Location and functional analysis of the Aspergillus nidulans Aurora kinase confirm mitotic functions and suggest non-mitotic roles.

Filamentous fungi have devastating negative impacts as pathogens and agents of food spoilage but also have critical ecological importance and are utilized for industrial applications. The characteristic multinucleate nature of filamentous fungi is facilitated by limiting if, when and where septation, the fungal equivalent of cytokinesis, occurs. In the model filamentous fungus Aspergillus nidulans septation does not occur immediately after mitosis and is an incomplete process resulting in the formation of a septal pore whose permeability is cell cycle regulated. How mitotic regulators, such as the Aurora kinase, contribute to the often unique biology of filamentous fungi is not well understood. The Aurora B kinase has not previously been investigated in any detail during hyphal growth. Here we demonstrate for the first time that Aurora displays cell cycle dependent locations to the region of forming septa, the septal pore and mature septa as well as the mitotic apparatus. To functionally analyze Aurora, we generated a temperature sensitive allele revealing essential mitotic and spindle assembly checkpoint functions consistent with its location to the kinetochore region and spindle midzone. Our analysis also reveals that cellular and kinetochore Aurora levels increase during a mitotic spindle assembly checkpoint arrest and we propose that this could be important for checkpoint inactivation when spindle formation is prevented. We demonstrate that Aurora accumulation at mature septa following mitotic entry does not require mitotic progression but is dependent upon a timing mechanism. Surprisingly we also find that Aurora inactivation leads to cellular swelling and lysis indicating an unexpected function for Aurora in fungal cell growth. Thus in addition to its conserved mitotic functions our data suggest that Aurora has the capacity to be an important regulator of septal biology and cell growth in filamentous fungi.

Identification of interphase functions for the NIMA kinase involving microtubules and the ESCRT pathway.

The Never in Mitosis A (NIMA) kinase (the founding member of the Nek family of kinases) has been considered a mitotic specific kinase with nuclear restricted roles in the model fungus Aspergillus nidulans. By extending to A. nidulans the results of a synthetic lethal screen performed in Saccharomyces cerevisiae using the NIMA ortholog KIN3, we identified a conserved genetic interaction between nimA and genes encoding proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. Absence of ESCRT pathway functions in combination with partial NIMA function causes enhanced cell growth defects, including an inability to maintain a single polarized dominant cell tip. These genetic insights suggest NIMA potentially has interphase functions in addition to its established mitotic functions at nuclei. We therefore generated endogenously GFP-tagged NIMA (NIMA-GFP) which was fully functional to follow its interphase locations using live cell spinning disc 4D confocal microscopy. During interphase some NIMA-GFP locates to the tips of rapidly growing cells and, when expressed ectopically, also locates to the tips of cytoplasmic microtubules, suggestive of non-nuclear interphase functions. In support of this, perturbation of NIMA function either by ectopic overexpression or through partial inactivation results in marked cell tip growth defects with excess NIMA-GFP promoting multiple growing cell tips. Ectopic NIMA-GFP was found to locate to the plus ends of microtubules in an EB1 dependent manner, while impairing NIMA function altered the dynamic localization of EB1 and the cytoplasmic microtubule network. Together, our genetic and cell biological analyses reveal novel non-nuclear interphase functions for NIMA involving microtubules and the ESCRT pathway for normal polarized fungal cell tip growth. These insights extend the roles of NIMA both spatially and temporally and indicate that this conserved protein kinase could help integrate cell cycle progression with polarized cell growth.

Regulated inactivation of the spindle assembly checkpoint without functional mitotic spindles.

The spindle assembly checkpoint (SAC) arrests mitosis until bipolar attachment of spindle microtubules to all chromosomes is accomplished. However, when spindle formation is prevented and the SAC cannot be satisfied, mammalian cells can eventually overcome the mitotic arrest while the checkpoint is still activated. We find that Aspergillus nidulans cells, which are unable to satisfy the SAC, inactivate the checkpoint after a defined period of mitotic arrest. Such SAC inactivation allows normal nuclear reassembly and mitotic exit without DNA segregation. We demonstrate that the mechanisms, which govern such SAC inactivation, require protein synthesis and can occur independently of inactivation of the major mitotic regulator Cdk1/Cyclin B or mitotic exit. Moreover, in the continued absence of spindle function cells transit multiple cell cycles in which the SAC is reactivated each mitosis before again being inactivated. Such cyclic activation and inactivation of the SAC suggests that it is subject to cell-cycle regulation that is independent of bipolar spindle function.

Double duty for nuclear proteins--the price of more open forms of mitosis.

During cell division, eukaryotic cells pass on their genetic material to the next generation by undergoing mitosis, which segregates their chromosomes. During mitosis, the nuclear envelope, nuclear pore complexes and nucleolus must also be segregated. Cells achieve this in a range of different forms of mitosis, from closed, in which these nuclear structures remain intact, to open, in which these nuclear structures are disassembled. In between lies a smorgasbord of intermediate forms of mitosis, displaying varying degrees of nuclear disassembly. Gathering evidence is revealing links between the extent of nuclear disassembly and the evolution of new roles for nuclear proteins during mitosis. We propose that proteins with such double duties help coordinate reassembly of the nucleus with chromosomal segregation.

Single-step affinity purification for fungal proteomics.

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

Partial nuclear pore complex disassembly during closed mitosis in Aspergillus nidulans.

BACKGROUND: Many organisms undergo closed mitosis and locate tubulin and mitotic kinases to nuclei only during mitosis. How this is regulated is unknown. Interestingly, the NIMA kinase of Aspergillus nidulans interacts with two nuclear pore complex (NPC) proteins and NIMA is required for mitotic localization of the Cdk1 kinase to nuclei. Therefore, we wished to define the mechanism by which the NPC is regulated during A. nidulans' closed mitosis. RESULTS: The structural makeup of the NPC is dramatically changed during A. nidulans' mitosis. At least five NPC proteins disperse throughout the cell during mitosis while at least three structural components remain at the NPC. These modifications correlate with marked changes in the function of the NPC. Notably, during mitosis, An-RanGAP is not excluded from nuclei, and five other nuclear or cytoplasmic proteins investigated fail to locate as they do during interphase. Mitotic modification of the NPC requires NIMA and Cdk1 kinase activation. NIMA appears to be particularly important. Most strikingly, ectopic induction of NIMA promotes mitotic-like changes in NPC structure and function during S phase. Furthermore, NIMA locates to the NPC during entry into mitosis, and a dominant-negative version of NIMA that causes G2 delay dwells at the NPC. CONCLUSIONS: We conclude that partial NPC disassembly under control of NIMA and Cdk1 in A. nidulans may represent a new mechanism for regulating closed mitoses. We hypothesize that proteins locate by their relative binding affinities within the cell during A. nidulans' closed mitosis, analogous to what occurs during open mitosis.

A point mutation in the Aspergillus nidulans sonBNup98 nuclear pore complex gene causes conditional DNA damage sensitivity.

The nuclear pore complex (NPC) is embedded in the nuclear envelope where it mediates transport between the cytoplasm and nucleus and helps to organize nuclear architecture. We previously isolated sonB1, a mutation encoding a single amino acid substitution within the Aspergillus nidulans SONBnNup98 NPC protein (nucleoporin). Here we demonstrate that this mutation causes marked DNA damage sensitivity at 42 degrees . Although SONBnNup98 has roles in the G2 transition, we demonstrate that the G2 DNA damage checkpoint is functional in the sonB1 mutant at 42 degrees . The MRN complex is composed of MRE11, RAD50, and NBS1 and functions in checkpoint signaling, DNA repair, and telomere maintenance. At 42 degrees we find that the DNA damage response defect of sonB1 mutants causes synthetic lethality when combined with mutations in scaANBS1, the A. nidulans homolog of NBS1. We provide evidence that this synthetic lethality is independent of MRN cell cycle checkpoint functions or MREAMRE11-mediated DNA repair functions. We also demonstrate that the single A. nidulans histone H2A gene contains the C-terminal SQE motif of histone H2AX isoforms and that this motif is required for the DNA damage response. We propose that the sonB1 nucleoporin mutation causes a defect in a novel part of the DNA damage response.

The SONB(NUP98) nucleoporin interacts with the NIMA kinase in Aspergillus nidulans.

The Aspergillus nidulans NIMA kinase is essential for mitotic entry. At restrictive temperature, temperature-sensitive nimA alleles arrest in G2, before accumulation of NIMA in the nucleus. We performed a screen for extragenic suppressors of the nimA1 allele and isolated two cold-sensitive son (suppressor of nimA1) mutants. The sonA1 mutant encoded a nucleoporin that is a homolog of yeast Gle2/Rae1. We have now cloned SONB, a second nucleoporin genetically interacting with NIMA. sonB is essential and encodes a homolog of the human NUP98/NUP96 precursor. Similar to NUP98/NUP96, SONB(NUP98/NUP96) is autoproteolytically cleaved to generate SONB(NUP98) and SONB(NUP96). SONB(NUP98) localizes to the nuclear pore complex and contains a GLEBS domain (Gle2 binding sequence) that binds SONA(GLE2). A point mutation within the GLEBS domain of SONB1(NUP98) suppresses the temperature sensitivity of the nimA1 allele and compromises the physical interaction between SONA(GLE2) and SONB1(NUP98). The sonB1 mutation also causes sensitivity to hydroxyurea. We isolated the histone H2A-H2B gene pair as a copy-number suppressor of sonB1 cold sensitivity and hydroxyurea sensitivity. The data suggest that the nucleoporins SONA(GLE2) and SONB(NUP98) and the NIMA kinase interact and regulate nuclear accumulation of mitotic regulators to help promote mitosis.

Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation.

Functional analysis of the Aspergillus nidulans kinome.

The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have made systematic approaches to study large gene sets possible. To enhance A. nidulans functional genomics we have created gene deletion constructs for 9851 genes representing 93.3% of the encoding genome. To illustrate the utility of these constructs, and advance the understanding of fungal kinases, we have systematically generated deletion strains for 128 A. nidulans kinases including expanded groups of 15 histidine kinases, 7 SRPK (serine-arginine protein kinases) kinases and an interesting group of 11 filamentous fungal specific kinases. We defined the terminal phenotype of 23 of the 25 essential kinases by heterokaryon rescue and identified phenotypes for 43 of the 103 non-essential kinases. Uncovered phenotypes ranged from almost no growth for a small number of essential kinases implicated in processes such as ribosomal biosynthesis, to conditional defects in response to cellular stresses. The data provide experimental evidence that previously uncharacterized kinases function in the septation initiation network, the cell wall integrity and the morphogenesis Orb6 kinase signaling pathways, as well as in pathways regulating vesicular trafficking, sexual development and secondary metabolism. Finally, we identify ChkC as a third effector kinase functioning in the cellular response to genotoxic stress. The identification of many previously unknown functions for kinases through the functional analysis of the A. nidulans kinome illustrates the utility of the A. nidulans gene deletion constructs.

Gamma-tubulin regulates the anaphase-promoting complex/cyclosome during interphase.

A cold-sensitive gamma-tubulin allele of Aspergillus nidulans, mipAD159, causes defects in mitotic and cell cycle regulation at restrictive temperatures that are apparently independent of microtubule nucleation defects. Time-lapse microscopy of fluorescently tagged mitotic regulatory proteins reveals that cyclin B, cyclin-dependent kinase 1, and the Ancdc14 phosphatase fail to accumulate in a subset of nuclei at restrictive temperatures. These nuclei are permanently removed from the cell cycle, whereas other nuclei, in the same multinucleate cell, cycle normally, accumulating and degrading these proteins. After each mitosis, additional daughter nuclei fail to accumulate these proteins, resulting in an increase in noncycling nuclei over time and consequent inhibition of growth. Extensive analyses reveal that these noncycling nuclei result from a nuclear autonomous, microtubule-independent failure of inactivation of the anaphase-promoting complex/cyclosome. Thus, gamma-tubulin functions to regulate this key mitotic and cell cycle regulatory complex.

Nucleolar separation from chromosomes during Aspergillus nidulans mitosis can occur without spindle forces.

How the nucleolus is segregated during mitosis is poorly understood and occurs by very different mechanisms during closed and open mitosis. Here we report a new mechanism of nucleolar segregation involving removal of the nucleolar-organizing regions (NORs) from nucleoli during Aspergillus nidulans mitosis. This involves a double nuclear envelope (NE) restriction which generates three NE-associated structures, two daughter nuclei (containing the NORs), and the nucleolus. Therefore, a remnant nucleolar structure can exist in the cytoplasm without NORs. In G1, this parental cytoplasmic nucleolus undergoes sequential disassembly releasing nucleolar proteins to the cytoplasm as nucleoli concomitantly reform in daughter nuclei. By depolymerizing microtubules and mutating spindle assembly checkpoint function, we demonstrate that a cycle of nucleolar "segregation" can occur without a spindle in a process termed spindle-independent mitosis (SIM). During SIM physical separation of the NOR from the nucleolus occurs, and NE modifications promote expulsion of the nucleolus to the cytoplasm. Subsequently, the cytoplasmic nucleolus is disassembled and rebuilt at a new site around the nuclear NOR. The data demonstrate the existence of a mitotic machinery for nucleolar segregation that is normally integrated with mitotic spindle formation but that can function without it.

The three fungal transmembrane nuclear pore complex proteins of Aspergillus nidulans are dispensable in the presence of an intact An-Nup84-120 complex.

In Aspergillus nidulans nuclear pore complexes (NPCs) undergo partial mitotic disassembly such that 12 NPC proteins (Nups) form a core structure anchored across the nuclear envelope (NE). To investigate how the NPC core is maintained, we affinity purified the major core An-Nup84-120 complex and identified two new fungal Nups, An-Nup37 and An-ELYS, previously thought to be vertebrate specific. During mitosis the An-Nup84-120 complex locates to the NE and spindle pole bodies but, unlike vertebrate cells, does not concentrate at kinetochores. We find that mutants lacking individual An-Nup84-120 components are sensitive to the membrane destabilizer benzyl alcohol (BA) and high temperature. Although such mutants display no defects in mitotic spindle formation, they undergo mitotic specific disassembly of the NPC core and transient aggregation of the mitotic NE, suggesting the An-Nup84-120 complex might function with membrane. Supporting this, we show cells devoid of all known fungal transmembrane Nups (An-Ndc1, An-Pom152, and An-Pom34) are viable but that An-ndc1 deletion combined with deletion of individual An-Nup84-120 components is either lethal or causes sensitivity to treatments expected to destabilize membrane. Therefore, the An-Nup84-120 complex performs roles, perhaps at the NPC membrane as proposed previously, that become essential without the An-Ndc1 transmembrane Nup.

Mlp1 acts as a mitotic scaffold to spatially regulate spindle assembly checkpoint proteins in Aspergillus nidulans.

During open mitosis several nuclear pore complex (NPC) proteins have mitotic specific localizations and functions. We find that the Aspergillus nidulans Mlp1 NPC protein has previously unrealized mitotic roles involving spatial regulation of spindle assembly checkpoint (SAC) proteins. In interphase, An-Mlp1 tethers the An-Mad1 and An-Mad2 SAC proteins to NPCs. During a normal mitosis, An-Mlp1, An-Mad1, and An-Mad2 localize similarly on, and around, kinetochores until telophase when they transiently localize near the spindle but not at kinetochores. During SAC activation, An-Mlp1 remains associated with kinetochores in a manner similar to An-Mad1 and An-Mad2. Although An-Mlp1 is not required for An-Mad1 kinetochore localization during early mitosis, it is essential to maintain An-Mad1 in the extended region around kinetochores in early mitosis and near the spindle in telophase. Our data are consistent with An-Mlp1 being part of a mitotic spindle matrix similar to its Drosophila orthologue and demonstrate that this matrix localizes SAC proteins. By maintaining SAC proteins near the mitotic apparatus, An-Mlp1 may help monitor mitotic progression and coordinate efficient mitotic exit. Consistent with this possibility, An-Mad1 and An-Mlp1 redistribute from the telophase matrix and associate with segregated kinetochores when mitotic exit is prevented by expression of nondegradable cyclin B.

Potential link between the NIMA mitotic kinase and nuclear membrane fission during mitotic exit in Aspergillus nidulans.

We have isolated TINC as a NIMA-interacting protein by using the yeast two-hybrid system and have confirmed that TINC interacts with NIMA in Aspergillus nidulans. The TINC-NIMA interaction is stabilized in the absence of phosphatase inhibitors and in the presence of kinase-inactive NIMA, suggesting that the interaction is enhanced when NIMA is not fully activated. TINC is a cytoplasmic protein. TINC homologues and a TINC-like protein (A. nidulans HETC) are conserved in other filamentous fungi. Neither deletion of tinC nor deletion of both tinC and A. nidulans hetC is lethal, but deletion of tinC does produce cold sensitivity as well as osmotic sensitivity. Expression of an amino-terminal-truncated form of TINC (DeltaN-TINC) inhibits colony growth in Aspergillus and localizes to membrane-like structures within the cell. Examination of cell cycle progression in these cells reveals that they progress through multiple defective mitoses. Many cells contain large polyploid single nuclei, while some appear to have separated masses of DNA. Examination of the nuclear envelopes of cells containing more than one DNA mass reveals that both DNA masses are contained within a single nuclear envelope, indicating that nuclear membrane fission is defective. The ability of these cells to separate DNA segregation from nuclear membrane fission suggests that this coordination is normally a regulated process in A. nidulans. Additional experiments demonstrate that expression of DeltaN-TINC results in premature NIMA disappearance in mitotic samples. We propose that TINC's interaction with NIMA and the cell cycle defects produced by DeltaN-TINC expression suggest possible roles for TINC and NIMA during nuclear membrane fission.

Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans.

A method to rapidly generate gene replacement constructs by fusion PCR is described for Aspergillus nidulans. The utility of the approach is demonstrated by green fluorescent protein (GFP) tagging of A. nidulans ndc80 to visualize centromeres through the cell cycle. The methodology makes possible large-scale GFP tagging, promoter swapping, and deletion analysis of A. nidulans.