Overexpression of Yeast Homologs of the Mammalian Checkpoint Gene Rcc1 Suppresses the Class of α-Tubulin Mutations That Arrest with Excess Microtubules

Microtubules in eukaryotic cells participate in a variety of nuclear and cytoplasmic structures, reflecting functional requirements and cell cycle position. We are studying the cellular regulation of microtubule assembly and organization in the yeast Saccharomyces cerevisiae. We screened for genes that when overexpressed suppress the growth phenotype of conditional mutants in α-tubulin that arrest with excess microtubules at the nonpermissive temperature (class 2 mutations). Here we describe one such suppressing element, called ATS1 (for Alpha Tubulin Suppressor). Overexpression of this gene rescues both the growth and microtubule phenotypes of all class 2 mutations, but not the cold-sensitive mutations that arrest with no microtubules (class 1 mutations). Deletion of ATS1 confers a modest slow growth phenotype which is slightly enhanced in strains containing both a deletion of ATS1 and a class 2 tub1 mutation. The predicted ATS1 protein contains 333 amino acids and has considerable structural homology to the products of both the mammalian mitotic control gene RCC1 and the S. cerevisiae gene SRM1/PRP20. Overexpression of SRM1/PRP20 also suppresses class 2 mutants. The results suggest that this family of genes may participate in regulatory interactions between microtubules and the cell cycle.     

The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization

In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)- Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Delta, pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin cytoskeleton functions and, of these, bni1Delta affected Kar9p localization most severely. Like kar9Delta, bni1Delta mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Delta, the bni1Delta mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Delta bni1Delta double mutants suggested that Kar9p retained some function in bni1Delta mitotic cells. Unlike the polarization mutants, kar9Delta shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Delta. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks.     

Microtubule stability in budding yeast: characterization and dosage suppression of a benomyl-dependent tubulin mutant.

To better understand the dynamic regulation of microtubule structures in yeast, we studied a conditional-lethal beta-tubulin mutation tub2-150. This mutation is unique among the hundreds of tubulin mutations isolated in Saccharomyces cerevisiae in that it appears to cause an increase in the stability of microtubules. We report here that this allele is a mutation of threonine 238 to alanine, and that tub2-150 prevents the spindle from elongating during anaphase, suggesting a nuclear microtubule defect. To identify regulators of microtubule stability and/or anaphase, yeast genes were selected that, when overexpressed, could suppress the tub2-150 temperature-sensitive phenotype. One of these genes, JSN1, encodes a protein of 125 kDa that has limited similarity to a number of proteins of unknown function. Overexpression of the JSN1 gene in a TUB2 strain causes that strain to become more sensitive to benomyl, a microtubule-destabilizing drug. Of a representative group of microtubule mutants, only one other mutation, tub2-404, could be suppressed by JSN1 overexpression, showing that JSN1 is an allele-specific suppressor. As tub2-404 mutants are also defective for spindle elongation, this provides additional support for a role for JSN1 during anaphase.     

Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes.

By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss. In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes. These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation. Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants. Null alleles of these genes caused cold sensitivity for viability. At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure. cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy). Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl. Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.     

ELECTROMAGNETIC ACTIVITY OF YEAST CELLS IN THE M PHASE

Electromagnetic activity around yeast mitotic cells (Saccharomyces cerevisiae) was measured in the frequency range 8–9 MHz and special care was taken to extract reliable information from the raw signals. The characteristic of cold-sensitive tubulin mutants tub2-401 and tub2-406, which come to arrest before mitosis at a restrictive temperature (14°C) and which re-enter mitosis upon a shift back to a permissive temperature (28°C), was used to prepare synchronized mitotic cells. Immunofluorescence microscopy using an antitubulin antibody was used to analyze microtubular structures. The arrested tub2-401 mutant that had back-shifted to permissive temperature displayed no microtubules and no electromagnetic activity around the cells. In contrast, the arrested cells of the mutant tub2-406 displayed developed, but aberrant, nonfunctional microtubules and a high electromagnetic activity around the cells. The electromagnetic activity around the arrested mutant tub2-401 back-shifted to permissive temperature peaks at four time points which may coincide with (i) formation of the mitotic spindle, (ii) binding of chromatids to kinetochore microtubules, (iii) elongation of the spindle in anaphase A, and (iv) elongation of the spindle in anaphase B. The profile of the electromagnetic activity around the synchronized mutant tub2-406 at permissive temperature seems to be delayed by the time required for aberrant nonfunctional microtubules to be depolymerized. Experimental results presented in this paper support Pohl's idea of existence of the electromagnetic field around yeast cells.     

Isolation and Characterization of Conditional-Lethal Mutations in the Tub1 α-Tubulin Gene of the Yeast Saccharomyces Cerevisiae

Microtubules in yeast are functional components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating. We have isolated 70 conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae using a plasmid replacement technique. Of the 70 mutations isolated, 67 resulted in cold-sensitivity, one resulted in temperature-sensitivity, and two resulted in both. Fine-structure mapping revealed that the mutations were located throughout the TUB1 gene. We characterized the phenotypes caused by 38 of the mutations after shifts of mutants to the nonpermissive temperature. Populations of temperature-shifted mutant cells contained an excess of large-budded cells with undivided nuclei, consistent with the previously determined role of microtubules in yeast mitosis. Several of the mutants arrested growth with a sufficiently uniform morphology to indicate that TUB1 has at least one specific role in the progression of the yeast cell cycle. A number of the mutants had gross defects in microtubule assembly at the restrictive temperature, some with no microtubules and some with excess microtubules. Other mutants contained disorganized microtubules and nuclei. There were no obvious correlations between these phenotypes and the map positions of the mutations. Greater than 90% of the mutants examined were hypersensitive to the antimicrotubule drug benomyl. Mutations that suppressed the cold-sensitive phenotypes of two of the TUB1 alleles occurred in TUB2, the single structural gene specifying beta-tubulin.     

STU1, a suppressor of a beta-tubulin mutation, encodes a novel and essential component of the yeast mitotic spindle

We have isolated a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in S. cerevisiae, that confers a specific defect in spindle microtubule function. At 14 degrees C, tub2-406 cells lack a normal bipolar spindle but do assemble functional cytoplasmic microtubules. In an attempt to identify proteins that are important for spindle assembly, we screened for suppressors of the cold-sensitivity of tub2-406 and obtained four alleles of a novel gene, STU1. Genetic interactions between stu1 alleles and alleles of TUB1 and TUB2 suggest that Stu1p specifically interacts with microtubules. STU1 is essential for growth and disruption of STU1 causes defects in spindle assembly that are similar to those produced by the tub2-406 mutation. The nucleotide sequence of the STU1 gene predicts a protein product of 174 kD with no significant similarity to known proteins. An epitope-tagged Stulp colocalizes with microtubules in the mitotic spindle of yeast. These results demonstrate that Stulp is an essential component of the yeast mitotic spindle.     

The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis

The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.     

A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae

A Saccharomyces cerevisiae gamma-tubulin-related gene, TUB4, has been characterized. The predicted amino acid sequence of the Tub4 protein (Tub4p) is 29-38% identical to members of the gamma-tubulin family. Indirect immunofluorescence experiments using a strain containing an epitope-tagged Tub4p indicate that Tub4p resides at the spindle pole body throughout the yeast cell cycle. Deletion of the TUB4 gene indicates that Tub4p is essential for yeast cell growth. Tub4p-depleted cells arrest during nuclear division; most arrested cells contain a large bud, replicated DNA, and a single nucleus. Immunofluorescence and nuclear staining experiments indicate that cells depleted of Tub4p contain defects in the organization of both cytoplasmic and nuclear microtubule arrays; such cells exhibit nuclear migration failure, defects in spindle formation, and/or aberrantly long cytoplasmic microtubule arrays. These data indicate that the S. cerevisiae gamma- tubulin protein is an important SPB component that organizes both cytoplasmic and nuclear microtubule arrays.     

The plant microtubule-associated protein AtMAP65-3/PLE is essential for cytokinetic phragmoplast function

Directional cell expansion in interphase and nuclear and cell division in M-phase are mediated by four microtubule arrays, three of which are unique to plants: the interphase array, the preprophase band, and the phragmoplast. The plant microtubule-associated protein MAP65 has been identified as a key structural component in these arrays. The Arabidopsis genome has nine MAP65 genes, and here we show that one, AtMAP65-3/PLE, locates only to the mitotic arrays and is essential for cytokinesis. The Arabidopsis pleiade (ple) alleles are single recessive mutations, and we show that these mutations are in the AtMAP65-3 gene. Moreover, these mutations cause C-terminal truncations that abolish microtubule binding. In the ple mutants the anaphase spindle is normal, and the cytokinetic phragmoplast can form but is distorted; not only is it wider, but the midline, the region where oppositely oriented microtubules overlap, is unusually expanded. Here we present data that demonstrate an essential role for AtMAP65-3/PLE in cytokinesis in plant cells.     

Single site alpha-tubulin mutation affects astral microtubules and nuclear positioning during anaphase in Saccharomyces cerevisiae: possible role for palmitoylation of alpha-tubulin

We generated a strain of Saccharomyces cerevisiae in which the sole source of alpha-tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle. During G1 of the cell cycle, microtubules in the C377S alpha-tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain. However, mitotic C377S tub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells. Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected. In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells. When the doubling time was increased by growing cells at a lower temperature (15 degrees C), we often found abnormally long mitotic spindles. No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally. Because cys-377 is a presumptive site of palmitoylation in alpha-tubulin in S. cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein. We detected palmitoylated alpha-tubulin in TUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated alpha-tubulin in C377S tub1 cells. Our results suggest that cys-377 of alpha-tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle.     

The Drosophila microtubule-associated protein mini spindles is required for cytoplasmic microtubules in oogenesis

The XMAP215/TOG family of proteins is a closely related set of MAPs (microtubule-associated proteins) found in animals, yeast, and plants . In yeast and animal cells, the XMAP215/TOG proteins are required for both mitosis and meiosis. Although effects of XMAP215/TOG proteins on cytoplasmic microtubules have not previously been shown in animal cells, in plants the Arabidopsis family member MOR1 is required for the organization of cortical microtubule arrays . The Drosophila family member, encoded by the mini spindles (msps) gene, is maternally expressed and loaded into the egg, where it is an essential component of meiotic and mitotic spindles . Here we show that msps is also required during oogenesis for the structure and function of cytoplasmic microtubules. Localization of bicoid (bcd) mRNA in the oocyte is a microtubule-mediated event . We show that bcd RNA localization is defective in msps mutants. We also identify defects in cytoplasmic microtubules in both the germ and follicle cells of mutant ovaries and determine the expression pattern of msps mRNA and protein in developing egg chambers. Our findings reveal a new role for msps in cell patterning and raise the possibility that other family members may perform similar functions.     

Gamma-tubulin is required for proper recruitment and assembly of Kar9-Bim1 complexes in budding yeast

Microtubule plus-end-interacting proteins (+TIPs) promote the dynamic interactions between the plus ends (+ends) of astral microtubules and cortical actin that are required for preanaphase spindle positioning. Paradoxically, +TIPs such as the EB1 orthologue Bim1 and Kar9 also associate with spindle pole bodies (SPBs), the centrosome equivalent in budding yeast. Here, we show that deletion of four C-terminal residues of the budding yeast gamma-tubulin Tub4 (tub4-delta dsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependent spindle positioning. Surprisingly, we find Kar9 localizes to microtubule +ends in tub4-delta dsyl cells, but these microtubules fail to position the spindle when targeted to the bud. Using cofluorescence and coaffinity purification, we show Kar9 complexes in tub4-delta dsyl cells contain reduced levels of Bim1. Astral microtubule dynamics is suppressed in tub4-delta dsyl cells, but it are restored by deletion of Kar9. Moreover, Myo2- and F-actin-dependent dwelling of Kar9 in the bud is observed in tub4-delta dsyl cells, suggesting defective Kar9 complexes tether microtubule +ends to the cortex. Overproduction of Bim1, but not Kar9, restores Kar9-dependent spindle positioning in the tub4-delta dsyl mutant, reduces cortical dwelling, and promotes Bim1-Kar9 interactions. We propose that SPBs, via the tail of Tub4, promote the assembly of functional +TIP complexes before their deployment to microtubule +ends.     

Yeast Num1p associates with the mother cell cortex during S/G2 phase and affects microtubular functions

The NUM1 gene is involved in the control of nuclear migration in Saccharomyces cerevisiae. The content of NUM1 mRNA fluctuates during the cell cycle, reaching a maximum at S/G2 phase, and the translation product Num1p associates with the cortex of mother cells mainly during S, G2, and mitosis, as seen by indirect immunofluorescence. The nuclear spindle in NUM1-deficient large-budded cells often fails to align along the mother/bud axis, while abnormally elongated astral microtubules emanate from both spindle pole bodies. A num1 null mutation confers temperature sensitivity to the cold-sensitive alpha-tubulin mutant tub1- 1, and shows synthetic lethality with the beta-tubulin mutant alleles tub2-402, tub2-403, tub2-404, and tub2-405. Deletion mapping has defined three functionally important Num1p regions: a potential EF hand Ca2+ binding site, a cluster of potential phosphorylation sites and a pleckstrin homology domain. The latter domain appears to be involved in targeting Num1p to the mother cell cortex. Our data suggest that the periodically expressed NUM1 gene product controls nuclear migration by affecting astral microtubule functions.     

A novel protein complex promoting formation of functional alpha- and gamma-tubulin.

We describe the identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with tub4-1, encoding a mutated yeast gamma-tubulin. The cytoplasmic Gim proteins encoded by these GIM genes are present in common complexes as judged by co-immunoprecipitation and gel filtration experiments. The disruption of any of these genes results in similar phenotypes: the gim null mutants are synthetically lethal with tub4-1 and super-sensitive towards the microtubule-depolymerizing drug benomyl. All except Deltagim4 are cold-sensitive and their microtubules disassemble at 14 degrees C. The Gim proteins have one function related to alpha-tubulin and another to Tub4p, supported by the finding that the benomyl super-sensitivity is caused by a reduced level of alpha-tubulin while the synthetic lethality with tub4-1 is not. In addition, GIM1/YKE2 genetically interacts with two distinct classes of genes, one of which is involved in tubulin folding and the other in microtubule nucleation. We show that the Gim proteins are important for Tub4p function and bind to overproduced Tub4p. The mammalian homologues of GIM1/YKE2 and GIM2/PAC10 rescue the synthetically lethal phenotype with tub4-1 as well as the cold-sensitivity and benomyl super-sensitivity of the yeast deletion mutants. We suggest that the Gim proteins form a protein complex that promotes formation of functional alpha- and gamma-tubulin.     

Regulatory ATPase sites of cytoplasmic dynein affect processivity and force generation

The heavy chain of cytoplasmic dynein contains four nucleotide-binding domains referred to as AAA1-AAA4, with the first domain (AAA1) being the main ATP hydrolytic site. Although previous studies have proposed regulatory roles for AAA3 and AAA4, the role of ATP hydrolysis at these sites remains elusive. Here, we have analyzed the single molecule motility properties of yeast cytoplasmic dynein mutants bearing mutations that prevent ATP hydrolysis at AAA3 or AAA4. Both mutants remain processive, but the AAA4 mutant exhibits a surprising increase in processivity due to its tighter affinity for microtubules. In addition to changes in motility characteristics, AAA3 and AAA4 mutants produce less maximal force than wild-type dynein. These results indicate that the nucleotide binding state at AAA3 and AAA4 can allosterically modulate microtubule binding affinity and affect dynein processivity and force production.     

Kar9p Is a Novel Cortical Protein Required for Cytoplasmic Microtubule Orientation in Yeast

kar9 was originally identified as a bilateral karyogamy mutant, in which the two zygotic nuclei remained widely separated and the cytoplasmic microtubules were misoriented (Kurihara, L.J., C.T. Beh, M. Latterich, R. Schekman, and M.D. Rose. 1994. J. Cell Biol. 126:911–923.). We now report a general defect in nuclear migration and microtubule orientation in kar9 mutants. KAR9 encodes a novel 74-kD protein that is not essential for life. The kar9 mitotic defect was similar to mutations in dhc1/dyn1 (dynein heavy chain gene), jnm1, and act5. kar9Δ dhc1Δ, kar9Δ jnm1Δ, and kar9Δ act5Δ double mutants were synthetically lethal, suggesting that these genes function in partially redundant pathways to carry out nuclear migration. A functional GFP-Kar9p fusion protein localized to a single dot at the tip of the shmoo projection. In mitotic cells, GFP-Kar9p localized to a cortical dot with both mother–daughter asymmetry and cell cycle dependence. In small-budded cells through anaphase, GFP-Kar9p was found at the tip of the growing bud. In telophase and G1 unbudded cells, no localization was observed. By indirect immunofluorescence, cytoplasmic microtubules intersected the GFP-Kar9p dot. Nocodazole experiments demonstrated that Kar9p's cortical localization was microtubule independent. We propose that Kar9p is a component of a cortical adaptor complex that orients cytoplasmic microtubules.     

KAR3, a kinesin-related gene required for yeast nuclear fusion

The KAR3 gene is essential for yeast nuclear fusion during mating, and its expression is strongly induced by alpha factor. The predicted KAR3 protein sequence contains two globular domains separated by an alpha-helical coiled coil. The carboxy-terminal domain is homologous to the amino-terminal mechanochemical domain of Drosophila kinesin heavy chain. Mutation of the putative ATP binding site produces a dominant "poison" of nuclear fusion. The mutant protein shows enhanced microtubule association in vivo, as predicted for a kinesin-like protein in a state of rigor binding. Localization of hybrid proteins to cytoplasmic microtubules in shmoos indicates that the amino-terminal domain also contains determinants for microtubule association. Thus, KAR3 is a member of a larger family of kinesin-like proteins characterized by the presence of the mechanochemical domain tethered to different protein binding domains. The phenotypes of kar3 mutants suggest that the protein mediates microtubule sliding during nuclear fusion and possibly mitosis.     

The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast

BACKGROUND: Two genetic 'pathways' contribute to the fidelity of nuclear segregation during the process of budding in the yeast Saccharomyces cerevisiae. An early pathway, involving Kar9p and other proteins, orients the mitotic spindle along the mother-bud axis. Upon the onset of anaphase, cytoplasmic dynein provides the motive force for nuclear movement into the bud. Loss of either pathway results in nuclear-migration defects; loss of both is lethal. Here, to visualize the functional steps leading to correct spindle orientation along the mother-bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions. RESULTS: Transport of Kar9p into the bud was found to require the myosin Myo2p. Kar9p interacted with microtubules through the microtubule-binding protein Bim1p and facilitated microtubule penetration into the bud. Once microtubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex. Kar9p was also observed at sites of microtubule shortening in the bud, suggesting that Kar9p couples microtubule shortening to nuclear migration. CONCLUSIONS: Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle. A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle. Later, Kar9p couples microtubule disassembly with nuclear migration.