SummaryDysfunction of the Neurospora crassa nuclear Dbf2-related kinase COT1 leads to cessation of tip extension and massive induction of new sites of growth. To determine the role phosphorylation plays in COT1 function, we mutated COT1 residues corresponding to positions of highly conserved nuclear Dbf2-related phosphorylation sites. Analyses of the point-mutation cot-1 strains (mimicking non-and constitutively phosphorylated states) indicate the involvement of COT1 phosphorylation in the regulation of hyphal elongation and branching as well as asexual development by altering cell wall integrity and actin organization. Phosphorylation of COT1's activation segment (at Ser417) is required for proper in vitro kinase activity, but has only a limited effect on hyphal growth. In marked contrast, even though phosphorylation of the C-terminal hydrophobic motif (at Thr589) is crucial for all COT1 functions in vivo, the lack of Thr589 phosphorylation did not significantly affect in vitro COT1 kinase activity. Nevertheless, its regulatory role has been made evident by the significant increase observed in COT1 kinase activity when this residue was substituted in a manner mimicking constitutive phosphorylation. We conclude that COT1 regulates elongation and branching in an independent manner, which is determined by its phosphorylation state.

Neurospora crassa dbf-2 encodes an NDR (nuclear Dbf2-related) protein kinase, homologous to LATS1, a core component of the Hippo pathway. This pathway plays important roles in restraining cell proliferation and promoting apoptosis in differentiating cells. Here, we demonstrate that DBF-2 is involved in three fundamental processes in a filamentous fungus: cell cycle regulation, glycogen biosynthesis, and conidiation. DBF-2 is predominantly localized to the nucleus, and most (approximately 60%) dbf-2 null mutant nuclei are delayed in mitosis, indicating that DBF-2 activity is required for properly completing the cell cycle. The dbf-2 mutant exhibits reduced basal hyphal extension rates accompanied by a carbon/nitrogen ratio-dependent bursting of hyphal tips, vast glycogen leakage, defects in aerial hypha formation, and impairment of all three asexual conidiation pathways in N. crassa. Our findings also indicate that DBF-2 is essential for sexual reproduction in a filamentous fungus. Defects in other Hippo and glycogen metabolism pathway components (mob-1, ccr-4, mst-1, and gsk-3) share similar phenotypes such as mitotic delay and decreased CDC-2 (cell division cycle 2) protein levels, massive hyphal swellings, hyphal tip bursting, glycogen leakage, and impaired conidiation. We propose that DBF-2 functions as a link between Hippo and glycogen metabolism pathways.The nuclear Dbf2-related (NDR) protein kinases are essential components of signaling networks that control cellular processes in various organisms, including morphogenesis, exit from mitosis, cytokinesis, proliferation, differentiation, and apoptosis (23). Based on structural and functional conservation over long evolutionary distances, NDR kinases can be ascribed to one of two subgroups. One is comprised of mammalian NDR1/2, and the other is comprised of LATS1/2 (large tumor suppressor 1/2), as well as their orthologs and related kinases in different organisms.The filamentous fungus Neurospora crassa has two NDR kinases, which representative both subgroups. These are encoded by cot-1 (colonial temperature sensitive 1; NCU07296.3) and dbf-2 (NCU09071.3). While cot-1 (an ortholog of human NDR1/2) is involved in apical hyphal cell elongation and polarity (71), the role of dbf-2 (an ortholog of human LATS1/2) is yet unknown. Additionally, significant sequence similarities (52.8%) between the catalytic domains of DBF-2 and COT-1 raise the question whether their functions or localizations overlap. COT-1 has been localized to several intracellular compartments, including the cytoplasm and nucleus, and in association with the plasma membrane and various proteins (18,19,54). The role that COT-1 plays in the formation of branched cellular structures as well as its cellular localization has been suggested to be preserved throughout evolution (54, 77). Similar conservation may also exist among DBF-2 and its orthologs, of which human LATS1 and Drosophila melanogaster WTS/LATS are, by far, the most extensively analyzed (13,45,53,74,75).Both human LATS1 and NDR1 are wide...

The new tetraglycosylceramide neurosporaside (1a) has been isolated from the fungus Neurospora crassa. Neurosporaside is a tetraglycosylated glycosphingolipid characterized by a sugar chain unprecedented among natural glycoconjugates. The structure of neurosporaside was elucidated by extensive spectroscopic analysis and microscale degradation analysis, which allowed full structure elucidation using less than 1 mg of compound.