Investigating the Antifungal Mechanism of Action of Polygodial by Phenotypic Screening in Saccharomyces cerevisiae.

Polygodial is a "hot" peppery-tasting sesquiterpenoid that was first described for its anti-feedant activity against African armyworms. Using the haploid deletion mutant library of Saccharomyces cerevisiae, a genome-wide mutant screen was performed to shed more light on polygodial's antifungal mechanism of action. We identified 66 deletion strains that were hypersensitive and 47 that were highly resistant to polygodial treatment. Among the hypersensitive strains, an enrichment was found for genes required for vacuolar acidification, amino acid biosynthesis, nucleosome mobilization, the transcription mediator complex, autophagy and vesicular trafficking, while the resistant strains were enriched for genes encoding cytoskeleton-binding proteins, ribosomal proteins, mitochondrial matrix proteins, components of the heme activator protein (HAP) complex, and known regulators of the target of rapamycin complex 1 (TORC1) signaling. WE confirm that polygodial triggers a dose-dependent vacuolar alkalinization and that it increases Ca2+ influx and inhibits glucose-induced Ca2+ signaling. Moreover, we provide evidence suggesting that TORC1 signaling and its protective agent ubiquitin play a central role in polygodial resistance, suggesting that they can be targeted by polygodial either directly or via altered Ca2+ homeostasis.

Decreased Vacuolar Ca2+ Storage and Disrupted Vesicle Trafficking Underlie Alpha-Synuclein-Induced Ca2+ Dysregulation in S. cerevisiae.

The yeast Saccharomyces cerevisiae is a powerful model to study the molecular mechanisms underlying α-synuclein (α-syn) cytotoxicity. This is due to the high degree of conservation of cellular processes with higher eukaryotes and the fact that yeast does not endogenously express α-synuclein. In this work, we focused specifically on the interplay between α-syn and intracellular Ca2+ homeostasis. Using temperature-sensitive SEC4 mutants and deletion strains for the vacuolar Ca2+ transporters Pmc1 and Vcx1, together with aequorin-based Ca2+ recordings, we show that overexpression of α-syn shifts the predominant temporal pattern of organellar Ca2+ release from a biphasic to a quasi-monophasic response. Fragmentation and vesiculation of vacuolar membranes in α-syn expressing cells can account for the faster release of vacuolar Ca2+. α-Syn further significantly reduced Ca2+ storage resulting in increased resting cytosolic Ca2+ levels. Overexpression of the vacuolar Ca2+ ATPase Pmc1 in wild-type cells prevented the α-syn-induced increase in resting Ca2+ and was able to restore growth. We propose that α-syn-induced disruptions in Ca2+ signaling might be an important step in initiating cell death.

Continuous cell injury promotes hepatic tumorigenesis in cdc42-deficient mouse liver.

BACKGROUND & AIMS: The Rho small guanosine triphosphatase Cdc42 is critical for diverse cellular functions, including regulation of actin organization, cell polarity, intracellular membrane trafficking, transcription, cell-cycle progression, and cell transformation. This implies that Cdc42 might be required for liver function. METHODS: Mice in which Cdc42 was ablated in hepatocytes and bile duct cells were generated by Cre-loxP technology. Livers were examined by histologic, immunohistochemical, ultrastructural, and serum analysis to define the effect of loss of Cdc42 on liver structure. RESULTS: Mice lacking Cdc42 in their hepatocytes were born at Mendelian ratios. They did not show increased mortality but showed chronic jaundice. They developed hepatomegaly soon after birth, and signs of liver transformation, such as formation of nodules and tumors, became visible macroscopically at age 6 months. Hepatocellular carcinoma was observed 8 months after birth. Tumors grew slowly and lacked expression of nuclear beta-catenin. Lung metastases were observed at the late stage of carcinogenesis. Immunofluorescent examination and electron microscopy revealed severe defects in the liver. At the age of 2 months, the canaliculi between hepatocytes were greatly enlarged, although the tight junctions flanking the canaliculi appeared normal. Regular liver plates were absent. E-cadherin expression pattern and gap junction localization were distorted. Analysis of serum samples indicated cholestasis. CONCLUSIONS: We describe a mouse model in which chronic liver disease leads to hepatocarcinogenesis.

Ca(2+) homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca(2+) storage.

Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca(2+) signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca(2+) signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca(2+) transporters, combined with manipulations to estimate intracellular Ca(2+) storage, we evaluated the contribution of individual transport systems to intracellular Ca(2+) homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca(2+) homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca(2+) uptake and Ca(2+) influx across the plasma membrane. In the pmr1Δ strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca(2+) channels and Pmc1 but required Vcx1. By contrast, in the cod1Δ strain increased vacuolar Ca(2+) uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca(2+) uptake and point to the existence of not-yet identified Ca(2+) influx pathways.

Plakophilin-3-deficient mice develop hair coat abnormalities and are prone to cutaneous inflammation.

We generated mice deficient in plakophilin-3 (PKP3), a member of the Armadillo-repeat family and a component of desmosomes and stress granules in epithelial cells. In these mice, several subsets of hair follicles (HFs) had morphological abnormalities, and the majority of awl and auchene hair shafts had fewer medullar air columns. Desmosomes were absent from the basal layer of the outer root sheath of HFs and from the matrix cells that are in contact with dermal papillae. In the basal layer of PKP3-null epidermis, densities of desmosomes and adherens junctions were remarkably altered. Compensatory changes in several junctional proteins were observed. PKP3-null mice housed in conventional facilities were prone to dermatitis. Our animal model provides in vivo evidence that PKP3 plays a critical role in morphogenesis of HFs and shafts and in limiting inflammatory responses in the skin.