Interactions between the kinetochore complex and the protein kinase A pathway in Saccharomyces cerevisiae.

The kinetochore is a large structure composed of multiple protein subcomplexes that connect chromosomes to spindle microtubules to enable accurate chromosome segregation. Significant advances have been made in the identification of kinetochore proteins and elucidation of kinetochore structure; however, comparatively little is known about how cellular signals integrate with kinetochore function. In the budding yeast Saccharomyces cerevisiae, the cyclic AMP protein kinase A signaling pathway promotes cellular growth in response to glucose. In this study, we find that decreasing protein kinase A activity, either by overexpressing negative regulators of the pathway or deleting the upstream effector Ras2, improves the viability of ipl1 and spc24 kinetochore mutants. Ipl1/Aurora B is a highly conserved kinase that corrects attachment of sister kinetochores that have attached to the same spindle pole, whereas Spc24 is a component of the conserved Ndc80 kinetochore complex that attaches directly to microtubules. Unexpectedly, we find that kinetochore mutants have increased phosphorylation levels of protein kinase A substrates, suggesting that the cyclic AMP protein kinase A signaling pathway is stimulated. The increase in protein kinase A activity in kinetochore mutants is not induced by activation of the spindle checkpoint or a metaphase delay because protein kinase A activity remains constant during an unperturbed cell cycle. Finally, we show that lowering protein kinase A activity can rescue the chromosome loss defect of the inner kinetochore ndc10 mutant. Overall, our data suggest that the increased protein kinase A activity in kinetochore mutants is detrimental to cellular growth and chromosome transmission fidelity.

Flow manipulation and cell immobilization for biochemical applications using thermally responsive fluids.

A flow redirection and single cell immobilization method in a microfluidic chip is presented. Microheaters generated localized heating and induced poly(N-isopropylacrylamide) phase transition, creating a hydrogel that blocked a channel or immobilized a single cell. The heaters were activated in sets to redirect flow and exchange the fluid in which an immobilized cell was immersed. A yeast cell was immobilized in hydrogel and a 4',6-diamidino-2-phenylindole (DAPI) fluorescent stain was introduced using flow redirection. DAPI diffused through the hydrogel and fluorescently labelled the yeast DNA, demonstrating in situ single cell biochemistry by means of immobilization and fluid exchange.

Genome-wide Fitness Profiles Reveal a Requirement for Autophagy During Yeast Fermentation.

The ability of cells to respond to environmental changes and adapt their metabolism enables cell survival under stressful conditions. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is particularly well adapted to the harsh conditions of anaerobic wine fermentation. However, S. cerevisiae gene function has not been previously systematically interrogated under conditions of industrial fermentation. We performed a genome-wide study of essential and nonessential S. cerevisiae gene requirements during grape juice fermentation to identify deletion strains that are either depleted or enriched within the viable fermentative population. Genes that function in autophagy and ubiquitin-proteasome degradation are required for optimal survival during fermentation, whereas genes that function in ribosome assembly and peroxisome biogenesis impair fitness during fermentation. We also uncover fermentation phenotypes for 139 uncharacterized genes with no previously known cellular function. We demonstrate that autophagy is induced early in wine fermentation in a nitrogen-replete environment, suggesting that autophagy may be triggered by other forms of stress that arise during fermentation. These results provide insights into the complex fermentation process and suggest possible means for improvement of industrial fermentation strains.

Molecular cloning of a pathogen/wound-inducible PR10 promoter from Pinus monticola and characterization in transgenic Arabidopsis plants.

In Pinus monticola (Dougl. ex D. Don), the class ten pathogenesis-related (PR10) proteins comprise a family of multiple members differentially expressed upon pathogen infection and other environmental stresses. One of them, PmPR10-1.13, is studied here by investigating its transcriptional regulation in transgenic Arabidopsis plants. For functional analyses of the PmPR10-1.13 promoter, a 1,316-bp promoter fragment and three 5' deletions were translationally fused to the ss-glucuronidase (GUS) reporter gene. The 1,316-bp promoter-driven GUS activity first appeared in hypocotyls and cotyledons in 2- to 3-day-old seedlings. As transgenic plants grew, GUS activity was detected strongly in apical meristems, next in stems and leaves. No GUS activity was detected in roots and in reproductive tissues of flower organs. In adult plants, the PmPR10-1.13 promoter-directed GUS expression was upregulated following pathogen infection and by wounding treatment, which generally mimic the endogenous expression pattern in western white pine. Promoter analysis of 5' deletions demonstrated that two regions between -1,316 and -930, and between -309 and -100 were responsible for the wound responsiveness. By structural and functional comparisons with PmPR10-1.14 promoter, putative wound-responsive elements were potentially identified in the PmPR10-1.13 promoter. In conclusion, PmPR10-1.13 showed properties of a defence-responsive gene, being transcriptionally upregulated upon biotic and abiotic stresses.

Cloning and characterization of a cDNA of cro rI from the white pine blister rust fungus Cronartium ribicola.

White pine blister rust (WPBR) is caused by the fungus Cronartium ribicola which has five spore stages on two unrelated hosts, the five-needle pines and Ribes spp. Recently, during the molecular analysis of the proteins and genes involved in host-pathogen interaction, the WPBR fungal protein Cro rI was identified in infected white pine tissues. To further characterize Cro rI, an expression cDNA library from poly(A)(+) mRNA of C. ribicola axenic mycelial culture was constructed and immunoscreened and the cDNA was cloned. Sequence analysis indicated an open reading frame of 462 bases, which encodes a protein of 153 amino acid residues with a molecular mass of 16.7 kDa and a predicted isoelectric point (pI) of 8.93. Based on the N-terminal amino acid sequences of Cro rI, the secreted portion of Cro rI protein should be 136 amino acids long with several putative posttranslational modification sites and a molecular mass of 14.8 kDa. The predicted pI for the secreted portion was 9.34. The predicted N-terminal signal peptide was 17 amino acids long. The N-terminal 42-amino acid sequence of the predicted mature protein (secreted portion) was identical to the amino terminal sequence of Cro rI that was previously determined. Southern blot hybridizations indicated that the C. ribicola genome contained at least two copies of the cro rI gene. Isolation of the genomic PCR fragment, which was approximately 400 bp longer than the cDNA, and subsequent cloning and sequencing analyses confirmed that there were three introns within the coding regions. Western immunoblot analyses revealed that Cro rI protein accumulated in large amounts only in the infected white pine tissues while no trace was detectable in the alternate Ribes stage or the five different spores, suggesting a critical role of Cro rI in the haploid stage of the fungus (in pine). The translocation of Cro rI was only found to occur in cankered trees, and not in the young infected seedlings. The implications of Cro rI in pathogenesis are discussed.