Calcium signaling is involved in dynein-dependent microtubule organization.

The microtubule cytoskeleton supports cellular morphogenesis and polar growth, but the underlying mechanisms are not understood. In a screen for morphology mutants defective in microtubule organization in the fungus Ustilago maydis, we identified eca1 that encodes a sarcoplasmic/endoplasmic calcium ATPase. Eca1 resides in the endoplasmic reticulum and restores growth of a yeast mutant defective in calcium homeostasis. Deletion of eca1 resulted in elevated cytosolic calcium levels and a severe growth and morphology defect. While F-actin and myosin V distribution is unaffected, Deltaeca1 mutants contain longer and disorganized microtubules that show increased rescue and reduced catastrophe frequencies. Morphology can be restored by inhibition of Ca(2+)/calmodulin-dependent kinases or destabilizing microtubules, indicating that calcium-dependent alterations in dynamic instability are a major cause of the growth defect. Interestingly, dynein mutants show virtually identical changes in microtubule dynamics and dynein-dependent ER motility was drastically decreased in Deltaeca1. This indicates a connection between calcium signaling, dynein, and microtubule organization in morphogenesis of U. maydis.

Genetic manipulation of the pathogenic yeast Candida parapsilosis.

Candida parapsilosis is an important human pathogen, responsible for severe cases of systemic candidiasis and one of the leading causes of mortality in neonates. In this report, we describe the first system for genetic manipulation of C. parapsilosis. We isolated and subsequently determined DNA sequences of genes encoding galactokinase ( CpGAL1) and orotidine-5'-phosphate decarboxylase ( CpURA3) from a genomic DNA library of C. parapsilosis by functional complementation of corresponding mutations in Saccharomyces cerevisiae. The predicted protein products, Gal1p and Ura3p, displayed a high degree of homology with corresponding sequences of C. albicans and S. cerevisiae, respectively. A collection of galactokinase-deficient ( gal1) strains of C. parapsilosis was prepared using direct selection of mutagenized cells on media containing 2-deoxy-galactose. Additionally, we constructed a plasmid vector carrying CpGAL1 as a selection marker and a genomic DNA fragment with an autonomously replicating sequence activity that transforms the C. parapsilosis gal1 mutant strain with high efficiency. This system for genetic transformation of C. parapsilosis may significantly advance the study of this human pathogen, greatly improving our understanding of its biology and virulence, with implications for drug development.

Isolation of genes coding for Ade2 and Ura3 homologues from the multinuclear yeast Dipodascus magnusii.

Dipodascus magnusii is a nonconventional yeast species with giant multinuclear cells. We constructed two genomic DNA libraries in plasmid vectors and isolated the first two D. magnusii protein-encoding genes, DmADE2 and DmURA3, coding for phosphoribosylaminoimidazole carboxylase and orotidine-5'-phosphate decarboxylase, respectively. Both genes represent functional orthologues, since they complement ade2 and ura3 mutations in Saccharomyces cerevisiae and their putative products possess conserved sequences important for enzymatic activities. Moreover, the results of Southern blot analysis indicate that the genome of D. magnusii contains additional, paralogous sequences of the DmADE2 and DmURA3 genes.

Cell cycle control of septin ring dynamics in the budding yeast.

Septins constitute a cytoskeletal structure that is conserved in eukaryotes. In Saccharomyces cerevisiae, the Cdc3, Cdc10, Cdc11, Cdc12 and Shs1/Sep7 septins assemble as a ring that marks the cytokinetic plane throughout the budding cycle. This structure participates in different aspects of morphogenesis, such as selection of cell polarity, localization of chitin synthesis, the switch from hyperpolar to isotropic bud growth after bud emergence and the spatial regulation of septation. The septin cytoskeleton assembles at the pre-bud site before bud emergence, remains there during bud growth and duplicates at late mitosis eventually disappearing after cell separation. Using a septin-GFP fusion and time-lapse confocal microscopy, we have determined that septin dynamics are maintained in budding zygotes and during unipolar synchronous growth in pseudohyphae. By means of specific cell cycle arrests and deregulation of cell cycle controls we show that septin assembly is dependent on G1 cyclin/Cdc28-mediated cell cycle signals and that the small GTPase Cdc42, but not Rho1, are essential for this event. However, during bud growth, the septin ring shapes a bud-neck-spanning structure that is unaffected by failures in the regulation of mitosis, such as activation of the DNA repair or spindle assembly checkpoints or inactivation of the anaphase-promoting complex (APC). At the end of the cell cycle, the splitting of the ring into two independent structures depends on the function of the mitotic exit network in which the protein phosphatase Cdc14 participates. Our data support a role of cell cycle control mechanisms in the regulation of septin dynamics to accurately coordinate morphogenesis throughout the budding process in yeast.

Cell integrity and morphogenesis in a budding yeast septin mutant.

The non-sporulating diploid strain V327 of Saccharomyces cerevisiae was previously isolated in a search for thermosensitive autolytic mutants. This strain is very efficient at releasing intracellular proteins into the medium when incubated at high temperatures. The expression of this lytic phenotype depends on a morphogenetic defect, consisting of the appearance of elongated chains of cells. Transmission electron microscopy revealed a mislocalization of septa at semi-permissive temperatures and a total lack of septation together with abnormal cell wall architecture at a non-permissive temperature. The septin-encoding CDC10 gene was cloned by complementation of the pleiotropic phenotype of the V327 mutant. Rescue and sequencing of CDC10 alleles from V327 revealed a point mutation that created a single amino acid change in a region which is well conserved among septins. This new allele was named cdc10-11. The construction of a cdc10-11 haploid strain by substituting the CDC10 gene with the rescued allele permitted further genetic analyses of the mutation and allowed the construction of new homozygous cdc10-11 diploid strains that showed a reduced ability to sporulate. Fusing both the wild-type and the cdc10-11 alleles to green fluorescent protein (GFP) demonstrated that the mutation does not affect the localization of this septin to the bud neck at the standard growth temperature of 24 degrees C, although the morphogenetic phenotype at 37 degrees C parallels the disappearance of Cdc10-GFP at the ring encircling the septum area.