Deletion of the dynein heavy-chain gene DYN1 leads to aberrant nuclear positioning and defective hyphal development in Candida albicans.
Cytoplasmic dynein is a microtubule-associated minus-end-directed motor protein. CaDYN1 encodes the single dynein heavy-chain gene of Candida albicans. The open reading frames of both alleles of CaDYN1 were completely deleted via a PCR-based approach. Cadyn1 mutants are viable but grow more slowly than the wild type. In vivo time-lapse microscopy was used to compare growth of wild-type (SC5314) and dyn1 mutant strains during yeast growth and after hyphal induction. During yeast-like growth, Cadyn1 strains formed chains of cells. Chromosomal TUB1-GFP and HHF1-GFP alleles were used both in wild-type and mutant strains to monitor the orientation of mitotic spindles and nuclear positioning in C. albicans. In vivo fluorescence time-lapse analyses with HHF1-GFP over several generations indicated defects in dyn1 cells in the realignment of spindles with the mother-daughter axis of yeast cells compared to that of the wild type. Mitosis in the dyn1 mutant, in contrast to that of wild-type yeast cells, was very frequently completed in the mother cells. Nevertheless, daughter nuclei were faithfully transported into the daughter cells, resulting in only a small number of multinucleate cells. Cadyn1 mutant strains responded to hypha-inducing media containing l-proline or serum with initial germ tube formation. Elongation of the hyphal tubes eventually came to a halt, and these tubes showed a defect in the tipward localization of nuclei. Using a heterozygous DYN1/dyn1 strain in which the remaining copy was controlled by the regulatable MAL2 promoter, we could switch between wild-type and mutant phenotypes depending on the carbon source, indicating that the observed mutant phenotypes were solely due to deletion of DYN1.
Martin
R
R
Hans-Knoell Institute for Natural Products Research e.V. and Friedrich-Schiller-University Department of Microbiology, Hans-Knoell Str.2, D-07745 Jena, Germany.
Walther
A
A
Wendland
J
J
D021581:Active Transport, Cell Nucleus; D000483:Alleles; D002176:Candida albicans / Q000235:genetics* / Q000378:metabolism; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004398:Dyneins / Q000235:genetics*; D017353:Gene Deletion; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006579:Heterozygote; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008938:Mitosis; D008957:Models, Genetic; D009154:Mutation; D016366:Open Reading Frames; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism; D013997:Time Factors
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004398:Dyneins