The NDR Kinase DBF-2 Is Involved in Regulation of Mitosis, Conidial Development, and Glycogen Metabolism in Neurospora crassa

Abstract

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...

Architecture and development of the Neurospora crassa hypha – a model cell for polarized growth

“…Deletion of the dbf-2 gene, encoding the final kinase of the SIN cascade, and of its co-activator mob-1 results in aseptate strains that are unable to produce macroconidia. Moreover, elongated nuclei are detected in vegetative hyphae, and abnormal meiotic progeny is observed in the two mutants, supporting weak cell cycle defects, but not a complete block in mitosis (M€ arz et al 2009;Dvash et al 2010). This is reminiscent of the situation observed in Schizosaccharomyces pombe, where mutations in positive SIN components lead to growth arrest after multiple rounds of mitosis in non-dividing cells (Krapp & Simanis 2008).…”

“…Recent studies confirm the presence of most components of the SIN in N. crassa (Dvash et al 2010;M€ arz et al 2009;. Deletion of the dbf-2 gene, encoding the final kinase of the SIN cascade, and of its co-activator mob-1 results in aseptate strains that are unable to produce macroconidia.…”

“…Mutants of all currently characterized proteins required for septum formation are aconidiate (e.g., the SIN mutants Δdbf-2 and Δmob-1, and RHO-4 module mutants Δrho-4, Δbud-3, Δbud-4 and Δrgf-3; M€ arz et al 2009;Rasmusen & Glass 2005;Justa-Schuch et al 2010;Dvash et al 2010). Thus the functionality of the SIN pathway and of RHO-4 signalling is absolutely required for conidiation.…”

Global Analysis of Serine-Threonine Protein Kinase Genes in Neurospora crassa

“…However, our chemical screening analysis revealed phenotypes for a total of 25 mutants, with 8 that did not exhibit defects in the growth and developmental assays. Of these eight, only one had been previously characterized, gsk-3 (20). That earlier study linked gsk-3 to glycogen metabolism and mitosis in Neurospora, but most of the phenotypes were either not analyzed in our study (arthro and microconidiation) or were relatively subtle.…”

“…Of the 107 S/T kinase genes, 32 have been previously analyzed ( Table 1). The list includes the nine components of the three MAPK cascades (24,42,49,53,56,68), PKA (5,32), PKC (23), checkpoint kinase 2 (73), ATM (Ataxia telangiectasia mutated; mus-21) (87), ATR (ATM and Rad3-related) homologs (mus-9) (87), the NDR (nuclear Dbf2-related) family of S/T kinases, cot-1 and dbf-2 (20,95), and others (see Table 1 for a complete list). Characterized Neurospora kinases have been shown to regulate a diversity of cellular functions, including polarized growth, hyphal fusion, asexual sporulation (conidiation), female sexual development, stress regulation, DNA damage responses, and the circadian clock (4,18,20,22,60,68,73,84,87,95).…”