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.

Fig. 1.

Mitosis is impaired in the dbf-2 mutant. In contrast to the round nuclear morphology in the wt and ku-70 control strains, the dbf-2 mutant nuclei are elongated, as determined by DAPI staining of hyphae (top panels). Round nuclei are also observed in conidia of the wt (middle panels; fluorescence and fluorescence with bright field, respectively), whereas nuclei in constricted proconidial chains of the dbf-2 strain are elongated. Scale bars represent 50 μm (wt and dbf-2 strains), 25 μm (ku-70 strain), and 10 μm (middle panels). The prevalence of elongated nuclei in the different strains is shown (bottom left panel). Data shown are based on the averages of three experiments, in which nuclei in 10 fields (of each strain) were counted. Bars indicate standard errors. DBF-2::GFP expressed in the nucleus of a pED4 dbf-2 transformant (G10) fully complements the dbf-2 mutant phenotype (bottom right panel).

Fig. 2.

The dbf-2 mutant is characterized by massive glycogen leakage. (A) Intact hyphal cells are characteristic of wt growth on standard media, while growth of the dbf-2 strain either on solid or in liquid media (middle and right panels, respectively) is accompanied by large cytoplasmic leakage foci at the hyphal tips (arrows). (B) Minimal iodine vapor staining is observed in the wt whereas intense iodine vapor staining is observed in the dbf-2 mutant grown in liquid (upper panel) or on solid (lower panel) media. Arrows indicate iodine vapor staining of leakage foci from hyphal tips of the dbf-2 strain. Images were obtained by light microscopy. (C) The predominant content of the cytoplasmic leakage is glycogen, as determined by ¹H NMR analysis of the droplet material collected from growing dbf-2 mutant hyphal tips, compared with a glycogen standard (upper and lower spectra, respectively).

Fig. 3.

The extent of glycogen leakage in the dbf-2 strain is dependent on the carbon/nitrogen (C/N) ratio in the growth medium. The strain was cultured on minimal glucose (1.5%) medium with various concentrations of NH₄NO₃. Arrows mark the locations of glycogen droplets.

Fig. 4.

Defects in the putative N. crassa dbf-2 (Hippo) and glycogen metabolism pathway components share common morphological consequences. Typically, all the genetic defects are accompanied by hyphal swelling, bursting of hyphal tips (arrows in the center column), and cytoplasmic leakage (arrows in the right column).

Fig. 5.

Various dbf-2 (Hippo) and glycogen metabolism pathway mutants exhibit altered aerial hypha production (as evident from the presence of fungal biomass on flask walls). All strains were photographed 2 weeks after inoculation.

Fig. 6.

Conidial production patterns in the dbf-2 mutant resemble those of other Hippo and glycogen metabolism pathway component mutants. The abundances of macroconidia (top panel), microconidia (middle panel), and arthroconidia (bottom panel) were determined in strains with mutations in genes involved in dbf-2 (Hippo) and glycogen signaling. While typical conidial separation can be observed in the wt strain, the process is impaired in the dbf-2 mutant (top panel). Microconidia are produced in abundance in the dbf-2 mutant and are at times engulfed by curling hyphae (middle panel). Typical production of arthroconidia in the wt strain is markedly lower than that observed in the dbf-2 mutant (bottom panel). Comparative quantification of the abundance of different conidial types is presented on the right side of each row.

Fig. 7.

Impaired mitosis accompanies defects in dbf-2 (Hippo) and glycogen pathway components. (A) Elongated nuclei (as visualized by DAPI staining) can be observed in the mst-1, ccr-4, mob-1, and gsk-3 strains. (B) Comparative quantification of the percentage of elongated nuclei in strains defective in putative components of N. crassa dbf-2 (Hippo) and glycogen metabolism pathways in comparison to the wt and ku-70 control strains. Bars indicate standard errors.

Fig. 8.

Altered cdc-2 expression in various mutants harboring defects in (dbf-2) Hippo and glycogen pathways. (A and B) Decreased cdc-2-encoded protein levels (detected at 34 and 35 kDa) in dbf-2, ccr-4, mob-1, and mst-1 mutants, compared to the wt and ku-70 control strains, as determined by Western blot analysis (A). A general protein stain demonstrating equal loading is shown as well (B). (C) Changes in cdc-2 expression in different mutants impaired in dbf-2 and glycogen pathway components as determined by real-time RT-PCR. The quantity of cdc-2 cDNA measured was normalized to that of actin cDNA, in extracts from each mutant. Data shown are the average expression levels based on three experiments. Bars indicate standard errors. The abundance of cDNA from the wt strain samples was assigned a value of 1.

Fig. 9.

Abnormal localization of DBF-2::GFP expressed in the mob-1, ppe-1, ccr-4, and rgb-1^RIP strains (but not in the mob-1 or ppe-1 mutant) as determined by fluorescent microscopy. Accumulation of DBF-2 in the cytoplasm and in large cytoplasmic organelles is marked by arrows. For normal localization of DBF-2::GFP, see the G10 panel in Fig. 1.