SummaryNuclear migration and nuclear positioning are fundamental processes in all eukaryotic cells. They are easily monitored during hyphal growth of filamentous fungi. We expressed the green fluorescent protein (GFP) as a fusion protein with the putative nuclear localization domain of the transcriptional activator stuA in nuclei of Aspergillus nidulans and visualized these organelles in living cells. Nuclear staining was observed in interphase nuclei but not during mitosis. Nuclear division, nuclear migration, septum formation and branching were analysed with time-lapse video microscopy during hyphal extension. Hyphae elongated at 0.1-1.2 m min ¹1 and nuclei moved with similar speeds towards the hyphal tip until they had reached a defined position. An individual regulation of nuclear mobility in a given hyphal compartment was observed. Some representative movies are available on the Internet (http:/ /www.blacksci.co.uk/products/journals/molextra.htm). Nuclear positioning was also studied at the molecular level. The ApsA protein, which regulates nuclear migration, was localized at the cytoplasmic membrane in germlings and hyphae by immunofluorescence and GFP tagging. A model of nuclear migration, nuclear positioning and the role of ApsA is presented.

SummaryFilamentous fungi are model microorganisms for studying nuclear migration in eukaryotic cells. Two genes, apsA and apsB (¼ anucleate primary sterigmata), were identified in Aspergillus nidulans that affect nuclear distribution in hyphae and specifically block conidiophore development at the metula stage when mutant. Here we describe the cloning, sequencing and molecular analysis of apsB. The gene encodes a 121 kDa coiled-coil, hydrophilic protein that was localized in the cytoplasm. No protein-protein interaction was detected between ApsB and ApsA, a membrane-associated, previously identified protein. An apsB null mutant was characterized by video epifluorescence microscopy using strains that express green fluorescent protein (GFP) in nuclei. With this novel approach, we have discovered a new mutant phenotype and have found that nuclei display an increased chaotic movement in older hyphal compartments that results in clustering and an uneven distribution of these organelles. These results suggest a regulatory role of ApsB in nuclear migration.

The fungal vacuole is an acidic organelle that is involved in a variety of physiological processes, such as protein turnover, ion and pH homeostasis and osmoregulation. The function of the vacuole largely depends on vesicle transport providing the organelle with enzymes and substrates. The process of vesicle transportation has been studied best in Saccharomyces cerevisiae, where several proteins that are crucial for intracellular vesicle sorting have been identified. One such protein is Pep3 (Vps18). In pep3 mutants vacuole function and vacuole morphology are affected. We cloned the gene for a potential homolog of Pep3 from the filamentous fungus Aspergillus nidulans. The gene, digA (for dichotomous growth), was identified in a screen for nuclear migration mutants. A. nidulans digA encodes a protein of 108.3 kDa, which includes a 122-amino acid clathrin repeat motif, two short coiled-coil regions, and a RING finger Zn-binding motif at the C-terminus. All three sequence motifs suggest interaction of DigA with other proteins. DigA is 25% identical to a homolog from Schizosaccharomyces pombe, 23% to a protein from human, 21% to the product of the Drosophila melanogaster gene deep orange (dor) and 18% to S. cerevisiae Pep3 (Vps18). We localized DigA as a HA-epitope-tagged protein in the cytoplasm of A. nidulans vegetative cells, by secondary immunofluorescence. A digA mutant displays a pleiotropic phenotype with clustered mitochondria, clustered nuclei and a defect in polarization of the actin cytoskeleton. These results suggest that vacuolar functions are required for organelle positioning and polarized growth.