Genetic evidence for a microtubule-destabilizing effect of conventional kinesin and analysis of its consequences for the control of nuclear distribution in Aspergillus nidulans.

Conventional kinesin is a microtubule-dependent motor protein believed to be involved in a variety of intracellular transport processes. In filamentous fungi, conventional kinesin has been implicated in different processes, such as vesicle migration, polarized growth, nuclear distribution, mitochondrial movement and vacuole formation. To gain further insights into the functions of this kinesin motor, we identified and characterized the conventional kinesin gene, kinA, of the established model organism Aspergillus nidulans. Disruption of the gene leads to a reduced growth rate and a nuclear positioning defect, resulting in nuclear cluster formation. These clusters are mobile and display a dynamic behaviour. The mutant phenotypes are pronounced at 37 degrees C, but rescued at 25 degrees C. The hyphal growth rate at 25 degrees C was even higher than that of the wild type at the same temperature. In addition, kinesin-deficient strains were less sensitive to the microtubule destabilizing drug benomyl, and disruption of conventional kinesin suppressed the cold sensitivity of an alpha-tubulin mutation (tubA4). These results suggest that conventional kinesin of A. nidulans plays a role in cytoskeletal dynamics, by destabilizing microtubules. This new role of conventional kinesin in microtubule stability could explain the various phenotypes observed in different fungi.

Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii.

We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network.

Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae.

We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosac-charomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1.4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0.9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2.4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter.