Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV Core DII protein

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV

Composition and Acidification of the Culture Medium Influences Chronological Aging Similarly in Vineyard and Laboratory Yeast

Chronological aging has been studied extensively in laboratory yeast by culturing cells into stationary phase in synthetic complete medium with 2% glucose as the carbon source. During this process, acidification of the culture medium occurs due to secretion of organic acids, including acetic acid, which limits survival of yeast cells. Dietary restriction or buffering the medium to pH 6 prevents acidification and increases chronological life span. Here we set out to determine whether these effects are specific to laboratory-derived yeast by testing the chronological aging properties of the vineyard yeast strain RM11. Similar to the laboratory strain BY4743 and its haploid derivatives, RM11 and its haploid derivatives displayed increased chronological life span from dietary restriction, buffering the pH of the culture medium, or aging in rich medium. RM11 and BY4743 also displayed generally similar aging and growth characteristics when cultured in a variety of different carbon sources. These data support the idea that mechanisms of chronological aging are similar in both the laboratory and vineyard strains

The propeptide of yeast cathepsin D inhibits programmed necrosis

The lysosomal endoprotease cathepsin D (CatD) is an essential player in general protein turnover and specific peptide processing. CatD-deficiency is associated with neurodegenerative diseases, whereas elevated CatD levels correlate with tumor malignancy and cancer cell survival. Here, we show that the CatD ortholog of the budding yeast Saccharomyces cerevisiae (Pep4p) harbors a dual cytoprotective function, composed of an anti-apoptotic part, conferred by its proteolytic capacity, and an anti-necrotic part, which resides in the protein's proteolytically inactive propeptide. Thus, deletion of PEP4 resulted in both apoptotic and necrotic cell death during chronological aging. Conversely, prolonged overexpression of Pep4p extended chronological lifespan specifically through the protein's anti-necrotic function. This function, which triggered histone hypoacetylation, was dependent on polyamine biosynthesis and was exerted via enhanced intracellular levels of putrescine, spermidine and its precursor S-adenosyl-methionine. Altogether, these data discriminate two pro-survival functions of yeast CatD and provide first insight into the physiological regulation of programmed necrosis in yeast

Induction of autophagy by spermidine promotes longevity.

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Deletion of Cryptococcus neoformans AIF Ortholog Promotes Chromosome Aneuploidy and Fluconazole-Resistance in a Metacaspase-Independent Manner

Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLCR) in vitro and in vivo. FLCR isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLCR subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance

Genomic Instability Is Associated with Natural Life Span Variation in Saccharomyces cerevisiae

Increasing genomic instability is associated with aging in eukaryotes, but the connection between genomic instability and natural variation in life span is unknown. We have quantified chronological life span and loss-of-heterozygosity (LOH) in 11 natural isolates of Saccharomyces cerevisiae. We show that genomic instability increases and mitotic asymmetry breaks down during chronological aging. The age-dependent increase of genomic instability generally lags behind the drop of viability and this delay accounts for ∼50% of the observed natural variation of replicative life span in these yeast isolates. We conclude that the abilities of yeast strains to tolerate genomic instability co-vary with their replicative life spans. To the best of our knowledge, this is the first quantitative evidence that demonstrates a link between genomic instability and natural variation in life span

SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes febrile disease, muscle and joint pain, which can become chronic in some individuals. The non-structural protein 3 (nsP3) plays essential roles during infection, but a complete understanding of its function is lacking. Here we used a microscopy-based approach to image CHIKV nsP3 inside human cells. The SNAP system consists of a self-labelling enzyme tag, which catalyses the covalent linking of exogenously supplemented synthetic ligands. Genetic insertion of this tag resulted in viable replicons and specific labelling while preserving the effect of nsP3 on stress granule responses and co-localisation with GTPase Activating Protein (SH3 domain) Binding Proteins (G3BPs). With sub-diffraction, three-dimensional, optical imaging, we visualised nsP3-positive structures with variable density and morphology, including high-density rod-like structures, large spherical granules, and small, low-density structures. Next, we confirmed the utility of the SNAP tag for studying protein turnover by pulse-chase labelling. We also revealed an association of nsP3 with cellular lipid droplets and examined the spatial relationships between nsP3 and the non-structural protein 1 (nsP1). Together, our study provides a sensitive, specific, and versatile system for fundamental research into the individual functions of a viral non-structural protein during infection with a medically important arthropod-borne virus (arbovirus)

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death

A genetic analysis of nitric oxide-mediated signaling during chronological aging in the yeast

In mammals, NO•, a signaling molecule is implicated in the regulation of vasodilation, neurotransmission and immune response. It is believed that NO• is a signaling molecule also in unicellular organism like yeast and may be involved in the regulation of apoptosis and sporulation. It has been reported that NO• is produced during chronological aging (CA) leading to an increase of the superoxide level, which in turn mediates apoptosis. Since this conclusion was based on indirect measurements of NO• by the Griess reaction, the role of NO• signaling during CA in the yeast remains uncertain. We investigated this issue more precisely using different genetic and biochemical methodologies. We used cells lacking the factors influencing nitrosative stress response like flavohemoglobin metabolizing NO•, S-nitrosoglutathione reductase metabolizing S-nitrosoglutathione and the transcription factor Fzf1p mediating NO• response. We measured the standard parameters describing CA and found an elevation in the superoxide level, percentage of death cells, the level of TUNEL positive cells and a decrease in proliferating potential. These observations showed no significant differences between wild type cells and the disruptants except for a small elevation of the superoxide level in the Δsfa1 mutant. The intracellular NO• level and flavohemoglobin expression decreased rather than increased during CA. Products of general nitrogen metabolism and protein tyrosine nitration were slightly decreased during CA, the magnitude of changes showing no differences between the wild type and the mutant yeast. Altogether, our data indicate that apoptosis during yeast CA is mediated by superoxide signaling rather than NO• signaling

Effects of calorie restriction on life span of microorganisms

Calorie restriction (CR) in microorganisms such as budding and fission yeasts has a robust and well-documented impact on longevity. In order to efficiently utilize the limited energy during CR, these organisms shift from primarily fermentative metabolism to mitochondrial respiration. Respiration activates certain conserved longevity factors such as sirtuins and is associated with widespread physiological changes that contribute to increased survival. However, the importance of respiration during CR-mediated longevity has remained controversial. The emergence of several novel metabolically distinct microbial models for longevity has enabled CR to be studied from new perspectives. The majority of CR and life span studies have been conducted in the primarily fermentative Crabtree-positive yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, but studies in primarily respiratory Crabtree-negative yeast and obligate aerobes can offer complementary insight into the more complex mammalian response to CR. Not only are microorganisms helping characterize a conserved cellular mechanism for CR-mediated longevity, but they can also directly impact mammalian metabolism as part of the natural gut flora. Here, we discuss the contributions of microorganisms to our knowledge of CR and longevity at the level of both the cell and the organism