and was frequently concentrated near the spindle midzone, the zone of overlap between microtubules from opposite poles
(65% of late mitotic cells where Kip3p spindle localization
was observed, n = 250; Fig.10, G-I). In the remaining late
mitotic cells, Kip3p staining was evenly distributed along
the length of the elongated spindle (Fig. 10, J-L). In addition to microtubule staining, expression of KIP3-6MYC
resulted in bright punctate staining of the nucleus and cytoplasm of all cells observed. This punctate staining was not observed over the vacuole and was specific to Kip3p-6myc because it was not observed in an untagged KIP3
control strain (Fig. 10, M-O). We were able to detect microtubule localization of Kip3p in 11% of the cells examined (n = 300). It is possible that microtubule staining in
many cells was obscured by the generalized staining.
The Complete Set of S. cerevisiae Kinesin-related Proteins
A search of the complete yeast genome sequence revealed
KIP3 as the single previously uncharacterized gene encoding a protein with strong sequence conservation with the
force-generating domain of kinesin. Thus, budding yeast
contains six genes encoding kinesin-related proteins (KIP1,
KIP2, KIP3, KAR3, CIN8, and SMY1), and it is the first
organism whose complete set of kinesin-related proteins has been identified. Three of the kinesin-related proteins
are members of known kinesin subfamilies defined by sequence conservation; KIP1 and CIN8 belong to the BimC
subfamily and KAR3 belongs to the COOH-terminal subfamily (Moore and Endow, 1996). Members of these two
subfamilies show functional conservation as well as sequence conservation, with members of the BimC subfamily playing roles in mitotic spindle assembly and members
of the COOH-terminal subfamily playing roles in spindle
pole function. The kinesin-related proteins encoded by
KIP2, KIP3, and SMY1 lack any strong subfamily homology.
The relatively small number of microtubule-mediated
movements that occur in yeast can now be ascribed to the
force generated by the six kinesins, dynein, and potentially
to forces generated by microtubule polymerization and
depolymerization. No single motor protein is essential for
viability, perhaps because of cooperation between different force-generating mechanisms, such as the overlap between KIP1 and CIN8 for mitotic spindle assembly (Hoyt
et al., 1992; Roof et al., 1992). Given the complete set of
motor proteins, it is now possible to systematically dissect
the contribution of each motor protein to a particular microtubule-based movement.
A Kinesin-related Protein Required for Nuclear Migration
We used two experimental approaches to characterize the
effect of loss of KIP3 function on nuclear migration. In the
first approach, we examined populations of fixed cells to
assess the position of the nucleus and orientation of the
mitotic spindle relative to the bud. Undivided nuclei in
budded wild-type cells had a strong tendency to be located
~1 µm from the neck, and the associated mitotic spindle
had a strong tendency to be oriented within 30 degrees of
the mother-daughter axis, consistent with previous studies
of S. cerevisiae morphology (Byers and Goetsch, 1975; Jacobs et al., 1988). Both position and orientation of the nucleus became essentially random upon loss of KIP3 function. Since these experiments relied on use of the bud as a
reference point to quantitate the position and orientation
of undivided nuclei, they define an essential role for KIP3
for directed nuclear migration and orientation during the
cell cycle interval from Start to just before anaphase. We
were unable to use fixed cells to quantitate nuclear movements in G1 phase cells because these cells lack a bud to
use as a reference point.
In the second experimental approach to characterize nuclear migration, we used live cell microscopy to follow
movement of the spindle pole body in individual cells. In
addition to defining the path and kinetics of movement
during nuclear migration, this approach enabled us to examine nuclear positioning during the G1 phase of the cell
cycle. By observing nuclear migration in a live cell through
the point of bud emergence, we could infer the site of bud
emergence for use as a reference point in G1 phase cells.
Bud emergence in haploid cells follows the axial pattern of
bud site selection, in which the new bud emerges adjacent
to the previous division site (Chant and Pringle, 1995). A
consequence of axial budding is that anaphase spindle
elongation places the nucleus at the edge of the cell opposite to the site of bud emergence.
The first spindle-independent movement of the nucleus
occurs immediately after spindle disassembly, when the
nucleus rapidly moves toward the cell center (Fig. 6 and
Yeh et. al., 1995). We observed this movement in wild-type, kip3, and dyn1 strains. In the subsequent period of
movement, the nucleus continued toward the prebud site,
with steady forward movement and no large reversals in
direction. The second period of movement sometimes occurred immediately after the initial movement, so that migration was complete within 15 min of spindle disassembly, before a bud could be seen. In other examples, there
was a delay of about 20 min before the second period of
movement occurred. The new bud can be detected only at
the end of G1, but actin, Spa2p, and neck filament proteins
localize to the prebud site before bud emergence (Kim et
al., 1991; Snyder et al., 1991; Lew and Reed, 1993). Although there are differences in the reported timing of assembly of proteins at the prebud site, the intersection of
astral microtubules with the prebud site provide a potential mechanism for directed nuclear migration during G1.
When the nucleus reached a site ~1 µm from the bud
neck, its position became relatively stable. We observed similar patterns of preanaphase movement in wild-type
and dyn1 strains.
Loss of KIP3 function caused a dramatically different course of phase 1 nuclear migration. After the kip3 mutant completed the rapid inward movement after spindle disassembly, nuclei exhibited large forward and reverse oscillations in position relative to the bud neck (see Fig. 6, C and D). The nucleus sometimes came within ~1 µm of the neck, only to reverse direction and move 1-2 µm in the opposite direction. Since the nuclei were located far from the neck for long time intervals, th
