Potassium and Sodium Transport in Yeast

[EN] As the proper maintenance of intracellular potassium and sodium concentrations is vital for cell growth, all living organisms have developed a cohort of strategies to maintain proper monovalent cation homeostasis. In the model yeast Saccharomyces cerevisiae, potassium is accumulated to relatively high concentrations and is required for many aspects of cellular function, whereas high intracellular sodium/potassium ratios are detrimental to cell growth and survival. The fact that S. cerevisiae cells can grow in the presence of a broad range of concentrations of external potassium (10 M–2.5 M) and sodium (up to 1.5 M) indicates the existence of robust mechanisms that have evolved to maintain intracellular concentrations of these cations within appropriate limits. In this review, current knowledge regarding potassium and sodium transporters and their regulation will be summarized. 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Mitochondrial Alterations in PINK1 Deficient Cells Are Influenced by Calcineurin-Dependent Dephosphorylation of Dynamin-Related Protein 1

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf-Hirschhorn syndrome

Human Wolf-Hirschhorn syndrome (WHS) is a multigenic disorder resulting from a hemizygous deletion on chromosome 4. LETM1 is the best candidate gene for seizures, the strongest haploinsufficiency phenotype of WHS patients. Here, we identify the Drosophila gene CG4589 as the ortholog of LETM1 and name the gene DmLETM1. Using RNA interference approaches in both Drosophila melanogaster cultured cells and the adult fly, we have assayed the effects of down-regulating the LETM1 gene on mitochondrial function. We also show that DmLETM1 complements growth and mitochondrial K(+)/H(+) exchange (KHE) activity in yeast deficient for LETM1. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues demonstrate that the depletion of DmLETM1 results in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of DmLETM1 results in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. Taken together our results demonstrate the function of DmLETM1 as a mitochondrial osmoregulator through its KHE activity and uncover a pathophysiological WHS phenotype in the model organism D. melanogaster

Mitochondria removal by autophagy

Mitochondrial dysfunction has severe cellular consequences and is linked with neurodegenerative diseases and aging. Maintaining a healthy population of mitochondria is thus essential for proper cellular homeostasis. Several strategies have evolved to prevent and limit mitochondria damage, and macroautophagy plays a role in degrading superfluous or severely damaged mitochondria. Selective removal of mitochondria by autophagy (termed mitophagy) has been extensively studied recently in both yeast and mammalian cells. In this review, we summarize our current knowledge of mitophagy. We also compare the molecular process of mitophagy with other types of specific autophagic pathways and discuss its biological importance

Elevated metallothionein expression in long-lived species mediates the influence of cadmium accumulation on aging

Cadmium (Cd) accumulates with aging and is elevated in long-lived species. Metallothioneins (MTs), small cysteine-rich proteins involved in metal homeostasis and Cd detoxification, are known to be related to longevity. However, the relationship between Cd accumulation, the role of MTs, and aging is currently unclear. Specifically, we do not know if long-lived species evolved an efficient metal stress response by upregulating their MT levels to reduce the toxic effects of environmental pollutants, such as Cd, that accumulate over their longer life span. It is also unknown if the number of MT genes, their expression, or both protect the organisms from potentially damaging effects during aging. To address these questions, we reanalyzed several cross-species studies and obtained data on MT expression and Cd accumulation in long-lived mouse models. We confirmed a relationship between species maximum life span in captive mammals and their Cd content in liver and kidney. We found that although the number of MT genes does not affect longevity, gene expression and protein amount of specific MT paralogs are strongly related to life span in mammals. MT expression rather than gene number may influence the high Cd levels and longevity of some species. In support of this, we found that overexpression of MT-1 accelerated Cd accumulation in mice and that tissue Cd was higher in long-lived mouse strains with high MT expression. We conclude that long-lived species have evolved a more efficient stress response by upregulating the expression of MT genes in presence of Cd, which contributes to elevated tissue Cd levels

The Lipid Metabolism as Target and Modulator of BOLD‐100 Anticancer Activity: Crosstalk with Histone Acetylation

Abstract The leading first‐in‐class ruthenium‐complex BOLD‐100 currently undergoes clinical phase‐II anticancer evaluation. Recently, BOLD‐100 is identified as anti‐Warburg compound. The present study shows that also deregulated lipid metabolism parameters characterize acquired BOLD‐100‐resistant colon and pancreatic carcinoma cells. Acute BOLD‐100 treatment reduces lipid droplet contents of BOLD‐100‐sensitive but not ‐resistant cells. Despite enhanced glycolysis fueling lipid accumulation, BOLD‐100‐resistant cells reveal diminished lactate secretion based on monocarboxylate transporter 1 (MCT1) loss mediated by a frame‐shift mutation in the MCT1 chaperone basigin. Glycolysis and lipid catabolism converge in the production of protein/histone acetylation substrate acetyl‐coenzymeA (CoA). Mass spectrometric and nuclear magnetic resonance analyses uncover spontaneous cell‐free BOLD‐100‐CoA adduct formation suggesting acetyl‐CoA depletion as mechanism bridging BOLD‐100‐induced lipid metabolism alterations and histone acetylation‐mediated gene expression deregulation. Indeed, BOLD‐100 treatment decreases histone acetylation selectively in sensitive cells. Pharmacological targeting confirms histone de‐acetylation as central mode‐of‐action of BOLD‐100 and metabolic programs stabilizing histone acetylation as relevant Achilles’ heel of acquired BOLD‐100‐resistant cell and xenograft models. Accordingly, histone gene expression changes also predict intrinsic BOLD‐100 responsiveness. Summarizing, BOLD‐100 is identified as epigenetically active substance acting via targeting several onco‐metabolic pathways. Identification of the lipid metabolism as driver of acquired BOLD‐100 resistance opens novel strategies to tackle therapy failure