Correspondence to: M. Andrew Hoyt, Department of Biology, Mudd Hall, Room 36, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Tel:(410) 516-7299 Fax:(410) 516-5213 E-mail:hoyt{at}jhu.edu.
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Abstract |
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The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.
Key Words: kinesin, dynein, motor proteins, microtubules, mitotic spindle
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Introduction |
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MICROTUBULES and their associated motor proteins accomplish many intracellular movements in eukaryotic cells. Two superfamilies of microtubule-based motor proteins have been identified, the kinesins and the dyneins. Members of each superfamily are defined by distinct force-producing domains that are conserved in amino acid sequence. Since all eukaryotic cells express multiple microtubule motors, a major research question concerns the cellular roles performed by each motor.
The budding yeast Saccharomyces cerevisiae has been particularly useful for the experimental dissection of microtubule-based motor roles (
The mitotic division of S. cerevisiae cells requires microtubule-based activities operating on both sides of the nuclear envelope. The nuclear microtubules assemble into a bipolar spindle structure that separates replicated chromosomes in anaphase, primarily by elongation (anaphase Btype movement). The major and essential role for the cytoplasmic microtubules is positioning the spindle within the cell at the neck between mother and bud. Since this process results in the movement of the entire nuclear envelope and its contents, it is often referred to as nuclear migration. Proper spindle positioning ensures that anaphase spindle elongation segregates one progeny nucleus to the mother cell and the other to the bud.
Two of the S. cerevisiae kinesin-related proteins, Cin8p and Kip1p, are members of the BimC sequence subclass. BimC motors are required for bipolar spindle assembly in S. cerevisiae and other eukaryotic cell types ( mutant cultures display many aberrant spindles (
Dynein is an important spindle positioning motor, but is not essential for cell viability (
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Materials and Methods |
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Yeast Strains and Media
The S. cerevisiae strains used in these experiments are derivatives of S288C and are listed in Table 1. The dyn1::HIS3 (10 allele was created by transforming a dyn1::URA3 strain with a DNA fragment of DYN1 that spanned the URA3 insertion but harbored an internal deletion. Ura- were selected on media containing 5-fluoro-orotic acid (5-FOA)1 (US Biological). The same strategy was used to create the unmarked kip2-
10 allele. The smy1-
10 unmarked deletion was created by PCR-targeted gene disruption (
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Multiple motor gene deletion mutants were constructed by standard genetic techniques. Strains with reduced spindle motor function often displayed evidence of aneuploidy (i.e., non-Mendelian segregation of genetic markers). Therefore, we routinely verified all seven microtubule motor gene alleles by PCR. For DYN1 and KIP2, we were able to use a primer set that flanked the genes and yielded distinct PCR products from both the wild-type and deletion alleles. For the remaining five microtubule motor genes, two PCR reactions were used to test each allelic form. Both reactions used an upstream primer with a sequence present in both wild-type and deletion mutant alleles. This was paired with downstream primers that were specific for either the wild-type allele DNA in one reaction or the deletion DNA in the other.
Rich (yeast extract, peptone, dextrose [YPD]) and minimal (synthetic dextrose [SD]) media were as described (-factor (Bachem Bioscience) was added to 4 µg/ml to log-phase cells in liquid YPD, pH 4.0, and incubated at 26°C until >80% of cells were unbudded. For arrest in S phase, hydroxyurea (Sigma Chemical Co.) was added to 0.1 M to log-phase cells in liquid YPD, pH 5.8, and incubated at 26°C until >70% of cells were large-budded.
DNA Manipulations
Temperature-sensitive alleles of KAR3 were generated by a mutagenic PCR procedure ( kip3
strains at 35°C, and was chosen for study in all subsequent experiments. pMA1428kar3-64 did not alter the temperature resistance of either wild-type or kip3
, dyn1
, and kar3
single mutant strains. Therefore, kar3-64 is a recessive and nonepistatic mutant.
Microscopy
To stain for DNA, cells were pelleted out of liquid media, resuspended in 70% ethanol, and stored on ice for 30 min. The ethanol-fixed cells were washed once with water and then resuspended in 0.3 µg/ml 4,6-diamidino-2-phenylindole (DAPI) containing 1 mg/ml p-phenylenediamine to prevent fading (Sigma Chemical Co.). To stain for chitin-containing bud scars (
Electron microscopic examination of thin sections was performed as described by
Quantitation of Nuclear Mislocalization
To quantitate nuclear mislocalization in cells, two assays were used. In the first, cells were arrested at 26°C with -factor as described above. Cells were then washed once with water to remove the
-factor and resuspended in YPD media prewarmed to 35°C. The cultures were incubated at 35°C and at regular intervals samples were removed, fixed with ethanol, and stained with DAPI. The percentage of total cells that were large-budded (bud diameter greater than three quarters the diameter of the mother cell) with the nucleus away from the neck was determined for each time point. "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 the diameter of the entire nuclear DNA mass, as judged by eye. In the second assay, cells were arrested at 26°C with hydroxyurea as described above. Next, cultures were shifted to 35°C, and at regular intervals samples were removed, fixed, and either stained with DAPI alone, or processed for both antitubulin immunofluorescence and DAPI staining. The percentage of large-budded cells with the nucleus away from the neck was determined for each time point.
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Results |
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Characterization of a Temperature-Sensitive KAR3 Allele
In a previous study we obtained evidence of overlap for an essential function between Kar3p, Kip3p, and dynein. Although neither KAR3, KIP3, nor DYN1 (encoding the dynein heavy chain) are individually essential for cell viability, the double deletion mutant combinations are inviable, or in the case of dyn1 kip3
, grow very slowly (
kip3
(pkar3-64) strain was inviable at 33°C and higher (Figure 1; compare row A10 to A9, 0 benomyl columns). kar3
dyn1
(pkar3-64) cells were also temperature-sensitive for growth, although the effect was not as extreme as for kip3
(Figure 1; compare row A6 to A5). pkar3-64 also failed to complement the slight temperature sensitivity caused by kar3
(Figure 1; compare row A4 to A3). In the absence of dynein, kinesin-related Kip2p acts antagonistically to Kar3p and Kip3p. Loss of Kip2p suppresses the growth defects of the dyn1
kip3
and dyn1
kar3
double mutants, but not that of kip3
kar3
(
dyn1
kip2
(pkar3-64) strain showed moderately improved growth at 35°C compared with the kar3
dyn1
(pkar3-64) strain (Figure 1; compare row A8 to A6). However, the kar3
kip3
kip2
(pkar3-64) strain was just as temperature-sensitive as the corresponding strain possessing the wild-type KIP2 allele (Figure 1; compare row A12 to A10).
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Log-phase cultures of kar3 kip3
(pkar3-64), kar3
dyn1
(pkar3-64), and wild-type cells were shifted to 35°C for 2 h and examined by microscopy. The kar3
kip3
(pkar3-64) and kar3
dyn1
(pkar3-64) showed evidence of reduced proficiency to accomplish mitosis; 57 and 52%, respectively, of the mutant cells were large-budded and mononucleate, relative to 26% of the wild-type (n = 200 cells for each). The kar3
dyn1
(pkar3-64) cells often displayed mislocalized nuclei indicative of a spindle positioning defect (see next section).
Cells lacking KAR3 display a bilateral karyogamy defect; kar3 cells will form diploids with normal efficiency with KAR3 cells of the opposite mating type, but not with kar3
cells (
strain, but not a KAR3 strain (data not shown).
A Role for Kar3p in Spindle Positioning Revealed in Cells Missing Dynein
In a previous study, the spindle positioning role of Kip3p was clearly revealed in cells missing dynein. Loss of Kip3 function in dynein-deficient cells caused spindle (and nuclear) mispositioning. The lethality of kar3 dyn1 and kar3 kip3 cells suggested that Kar3p may also participate in spindle positioning. To investigate this possibility, we assessed the proficiency of kar3-64 cells at positioning the nucleus at the motherbud neck (a consequence of proper spindle positioning). Two related assays were used. In the first, cells were arrested in G1 with -factor at 26°C and released to media at 35°C. The ability of the cells to translocate their nuclei to its proper position at the neck between mother and bud cell bodies was then assessed. In the second, spindle positioning was allowed to first occur at permissive temperature (26°C) in the presence of the DNA synthesis inhibitor hydroxyurea. Yeast cells treated with this inhibitor are still able to accomplish bipolar spindle assembly and spindle positioning (
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Loss of Kar3p function alone did not cause nuclear mislocalization. In both assays, the kar3 (pkar3-64) strain behaved similar to wild-type (Figure 3, top panels). This agrees with previous reports, in which the nuclear migration index (the shortest distance between the nucleus and the bud neck divided by the mother cell diameter) for a kar3
strain was found to be comparable to the nuclear migration index of a wild-type strain (
(pkar3-64) strain did not exhibit nuclear mislocalization, this strain did accumulate a large percentage (~50%) of large-budded mononucleate cells at 35°C, as reported previously for strains lacking Kar3p (
dyn1
[pkar3-64] strain in Figure 2 A and Figure 3, second row of panels). The extent of this phenotype was always greater than loss of dynein (kar3
dyn1
[pKAR3] strain) or Kar3p (kar3
[pkar3-64] strain) alone. As was observed for loss of both Kip3p and dynein activities (
dyn1
(pkar3-64) cells exhibited mislocalized nuclei or two nuclei in one cell body, compared with 6, 8, and 0% of kar3
(pkar3-64), dyn1
, and wild-type, respectively (n = 200 cells for each).
The ability of the deletion of KIP2 to suppress the growth defect of kar3 dyn1 cells (see above) was also reflected in its effects on spindle positioning proficiency. In both assays, the kar3 dyn1
kip2
(pkar3-64) strain displayed a greatly attenuated nuclear positioning defect relative to the kar3
dyn1
(pkar3-64) strain (Figure 2 B and Figure 3, second row of panels). This indicates that the spindle mislocalization observed in the absence of Kar3p and dynein was caused by the action of Kip2p. kar3
dyn1
kip2
(pkar3-64) cells were found to have significantly shorter cytoplasmic microtubules compared with the kar3
dyn1
(pkar3-64) strain (Figure 2). This is consistent with the previous observation that loss of Kip2p causes a reduction in cytoplasmic microtubule length (
The spindle positioning defect caused by the absence of Kar3p and dynein, and its suppression by kip2, was very similar to the defect observed previously for cells missing Kip3p and dynein (
kip3
(pkar3-64) strain. Both assays demonstrated that loss of Kar3p in the absence of Kip3p caused only relatively mild nuclear mislocalization (Figure 3, third row of panels). However, this effect was greater than those observed in the absence of either Kar3p or Kip3p alone. Similar findings were obtained in experiments using a temperature-sensitive KIP3 allele in a kar3
strain (Cottingham, F.R., and M.A. Hoyt, unpublished observations). The minor nuclear position defect observed in the absence of Kar3p and Kip3p did not appear to be affected by the presence or absence of Kip2p, i.e., the kar3
kip3
kip2
(pkar3-64) strain behaved similarly to the kar3
kip3
(pkar3-64) strain in both nuclear position assays. These findings indicate that Kar3p and Kip3p together make only a minor contribution to spindle positioning when dynein is present. However, when dynein is absent, the contributions of both Kip3p (
, suggests that they overlap for a different essential process. The nature of this process is addressed in the following section.
The findings presented here and elsewhere ( dyn1
kip3
kip2
(pkar3-64) strain exhibited a nuclear positioning defect at 35°C; it was not able to move the nucleus to the neck efficiently or maintain it there following hydroxyurea synchronization. The movement of nuclei away from the neck after hydroxyurea treatment was found to occur independent of microtubule function (Cottingham, F.R., and M.A. Hoyt, unpublished observations). Therefore, in this experiment, Kar3p was acting as a solitary and essential spindle positioning motor required to resist a microtubule-independent force that operates on the nucleus. In the last Results section, we demonstrate that either Kar3p or Kip3p alone is sufficient to perform spindle positioning.
Spindle Assembly and Integrity Requires Either Kar3p or Kip3p
As demonstrated above, loss of function of both Kar3p and Kip3p caused only a mild spindle positioning defect. Therefore, the inviability of kar3 kip3 double mutant cells may be due to a defect in another essential cellular process. In the course of our spindle positioning experiments, microscopy revealed frequent abnormal spindle morphologies in kar3 cells, as has also been observed in kar3 cells by others (e.g., -factor at 26°C and released into media at 35°C. Figure 4 shows examples of spindles fixed and stained 1 h after release from the
-factor block. Most wild-type cells possessed spindles that were clearly bipolar; a bright bar of spindle microtubules was visible in the nuclei (in some cases the bar was long, indicating anaphase had initiated). Fewer bar structures were found in kar3
(pkar3-64) cells and even fewer in kar3
kip3
(pkar3-64) cells. Instead of bars of microtubules, kar3
kip3
(pkar3-64) cells often had a bright small mass of nuclear microtubules from which the cytoplasmic microtubules radiated. As described previously for cells deficient for Kar3p and Dyn1p or Kip3p and Dyn1p, the cytoplasmic microtubules of cells missing the functions of both Kar3p and Kip3p grew to much longer lengths than wild-type.
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To quantitate the effects of loss of function of Kar3p and Kip3p on bipolar structure, we examined cells whose spindle poles were marked with the GFP. A construct encoding a GFP-tagged SPB protein, Nuf2p, was integrated into our motor mutant strains (-factor block into 35°C, live cells were observed under the microscope and the percentage of cells with two clearly separated GFP dots was determined (Figure 5a and Figure b). Relative to the wild-type, the kar3
kip3
(pkar3-64) strain was severely compromised for its ability to generate cells with two distinct GFP dots. The majority of kar3
kip3
(pkar3-64) cells were large-budded, with a single GFP dot located at the bud neck (Figure 5 A, bottom row). Therefore, Kip3p makes an important contribution to spindle assembly that is revealed when Kar3p activity is compromised. However, the activity provided by Kip3p cannot completely compensate for loss of Kar3p, since kar3-64 and kar3
(not shown) cells exhibited an intermediate reduction in spindle assembly proficiency, even when Kip3p was present. Dynein, on the other hand, does not appear to overlap with Kar3p for establishing spindle structure, since the kar3
dyn1
(pkar3-64) strain was no more severely affected than the kar3
(pkar3-64).
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To assess the ability of bipolar spindles to maintain their integrity after motor loss, we arrested the mutants with hydroxyurea at 26°C (a condition that allows bipolar spindle assembly) and then shifted to 35°C. Observation of the Nuf2p-GFP dots as a function of time revealed that wild-type and kip3-14 (a temperature-sensitive allele) cells maintained bipolar spindle structure (Figure 5 C). In contrast, ~80% of the kar3 (pkar3-64) cells displayed two Nuf2p-GFP dots before the temperature shift, but only ~45% displayed two dots after 3 h at 35°C. The presence or absence of dynein in the kar3-64 cells made no apparent difference. Spindles also did not lose bipolarity when both Kip3p and dynein were inactivated (
kip3
(pkar3-64) cells had two clearly separated dots. Therefore, Kar3p and Kip3p overlap for a function required to maintain bipolar spindle integrity.
Thin-section electron microscopy was used to examine the spindle morphology of kar3 kip3
(pkar3-64) and wild-type cells treated with hydroxyurea and shifted to 35°C for 3 h (Figure 6). The wild-type spindles were short and bipolar, with parallel SPBs separated by ~1.5 µm and joined by a bundle of microtubules. No normal-appearing spindles were found in the kar3
kip3
(pkar3-64) culture. Of the spindles in which two poles could be visualized in one section, nine had SPBs located adjacent to one another. Six spindles had SPBs that were separated from between 0.2 and 1.2 µm (average = 0.53 ± 0.37 µm, SD), but were clearly defective. The poles of these spindles were not parallel. These findings agree with those from the Nuf2p-GFP analysis. Loss of Kar3p and Kip3p function caused preformed bipolar spindles to break with SPBs frequently moving close together.
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Genetic Interactions with Kar3p Accessory Factors
Kar3p forms distinct complexes with two homologous accessory factors, Cik1p and Vik1p ( mutant (
is lethal in combination with dyn1
, and this lethality is suppressed by kip2
. Viable cik1
dyn1
cells could only be created when KIP2 was deleted. Figure 7 demonstrates that introduction of KIP2 by transformation into a cik1
dyn1
kip2
triple mutant prevented colony formation, indicating that lethality is due to the activity of KIP2. In this case, the similarity in genetic behavior of cik1
to kar3
suggests that it is the Cik1pKar3p complex that overlaps with dynein for an essential spindle positioning role. However, unlike kar3
, cik1
could be combined with kip3
yielding viable, healthy cells. Therefore, Cik1pKar3p complexes do not uniquely perform the function required for spindle integrity that overlaps with Kip3p. This supports the hypothesis that Cik1p is only required for a subset of Kar3p's roles (
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vik1 dyn1
and vik1
kip3
double mutant cells were also viable and healthy. Therefore, Vik1pKar3p complexes do not uniquely overlap for essential functions with either Dyn1p or Kip3p. However, cik1
vik1
kip3
triple mutants were inviable. Strain MAY6417 (cik1
vik1 kip3
[pKIP3 URA3]) could not survive loss of the KIP3 URA3 plasmid, as evidenced by its inability to segregate cells capable of growth on 5-FOA. This may indicate that the essential spindle integrity function that overlaps with Kip3p is performed by both Cik1pKar3p and Vik1pKar3p complexes, and that either alone is sufficient. Alternatively, the lethality of the cik1
vik1
kip3
triple mutant may reflect a nonspecific additive effect of combining three deleterious mutations.
Benomyl Can Partially Substitute for the Essential Function Performed by Either Kar3p or Kip3p
Kar3p can act as a microtubule destabilizer in vitro ( vegetative growth defects can be suppressed by the presence of the microtubule-destabilizing reagent benomyl (
kip3
(pkar3-64) cells could be partially suppressed by the presence of 510 µg/ml benomyl in the media (Figure 1, rows A10 and B9). The temperature sensitivity of kar3
kip3
(pkip3-14) cells could be suppressed by the same treatment as well (Figure 1, row B7), similar to the suppression of kip3-30 (temperature-sensitive) kar3
reported by others (
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Characterization of Minimal Microtubule-based Motor Strains
The S. cerevisiae genome encodes six kinesin-related motor genes and a single dynein heavy chain. In this and a previous study (
From the two viable triple mutants described above (dyn1 kip3
kip2
and dyn1
kar3
kip2
) we were able to delete an additional two genes encoding kinesin-related proteins, KIP1 and SMY1. KIP1 encodes the BimC family motor that is phenotypically less important (the more important BimC motor is encoded by CIN8) (
Cells expressing only CIN8 and KAR3 were quite healthy relative to wild-type, as judged by doubling time and other cell cycle criteria (Table 2). Some difficulty in progression through mitosis was evident from the approximately twofold elevation of large-budded, mononucleate cells in log-phase cultures. Spindle positioning errors were also evident (by the appearance of bi and anucleate cell bodies), but at a level no higher than a dyn1 single mutant (
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To determine if Cin8p Kar3p or Cin8p Kip3p represent the lowest possible motor complements, we created strains in which one of the two active motor genes was replaced with a temperature-sensitive allele. While cells surviving with only CIN8 KAR3 or CIN8 KIP3 were able to grow at 35°C, neither CIN8 kar3-64, CIN8 kip3-14, nor cin8-3 KAR3 cells grew at this elevated temperature (Figure 8). Therefore, under normal growth conditions, Cin8pKar3p or Cin8pKip3p were the minimal motor sets that supported cell viability (but see below). We examined the effects on spindle integrity and positioning caused by elimination of the function of one of the two active motors in the two-motor strains. Wild-type, CIN8 kar3-64, CIN8 kip3-14, and cin8-3 KAR3 cells were arrested with hydroxyurea at 26°C and then shifted to 35°C for 3 h, maintaining the hydroxyurea block. The motor mutant strains exhibited greatly reduced numbers of bipolar spindles as judged by antitubulin immunofluorescence microscopy (78% for wild-type; 17% for CIN8 kar3-64; 9% for CIN8 kip3-14; and 7% for cin8-3 KAR3). This was an expected finding, because a BimC motor (either Cin8p or Kip1p) plus either Kar3p or Kip3p is required for bipolar spindle integrity. However, examination of the nuclear positioning proficiency of these strains did reveal differences. After hydroxyurea synchronization, the shift to 35°C caused nuclei in CIN8 kar3-64 and CIN8 kip3-14 cells, but not cin8-3 KAR3 cells, to mislocalize (Figure 9). Therefore, it is reasonable to conclude that Kar3p or Kip3p are the only motors performing spindle positioning in their respective two-motor strains, and that Cin8p makes no detectable contribution to this process.
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Finally, we found further support for a microtubule-destabilizing role for Kar3p and Kip3p in vivo. Addition of benomyl to the media could suppress the temperature-sensitive growth of cells surviving with only CIN8 kar3-64 or CIN8 kip3-14 (Figure 8). The slow growth of the CIN8 KIP3 strain was exacerbated at elevated temperature, a phenotype that was also suppressed by benomyl. In contrast, the temperature sensitivity of the cin8-3 KAR3 strain was not relieved by benomyl. Therefore, although microtubule destabilization may be an important function of Kar3p and Kip3p, this does not appear to be a role for the BimC motor Cin8p.
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Discussion |
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Kar3p and Kip3p Overlap for Spindle Structural and Positioning Functions
Our findings demonstrate that S. cerevisiae Kar3p and Kip3p motors perform overlapping roles contributing to both mitotic spindle positioning and spindle structural integrity. These two activities are primarily accomplished by distinct sets of microtubules, cytoplasmic and nuclear, respectively. Therefore, we conclude that Kar3p and Kip3p act on both sides of the nuclear envelope, a property that may be unique among the seven S. cerevisiae microtubule-based motors. We suggest that a major aspect of the nuclear and cytoplasmic functions of both of these motors is to destabilize microtubules.
In the absence of dynein, both Kar3p and Kip3p are required for spindle positioning and vigorous cell growth. Similar to cells in which dynein and Kip3p were eliminated (
In previous studies, Kar3p was visualized only upon microtubules in the nucleus of mitotic cells (
Our studies also demonstrate a role for Kar3p and Kip3p in the nucleus, overlapping for a function essential for bipolar spindle assembly and structural integrity. Previous observations of spindle abnormalities in kar3 cells suggested a spindle integrity role for Kar3p (
cells (
Kar3p forms functionally distinct complexes with two homologous accessory factors, Cik1p and Vik1p ( mutant, cik1
is lethal in combination with dyn1
, and this lethality is suppressed by kip2
. This overlap in function between Cik1p and dynein suggests that Kar3p complexed to Cik1p performs the cytoplasmic spindle positioning function. Our genetic studies indicated that neither Cik1pKar3p nor Vik1pKar3p complexes uniquely perform the function essential for spindle integrity that overlaps with Kip3p. However, since cik1
vik1
kip3
triple mutants were inviable, it remains possible that these two complexes redundantly provide this activity. Our findings lend further support to the hypothesis that Cik1p and Vik1p target the Kar3p motor to distinct cellular functions (
Spindle Motors Regulate Microtubule Dynamics
The molecular nature of the spindle positioning and structural defects caused by loss of Kar3p and Kip3p is likely to involve a defect in microtubule polymer length regulation. Recent studies have implicated microtubule motors as important regulators of microtubule dynamics. Notably, members of the XKCM1/MCAK family of vertebrate kinesins have been demonstrated to cause microtubule instability by promoting catastrophe events (
It is not clear how increased microtubule stability or length caused the observed kar3 kip3 mutant spindle structural and positioning defects. The dynamic behavior of the ends of microtubules is probably important for functions such as interactions with kinetochores, antiparallel microtubules, and the cell cortex. Any of these processes may be disrupted by the inability to control the growth of microtubules. A less direct possibility for the kar3 kip3 spindle structural defect is that the longer or less dynamic spindle microtubules do not form proper interactions with the BimC motors, leading ultimately to spindle collapse. For the spindle positioning defect, it is possible that the extremely long cytoplasmic microtubules act to prohibit migration of the nucleus up to the cell neck or actually push the nucleus away.
The functional overlap between Kar3p and Kip3p could not have been predicted from the amino acid sequence of these gene products, since each is distinct. Kar3p defines a unique subclass of kinesin-related proteins characterized by their COOH terminuslocated motor domain and their minus enddirected force production on microtubules (
In contrast to Kar3p, Kip3p, and dynein, the Kip2p motor acts to increase the length of cytoplasmic microtubules. Loss of function causes extremely short cytoplasmic microtubules ( also suppresses their lethal spindle positioning defect (this study;
The Minimal Motor Requirement for S. cerevisiae
Cytological descriptions of mitotic spindle function have revealed a complex series of motility events that affect the segregation of replicated chromatids. These events include bipolar spindle assembly, kinetochore capture and congression, and two chromosome-separating movements, anaphase A (movement of kinetochores towards spindle poles) and anaphase B (separation of spindle poles). In considering the minimal microtubule-based motor set required for S. cerevisiae viability, it first must be recognized that unlike higher eukaryotic cells, this yeast requires microtubules for only one essential role, mitotic spindle function. Nonetheless, our finding that S. cerevisiae can survive with only two microtubule-based motors is unexpected in light of the observed complexities of spindle motile behavior.
We were able to create viable strains expressing only two of seven microtubule-based motors; the BimC-type motor Cin8p combined with either Kar3p or Kip3p. Attempts to reduce motor function further caused expected deleterious phenotypic consequences and revealed that these represented the minimal sets required for viability. Removing the function of Kar3p (from Cin8p Kar3p-ts cells) or Kip3p (from Cin8p Kip3-ts cells) caused spindles to both collapse and misposition. Removing the function of Cin8p caused spindle collapse, an expected consequence since these cells were missing the other BimC motor, Kip1p (
A few caveats must be considered regarding the conclusion that only two motors are required for viability. First, we cannot exclude the possibility that the observed vigorous growth of the two-motor cells could have been aided by suppressing mutations. The genotypes of created strains were verified with respect to the seven motor loci, so suppressors would necessarily affect genes that do not encode dyneins or kinesins. Second, we must strongly consider a contribution from microtubule-based motility mechanisms that do not include dyneins and kinesins. We are not aware of any force-producing microtubule-based ATPases outside of the dynein and kinesin families. However, an undiscovered class of motors cannot be excluded. All kinesins, dyneins, and myosins (as well as many other proteins) possess a domain with a nucleotide-binding sequence (G-X-X-X-X-G-K-T/S; (-helical coil sequence. An examination of the S. cerevisiae genome reveals gene products of unknown function that contain both of these sequence motifs (i.e., the gene products specified by YLR106C and YPL217C). Another possibility is a motility mechanism that uses a different form of force generation. For example, a protein that is able to couple the dynamic behavior at the ends of microtubules to force production (
The presence of benomyl in the media partially suppressed the temperature-sensitive growth defects of the Cin8p Kar3p-ts and Cin8p Kip3p ts two-motor strains. However, we have been unable to find a condition allowing vigorous growth in the complete absence of Kar3p and Kip3p (cells surviving on Cin8p alone). Therefore, the temperature-sensitive forms of these motors may retain some activity at the elevated temperature that is required in addition to the microtubule-destabilizing activity of benomyl. Nonetheless, these findings further indicate that microtubule-destabilization is an important activity of Kar3p and Kip3p.
In summary, S. cerevisiae cells only require two microtubule-based motors to accomplish mitosis. The BimC motor Cin8p is required to assemble and elongate the bipolar spindle, probably by virtue of its ability to cross-link and slide microtubules. The second motor, either Kar3p or Kip3p, acts to promote microtubule shortening both within the nucleus and in the cytoplasm. Until the possible discovery of other spindle motility-producing mechanisms, we must entertain models in which all essential spindle motile activities can be accomplished by this limited set of motor proteins.
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Footnotes |
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The first two authors contributed equally to this study.
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Acknowledgements |
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The authors wish to thank Laura Totis for technical assistance; Michael McCaffery for performing electron microscopy; Brendan Manning and Michael Snyder for strains; and Cindy Dougherty, Emily Hildebrandt, and Kevin McCabe for comments on the manuscript.
These experiments were supported by National Institutes of Health grant GM40714, awarded to M.A. Hoyt.
Submitted: 12 July 1999
Revised: 2 September 1999
Accepted: 13 September 1999
1.Abbreviations used in this paper: DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; SPB, spindle pole body; 5-FOA, 5-fluoro-orotic acid
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References |
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