Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218
Proper positioning of the mitotic spindle is
often essential for cell division and differentiation processes. The asymmetric cell division characteristic of
budding yeast, Saccharomyces cerevisiae, requires that
the spindle be positioned at the mother-bud neck and
oriented along the mother-bud axis. The single dynein
motor encoded by the S. cerevisiae genome performs an
important but nonessential spindle-positioning role.
We demonstrate that kinesin-related Kip3p makes a
major contribution to spindle positioning in the absence of dynein. The elimination of Kip3p function in
dyn1 cells severely compromised spindle movement
to the mother-bud neck. In dyn1
cells that had completed positioning, elimination of Kip3p function
caused spindles to mislocalize to distal positions in
mother cell bodies. We also demonstrate that the spindle-positioning defects exhibited by dyn1 kip3 cells are
caused, to a large extent, by the actions of kinesin-
related Kip2p. Microtubules in kip2
cells were shorter
and more sensitive to benomyl than wild-type, in contrast to the longer and benomyl-resistant microtubules
found in dyn1
and kip3
cells. Most significantly, the
deletion of KIP2 greatly suppressed the spindle localization defect and slow growth exhibited by dyn1 kip3
cells. Likewise, induced expression of KIP2 caused
spindles to mislocalize in cells deficient for dynein and
Kip3p. Our findings indicate that Kip2p participates in
normal spindle positioning but antagonizes a positioning mechanism acting in dyn1 kip3 cells. The observation that deletion of KIP2 could also suppress the inviability of dyn1
kar3
cells suggests that kinesin-related
Kar3p also contributes to spindle positioning.
MITOTIC spindles commonly segregate chromosomes at specific positions within eukaryotic
cells. In a cell type-specific fashion, spindles undergo movements and orient in response to spatial cues originating from cell cortical regions (for reviews see Rhyu
and Knoblich, 1995 The asymmetric cell division characteristic of the budding yeast Saccharomyces cerevisiae makes spindle positioning essential for propagation. In the G1 phase of the
cell cycle, the S. cerevisiae spindle is located at a position
near the middle of the mother cell body. Proper segregation of progeny nuclei requires that the spindle translocate
to the neck separating the mother and bud cell bodies and
orient along the long mother-bud cell axis before anaphase.
Since this process results in the movement of the entire
nuclear contents to the neck region, it is often referred to
as nuclear migration. In addition to budding yeast mitotic division, nuclear migration events are essential for gamete
fusion processes, insect embryonic development, and the
growth of the vegetative mycelium of filamentous fungi
(Morris et al., 1995 Spindle positioning in S. cerevisiae requires the actions
of the cytoplasmic microtubules that extend from the spindle pole bodies out towards the cell cortex (Palmer et al.,
1992 The observation that dynein is not essential for S. cerevisiae spindle positioning indicates that another motor
mechanism(s) contributes to this process. In addition to a
single dynein heavy chain, the S. cerevisiae genome encodes six kinesin-related proteins (KRPs).1 Studies, including those described here, have demonstrated that none of these seven motors is individually essential for S. cerevisiae viability; all seven motor gene single deletion
mutants are viable (Meluh and Rose, 1990 Yeast Strains and Media
The S. cerevisiae strains used in these experiments are derivatives of
S288C and are listed in Table I. The cin8::LEU2, dyn1::HIS3, dyn1::
URA3, kar3::LEU2, kip1::HIS3, kip2::URA3, and smy1::LEU2 alleles
were described previously (Meluh and Rose, 1990 Table I.
Yeast Strains and Plasmids
; Gönczy and Hyman, 1996
). Spindle-positioning events are often essential for cell propagation
or the asymmetric cell divisions required for differentiation. For example, the distinct developmental programs
followed by the progeny of the first two cells of the Caenorhabditis elegans embryo require that the cleavage
planes of these cells occur perpendicular to each other. In
these cells, the plane of cell cleavage is determined by
spindle position. The perpendicular cleavage arrangement
is achieved by a 90° rotation of the spindle poles, in the
posterior cell only, in response to a cortically located structure (Hyman and White, 1987
; Hyman, 1989
; Waddle et
al., 1994
). A similar effect has been observed during mammalian brain development. The developmental fate of columnar epithelial progenitor cells is predicted by spindle
orientation and the subsequent position of the cleavage
plane (Chenn and McConnell, 1995
).
). Nuclear migration events have also
been correlated with essential mammalian brain development processes (Book and Morest, 1990
; Book et al., 1991
;
Hager et al., 1995
). The mechanisms of many of these processes are not well understood, but all appear to rely upon
microtubules and cortically derived signals to guide the
movement of nuclei. In addition, nuclear migration processes in S. cerevisiae, Aspergillus nidulans, and Neurospora crassa share in common the participation of the microtubule-based cytoplasmic dynein motor (Eshel, 1993;
Li et al., 1993
; Plamann et al., 1994
; Xiang et al., 1994
).
; Sullivan and Huffaker, 1992
). Interactions of the distal ends of the cytoplasmic microtubules with cortical sites
have recently been shown to result in spindle movements
(Carminati and Stearns, 1997
). In mutants specifically defective for cytoplasmic microtubules, nuclei do not migrate
to the mother-bud neck, and spindles undergo anaphase chromosome segregation at inappropriate positions in
mother cell bodies. The single dynein heavy chain encoded
by the S. cerevisiae genome performs an important but nonessential role in spindle positioning (Eshel et al., 1993
; Li
et al., 1993
; Geiser et al., 1997
). DYN1 deletion mutants frequently display mislocalized spindles but nonetheless grow
at rates indistinguishable from wild-type at room temperature. At lower incubation temperatures, however, the spindle-positioning defect is greatly exacerbated and cell propagation is inhibited. Collected evidence suggests that dynein is contributing to spindle positioning by acting upon the cytoplasmic microtubules from cortical sites (Yeh et al., 1995
).
; Hoyt et al.,
1992
; Lillie and Brown, 1992
; Roof et al., 1992
; Eshel et al.,
1993
; Li et al., 1993
). This is due to overlap in function between the motors such that each essential mitotic spindle
movement is accomplished by at least two motors. In this
study, we demonstrated that the spindle-positioning function of Dyn1p overlaps with that of Kip3p (encoded by
open reading frame YGL216W, also known as RRC805;
these sequence data are available from GenBank/EMBL/
DDBJ under accession number Z72739), a KRP revealed
by the Saccharomyces Genome Sequencing Project. (A
role for Kip3p in spindle positioning was also recently described by DeZwaan et al. [1997].) In the absence of both
Dyn1p and Kip3p, spindle positioning was dramatically inhibited by a mechanism that required the Kip2p KRP.
dyn1
kip2
kip3
triple mutants were viable and healthy,
indicating that yet another motor contributes to spindle
positioning. Genetic evidence presented suggests that this
other spindle positioning motor is Kar3p.
Materials and Methods
; Hoyt et al., 1992
; Lillie
and Brown, 1992
; Roof et al., 1992
; Eshel et al., 1993
; Geiser et al., 1997
).
The kip3::kan allele is a complete disruption of the KIP3 open reading
frame (YGL216W) located on chromosome VII and was constructed using the PCR-mediated method described in Wach et al. (1994)
. Briefly,
the kanamycin resistance gene (kanMX) from plasmid pFA6-kanMX4
was amplified with flanking ends homologous to 40 bp upstream and 40 bp
downstream from the KIP3 open reading frame. kanMX confers resistance to the antibiotic G418. The PCR product was transformed into
strain MAY591, and colonies were selected on rich medium containing
200 mg/liter G418 (Life Technologies, Gaithersburg, MD). PCR primers
corresponding to the upstream region of the KIP3 open reading frame
and to an internal segment of either KIP3 or kanMX were used to confirm that the kanMX gene had replaced the entire KIP3 open reading frame in
G418-resistant cells.
Rich (YPD) and minimal (SD) media were as described (Sherman et
al., 1983). Benomyl (DuPont, Wilmington, DE) was added to solid YPD
medium from a 10 mg/ml stock in dimethyl sulfoxide. For G1 synchronization,
-factor (Bachem Bioscience, King of Prussia, PA) was added to 4 µg/ml to log-phase cells in liquid YPD, pH 4.0, and incubated until >80%
of cells were unbudded. For arrest in S phase, hydroxyurea (Sigma Chemical Co., St. Louis, MO) was added to 0.1 M to log-phase cells in liquid
YPD, pH 5.8, and incubated until >70% of cells were large budded.
DNA Manipulations
The shuttle vectors pRS316, pRS317, and pRS318 are described in Sikorski and Hieter (1989). KIP2 was obtained on a phage
clone (No. 70186)
from the American Type Culture Collection (Rockville, MD). pFC50
(KIP2 LYS2 CEN) is a 3.0-kb subclone containing KIP2 bounded by ClaI
and KpnI. To put KIP2 under the control of a galactose-inducible promoter, the KIP2 gene was amplified such that an SpeI site was introduced
6 bp upstream of its open reading frame. The sequence of the 5
primer is
GAATCATCACTAGTGGTATTATGG. The resulting PCR product
was subcloned into p415GALS (Mumberg et al., 1994
) to create pFC56.
KIP3 was obtained as a phage
clone (No. 70127) from the American
Type Culture Collection. pFC51 (KIP3 LYS2 CEN) is a 4.8-kb subclone
containing KIP3 bounded by EcoRI and SpeI. pMA1223 (KAR3 URA3
CEN) was constructed by subcloning a 3.4-kb BamHI-ClaI fragment containing KAR3 from pMR1350 (a gift from Mark Rose, Princeton University, Princeton, NJ) into pRS316.
Temperature-sensitive alleles of KIP3 were generated by a mutagenic
PCR-based procedure (Staples and Dieckmann, 1993). Primers corresponding to the polylinker region of pRS316 were used to amplify the entire KIP3 gene, plus 1.4 kb of upstream sequence and 1.0 kb of downstream sequence, under mutagenic PCR conditions. We used taq
polymerase (Stratagene, La Jolla, CA) according to the manufacturer's instructions, except that Mn2+ and Mg2+ were added to final concentrations
of 0.25 and 4.5 mM, respectively. The PCR products were concentrated
using QIAquick spin columns (Qiagen, Chatsworth, CA). pFC51 was digested with MscI to create a gapped construct in which all of the KIP3
coding sequence was removed except for the last 18 bp at the 3
end. The
gapped pFC51 construct was gel purified and cotransformed with the concentrated PCR products into strain MAY4619 (kip3
kar3
lys2 ura3
[pMA1223 = KAR3 URA3]). This strain is unable to survive loss of
pMA1223 and is therefore rendered sensitive to 5-fluoro-orotic acid
(5-FOA; from US Biological, Swampscott, MA). Approximately 6,000 Lys+
transformants were selected at 26°C and then replica plated to 5-FOA-
containing media at 26 and 35°C. 21 colonies were selected that were viable on 5-FOA at 26 but not at 35°C. Plasmids were isolated from the 21 temperature-sensitive transformants and retransformed into MAY 4619. After growth on 5-FOA at 26°C, 18 of the 21 retransformed strains displayed reduced growth on YPD at 35°C. These 18 plasmids were designated pFC51kip3-2 through pFC51kip3-19.
Microscopic Analysis of Cells
To stain for DNA, cells were pelleted out of liquid media, resuspended in
70% ethanol, and stored on ice for 30 min. The fixed cells were washed
once with water and then resuspended in 0.32 µg/ml 4,6-diamidino-2-phenylindole (DAPI) plus 1 mg/ml p-phenylenediamine to prevent fading (both
from Sigma Chemical Co.). For staining of chitin-containing bud scars
(Pringle, 1991), cells were fixed as above, washed once with water, resuspended in 1 mg/ml calcofluor (Sigma Chemical Co.) solution, and stored
at room temperature in the dark for 5 min. The cells were washed four
times with water and then resuspended in 0.32 µg/ml DAPI plus 1 mg/ml
p-phenylenediamine before viewing. For antitubulin immunofluorescence
microscopy, cells were fixed by adding formaldehyde directly to the medium to a final concentration of 3.7%. Microtubules were visualized by
the procedure described in Pringle et al. (1991)
using the rat anti-
-tubulin antibody YOL1/34 (Harlan Bioproducts for Science, Indianapolis, IN)
and rhodamine-conjugated goat anti-rat secondary antibodies (Jackson
ImmunoResearch, West Grove, PA). Stained cells were examined with an
inverted microscope (model Axiovert 135; Carl Zeiss, Inc., Thornwood,
NY) equipped with epifluorescent optics using a 100× objective. Digital
images were captured with a cooled, slow scan CCD camera.
For microtubule and spindle length measurements, cells were arrested with hydroxyurea at 26°C and stained for microtubule structures as described above. Images of antitubulin-stained cells were captured electronically, and the cursor-based measuring tool of the NIH Image 1.61 software program, calibrated with a stage micrometer, was used to measure the lengths of cytoplasmic microtubules and spindles. Spindle length was determined by measuring the length of the bright bar of antitubulin staining whose ends were coincident with the edges of the DAPI-stained mass. Cytoplasmic microtubules were defined as the less intensely stained fibers emanating from the spindle poles.
To quantitate binucleate cells, cultures were grown to log phase in liquid YPD at 26°C. The cells were either stained with DAPI immediately or shifted to 12°C for 24 h and then stained with DAPI. Binucleate cells were defined as those in which a single cell body contained two (or more) distinguishable DAPI-staining masses. Mother cell bodies were defined as those which contained at least two calcofluor-stained chitin rings on their surface. In our studies of DAPI-stained cells, two aberrant nuclear morphologies were quantitated: (a) Large-budded cells with a single nuclear DNA mass located away from the neck. Nucleus away-from-the-neck cells were defined as those in which the closest distance between the nucleus and the neck was greater than one half of the diameter of the entire nuclear DNA mass, as judged by eye. (b) Large-budded cells with two DAPI-staining masses located within one cell compartment.
Galactose-induced Expression of KIP2
It was discovered that fully induced expression of KIP2 (in 2% galactose)
from the GALS promoter on pFC56 resulted in slow growth of wild-type
cells. Note that GALS promoter activity has already been weakened by
mutation (Mumberg et al., 1994). We empirically determined that a mixture of 2% galactose plus 0.05% glucose reduced the expression of KIP2
sufficiently such that growth of dyn1
kip2
kip3
cells was inhibited but
that of wild-type cells was not. Cells to be tested were grown to log phase
in liquid synthetic raffinose (2%) media lacking leucine, pH 5.8, and
treated with hydroxyurea until >70% of cells were large budded. A sample was removed and fixed with ethanol. The remainder of the culture was transferred to synthetic minus leu media, pH 5.8, containing 0.1 M hydroxyurea plus 2% galactose and 0.05% glucose to induce expression of KIP2.
Samples were fixed with ethanol and stained with DAPI, and the percent
of large-budded cells with the nucleus away from the neck was determined
as described above.
Genetic Interactions between DYN1 and Other Motor Genes
S. cerevisiae cells deleted for the single dynein heavy
chain-encoding gene DYN1 exhibit spindle-positioning
defects but grow at wild-type rates at 26°C (Eshel et al.,
1993; Li et al., 1993
). Previous studies from this laboratory
and others revealed that five S. cerevisiae KRP genes can
also be individually deleted without affecting cell viability
(Meluh and Rose, 1990
; Hoyt et al., 1992
; Lillie and
Brown, 1992
; Roof et al., 1992
). For this study, we created
a deletion allele of KIP3, the sixth and final KRP gene revealed by the Saccharomyces Genome Sequencing Project. The entire KIP3 open reading frame was replaced with a
bacterial kanamycin resistance gene (see Materials and
Methods). Yeast cells deleted for KIP3 were viable and
did not exhibit any obvious growth defects at incubation
temperatures between 11 and 37°C. Mild microtubule-related phenotypes for kip3
cells were observed and are
described below.
In an attempt to define the motor(s) that overlap in
function with dynein, we created double mutants between
dyn1 and deletion alleles of the six KRP motor genes.
For kip1
, kip2
, and smy1
, viable double mutant cells
were recovered that grew at rates comparable to the wild-type at 26°C. In contrast, we were unable to recover viable
combinations of dyn1
with cin8
(Saunders et al., 1995
) and kar3
. dyn1
kip3
mutant cells were viable, but
grew into colonies at very slow rates (see Figs. 2 C and 5).
dyn1
kip3
cultures also contained high numbers of dead
cells detected by both microscopy and by a 48% reduced
plating efficiency relative to wild-type (plating efficiency
= colony forming units/cells counted in microscope).
These findings suggest that Cin8p, Kar3p, and Kip3p overlap with dynein for an essential or important function.
In our genetic studies, we noticed an interesting property of the KIP2 deletion allele. kip2 single mutant cells
exhibited a nuclear migration defect almost as severe as
that displayed by dyn1
cells (see below). Unexpectedly,
the deletion of KIP2 was found to suppress the deleterious
effects of the dyn1
kip3
and dyn1
kar3
genotypes
(Table II). Although dyn1
kip3
cells exhibited extremely poor colony-forming ability, the dyn1
kip3
kip2
triple mutants formed colonies of wild-type size.
dyn1
kar3
double mutants were inviable. Strikingly,
dyn1
kar3
kip2
triple mutants were viable, but formed
colonies slightly smaller than wild-type. A demonstration
of these antagonistic actions of KIP2 is shown in Fig. 1. A
vector plasmid and a plasmid expressing KIP2 were both
able to transform wild-type cells with high efficiency. In
contrast, dyn1
kip3
kip2
cells and dyn1
kar3
kip2
cells were transformed well by the vector plasmid, but not
the KIP2 plasmid. In contrast to the dyn1
kip3
and
dyn1
kar3
combinations, elimination of KIP2 was unable to suppress the growth defect of kip3
kar3
cells;
both kip3
kar3
and kip3
kar3
kip2
spores were unable to grow into colonies. Table II summarizes the growth properties of all the dyn1
, kip3
, kar3
, and kip2
mutant combinations.
Table II. Growth Properties of Relevant Motor Mutants |
The suppression by kip2 of combinations of DYN1,
KIP3, and KAR3 alleles functionally links the products of
these genes. In contrast, the lethality of dyn1
cin8
could
not be suppressed by deletion of KIP2. In addition, cin8
kip2
mutants, as well as dyn1
cin8
and dyn1
cin8
kip2
, were found to be inviable. The cause of the lethality of these cin8
combinations is not known. We have
previously suggested that the lethality of the Cin8p
Dyn1p-deficient combination reflects the contribution of both of these motors to anaphase spindle elongation
(Saunders et al., 1995
).
In summary, the deleterious effects of dyn1 kip3
and
dyn1
kar3
, but not kip3
kar3
, were strongly suppressed by the deletion of KIP2. These findings indicate
that the loss of Dyn1p plus either Kip3p or Kar3p can be
tolerated, providing that an antagonistically acting function of Kip2p is eliminated. In the remainder of this article, we characterize the spindle positioning and other microtubule phenotypes associated with loss of function of
Dyn1p, Kip3p, and Kip2p. The role of Kar3p in spindle
positioning has been harder to discern, probably because
this motor has more than one mitotic function (see Discussion).
dyn1, kip2
, and kip3
Cause
Spindle-positioning Defects
S. cerevisiae spindle-positioning errors lead to anaphase
nuclear division occurring exclusively in the mother cell
body. This produces a binucleate mother cell body and an
anucleate bud (Fig. 2 A). We examined the dyn1, kip2
,
and kip3
single and multiple mutant combinations for
the production of binucleate cell bodies at 26 and 12°C
(Fig. 2 B). As previously reported (Eshel et al., 1993
; Li et
al., 1993
; Geiser et al., 1997
), dyn1
cultures accumulated
elevated binucleate cells in a temperature-dependent fashion; very high levels were observed after incubation at
12°C. The kip2
mutant behaved similarly, although to a
lesser extent. The kip3
mutant was only slightly elevated
for binucleate cell production at 12°C. However, a role for
KIP3 in spindle positioning was indicated by the elevated
number of binucleate cells in cultures of the slow-growing
dyn1
kip3
double mutant. This was the only mutant
that displayed high levels (>20%) of binucleate cells at 26°C. dyn1
kip3
double mutant cultures also contained
significant numbers of cells with unusual nuclear morphologies (arrow in Fig. 2 A) and anucleate or dead cells.
Therefore, the binucleate percentage values determined
for this genotype might underrepresent the extent of the
defect. The dyn1
kip2
and kip2
kip3
double mutants also displayed binucleate cell levels that were greatly elevated over the wild-type, but only at 12°C. Note that the
levels exhibited by dyn1
kip2
and kip2
kip3
were intermediate to that exhibited by the single mutants. The deletion of KIP2 also partially relieved the cold-sensitive
growth defect caused by dyn1
(Fig. 2 C). The dyn1
kip2
kip3
triple mutant was reduced for binucleate cell production at 26°C relative to the dyn1
kip3
double
mutant. This reduction corresponded to the greatly improved growth rate of the triple mutant relative to the
double mutant (Fig. 2 C).
We examined the mutants to determine if the cell bodies
containing two nuclear DNA masses corresponded to the
mother or the bud. The mother cell was distinguished from
the bud by the presence of chitin-containing bud scars revealed by calcofluor staining. For the dyn1, kip2
, and
kip3
single mutants grown at 12°C, the binucleate bodies
were always mother cells (n = 104, 54, and 12 cells, respectively).
Cytoplasmic Microtubules Are Longer than Wild-Type
in dyn1 and kip3
Cells but Shorter in kip2
Cells
To examine the causes of the spindle-positioning defects
exhibited by the single and multiple mutants, microtubules
were visualized by antitubulin immunofluorescence microscopy (Figs. 3 and 4). Before fixation with formaldehyde, the DNA synthesis inhibitor hydroxyurea was added
to the cultures to synchronize cells at the preanaphase
short spindle stage of the mitotic cycle (Pringle and
Hartwell, 1981). Using this technique, stained wild-type
spindles appeared as a bright bar of nuclear microtubules
with whisker-like cytoplasmic microtubules attached to
the ends of the bars (the spindle poles). Striking differences were observed in the length and number of cytoplasmic microtubules in the various mutant genotypes. dyn1
and kip3
cells exhibited longer cytoplasmic microtubules than wild-type. The slow-growing dyn1
kip3
double
mutant exhibited extremely elongated cytoplasmic microtubules that were on average fourfold longer than the
wild-type. In contrast, cytoplasmic microtubules in kip2
cells were shorter than wild-type. The kip2
culture also
exhibited much higher numbers of cells for which we could not visualize a single cytoplasmic microtubule that survived fixation (Fig. 4 A). Combining the short microtubule
kip2
with either dyn1
or kip3
caused cytoplasmic microtubule phenotypes resembling those caused by kip2
alone (shortened and reduced in number). By both criteria, however, these doubles were less severely affected than the kip2
single mutant, consistent with an intermediate phenotype. The elimination of KIP2 in dyn1
kip3
cells, to create the triple mutant, suppressed the extremely
long cytoplasmic microtubule phenotype of this double
mutant, resulting in cytoplasmic microtubules intermediate in length between wild-type and kip2
. In these experiments, we also detected small but reproducible differences in spindle lengths (pole-to-pole distance; Fig. 4 B).
In particular, spindles were longer than wild-type in all
strains deleted for KIP3. We note that while formaldehyde
fixation may not preserve all yeast microtubule structure
(see Carminati and Stearns, 1997
), the differences observed here probably reflect actual differences in microtubule lengths and/or stabilities in vivo (see next section).
dyn1 and kip3
Increase Resistance to Benomyl while
kip2
Decreases Resistance
The observed differences in cytoplasmic microtubule
lengths suggested differences in microtubule stabilities
among the various motor mutants. The resistance of yeast
cells to compounds that promote microtubule depolymerization, such as benomyl, reflects the intrinsic stability of
cellular microtubules. We determined resistance to benomyl
by spotting cells onto solid media containing increasing
concentrations of the inhibitor (Fig. 5). We found that the
dyn1, kip2
, and kip3
genotypic combinations exhibited opposing and suppressing phenotypes that corresponded to the microtubule length phenotypes described
above. kip3
cells displayed higher resistance to benomyl
than the wild-type. dyn1
cells displayed resistance that
was only slightly but reproducibly elevated over wild-type.
In contrast, kip2
cells displayed markedly decreased benomyl resistance. It was not possible to accurately assess
the benomyl resistance of the dyn1
kip3
double mutant
because of its extreme slow growth. The two other double
mutant combinations, both of which involved kip2
, displayed resistances intermediate between those observed
for the single mutants. Deletion of either dyn1
or kip3
was able to restore near wild-type levels of resistance to
kip2
cells. The dyn1
kip2
kip3
triple mutant also displayed resistance near that of the wild-type.
Dyn1p and Kip3p Are Required to Achieve and Maintain Proper Spindle Positioning in the Presence of Kip2p
To determine the effects of loss of Kip3p function in various genetic backgrounds, 18 temperature-sensitive alleles
of KIP3 were generated using a mutagenic PCR-based approach (see Materials and Methods). A representative
temperature-sensitive allele, kip3-14, was selected for further study. Plasmids carrying kip3-14 or KIP3 were introduced into a dyn1 kip3
double mutant strain, as well as
into a dyn1
kip2
kip3
triple mutant strain. The resulting strains, along with a wild-type control, were synchronized in G1 with the
-factor mating pheromone at the
permissive temperature of 26°C. The G1 cells were released from the
-factor block, shifted to the nonpermissive temperature (37°C), and assayed for their ability to
proceed through mitosis by observation of stained nuclear DNA. As wild-type cells reached a large-budded morphology (bud diameter ~3/4 that of the mother), nuclear DNA
masses were either positioned in the neck or had undergone anaphase division with a single DNA mass positioned in each progeny cell body. For the dyn1
kip3
(pkip3-14) genotype only, cells with two morphologies that were rarely observed for wild-type accumulated after
the shift to 37°C (Fig. 6): large-budded cells with nuclear
DNA at a position away from the neck (top) and large-budded cells with two nuclear DNA masses in one cell
body (bottom). This finding indicates that Kip3p function
is required for efficient spindle positioning in the absence
of dynein. The nucleus-away-from-the-neck morphology
was somewhat elevated over the wild-type for the three other strains that were deleted for DYN1 (Fig. 6, top).
However, as previously described for dyn1
mutants (Yeh
et al., 1995
), these did not lead to the formation of binucleate cell bodies (Fig. 6, bottom), presumably because of the
actions of other motors that can resolve dyn1
spindle-
positioning errors (i.e., Kip3p). Notably, the dyn1
kip2
kip3
(pkip3-14) cells did not accumulate the aberrant
forms, indicating that elimination of Kip2p could suppress
the spindle-positioning defect exhibited by dyn1
kip3
(pkip3-14). This also demonstrates that another mechanism acts to position spindles efficiently in the absence of
dynein, Kip3p, and Kip2p.
We next examined the consequence of loss of Kip3p
function in cells that had completed spindle positioning
but had not entered anaphase. Cells of the genotypes described above were grown and treated with hydroxyurea
at 26°C. At the hydroxyurea arrest point, the majority of
cells had short preanaphase spindles and DNA masses that were positioned in or up against the mother-bud neck
(Figs. 7 and 8 A). For all dyn1 mutant genotypes, the
number of cells with nuclei located away from the neck
was slightly elevated over wild-type because of the spindle-positioning defect caused by loss of dynein. Maintaining the presence of hydroxyurea, the cells were then
shifted to 37°C. After the shift, the number of nucleus-away-from-neck cells remained fairly constant for all genotypes with the exception of dyn1
kip3
(pkip3-14). The
temperature-induced loss of Kip3p function in the dynein
mutant background caused spindles and DNA masses that
previously had been positioned at the neck to move to a
position distal to the neck. Calcofluor staining of bud scars indicated that the DNA masses had moved exclusively
back into the mother cell body (n = 200 cells). The spindles and associated DNA were usually positioned at the
peripheral region of the mother cell distal to the neck, with
extensive cytoplasmic microtubule arrays directed toward
the bud (Fig. 7).
The spindle mislocalization phenotype exhibited by
dyn1 kip3
(pkip3-14) was dependent upon Kip2p activity. The deletion of KIP2 suppressed the temperature-
induced loss of spindle positioning (see dyn1
kip2
kip3
(pkip3-14) genotype in Fig. 8 A). This strongly suggests that in the absence of Dyn1p and Kip3p activity, Kip2p function directly leads to spindle mislocalization.
Additional evidence that Kip2p is responsible for this effect was obtained in an experiment in which KIP2 expression was placed under the control of a galactose-inducible
promoter (Fig. 8 B). PGAL-KIP2 or PGAL control plasmids
were transformed into wild-type and dyn1
kip2
kip3
strains. After synchronization with hydroxyurea, galactose was added to induce expression from the galactose promoter. Expression of KIP2 in cells deficient for both
DYN1 and KIP3 caused properly positioned spindles to
mislocalize to sites away from the neck. Neither wild-type
cells expressing the same PGAL-KIP2 construct nor cells
carrying the PGAL vector displayed nuclear mislocalization when galactose was added.
Dynein motor function is important but not essential for
spindle positioning in S. cerevisiae. Although spindle-positioning errors can be detected in dyn1 cells growing at
room temperature, the extent of this defect is not sufficient to reduce the rate of cell division. Presumably, other
motor mechanisms contribute to spindle positioning in the
absence of dynein. The experiments described here demonstrated that the Kip3p KRP provides an important spindle-positioning function in the absence of dynein. The
elimination of Kip3p function in dynein-deficient cells severely compromised spindle movement to the mother-bud
neck. Elimination of Kip3p function in dyn1
cells that
had completed positioning caused spindles to mislocalize
into mother cell bodies. We also demonstrated that the
spindle-positioning defects exhibited by dyn1 kip3 cells are
caused, to a large extent, by the actions of the Kip2p KRP. kip2
cells exhibited microtubule phenotypes that were
opposite to those exhibited by dyn1
and kip3
cells. Microtubules in kip2
cells were shorter and more sensitive
to the action of benomyl than wild-type microtubules,
while those of dyn1
and kip3
cells were longer and
more resistant. kip2
dyn1
and kip2
kip3
double mutants displayed intermediate microtubule phenotypes.
Most significantly, elimination of KIP2 function greatly
suppressed the spindle localization defect and slow growth
exhibited by dyn1 kip3 cells. Likewise, induced expression
of KIP2 caused spindles to mislocalize in cells deficient for
dynein and Kip3p.
Although Kip2p normally contributes to spindle positioning, its actions can also antagonize a positioning mechanism. In the absence of dynein and Kip3p function, Kip2p
activity caused spindles to move to a location in the
mother cell distal to the neck. Since kip2 suppressed the
growth defect of the dyn1
kip3
double deletion mutant,
the antagonistic actions of Kip2p must be directed against whatever is accomplishing spindle positioning in the absence of dynein and Kip3p. Our genetic findings suggest
that this activity is provided by the Kar3p KRP. This
would be a novel role for the minus end-directed Kar3p
motor whose characterized mitotic functions include pulling spindle poles inwardly prior to anaphase and contributing to bipolar spindle structure (Meluh and Rose, 1990
;
Saunders and Hoyt, 1992
; Hoyt et al., 1993
; Endow et al.,
1994
; Saunders et al., 1997b
). Kar3p has also been associated with kinetochore movement along microtubules in
vitro (Middleton and Carbon, 1994
). Participation of
Kar3p in spindle positioning was suggested by the observation that deletion of KIP2 suppressed the lethality of the
dyn1
kar3
combination. A simple hypothesis consistent
with these findings is that Kip2p antagonizes the spindle-positioning activities of both Kip3p and Kar3p (Fig. 9). In
the absence of dynein, both Kip3p and Kar3p are required
to position spindles. If the antagonistic actions of Kip2p
are removed, however, then either Kip3p or Kar3p alone
is sufficient. kip3
kar3
cells are inviable and are not suppressed by kip2
. This suggests that Kip3p and Kar3p
overlap for an essential function that dynein alone cannot
provide. In mitotic cells, most Kar3p is concentrated near
the spindle poles with a smaller but detectable amount
spreading onto the nuclear microtubules (Page et al., 1994
;
Saunders et al., 1997a
). It is possible that some Kar3p also
acts upon the cytoplasmic microtubules to affect spindle
positioning. We note that Kar3p is essential for the nuclear
migration event that occurs during karyogamy (nuclear fusion during mating). In this capacity, Kar3p acts on the cytoplasmic microtubules to move haploid nuclei towards
each other before their fusion (Meluh and Rose, 1990
).
Kip3p does not appear to overlap with Kar3p for a karyogamy role since Kar3p is essential for this process and
kip3
cells did not display a defect in karyogamy proficiency (Cottingham, F.R., and M.A. Hoyt, unpublished
observations).
The molecular roles and mechanisms for the four spindle-positioning motors described here are currently not
clear. Below, we describe two general models for the roles
and interactions of these motors. These models are not
mutually exclusive. In the first model, spindle position is
determined by directly antagonistic motor activities. The
cooperative actions of dynein, Kip3p, and Kar3p exert a
force on one spindle pole, pulling it into the bud cell body.
At the same time, Kip2p is acting to pull the other pole
back towards the mother cell. This antagonism would result in proper positioning and orientation of the spindle
along the mother-bud axis. As noted above, we do not
view the three bud-directed activities as equivalent. Dynein is unable to perform some activity provided by either
Kip3p or Kar3p. Yeh et al. (1995) found that dyn1
cells
undergo nuclear division that appears different from DYN1 cells. The dynein-deficient cells often performed
anaphase at an incorrect position in the mother cell, followed by a phase in which a daughter nucleus was translocated to the bud. It is possible that dynein is primarily responsible for achieving proper spindle orientation before
anaphase onset while Kip3p and Kar3p act after anaphase
onset in a manner that can correct preanaphase positioning errors. Not all of our findings are easily accommodated by this simple model, however. Although Kip2p was responsible for aberrant spindle movement into the mother
in cells deficient for dynein and Kip3p, spindle mislocalization in kip2
mutants, similar to dyn1
mutants, occurred
exclusively in the mother. This finding does not exclude
the possibility that the main role of Kip2p is to pull the
spindle towards the mother cell, but it does require a more
complicated mechanism. Perhaps proper cytoplasmic microtubule connection on the bud side requires that a tension-producing force be exerted from the mother side. In
the absence of the mother-directed force supplied by
Kip2p, connections in the bud are made improperly, and
the spindle remains in the mother cell. A similar tension-based mechanism has been proposed for the attachment of
sister kinetochores to spindle fibers (Nicklas, 1997
). Spindle fiber-kinetochore connections are unstable in the absence of tension caused by a proper bipolar attachment. A
second finding that is difficult to explain by this model is
that dyn1 kip3 cells occasionally possessed cytoplasmic microtubules extending towards the bud from both poles of
mislocalized spindles (Figs. 3 and 7). This morphology is
unexpected if the spindle is being pulled back into the
mother cell body by motors operating on a set of cytoplasmic microtubules.
The second model is based upon the opposing microtubule phenotypes exhibited by the motor mutants. dyn1
and kip3
cells exhibited cytoplasmic microtubules that
were longer and more resistant to benomyl treatment than
wild-type. Another group has also observed longer and
less dynamic microtubules in dyn1
cells (Carminati and
Stearns, 1997
). It has recently been reported that kar3
cells also display longer cytoplasmic microtubules (Saunders et al., 1997a
). Coupled with the observation that
Kar3p can contribute to microtubule depolymerization in
vitro (Endow et al., 1994
), it was proposed that Kar3p contributes to microtubule shortening in vivo (Saunders et al.,
1997a
). The ability to depolymerize microtubules has also
been observed for the XKCM1 KRP motor of Xenopus
(Walczak et al., 1996
). The observation that dyn1
and kip3
cells exhibited microtubule phenotypes similar to
kar3
cells suggests that dynein and Kip3p also contribute
to microtubule shortening processes. In contrast, our observation of shorter and benomyl-hypersensitive microtubules in kip2
cells suggests that Kip2p actions are required to stabilize microtubules against depolymerization.
Perhaps the motor actions of Kip2p contribute in some
manner to microtubule polymerization, or the binding of Kip2p to sites on the microtubule lattice increases its stability. For our second model, we propose that the observed spindle-positioning effects were caused by changes
in cytoplasmic microtubule polymerization/depolymerization properties. For example, the extremely long cytoplasmic microtubules created in dyn1 kip3 cells may physically
mislocalize the spindle. The suppression by kip2
may be
the result of lowered microtubule stability caused by loss
of Kip2p. We note, however, that benomyl treatment did
not suppress the dyn1 kip3 growth defect (Fig. 5). Another
possibility is that the aberrant polymerization properties of the cytoplasmic microtubules precluded their ability to
form the proper cortical connections required to move nuclei. However, it is also possible that the microtubule phenotypes we observed were indirect effects caused by defects in the interactions of microtubules, motors, and the
cell cortex.
Both models proposed here require that the spindle-positioning motors act upon the cytoplasmic microtubules.
It has been reported that some of the cellular Dyn1p, Kip3p,
and Kip2p molecules can be detected in association with
cytoplasmic microtubules in mitotic cells (Miller, R.K.,
and M.D. Rose. 1995. Mol. Biol. Cell. 65:256a; Yeh et al.,
1995; DeZwaan et al., 1997
). As described above, Kar3p has also been detected on cytoplasmic microtubules, but
only in cells undergoing karyogamy (Meluh and Rose,
1990
; Page et al., 1994
).
Although the mechanisms leading to spindle positioning
are currently unclear, we note with interest that both bipolar spindle assembly and spindle positioning are accomplished by antagonistically acting motor proteins. Assembly of the S. cerevisiae preanaphase spindle requires the
actions of two KRPs from the BimC family, Cin8p and
Kip1p (Hoyt et al., 1992; Roof et al., 1992
). The spindle pole-separating actions of the BimC KRPs are antagonized before anaphase by the inwardly directed force produced by Kar3p (Saunders and Hoyt, 1992
; Hoyt et al.,
1993
; Saunders et al., 1997b
). Proper spindle structure is
achieved by the balance between the outwardly and inwardly directed forces acting upon the spindle poles. At roughly the same period of mitosis, dynein, Kip3p, and
Kar3p, acting antagonistically to Kip2p, are moving and
orienting the spindle to permit proper chromosome segregation to progeny cells.
Received for publication 19 June 1997 and in revised form 14 July 1997.
Address all correspondence to M. Andrew Hoyt, Department of Biology, The Johns Hopkins University, Baltimore, MD 21218. Tel.: (410) 516-7299. Fax: (410) 516-5213. E-mail: hoyt{at}jhu.eduThe authors wish to thank Mark Rose, and Susan Brown and Sue Lillie (University of Michigan, Ann Arbor, MI) for gifts of mutant strains and Cindy Dougherty, Katie Farr, John Geiser, and Emily Hildebrandt for their comments on the manuscript.
5-FOA, 5-fluoro-orotic acid; DAPI, 4,6-diamidino-2-phenylindole; KRP, kinesin-related protein.
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