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.

Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast.D URING the transition from interphase into mitosis, chromatin undergoes dramatic global restructuring to go from a relaxed interphase configuration amenable to gene expression to a condensed form characteristic of mitotic chromosomes. Chromatin condensation not only marks mitosis but is also essential for the normal segregation of the duplicated sister chromatids into daughter nuclei. On the other hand, during mitotic exit, decondensation of chromatin needs to be triggered in a correct temporal manner to enable successful transition into interphase. In Aspergillus nidulans, a model filamentous fungus that enabled discovery of cell cycle-specific genes, mitotic initiation requires the function of the essential NIMA mitotic kinase (Oakley and Morris 1983;Osmani et al. 1987). Early evidence pointed to a role for NIMA in regulating chromatin compaction through the cell cycle since overexpression of NIMA was sufficient to induce chromatin condensation independent of cell cycle phase (Osmani et al. 1988;. Importantly, NIMA is required for, and can promote, the phosphorylation of histone H3 at serine 10 (De Souza et al. 2000), a universal marker of mitotic chromatin. In addition, NIMA has been shown to regulate the partial disassembly of nuclear pore complexes, allowing the access of mitotic regulators to the nucleus (Wu et al. 1998;De S...

Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein-protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2 cdc2 that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2 cdc2 , nimE6 cyclinB , and nimT23 cdc25 , but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10 cyclinB mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxA Hrb1 is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and DsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXA HRB1 is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2 CDC2 /NIME CYCLINB kinase activity and that snxA1 or DsnxA alter localization patterns of NIME CYCLINB at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway. CONTROL of the eukaryotic G2/M transition by protein kinases has been widely studied and is highly conserved among all eukaryotes from the budding and fission yeasts and filamentous fungi to metazoans (for review, see Ma and Poon 2011). The CDK1/CYCLINB protein kinase complex is a major regulator of this transition in all eukaryotes and is responsible for the phosphorylations of numerous proteins, leading to massive nuclear and cytoplasmic reorganizations that regulate mitosis (for review, see Lindqvist et al. 2009). The complex itself is tightly regulated, both temporally and spatially, to allow mitotic entry.Although CDK1/CYCLINB activity is essential for mitotic entry in all eukaryotes, structural differences in the nucleus in various organisms result in "open" mitosis (more complex eukaryotes) or "closed" mitosis (budding yeasts); these differences likely affect the temporo-spatial functioning of CDK1/ CYCLINB. The partially closed mitosis of the filamentous fungus Aspergillus nidulans is an evolutionary intermediate between open and closed mitoses and provides a system for studying mitotic entry in organisms intermediate between budding ...

Aspergillus nidulans snxA, an ortholog of Saccharomyces cerevisiae Hrb1/Gbp2 messenger RNA shuttle proteins, is – in contrast to budding yeast – involved in cell cycle regulation, in which snxA1 and snxA2 mutations as well as a snxA deletion specifically suppress the heat-sensitivity of mutations in regulators of the CDK1 mitotic induction pathway. snxA mutations are strongly cold-sensitive, and at permissive temperature snxA mRNA and protein expression are strongly repressed. Initial attempts to identify the causative snxA mutations revealed no defects in the SNXA protein. Here we show that snxA1/A2 mutations resulted from an identical chromosome I – II reciprocal translocation with breakpoints in the snxA first intron and the fourth exon of a GYF-domain gene, gyfA. Surprisingly, a gyfA deletion and a reconstructed gyfA translocation allele suppressed the heat-sensitivity of CDK1 pathway mutants in a snxA+ background, demonstrating that two unrelated genes, snxA and gyfA, both act through the CDK1-CyclinB axis to restrain the G2-M transition, and for the first time identifying a role in G2-M regulation for a GYF-domain protein. To better understand snxA1/A2 reduced expression, we generated suppressors of snxA cold-sensitivity in two genes: (1) loss of the abundant nucleolar protein nsr1/nucleolin bypassed the requirement for snxA; and (2) loss of the Set2 histone H3 lysine36 (H3K36) methyltransferase or a non-methylatable histone H3K36L mutant rescued hypomorphic snxA mutants by restoring full transcriptional proficiency, indicating that methylation of H3K36 acts normally to repress snxA transcription. These observations are in line with known set2 functions in preventing excessive and cryptic transcription of active genes.