The human homologue of the Aspergillus nuclear migration gene nudC is preferentially expressed in dividing cells and ciliated epithelia.

We recently identified a novel human gene, HnudC, homologous to an Aspergillus nidulans gene coding for a protein crucial to nuclear migration, cell wall morphogenesis, and cell growth. While mRNA for this gene is expressed in most tissues, HNUDC protein expression is highly regulated. To provide insight into the function of this protein, we performed immunohistochemical analysis of the distribution of HNUDC in 19 different human tissues. Intense immunolabeling was observed in proliferating cells, including spermatocytes at all stages, early hematopoietic cells, cortical thymocytes, immunoblasts, and basal colonic and esophageal mucosa. Within a given tissue, cells with different proliferative capacities demonstrated different levels of HNUDC expression. HNUDC was also highly expressed in ciliated epithelia including those found in ependyma, bronchial mucosa, and fallopian tubes. Immunolabeling was moderate in several non-proliferating tissues, but little or no labeling was observed in most other tissues examined. We also demonstrated by western blotting that most cell lines express extremely high levels of HNUDC compared to their normal counterparts. While this supports a role for HnudC in cell proliferation, these data indicate that cell lines are not a reliable measure of HNUDC protein expression in normal tissues. We conclude that HNUDC is highly expressed in cell lines and the proliferating cells of normal tissues, consistent with our hypothesis that HNUDC is conserved throughout evolution for a crucial function in cell division. In addition, the high level in ciliated cells suggests an important role in ciliary motility or assembly, analogous to its role in A. nidulans nuclear movement.

Mitotic histone H3 phosphorylation by the NIMA kinase in Aspergillus nidulans.

Phosphorylation of histone H3 serine 10 correlates with chromosome condensation and is required for normal chromosome segregation in Tetrahymena. This phosphorylation is dependent upon activation of the NIMA kinase in Aspergillus nidulans. NIMA expression also induces Ser-10 phosphorylation inappropriately in S phase-arrested cells and in the absence of NIMX(cdc2) activity. At mitosis, NIMA becomes enriched on chromatin and subsequently localizes to the mitotic spindle and spindle pole bodies. The chromatin-like localization of NIMA early in mitosis is tightly correlated with histone H3 phosphorylation. Finally, NIMA can phosphorylate histone H3 Ser-10 in vitro, suggesting that NIMA is a mitotic histone H3 kinase, perhaps helping to explain how NIMA promotes chromatin condensation in A. nidulans and when expressed in other eukaryotes.

The nuclear migration gene NudC and human hematopoiesis.

The filamentous fungus Aspergillus nidulans nudC (nuclear distribution C) gene is required for movement of nuclei following mitosis and for normal colony growth. It is highly conserved, structurally and functionally, throughout most of evolution. The human homolog, called HnudC, has been cloned and has an important role in cell proliferation. In hematopoiesis, HNUDC is highly expressed in early hematopoietic precursors and declines during normal differentiation. Stimulation of proliferation of the erythroleukemia cell line TF-1 with GM-CSF enhances HnudC protein and mRNA expression and treatment with antisense (but not sense) oligonucleotides to HnudC mRNA significantly reduces cell division. A significant increase in HNUDC is present in bone marrow aspirates from patients with acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML) compared to the level in normal cellular counterparts, demonstrating dysregulated expression in leukemia. These data support the conclusion that HnudC plays a functional role in promoting hematopoietic cell growth and that it is involved in leukemogenesis.

Interaction between developmental and cell cycle regulators is required for morphogenesis in Aspergillus nidulans.

In Aspergillus nidulans, mutation of the transcriptional regulator brlA arrests formation of asexual spore-forming structures called conidiophores but does not hinder vegetative hyphal growth. During conidiophore development a 6-fold, brlA-dependent increase in the kinase activities of NIMX(cdc2) and NIMA occurs. A similar level of kinase induction was promoted by ectopic expression of brlA. Northern and Western analysis revealed marked induction of nimX(cdc2) mRNA after ectopic expression of brlA and increased amounts of NIMX(cdc2). Therefore, nimX(cdc2) is developmentally regulated by brlA indicating a direct role for brlA in the regulation of cell cycle genes. That correct regulation of nimX(cdc2) is important for normal development was further supported by analysis of conidiophore development and septation in cell cycle specific mutants. Most noticeably, the nimX(cdc2AF) mutation promoted inappropriate septation and hindered the switch from filamentous growth to budding growth seen during conidiophore development. Therefore, in contrast to the situation previously reported for other multicellular eukaryotes, interaction between developmental regulators and cell cycle regulators is essential for normal morphogenesis in A.nidulans.

Checkpoint defects leading to premature mitosis also cause endoreplication of DNA in Aspergillus nidulans.

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMX(cdc2) in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsB(rad3) and uvsD(rad26) have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIME(cyclinB), but ectopic expression of active nondegradable NIME(cyclinB) does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMX(cdc2) and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.

The extremely conserved pyroA gene of Aspergillus nidulans is required for pyridoxine synthesis and is required indirectly for resistance to photosensitizers.

Numerous disparate studies in plants, filamentous fungi, yeast, Archaea, and bacteria have identified one of the most highly conserved proteins (SNZ family) for which no function was previously defined. Members have been implicated in the stress response of plants and yeast and resistance to singlet oxygen toxicity in the plant pathogen Cercospora. However, it is found in some anaerobic bacteria and is absent in some aerobic bacteria. We have cloned the Aspergillus nidulans homologue (pyroA) of this highly conserved gene and define this gene family as encoding an enzyme specifically required for pyridoxine biosynthesis. This realization has enabled us to define a second pathway for pyridoxine biosynthesis. Some bacteria utilize the pdx pyridoxine biosynthetic pathway defined in Escherichia coli and others utilize the pyroA pathway. However, Eukarya and Archaea exclusively use the pyroA pathway. We also found that pyridoxine is destroyed in the presence of singlet oxygen, helping to explain the connection to singlet oxygen sensitivity defined in Cercospora. These data bring clarity to the previously confusing data on this gene family. However, a new conundrum now exists; why have highly related bacteria evolved with different pathways for pyridoxine biosynthesis?

A homolog of the fungal nuclear migration gene nudC is involved in normal and malignant human hematopoiesis.

The filamentous fungus Aspergillus nidulans nudC gene has an essential function in movement of nuclei following mitosis and is required for normal colony growth. Here, the molecular cloning and role in hematopoiesis of a human gene (designated HnudC) homologous to A. nidulans nudC is reported. The amino terminus of the larger human protein (HNUDC = 45 kDa) does not overlap with A. nidulans NUDC (22 kDa). However, NUDC and the C-terminal 94 amino acids of HNUDC are 67% identical. The C-terminal region of the HnudC gene fully complements the A. nidulans temperature-sensitive nudC3 mutation, suggesting that nudC has an essential function in cell growth that is conserved from filamentous fungi to humans. In initial studies, HNUDC levels were much higher in erythroid precursors compared to most other human tissues. Therefore, the potential role of HnudC in hematopoiesis was explored. In normal human bone marrow, HNUDC protein and mRNA are highly expressed in early myeloid and erythroid precursors and decline as these cells terminally differentiate. To determine whether hematopoietic growth factors induce HnudC expression, TF-1 cells were stimulated by granulocyte-macrophage colony-stimulating factor. This induced a significant increase in HNUDC protein and HnudC mRNA, suggesting that enhancement of HnudC expression in response to growth factor stimulation may be mediated at the transcription level. Furthermore, HNUDC was significantly enhanced in lysates of bone marrow aspirates from patients with acute myelogenous and acute lymphoblastic leukemia compared to aspirates from normal controls, suggesting that HnudC is involved in malignant hematopoietic cell growth as well. These data demonstrate that HNUDC is highly expressed in normal and malignant human hematopoietic precursors and suggest it is of functional importance in the proliferation of these cells.

A mitogen-activated protein kinase (MPKA) is involved in polarized growth in the filamentous fungus, Aspergillus nidulans.

An Aspergillus nidulans kinase gene, which encodes a protein kinase with high similarity to mitogen-activated protein kinases involved in cell wall construction and morphogenesis in yeast species, was cloned and sequenced. Targeted deletion of the Aspergillus nidulans kinase gene indicates that this kinase is involved in germination of conidial spores and polarized growth. These defects were largely remedied on complex high osmolarity media, although abnormal swellings of hyphal tips were still observed. Glycerol (1 M) only supported the growth of compact colonies. The Aspergillus nidulans kinase gene is, thus, required for normal polarized growth at several stages of colony formation in the filamentous fungus A. nidulans.

Regulation of the anaphase-promoting complex/cyclosome by bimAAPC3 and proteolysis of NIMA.

Surprisingly, although highly temperature-sensitive, the bimA1(APC3) anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1(APC3) is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34(cdc2) kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1(APC3)-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1(APC3) double mutant arrests in a mitotic state with very high p34(cdc2) H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1(APC3) mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimAAPC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1(APC3). The bimA1(APC3) mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimAAPC3 regulates the APC/C in a NIMA-dependent manner.

A role for NIMA in the nuclear localization of cyclin B in Aspergillus nidulans.

NIMA promotes entry into mitosis in late G2 by some mechanism that is after activation of the Aspergillus nidulans G2 cyclin-dependent kinase, NIMXCDC2/NIMECyclin B. Here we present two independent lines of evidence which indicate that this mechanism involves control of NIMXCDC2/NIMECyclin B localization. First, we found that NIMECyclin B localized to the nucleus and the nucleus-associated organelle, the spindle pole body, in a NIMA-dependent manner. Analysis of cells from asynchronous cultures, synchronous cultures, and cultures arrested in S or G2 showed that NIMECyclin B was predominantly nuclear during interphase, with maximal nuclear accumulation in late G2. NIMXCDC2 colocalized with NIMECyclin B in G2 cells. Although inactivation of NIMA using either the nimA1 or nimA5 temperature-sensitive mutations blocked cells in G2, NIMXCDC2/NIMECyclin B localization was predominantly cytoplasmic rather than nuclear. Second, we found that nimA interacts genetically with sonA, which is a homologue of the yeast nucleocytoplasmic transporter GLE2/RAE1. Mutations in sonA were identified as allele-specific suppressors of nimA1. The sonA1 suppressor alleviated the nuclear division and NIMECyclin B localization defects of nimA1 cells without markedly increasing NIMXCDC2 or NIMA kinase activity. These results indicate that NIMA promotes the nuclear localization of the NIMXCDC2/ NIMECyclin B complex, by a process involving SONA. This mechanism may be involved in coordinating the functions of NIMXCDC2 and NIMA in the regulation of mitosis.

A cyclin-dependent kinase family member (PHOA) is required to link developmental fate to environmental conditions in Aspergillus nidulans.

We addressed the question of whether Aspergillus nidulans has more than one cyclin-dependent kinase gene and identified such a gene, phoA, encoding two PSTAIRE-containing kinases (PHOAM1 and PHOAM47) that probably result from alternative pre-mRNA splicing. PHOAM47 is 66% identical to Saccharomyces cerevisiae Pho85. The function of this gene was studied using phoA null mutants. It functions in a developmental response to phosphorus-limited growth but has no effect on the regulation of enzymes involved in phosphorus acquisition. Aspergillus nidulans shows both asexual and sexual reproduction involving temporal elaboration of different specific cell types. We demonstrate that developmental decisions in confluent cultures depend upon both the initial phosphorus concentration and the inoculation density and that these factors influence development through phoA. In the most impressive cases, absence of phoA resulted in a switch from asexual to sexual development (at pH 8), or the absence of development altogether (at pH 6). The phenotype of phoA deletion strains appears to be specific for phosphorus limitation. We propose that PHOA functions to help integrate environmental signals with developmental decisions to allow ordered differentiation of specific cell types in A.nidulans under varying growth conditions. The results implicate a putative cyclin-dependent kinase in the control of development.

MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble kinases.

The MPM-2 antibody labels mitosis-specific and cell cycle-regulated phosphoproteins. The major phosphoproteins of mitotic chromosomes recognized by the MPM-2 antibody are DNA topoisomerase II (topoII) alpha and beta. In immunofluorescence studies of PtK1 cytoskeletons, prepared by detergent lysis in the presence of potent phosphatase inhibitors, the MPM-2 antibody labels phosphoproteins found at kinetochores, chromosome arms, midbody and spindle poles of mitotic cells. In cells extracted without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished. However, in cytoskeletons this epitope can be regenerated through the action of kinases stably bound at the kinetochore. Various kinase inhibitors were tested in order to characterize the endogenous kinase responsible for these phosphorylations. We found that the MPM-2 epitope will not rephosphorylate in the presence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the rephosphorylation indicating that the endogenous MPM-2 kinase at kinetochores is not p34cdc2, casein kinase II, MAP kinase, protein kinase A or protein kinase C. The addition of N-ethylmaleimide inactivated the endogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34cdc2-cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosphoepitope. However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores. Furthermore, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are distinct. Our results suggest that multiple kinases (either soluble or kinetochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope. The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in key events such as mitotic kinetochore assembly and sister chromatid separation at anaphase.

Targets of checkpoints controlling mitosis: lessons from lower eukaryotes.

Prevention of mitosis if DNA is damaged, or not fully replicated, is a widespread mechanism used by eukaryotic cells to maintain their ploidy and prevent accumulation of mutations. Such 'checkpoints' must inhibit mitotic regulators to prevent mitotic progression when DNA is not ready for segregation. The mitotic regulators targeted for negative regulation by these checkpoints differ among cell types, but two conserved targets have emerged, the anaphase-promoting complex (APC) and tyrosine phosphorylation of p34(cdc2). One potential downstream target of both these regulators has also been identified, the mitosis-promoting NIMA kinase.

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.

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.

Regulation of p34cdc2/cyclinB H1 and NIMA kinases during the G2/M transition and checkpoint responses in Aspergillus nidulans.

In A. nidulans, activation of both p34cdc2/cyclinB H1 and NIMA kinases is required to initiate mitosis. These two kinases are regulated at several levels during interphase and are activated independently as protein kinases during G2. They are also targeted for negative regulation, to prevent mitosis by mitotic entry checkpoint controls, when DNA is not replicated or is damaged. Then, to initiate mitosis, they promote each other's mitotic functions to coordinately promote mitosis upon completion of interphase events. In addition, inactivation of both kinases by mitotic specific proteolysis is also required for progression through mitosis into G1.

Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2.

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.

Cell cycle regulation in Aspergillus by two protein kinases.

Great progress has recently been made in our understanding of the regulation of the eukaryotic cell cycle, and the central role of cyclin-dependent kinases is now clear. In Aspergillus nidulans it has been established that a second class of cell-cycle-regulated protein kinases, typified by NIMA (encoded by the nimA gene), is also required for cell cycle progression into mitosis. Indeed, both p34cdc2/cyclin B and NIMA have to be correctly activated before mitosis can be initiated in this species, and p34cdc2/cyclin B plays a role in the mitosis-specific activation of NIMA. In addition, both kinases have to be proteolytically destroyed before mitosis can be completed. NIMA-related kinases may also regulate the cell cycle in other eukaryotes, as expression of NIMA can promote mitotic events in yeast, frog or human cells. Moreover, dominant-negative versions of NIMA can adversely affect the progression of human cells into mitosis, as they do in A. nidulans. The ability of NIMA to influence mitotic regulation in human and frog cells strongly suggests the existence of a NIMA pathway of mitotic regulation in higher eukaryotes. A growing number of NIMA-related kinases have been isolated from organisms ranging from fungi to humans, and some of these kinases are also cell-cycle-regulated. How NIMA-related kinases and cyclin-dependent kinases act in concert to promote cell cycle transitions is just beginning to be understood. This understanding is the key to a full knowledge of cell cycle regulation.

Two S-phase checkpoint systems, one involving the function of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit NIMA and prevent premature mitosis.

We demonstrate that there are at least two S-phase checkpoint mechanisms controlling mitosis in Aspergillus. The first responds to the rate of DNA replication and inhibits mitosis via tyrosine phosphorylation of p34cdc2. Cells unable to tyrosine phosphorylate p34cdc2 are therefore viable but are unable to tolerate low levels of hydroxyurea and prematurely enter lethal mitosis when S-phase is slowed. However, if the NIMA mitosis-promoting kinase is inactivated then non-tyrosine-phosphorylated p34cdc2 cannot promote cells prematurely into mitosis. Lack of tyrosine-phosphorylated p34cdc2 also cannot promote mitosis, or lethality, if DNA replication is arrested, demonstrating the presence of a second S-phase checkpoint mechanism over mitotic initiation which we show involves the function of BIME. In order to overcome the S-phase arrest checkpoint over mitosis it is necessary both to prevent tyrosine phosphorylation of p34cdc2 and also to inactivate BIME. Lack of tyrosine phosphorylation of p34cdc2 allows precocious expression of NIMA during S-phase arrest, and lack of BIME then allows activation of this prematurely expressed NIMA by phosphorylation. The mitosis-promoting NIMA kinase is thus a target for S-phase checkpoint controls.