Colin P. De Souza

Rapid Production of Gene Replacement Constructs and Generation of a Green Fluorescent Protein-Tagged Centromeric Marker in Aspergillus nidulans

ABSTRACT 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.

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.

Mitosis, Not Just Open or Closed†

During cell division, eukaryotic cells must faithfully pass on their genetic material to the next generation during mitosis. It has long been known that lower eukaryotes and higher eukaryotes achieve this in strikingly different ways. Higher eukaryotes undergo an open mitosis in which the nuclear

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.

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.

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.

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.

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.

γ-Tubulin regulates the anaphase-promoting complex/cyclosome during interphase

Activation of the APC/C requires microtubule-nucleating independent aspects of γ-tubulin function.

Single-step affinity purification for fungal proteomics.

ABSTRACT 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.