The yeast
is successfully used as a model organism to find genes responsible for lifespan control of higher organisms. As functional decline of higher eukaryotes can start as early as one quarter of ...the average lifespan, we asked whether
can be used to model this manifestation of aging. While the average replicative lifespan of
mother cells ranges between 15 and 30 division cycles, we found that resistances to certain stresses start to decrease much earlier. Looking into the mechanism, we found that knockouts of genes responsible for mitochondria-to-nucleus (retrograde) signaling,
or
significantly decrease the resistance of cells that generated more than four daughters, but not of the younger ones. We also found that even young mother cells frequently contain mitochondria with heterogeneous transmembrane potential and that the percentage of such cells correlates with replicative age. Together, these facts suggest that retrograde signaling starts to malfunction in relatively young cells, leading to accumulation of heterogeneous mitochondria within one cell. The latter may further contribute to a decline in stress resistances.
Abstract
Yeasts growing limited for nitrogen source or treated with fusel alcohols form elongated cells – pseudohyphae. Absence of mitochondrial DNA or anaerobic conditions inhibits this process, but ...the precise role of mitochondria is not clear. We found that a significant percentage of pseudohyphal cells contained mitochondria with different levels of membrane potential within one cell. An uncoupler carbonyl cyanide p (trifluoromethoxy) phenylhydrazone (FCCP), but not the ATP synthase inhibitor oligomycin D, prevented pseudohyphal growth. Interestingly, repression of the MIH1 gene encoding phosphatase activator of the G2/M transition partially restores the ability of yeast to form pseudohyphal cells in the presence of FCCP or in the absence of mitochondrial DNA. At the same time, retrograde signaling (the one triggered by dysfunctional mitochondria) appeared to be a positive regulator of butanol induced pseudohyphae formation: the deletion of any of the retrograde signaling genes (RTG1, RTG2, or RTG3) partially suppressed pseudohyphal growth. Together, our data suggest that two subpopulations of mitochondria are required for filamentous growth: one with high and another with low transmembrane potential. These mitochondria activated signaling pathways appear to converge at Mih1p level.
Eukaryotic cells contain dynamic mitochondrial filaments: they fuse and divide. Here we summarize data on the protein machinery driving mitochondrial dynamics in yeast and also discuss the factors ...that affect the fusion-fission balance. Fission is a general stress response of cells, and in the case of yeast this response appears to be prosurvival. At the same time, even under normal conditions yeast mitochondria undergo continuous cycles of fusion and fission. This seems to be a futile cycle and also expensive from the energy point of view. Why does it exist? Benefits might be the same as in the case of sexual reproduction. Indeed, mixing and separating of mitochondrial content allows mitochondrial DNA to segregate and recombine randomly, leading to high variation in the numbers of mutations per individual mitochondrion. This opens a possibility for effective purifying selection-elimination of mitochondria highly contaminated by deleterious mutations. The beneficial action presumes a mechanism for removal of defective mitochondria. We argue that selective mitochondrial autophagy or asymmetrical distribution of mitochondria during cell division could be at the core of such mechanism.
Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns ...centrifugally and centripetally in a pH- and microtubule-dependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with "late" phagosomes moving threefold more frequently than "early" ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dyneinmediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.
The existence of cell death program in unicellular organisms has been reported for a number of species. Nevertheless, the question why the ability to commit suicide has been maintained throughout ...evolution is far from being solved. While it is believed that altruistic death of individual yeast cells could be beneficial for the population, it is generally not known (i) what is wrong with the individuals destined for elimination, (ii) what is the critical value of the parameter that makes a cell unfit and (iii) how the cell monitors this parameter. Studies performed on yeast
Saccharomyces cerevisiae allow us to hypothesize on ways of possible solutions of these problems. Here we argue that (a) the main parameter for life-or-death decision measured by the cell is the degree of damage to the genetic material, (b) its critical value is dictated by quorum sensing machinery, and (c) it is measured by monitoring delays in cell division.
Stressed Saccharomyces cerevisiae cells easily lose respiratory function due to deletions in mitochondrial DNA, and this increases their general stress resistance. Is the loss active? We found that ...erythromycin (an inhibitor of mitochondrial translation) prevents the loss in control cells but not in the ones expressing mitochondrially-encoded protein Var1 in the nucleus. Var1 is a component of mitochondrial ribosomes; it is hydrophilic, positively charged, and prone to aggregation. Addition of DNase altered Var1 content in a preparation of mitochondrial nucleoids. Our data indicate that Var1 physically interacts with mitochondrial DNA and under stress negatively regulates its maintenance.
Glycolysis lies at the basis of metabolism and cell energy supply. The disregulation of glycolysis is involved in such pathological processes as cancer proliferation, neurodegenerative diseases, and ...amplification of ischemic damage. Phosphofructokinase-2 (PFK-2), a bifunctional enzyme and regulator of glycolytic flux, has recently emerged as a promising anticancer target. Herein, the computer-aided design of a new class of aminofurazan-triazole regulators of PFK-2 is described along with the results of their in vitro evaluation. The aminofurazan-triazoles differ from other recently described inhibitors of PFK-2 and demonstrate the ability to modulate glycolytic flux in rat muscle lysate, producing a twofold decrease by inhibitors and fourfold increase by activators. The most potent compounds in the series were shown to inhibit the kinase activity of the hypoxia-inducible form of PFK-2, PFKFB3, as well as proliferation of HeLa, lung adenocarcinoma, colon adenocarcinoma, and breast cancer cells at concentrations in the low micromolar range.
It was shown earlier that DNA damage induced by alkylating agent MMS (methyl methanesulfonate) results in formation of ROS (reactive oxygen species) in yeast cells. Here, we asked whether this ROS ...generation is favourable for the cells. It appeared that prooxidants rather than antioxidants stimulate the survival after MMS treatment. We found that positively charged detergents increase the survival via induction of H2O2 formation in the cells. Interestingly, prooxidants protected yeast cells from the moderate doses of MMS and enhanced the toxicity of relatively high ones. Prooxidants also protect the cells arrested in mitosis (nocodazole treatment), indicating that the protection is mostly due to ROS‐mediated transcriptional stress—response rather than due to enrichment of cell culture with highly MMS‐resistant G2/M cells. The comparison of the published expression profile responses to prooxidant and MMS treatments identifies a set of ROS‐activated genes, which are likely to protect cells from the genotoxic stress.
The yeast cell wall is constantly remodeled to enable cell growth and division. In this study, we describe a novel type of cell wall modification. We report that the drug amiodarone induces rapid ...channel formation within the cell wall of the yeast
Hansenula polymorpha
. Light microscopy shows that shortly after adding amiodarone, spherical structures, which can be stained with DNA binding dyes, form on the cell surface. Electron microphotographs show that amiodarone induces the formation of channels 50–80 nm in diameter in the cell wall that appear to be filled with intracellular material. Using fluorescent microscopy, we demonstrate MitoTracker-positive DNA-containing structures visibly extruded from the cells through these channels. We speculate that the observed channel formation acts to enable the secretion of mitochondrial material from the cell under stressful conditions, thus enabling adaptive changes to the extracellular environment.
Hydrophobic cations with delocalized charge are used to deliver drugs to mitochondria. However, micromolar concentrations of such compounds could be toxic due to their excessive accumulation in ...mitochondria. We studied possible pathophysiological effects of one such cation, i.e. dodecyltriphenylphosphonium (C
12
-TPP), in the yeast
Saccharomyces cerevisiae
. First, we found that C
12
-TPP induces high-amplitude mitochondrial swelling. The swelling can be prevented by addition of protonophorous uncoupler FCCP or antioxidant alpha-tocopherol, but not other tested antioxidants (N-acetylcysteine and Trolox). Second, FCCP prevents ROS-sensitive fluorescent dye (dichlorofluorescein diacetate) staining of yeast treated with C
12
-TPP. We also showed that all tested antioxidants partially restore the growth inhibited by C
12
-TPP. The latter points that ROS rather than the mitochondria swelling limit the growth rate.
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