Cyclins in aspergilli: Phylogenetic and functional analyses of group I cyclins.

We have identified the cyclin domain-containing proteins encoded by the genomes of 17 species of Aspergillus as well as 15 members of other genera of filamentous ascomycetes. Phylogenetic analyses reveal that the cyclins fall into three groups, as in other eukaryotic phyla, and, more significantly, that they are remarkably conserved in these fungi. All 32 species examined, for example, have three group I cyclins, cyclins that are particularly important because they regulate the cell cycle, and these are highly conserved. Within the group I cyclins there are three distinct clades, and each fungus has a single member of each clade. These findings are in marked contrast to the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, which have more numerous group I cyclins. These results indicate that findings on cyclin function made with a model Aspergillus species, such as A. nidulans, are likely to apply to other Aspergilli and be informative for a broad range of filamentous ascomycetes. In this regard, we note that the functions of only one Aspergillus group I cyclin have been analysed (NimECyclin B of A. nidulans). We have consequently carried out an analysis of the members of the other two clades using A. nidulans as our model. We have found that one of these cyclins, PucA, is essential, but deletion of PucA in a strain carrying a deletion of CdhA, an activator of the anaphase promoting complex/cyclosome (APC/C), is not lethal. These data, coupled with data from heterokaryon rescue experiments, indicate that PucA is an essential G1/S cyclin that is required for the inactivation of the APC/C-CdhA, which, in turn, allows the initiation of the S phase of the cell cycle. Our data also reveal that PucA has additional, non-essential, roles in the cell cycle in interphase. The A. nidulans member of the third clade (AN2137) has not previously been named or analyzed. We designate this gene clbA. ClbA localizes to kinetochores from mid G2 until just prior to chromosomal condensation. Deletion of clbA does not affect viability. However, by using a regulatable promoter system new to Aspergillus, we have found that expression of a version of ClbA in which the destruction box sequences have been removed is lethal and causes a mitotic arrest and a high frequency of non-disjunction. Thus, although ClbA is not essential, its timely destruction is essential for viability, chromosomal disjunction, and successful completion of mitosis.

Gamma-tubulin and the C-terminal motor domain kinesin-like protein, KLPA, function in the establishment of spindle bipolarity in Aspergillus nidulans.

Previous research has found that a gamma-tubulin mutation in Schizosaccharomyces pombe is synthetically lethal with a deletion of the C-terminal motor domain kinesin-like protein gene pkl1, but the lethality of the double mutant prevents a phenotypic analysis of the synthetic interaction. We have investigated interactions between klpA1, a deletion of an Aspergillus nidulans homolog of pkl1, and mutations in the mipA, gamma-tubulin gene. We find that klpA1 dramatically increases the cold sensitivity and slightly reduces the growth rate at all temperatures, of three mipA alleles. In synchronized cells we find that klpA1 causes a substantial but transient inhibition of the establishment of spindle bipolarity. At a restrictive temperature, mipAD123 causes a slight, transient inhibition of spindle bipolarity and a more significant inhibition of anaphase A. In the mipAD123/klpA1 strain, formation of bipolar spindles is more strongly inhibited than in the klpA1 single mutant and many spindles apparently never become bipolar. These results indicate, surprisingly, that gamma-tubulin and the klpA kinesin have overlapping roles in the establishment of spindle bipolarity. We propose a model to account for these data.

Alanine-scanning mutagenesis of Aspergillus gamma-tubulin yields diverse and novel phenotypes.

We have created 41 clustered charged-to-alanine scanning mutations of the mipA, gamma-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of gamma-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that gamma-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of gamma-tubulin and these data give a good initial indication of the functionally important regions of the molecule.

An abundance of tubulins.

Recent data have revealed that the tubulin superfamily of proteins is much larger than was thought previously. Six distinct families within the tubulin superfamily have been discovered and more might await discovery. alpha-, beta- and gamma-tubulins are ubiquitous in eukaryotes. alpha- and beta-tubulins are the major components of microtubules, and gamma-tubulin plays a major role in the nucleation of microtubule assembly. delta- and epsilon-tubulins are widespread but not ubiquitous, and zeta-tubulin has been found so far only in kinetoplastid protozoa. delta-Tubulin has an important role in flagellar assembly in Chlamydomonas, but its role in other organisms is just beginning to be investigated, as are the functions of the recently discovered epsilon- and zeta-tubulins.

The gamma-tubulin gene family in humans.

Despite the central role of gamma-tubulin in the organization of the microtubule cytoskeleton, the gamma-tubulin gene family in humans has not been characterized. We now report the identification of a second expressed human gamma-tubulin gene (TUBG2) and a gamma-tubulin pseudogene (TUBG1P) in addition to the previously identified gamma-tubulin gene (TUBG1). Evidence from Southern hybridizations suggests that there are probably no additional gamma-tubulin sequences in the human genome. TUBG1 and TUBG2 are within 20 kb of each other in region q21 of chromosome 17, and TUBG1P is on chromosome 7. The proteins encoded by TUBG1 and TUBG2 share 97.3% amino acid identity, and the two genes are coexpressed in a variety of tissues. Previous studies of gamma-tubulin in human tissues and cell lines have been based on the tacit assumption that a single gamma-tubulin (the gamma-tubulin encoded by TUBG1) was present. While this assumption is not correct, the similarity of the products of TUBG1 and TUBG2 suggests that results of previous immunolocalization and immunoprecipitation studies in human cells and tissues are likely to be valid. In addition, any pharmacological agents that target one human gamma-tubulin are likely to target both.

A mutation in gamma-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein pkl1p.

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. gamma-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in gamma-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30 degrees C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant gamma-tubulin is like the wild-type protein. Prediction of gamma-tubulin structure indicates that non-alpha/beta-tubulin protein-protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the gamma-tubulin mutant and in multicopy for normal cell morphology at 30 degrees C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for gamma-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of gamma-tubulin that involves non-tubulin protein-protein interactions, presumably with a second motor, MAP, or MTOC component.

Centrosome-independent mitotic spindle formation in vertebrates.

BACKGROUND: In cells lacking centrosomes, the microtubule-organizing activity of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pathway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labeling with green fluorescent protein (GFP) and laser microsurgery we have been able to selectively destroy centrosomes in living mammalian cells as they enter mitosis. RESULTS: We have established a mammalian cell line in which the boundaries of the centrosome are defined by the constitutive expression of gamma-tubulin-GFP. This feature allows us to use laser microsurgery to selectively destroy the centrosomes in living cells. Here we show that this method can be used to reproducibly ablate the centrosome as a functional entity, and that after destruction the microtubules associated with the ablated centrosome disassemble. Depolymerization-repolymerization experiments reveal that microtubules form in acentrosomal cells randomly within the cytoplasm. When both centrosomes are destroyed during prophase these cells form a functional bipolar spindle. Surprisingly, when just one centrosome is destroyed, bipolar spindles are also formed that contain one centrosomal and one acentrosomal pole. Both the polar regions in these spindles are well focused and contain the nuclear structural protein NuMA. The acentrosomal pole lacks pericentrin, gamma-tubulin, and centrioles, however. CONCLUSIONS: These results reveal, for the first time, that somatic cells can use a centrosome-independent pathway for spindle formation that is normally masked by the presence of the centrosome. Furthermore, this mechanism is strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.

Unusual antimicrotubule activity of the antifungal agent spongistatin 1.

Spongistatin 1, a macrocyclic lactone from the marine sponge Hyrtios erecta, has broad-spectrum antifungal activity. Since this compound is a potent antimicrotubule agent in mammalian cells, we examined its effects on the filamentous fungus Aspergillus nidulans to determine if its antifungal effects are due to antimicrotubule activity. At 25 microg/ml (twice the MIC), spongistatin 1 caused a greater-than-twofold elevation of the chromosome and spindle mitotic indices. Immunofluorescence microscopy revealed that mitotic spindles were smaller and shorter than in control germlings. However, late-anaphase and telophase nuclei were seen occasionally, and this suggests that the spindles are capable of segregating chromosomes. Spongistatin 1 had more dramatic effects on cytoplasmic microtubules. At 30 min after initiation of treatment, 83% of germlings contained fragmented microtubules and after 2 h of treatment, microtubules had disappeared completely from 82% of germlings. In contrast, microtubules disappeared rapidly and completely from germlings treated with benomyl. We conclude that spongistatin 1 has antimicrotubule activity in A. nidulans and that its mechanism of action may involve a novel microtubule-severing activity.

Gamma-tubulin at ten: progress and prospects.

The existence of gamma-tubulin was first reported approximately ten years ago, and it is appropriate to review the progress that has been made in gamma-tubulin research and to discuss some of the unanswered questions about gamma-tubulin function. gamma-Tubulin is ubiquitous in eukaryotes and is generally quite conserved. Two highly divergent gamma-tubulins have been discovered, however, one in Saccharomyces cerevisiae and one in Caenorhabditis elegans. Several organisms have two gamma-tubulin genes. In Drosophila melanogaster, the two gamma-tubulins differ significantly in sequence and expression pattern. In other organisms the two gamma-tubulins are almost identical and expression patterns have not been determined. gamma-Tubulin is located at microtubule organizing centers in many organisms, and it is also frequently associated with the mitotic spindle. gamma-Tubulin is essential for the formation of functional mitotic spindles in all organisms that have been examined to date. In animal cells, complexes containing gamma-tubulin are located at microtubule organizing centers where they nucleate the assembly of microtubules. In spite of the considerable progress that has been made in gamma-tubulin research important questions remain to be answered. The exact mechanisms of microtubule nucleation by gamma-tubulin complexes remain to be resolved as do the mechanisms by which microtubule nucleation from gamma-tubulin complexes is regulated. Finally, there is evidence that gamma-tubulin has important functions in addition to microtubule nucleation, and these functions are just beginning to be investigated.

Mitosis in wild-type and beta-tubulin mutant strains of Aspergillus nidulans.

We review and illustrate the wild-type mitotic cycle of Aspergillus nidulans and report the sequence alterations in six mutant alleles of the A. nidulans benA, beta-tubulin, gene. These alleles confer heat sensitivity and resistance to the antifungal, antimicrotubule compound benomyl, and they have been very important in the study of mitosis and microtubule function in A. nidulans. The mutations are novel and fall at amino acids 50, 134, and 257. We have examined the phenotypes conferred by the mutations at restrictive temperatures. None blocks the assembly of microtubules. One allele, benA33, blocks anaphase A and partially inhibits the disassembly of cytoplasmic microtubules in mitosis. We also often observe abnormal spindle morphologies in strains carrying benA33. Another allele, benA31, causes arrest in mitosis with short mitotic spindles and, thus, appears to inhibit spindle elongation.

The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds.

The cycle of spindle pole body (SPB) duplication, differentiation, and segregation in Schizosaccharomyces pombe is different from that in some other yeasts. Like the centrosome of vertebrate cells, the SPB of S. pombe spends most of interphase in the cytoplasm, immediately next to the nuclear envelope. Some gamma-tubulin is localized on the SPB, suggesting that it plays a role in the organization of interphase microtubules (MTs), and serial sections demonstrate that some interphase MTs end on or very near to the SPB. gamma-Tubulin is also found on osmiophilic material that lies near the inner surface of the nuclear envelope, immediately adjacent to the SPB, even though there are no MTs in the interphase nucleus. Apparently, the MT initiation activities of gamma-tubulin in S. pombe are regulated. The SPB duplicates in the cytoplasm during late G2 phase, and the two resulting structures are connected by a darkly staining bridge until the mitotic spindle forms. As the cell enters mitosis, the nuclear envelope invaginates beside the SPB, forming a pocket of cytoplasm that accumulates dark amorphous material. The nuclear envelope then opens to form a fenestra, and the duplicated SPB settles into it. Each part of the SPB initiates intranuclear MTs, and then the two structures separate to lie in distinct fenestrae as a bipolar spindle forms. Through metaphase, the SPBs remain in their fenestrae, bound to the polar ends of spindle MTs; at about this time, a small bundle of cytoplasmic MTs forms in association with each SPB. These MTs are situated with one end near to, but not on, the SPBs, and they project into the cytoplasm at an orientation that is oblique to the simple axis. As anaphase proceeds, the nuclear fenestrae close, and the SPBs are extruded back into the cytoplasm. These observations define new fields of enquiry about the control of SPB duplication and the dynamics of the nuclear envelope.

Differential expression of two gamma-tubulin isoforms during gametogenesis and development in Drosophila.

Previous work identified a gamma-tubulin gene, gamma Tub23C, in Drosophila (Zheng et al., 1991). We now report identification of a second gamma-tubulin gene, gamma Tub37CD. Immunoblot analysis and immunolocalization show that gamma Tub37CD and gamma Tub23C are differentially expressed during gametogenesis and development. During oogenesis, gamma Tub23C was detected at centrosomes and in the cytoplasm of mitotic germ cells, but was not detected in germ cells following completion of mitosis. Conversely, gamma Tub37CD was not detected in proliferating germ cells, but appeared to accumulate in germ cells during egg chamber development. Neither gamma-tubulin isoform was detected at the anterior or posterior poles of developing oocytes. During spermatogenesis, only gamma Tub23C was detected at centrosomes, where it showed cell cycle- and differentiation-dependent organization. During the transition into the first meiotic division, gamma Tub23C became organized as a corpuscular focus at centrioles until completion of meiosis II. During postmeiotic spermatid differentiation, gamma Tub23C was detected first as a rod and then as a collar-like structure near the juncture of the nucleus and the elongating flagellum, but was not detected in bundles of mature sperm. The germline-specific CDC25 encoded by twine is required for organization of gamma Tub23C into corpuscular focus in spermatocytes, but not for separation of centriole pairs in M-phase or postmeiotic organization of gamma Tub23C at centrioles. Following reconstitution of a canonical centrosome at fertilization, only gamma Tub37CD was detected at centrosomes in syncytial embryos, but both gamma Tub37CD and gamma Tub23C were detected at centrosomes in cellularized embryos. Colocalization of these two isoforms suggests that gamma Tub23C and gamma Tub37CD both contain structural features of gamma-tubulins essential for localization to centrosomes.

The role of gamma-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans.

gamma-Tubulin has been hypothesized to be essential for the nucleation of the assembly of mitotic spindle microtubules, but some recent results suggest that this may not be the case. To clarify the role of gamma-tubulin in microtubule assembly and cell-cycle progression, we have developed a novel variation of the gene disruption/heterokaryon rescue technique of Aspergillus nidulans. We have used temperature-sensitive cell-cycle mutations to synchronize germlings carrying a gamma-tubulin disruption and observe the phenotypes caused by the disruption in the first cell cycle after germination. Our results indicate that gamma-tubulin is absolutely required for the assembly of mitotic spindle microtubules, a finding that supports the hypothesis that gamma-tubulin is involved in spindle microtubule nucleation. In the absence of functional gamma-tubulin, nuclei are blocked with condensed chromosomes for about the length of one cell cycle before chromatin decondenses without nuclear division. Our results indicate that gamma-tubulin is not essential for progression from G1 to G2, for entry into mitosis nor for spindle pole body replication. It is also not required for reactivity of spindle pole bodies with the MPM-2 antibody which recognizes a phosphoepitope important to mitotic spindle formation. Finally, it does not appear to be absolutely required for cytoplasmic microtubule assembly but may play a role in the formation of normal cytoplasmic microtubule arrays.

Characterization of gamma-tubulin complexes in Aspergillus nidulans and detection of putative gamma-tubulin interacting proteins.

gamma-Tubulin is central to the nucleation of microtubule assembly in vivo. Although it is most obviously located at microtubule organizing centers, it is also found in soluble cytoplasmic complexes. Characterizing these complexes and identifying proteins that interact with gamma-tubulin in vivo will be necessary if gamma-tubulin function is to be understood fully. We have begun to investigate soluble complexes of gamma-tubulin in Aspergillus nidulans, the organism in which gamma-tubulin was discovered and in which a great deal of genetic and molecular genetic analysis of gamma-tubulin has been carried out. We find that approximately 32% of the gamma-tubulin in A. nidulans is soluble. Sucrose density gradients revealed that the soluble gamma-tubulin is in 8-20S complexes with little or no monomeric gamma-tubulin present. In the presence of 0.5 M KCl the average size of the complexes decreased and a peak was present between 4S and 11S. Cross-linking experiments with a zero-length cross-linker suggest that gamma-tubulin in isolated nuclei and in intact hyphae interacts physically with three proteins with molecular weights of approximately 105, 95, and 80 kDa.

Purification and characterization of assembly-competent tubulin from Aspergillus nidulans.

We have developed a procedure for purifying assembly-competent tubulin from Aspergillus nidulans. To our knowledge, this is the first report of the purification of assembly-competent tubulin from a filamentous fungus, and the procedure should be of great value in analyzing the large number of alpha- and beta-tubulin mutations that have been isolated and characterized in A. nidulans. Our procedure consists of overproduction of alpha- and beta-tubulin, partial purification by ion-exchange chromatography, and final purification by rounds of assembly and disassembly. We have found that taxol promotes the assembly of A. nidulans tubulin into microtubules, but a higher concentration of taxol is required for maximal assembly of A. nidulans tubulin than is required for brain tubulin. The critical concentration for assembly in the presence of taxol is also significantly higher for A. nidulans tubulin than for brain tubulin. In addition, A. nidulans microtubules that were assembled and maintained in the presence of taxol depolymerized in conditions in which taxol-stabilized mammalian microtubules remain intact. These results suggest that A. nidulans tubulin has a lower affinity for taxol than mammalian tubulin.

Human gamma-tubulin functions in fission yeast.

gamma-Tubulin is a phylogenetically conserved component of microtubule-organizing centers that is essential for viability and microtubule function. To examine the functional conservation of gamma-tubulin, we have tested the ability of human gamma-tubulin to function in the fission yeast Schizosaccharomyces pombe. We have found that expression of a human gamma-tubulin cDNA restores viability and a near-normal growth rate to cells of S. pombe lacking endogenous gamma-tubulin. Immunofluorescence microscopy showed that these cells contained normal mitotic spindles and interphase microtubule arrays, and that human gamma-tubulin, like S. pombe gamma-tubulin, localized to spindle pole bodies, the fungal microtubule-organizing centers. These results demonstrate that human gamma-tubulin functions in fission yeast, and they suggest that in spite of the great morphological differences between the microtubule-organizing centers of humans and fission yeasts, gamma-tubulin is likely to perform the same tasks in both. They suggest, moreover, that the proteins that interact with gamma-tubulin, including, most obviously, microtubule-organizing center proteins, must also be conserved. We have also found that a fivefold overexpression of S. pombe gamma-tubulin causes no reduction in growth rates or alteration of microtubule organization. We hypothesize that the excess gamma-tubulin is maintained in the cytoplasm in a form incapable of nucleating microtubule assembly. Finally, we have found that expression of human gamma-tubulin or overexpression of S. pombe gamma-tubulin causes no significant alteration of resistance to the antimicrotubule agents benomyl, thiabendazole and nocodazole.