Yeast Mutants Sensitive to Antimicrotubule Drugs Define Three Genes That Affect Microtubule Function

Three new genes affecting microtubule function in Saccharomyces cerevisiae were isolated by screening for mutants displaying supersensitivity to the antimicrotubule drug benomyl. Such mutants fall into six complementation groups: TUB1, TUB2 and TUB3, the three tubulin genes of yeast, and three new genes, which we have named CIN1, CIN2 and CIN4. Mutations in each of the CIN genes were also independently isolated by screening for mutants with increased rates of chromosome loss. Strains bearing mutations in the CIN genes are approximately tenfold more sensitive than wild type to both benomyl and to the related antimicrotubule drug, nocodazole. This phenotype is recessive for all alleles isolated. The CIN1, CIN2 and CIN4 genes were cloned by complementation of the benomyl-supersensitive phenotype. Null mutants of each of the genes are viable, and have phenotypes similar to those of the point mutants. Genetic evidence for the involvement of the CIN gene products in microtubule function comes from the observation that some tubulin mutations are suppressed by cin mutations, while other tubulin mutations are lethal in combination with cin mutations. Additional genetic experiments with cin mutants suggest that the three genes act together in the same pathway or structure to affect microtubule function.     

Saccharomyces Cerevisiae Pac2 Functions with Cin1, 2 and 4 in a Pathway Leading to Normal Microtubule Stability

The products of the Saccharomyces cerevisiae CIN1, CIN2 and CIN4 genes participate in a nonessential pathway required for normal microtubule function. In this article, we demonstrate that the product of PAC2 also functions in this pathway. PAC2 deletion mutants displayed phenotypes and genetic interactions similar to those caused by cin1Δ, cin2Δ and cin4Δ. These include cold-sensitive microtubule structures and sensitivity to the microtubule depolymerizing agent benomyl. Involvement in a common functional pathway is indicated by the observation that all double mutant combinations are viable and no more affected than any single mutant. In addition, extra copies of CIN1 were found to suppress the benomyl sensitivity of pac2Δ, cin2Δ and cin4Δ, but not that caused by other mutations that affect microtubule function. Cin1p and Pac2p were found to be related in sequence to mammalian proteins that aid in the folding of β-tubulin into an assembly-competent state. Alleles of CIN1 were identified that could suppress the benomyl sensitivity of cin4-4 in a highly specific fashion. Our findings suggest that the guanine nucleotide-binding Cin4p interacts with Cin1p and regulates its tubulin folding activity.     

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.     

Mutations in the yeast kinesin-like Cin8p are alleviated by osmotic support

Loss of function of Cin8p (a yeast kinesin-like motor protein) in the absence of either Kip1p (a motor of the same family) or Dyn1p (the dynein heavy chain) is lethal. We report that cin8 mutants are sensitive to the cell wall disrupting agents calcofluor white and SDS. Conditionally lethal double mutants containing the temperature sensitive allele cin8-3 in a background deletion of either kip1 or dyn1 grew normally at the restrictive temperature when osmolytes such as sorbitol were added to the medium. Sorbitol could not alleviate the sensitivity of cin8 mutants to calcofluor and SDS. However, it rendered cells more resistant to the microtubule depolymerizing drugs benomyl and thiabendazole (TBZ). Our findings reveal a novel interaction between mitotic motor proteins and the cell wall and suggest that the induction of signaling pathways aimed at maintaining the cell wall suppresses phenotypes of mutations in microtubule-associated motor proteins through stabilization of microtubules.     

The complete spectrum of yeast chromosome instability genes identifies candidate CIN cancer genes and functional roles for ASTRA complex components

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ～ 2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease.     

Yaf9, a novel NuA4 histone acetyltransferase subunit,is required for the cellular response to spindle stress in yeast

Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9 Delta mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Delta mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2 Delta) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.     

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.     

Mutability and mutational spectrum of chromosome transmission fidelity genes

It has been more than two decades since the original chromosome transmission fidelity (Ctf) screen of Saccharomyces cerevisiae was published. Since that time the spectrum of mutations known to cause Ctf and, more generally, chromosome instability (CIN) has expanded dramatically as a result of systematic screens across yeast mutant arrays. Here we describe a comprehensive summary of the original Ctf genetic screen and the cloning of the remaining complementation groups as efforts to expand our knowledge of the CIN gene repertoire and its mutability in a model eukaryote. At the time of the original screen, it was impossible to predict either the genes and processes that would be overrepresented in a pool of random mutants displaying a Ctf phenotype or what the entire set of genes potentially mutable to Ctf would be. We show that in a collection of 136 randomly selected Ctf mutants, >65% of mutants map to 13 genes, 12 of which are involved in sister chromatid cohesion and/or kinetochore function. Extensive screening of systematic mutant collections has shown that ~350 genes with functions as diverse as RNA processing and proteasomal activity mutate to cause a Ctf phenotype and at least 692 genes are required for faithful chromosome segregation. The enrichment of random Ctf alleles in only 13 of ~350 possible Ctf genes suggests that these genes are more easily mutable to cause genome instability than the others. These observations inform our understanding of recurring CIN mutations in human cancers where presumably random mutations are responsible for initiating the frequently observed CIN phenotype of tumors.     

Function of tubulin binding proteins in vivo

Overexpression of the beta-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant alpha-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes-CIN1, CIN2, CIN4, and PAC2-as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products-Cin1p/cofactor D and Pac2p/cofactor E-can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-beta-tubulin complex, as it does in the alpha-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant alpha-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free beta-tubulin.     

A role of Sep1 (= Kem1, Xrn1) as a microtubule-associated protein in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells lacking the SEP1 (also known as XRN1, KEM1, DST2, RAR5) gene function exhibit a number of phenotypes in cellular processes related to microtubule function. Mutant cells show increased sensitivity to the microtubule-destabilizing drug benomyl, increased chromosome loss, a karyogamy defect, impaired spindle pole body separation, and defective nuclear migration towards the bud neck. Analysis of the arrest morphology and of the survival during arrest strongly suggests a structural defect accounting for the benomyl hypersensitivity, rather than a regulatory defect in a checkpoint. Biochemical analysis of the purified Sep1 protein demonstrates its ability to promote the polymerization of procine brain and authentic S.cerevisiae tubulin into flexible microtubules in vitro. Furthermore, Sep1 co-sediments with these microtubules in sucrose cushion centrifugation. Genetic analysis of double mutant strains containing a mutation in SEP1 and in one of the genes coding for alpha- or beta-tubulin further suggests interaction between Sep1 and microtubules. Taken together these three lines of evidence constitute compelling evidence for a role of Sep1 as an accessory protein in microtubule function in the yeast S.cerevisiae.     

Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans

We have examined the effects of the antimicrotubule agent benomyl and several mutations on nuclear and mitochondrial movement in germlings of the filamentous fungus Aspergillus nidulans. While, as previously reported, benomyl inhibited nuclear division and movement, it did not inhibit mitochondrial movement. To test the effects of benomyl more rigorously, we germinated two benomyl super-sensitive, beta-tubulin mutants at a benomyl concentration 50-100 times greater than that required to inhibit colony formation completely. Again nuclear division and movement were inhibited, but mitochondrial movement was not. We also examined conditionally lethal beta-tubulin mutations that disrupt microtubule function under restrictive conditions. Nuclear division and movement were inhibited but, again, mitochondrial movement was not. Finally we examined the effects of five heat-sensitive mutations that inhibit nuclear movement but not nuclear division at restrictive temperatures. These mutations strongly inhibited nuclear movement at a restrictive temperature but did not inhibit mitochondrial movement. These data demonstrate that the mechanisms of nuclear and mitochondrial movement in Aspergillus nidulans are not identical and suggest that mitochondrial movement does not require functional microtubules.     

Kinesin-related proteins required for assembly of the mitotic spindle

We identified two new Saccharomyces cerevisiae kinesin-related genes, KIP1 and KIP2, using polymerase chain reaction primers corresponding to highly conserved regions of the kinesin motor domain. Both KIP proteins are expressed in vivo, but deletion mutations conferred no phenotype. Moreover, kip1 kip2 double mutants and a triple mutant with kinesin-related kar3 had no synthetic phenotype. Using a genetic screen for mutations that make KIP1 essential, we identified another gene, KSL2, which proved to be another kinesin-related gene, CIN8. KIP1 and CIN8 are functionally redundant: double mutants arrested in mitosis whereas the single mutants did not. The microtubule organizing centers of arrested cells were duplicated but unseparated, indicating that KIP1 or CIN8 is required for mitotic spindle assembly. Consistent with this role, KIP1 protein was found to colocalize with the mitotic spindle.     

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.     

Chromosome instability in colorectal tumor cells is associated with defects in microtubule plus-end attachments caused by a dominant mutation in APC

The attachment of microtubule plus ends to kinetochores and to the cell cortex is essential for the fidelity of chromosome segregation. Here, we characterize the causes underlying the high rates of chromosome instability (CIN+) observed in colorectal tumor cells. We show that CIN+ tumor cells exhibit inefficient microtubule plus-end attachments during mitosis, accompanied by impairment of chromosome alignment in metaphase. The mitotic abnormalities associated with CIN+ tumor cells correlated with status of adenomatous polyposis coli (APC). Importantly, we have shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and to cause a dramatic increase in mitotic abnormalities. We propose that APC functions to modulate microtubule plus-end attachments during mitosis, and that a single mutant APC allele predisposes cells to increased mitotic abnormalities, which may contribute to tumor progression.     

Diverse effects of beta-tubulin mutations on microtubule formation and function

We have used in vitro mutagenesis and gene replacement to construct five new cold-sensitive mutations in TUB2, the sole gene encoding beta-tubulin in the yeast Saccharomyces cerevisiae. These and one previously isolated tub2 mutant display diverse phenotypes that have allowed us to define the functions of yeast microtubules in vivo. At the restrictive temperature, all of the tub2 mutations inhibit chromosome segregation and block the mitotic cell cycle. However, different microtubule arrays are present in these arrested cells depending on the tub2 allele. One mutant (tub2-401) contains no detectable microtubules, two (tub2-403 and tub2-405) contain greatly diminished levels of both nuclear and cytoplasmic microtubules, one (tub2-104) contains predominantly nuclear microtubules, one (tub2-402) contains predominantly cytoplasmic microtubules, and one (tub2-404) contains prominent nuclear and cytoplasmic microtubule arrays. Using these mutants we demonstrate here that cytoplasmic microtubules are necessary for nuclear migration during the mitotic cell cycle and for nuclear migration and fusion during conjugation; only those mutants that possess cytoplasmic microtubules are able to perform these functions. We also show that microtubules are not required for secretory vesicle transport in yeast; bud growth and invertase secretion occur in cells which contain no microtubules.     

Selection for Early Meiotic Mutants in Yeast

In the yeast Saccharomyces cerevisiae, only a/alpha cells can enter meiosis; a and alpha cells cannot. Because a/alpha cells are typically diploid and a and alpha cells are typically haploid, this cell type restriction ensures that only diploid cells enter meiosis. Entry into meiosis is accompanied by an increase in expression of the IME1 gene; the IME1 product (IME1) then activates IME2 and other meiotic genes. We have found that IME1 expression is toxic to starved haploid cells, presumably because IME1 directs them into meiosis. IME1 toxicity is greater in rad52 mutants, in which meiotic recombination causes lethal damage. Suppressors of IME1 toxicity include recessive mutations in two genes, RIM11 and RIM16 (Regulator of Inducer of Meiosis), that are required for IME1 to activate IME2 expression. RIM11 maps near CIN4 on chromosome XIII.     

Cold-sensitive nuclear division arrest mutants of the fission yeast Schizosaccharomyces pombe

Thirteen recessive cold sensitive nuclear division arrest mutants were isolated from the fission yeast Schizosaccharomyces pombe. Twelve unlinked genes were defined; six in chromosome I, three in chromosome II and two in chromosome III. The map positions of three nuclear division arrest genes (nda1, nda2 and nda3) in chromosome II were determined precisely. Together with the previously obtained temperature-sensitive cell division cycle mutations, at least 20 genes appear to control the nuclear division of the fission yeast. Physiological studies indicated that most cold sensitive nda mutants incubated previously at 22 degrees C proceeded with a synchronously normal cell-cycle after temperature shift-up. The morphology of the nuclei and nuclear chromatin region was studied by the 4',6-diamidino-2-phenylindole staining method and by electron microscopy. Each mutant exhibited characteristic nuclear morphology at 22 degrees C, showing the specific blockages. The nda genes seem to control a pathway of structural alterations in the nuclear chromatin region with the order hemisphere, condensed ellipsoid, segregating U-form and separating hemispheres. Two genes, nda2 and nda3, pleiotropically control nuclear division, nuclear location and cell shape. The terminal phenotype of nda2-KM52 is characterized by the nuclear displacement, the absence of a spindle and abnormal locations of spindle pole bodies. The cells of nda3-KM311 were aberrant in shape and contained a partially separated chromatin region with a long spindle. Together with the results of the accompanying paper, we conclude that nda2 and nda3 genes control nuclear and cytoplasmic microtubular organization.     

Chromosome instability and tumor lethality suppression in carcinogenesis

The maintenance and survival of each organism depends on its genome integrity. Alterations of essential genes, or aberrant chromosome number and structure lead to cell death. Paradoxically, cancer cells, especially in solid tumors, contain somatic gene mutations and are chromosome instability (CIN), suggesting a mechanism that cancer cells have acquired to suppress the lethal mutations and/or CIN. Herein we will discuss a tumor lethality suppression concept based on the studies of yeast genetic interactions and transgenic mice. During the early stages of the multistep process of tumorigenesis, incipient cancer cells probably have adopted genetic and epigenetic alterations to tolerate the lethal mutations of other genes that ensue, and to a larger extent CIN. In turn, CIN mediated massive gain and loss of genes provides a wider buffer for further genetic reshuffling, resulting in cancer cell heterogeneity, drug resistance and evasion of oncogene addiction, thus CIN may be both the effector and inducer of tumorigenesis. Accordingly, interfering with tumor lethality suppression could lead to cancer cell death or growth defects. Further validation of the tumor lethality suppression concept would help to elucidate the role of CIN in tumorigenesis, the relationship between CIN and somatic gene mutations, and would impact the design of anticancer drug development.     

Diversity of TITAN functions in Arabidopsis seed development.

The titan mutants of Arabidopsis exhibit striking defects in seed development. The defining feature is the presence of abnormal endosperm with giant polyploid nuclei. Several TTN genes encode structural maintenance of chromosome proteins (condensins and cohesins) required for chromosome function at mitosis. Another TTN gene product (TTN5) is related to the ARL2 class of GTP-binding proteins. Here, we identify four additional TTN genes and present a general model for the titan phenotype. TTN1 was cloned after two tagged alleles were identified through a large-scale screen of T-DNA insertion lines. The predicted gene product is related to tubulin-folding cofactor D, which interacts with ARL2 in fission yeast (Schizosaccharomyces pombe) and humans to regulate tubulin dynamics. We propose that TTN5 and TTN1 function in a similar manner to regulate microtubule function in seed development. The titan phenotype can therefore result from disruption of chromosome dynamics (ttn3, ttn7, and ttn8) or microtubule function (ttn1 and ttn5). Three other genes have been identified that affect endosperm nuclear morphology. TTN4 and TTN9 appear to encode plant-specific proteins of unknown function. TTN6 is related to the isopeptidase T class of deubiquitinating enzymes that recycle polyubiquitin chains following protein degradation. Disruption of this gene may reduce the stability of the structural maintenance of chromosome complex. Further analysis of the TITAN network should help to elucidate the regulation of microtubule function and chromosome dynamics in seed development.