(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Selective retention of dysfunctional mitochondria during asymmetric cell division in yeast [1]

['Xenia Chelius', 'Zellbiologie', 'Universität Bayreuth', 'Bayreuth', 'Veronika Bartosch', 'Nathalie Rausch', 'Magdalena Haubner', 'Jana Schramm', 'Ralf J. Braun', 'Department Medizin']

Date: 2023-09

Decline of mitochondrial function is a hallmark of cellular aging. To counteract this process, some cells inherit mitochondria asymmetrically to rejuvenate daughter cells. The molecular mechanisms that control this process are poorly understood. Here, we made use of matrix-targeted D-amino acid oxidase (Su9-DAO) to selectively trigger oxidative damage in yeast mitochondria. We observed that dysfunctional mitochondria become fusion-incompetent and immotile. Lack of bud-directed movements is caused by defective recruitment of the myosin motor, Myo2. Intriguingly, intact mitochondria that are present in the same cell continue to move into the bud, establishing that quality control occurs directly at the level of the organelle in the mother. The selection of healthy organelles for inheritance no longer works in the absence of the mitochondrial Myo2 adapter protein Mmr1. Together, our data suggest a mechanism in which the combination of blocked fusion and loss of motor protein ensures that damaged mitochondria are retained in the mother cell to ensure rejuvenation of the bud.

Funding: This work was funded by the Deutsche Forschungsgemeinschaft, project numbers 433461293 (to BW) and 491183248 (Open Access Publishing Fund of the University of Bayreuth), and by Elitenetzwerk Bayern through the "Biological Physics" program (to BW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Chelius et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The mechanisms governing asymmetric distribution of mitochondria between the mother cell and the bud are poorly understood. Here, we have investigated how oxidative damage affects the inheritance of mitochondria during asymmetric division of yeast cells. We find that ROS produced in mitochondria trigger selective fragmentation and retention of dysfunctional organelles in the mother, while healthy mitochondria that are present in the same cell continue to move towards the bud. We provide evidence that the selection of fit mitochondria for inheritance occurs directly at the organellar level. Our results demonstrate that selection of mitochondria for bud-directed transport is achieved by Myo2 motor recruitment and Mmr1 plays a crucial role in this process.

During the cell cycle, mitochondria enter the bud immediately upon its emergence and exhibit continuous anterograde and retrograde movements concomitant with frequent fusion and fission [ 27 , 28 ]. Mitochondria are transported along actin cables towards the bud by the myosin motor, Myo2 [ 29 – 31 ]. Binding of Myo2 to mitochondria is promoted by Ypt11, a small rab GTPase, and Mmr1, a protein peripherally associated with the mitochondrial outer membrane [ 29 , 32 – 34 ]. Δmmr1 and Δypt11 single mutants have only slight mitochondrial inheritance defects while the Δmmr1 Δypt11 double mutant is inviable [ 33 , 34 ] or severely sick [ 35 ]. Simultaneous loss of active Mmr1 and Ypt11 results in a synthetic mitochondrial inheritance defect [ 33 – 36 ] indicating that Ypt11 and Mmr1 have partially redundant functions in Myo2 recruitment. Mitochondrial cortex tethers ensure that a part of the mitochondrial network is retained in the mother cell upon cytokinesis [ 37 – 40 ]. Thus, multiple activities of the transport, retention, fusion, and fission machineries have to be orchestrated to promote ordered mitochondrial inheritance [ 41 – 43 ].

Several observations suggest that mitochondria are asymmetrically inherited in yeast [ 13 , 20 ]. A mutation in the ATP2 gene, encoding a subunit of the F 1 part of the mitochondrial ATP synthase, leads to loss of mother-daughter age asymmetry and accumulation of dysfunctional mitochondria [ 21 ]. Aconitase, an enzyme of the citric acid cycle, is asymmetrically distributed in mitochondria of aging mothers and their daughters [ 22 ]. Fluorescent biosensors revealed that mitochondria in the bud have lower levels of reactive oxygen species (ROS) and are more reducing than their counterparts in the mother [ 23 , 24 ]. The mitochondria-to-cell size ratio is tightly controlled during inheritance in the bud, whereas it continually decreases in aging mothers [ 25 ]. Furthermore, protein aggregates in the mitochondrial matrix are sequestered to a specific deposit site, which is retained in mother cells [ 26 ].

Budding yeast Saccharomyces cerevisiae is a powerful model organism to study the asymmetric inheritance of organelles [ 13 – 16 ]. Each cell division produces a virgin daughter cell that buds off from its aging mother. During replicative aging, the mother accumulates aging factors and dies after about 20 to 30 cell divisions. Remarkably, daughter cells are born with full replicative potential, even if they bud off from old mother cells. To ensure rejuvenation of their daughters, yeast mother cells retain protein aggregates, damaged organelles, and other harmful materials and avoid their transport into the bud [ 17 – 19 ].

Decline of mitochondrial function and accumulation of mutations in the mitochondrial DNA (mtDNA) are hallmarks of aging [ 10 , 11 ]. Therefore, the proper distribution of intact mitochondria during cell division is crucial for cell homeostasis, and their asymmetric inheritance is thought to contribute to the rejuvenation of progeny cells. An analysis of stem-like cells revealed that young mitochondria are preferentially apportioned to daughter cells that maintain stem cell traits, whereas daughter cells destined to differentiate receive a larger share of old mitochondria [ 12 ]. Thus, it appears that young and healthy mitochondria are preferentially apportioned to the daughter cell that has to maintain a high replicative potential.

Mitochondria cannot be made de novo but have to be passed on to daughter cells during cell division. Depending on the organism and cell type, mitochondrial inheritance involves stochastic or ordered partitioning strategies [ 1 – 4 ]. In many mammalian cell types, mitochondria fragment during mitosis. This activity in concert with cytoskeleton-dependent motility contributes to stochastic partitioning of mitochondria to the daughter cells [ 5 – 7 ]. However, certain cell types divide asymmetrically and actively control uneven distribution of their cell organelles. This results in distinct fates of progeny cells and is important, e.g., for the maintenance of stem cells in metazoans or as a defense against aging in microorganisms [ 8 , 9 ].

Results

Mitochondria-targeted D-amino acid oxidase affects mitochondrial morphology and fusion Next, we employed a genetically encoded ROS stressor to analyze mitochondrial behavior in response to elevated H 2 O 2 levels in the mitochondrial matrix. D-amino acid oxidase (DAO) is an enzyme that deaminates D-amino acids and concomitantly reduces FAD. Subsequent re-oxidation of FADH 2 by molecular oxygen produces H 2 O 2 [54]. Heterologous expression of DAO can be used to induce D-alanine-controlled production of H 2 O 2 in cells [55,56]. To induce H 2 O 2 stress in the mitochondrial matrix, we used the previously described Su9-DAO construct [57]. It consists of the mitochondrial presequence of subunit 9 of the ATP synthase of Neurospora crassa fused to the DAO enzyme from Rhodotorula gracilis expressed from the constitutive TEF promoter. Su9-DAO was shown to produce deleterious levels of H 2 O 2 in mitochondria of yeast and mammalian cells upon addition of D-alanine, but not L-alanine [57,58]. We integrated the Su9-DAO coding sequence into the genome and observed that growth of Su9-DAO-expressing cells on agar plates was impaired by the addition of D-alanine in a dose-dependent manner. Growth was not affected by L-alanine (Fig 2A). This suggests that Su9-DAO produces H 2 O 2 in sufficient amounts to become toxic to the cells. We also observed that cells were more sensitive to Su9-DAO activity when grown on non-fermentable carbon sources (Fig 2A), suggesting that mitochondrial respiration is particularly affected. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Mitochondrially produced H 2 O 2 induces loss of membrane potential, mitochondrial fragmentation, and block of fusion. (A) Ten-fold serial dilutions of wild-type cells without (control) and with Su9-DAO were spotted on agar plates containing minimal complete medium with glucose (SCD) or glycerol and ethanol (SCGE) as carbon source and incubated at 30°C. (B) Wild-type cells expressing Su9-DAO were incubated in the presence of L-alanine or D-alanine for 3 h and then stained with TMRM. Fluorescence microscopy images are maximum intensity projections of z stacks using identical camera settings. Additional images are shown in S2A Fig. (C and D) Wild-type cells expressing Su9-DAO and mtGFP were incubated for 3 h in the presence of L-alanine or D-alanine. Fluorescence images are z stacks subjected to deconvolution. Mitochondrial morphology was quantified in 100 cells per sample (triplicate experiments ± SD). (E and F) Wild-type and Δmip1 cells expressing Su9-DAO were analyzed as in (C and D) (3 biological replicates ± SD). (G) Ten-fold serial dilutions of wild-type or Δmip1 cells were spotted on agar plates containing minimal complete medium with glucose and incubated at 30°C. (H and I) Wild-type cells expressing either Su9-DAO together with mtGFP (left) or only mtGFP (right) and wild-type cells expressing Su9-DAO together with mtERFP were incubated for 30 min with L-alanine or D-alanine, mixed, and incubated for another 3 h to allow mating and zygote formation. Fluorescence images are z stacks subjected to deconvolution and were merged with their corresponding DIC image. Medial buds of zygotes are marked with an asterisk. Mitochondrial fusion was quantified in 50 zygotes per sample (triplicate experiments, error bars indicate SD calculated from the sum of zygotes showing complete or partial fusion). Bars, 5 μm. Data pooling and statistics are detailed in S2 Table. The data underlying this figure can be found in S1 Datasheet. DAO, D-amino acid oxidase; DIC, differential interference contrast; mtERFP, mitochondria-targeted enhanced red fluorescent protein; mtGFP, mitochondria-targeted GFP; SCD, synthetic complete dextrose medium; SD, standard deviation; TMRM, tetramethylrhodamine methyl ester. https://doi.org/10.1371/journal.pbio.3002310.g002 To confirm that H 2 O 2 production by Su9-DAO compromises mitochondrial function, we stained yeast cells with tetramethylrhodamine methyl ester (TMRM), a membrane potential-sensitive dye. Incubation of Su9-DAO-expressing cells for 3 h in D-alanine-containing medium was sufficient to break down the mitochondrial membrane potential. These cells were indiscernible from respiratory-deficient rho0 cells lacking mtDNA (Figs 2B and S2A). We conclude that DAO activity in the mitochondrial matrix severely impairs mitochondrial function. Fluorescence microscopy of Su9-DAO-expressing cells grown in liquid culture revealed that mitochondria became highly fragmented and aggregated when mitochondrial H 2 O 2 production was induced by the addition of D-alanine to the medium (Fig 2C and 2D and S4 and S5 Videos). PI staining confirmed that cells were viable under these conditions (S2B Fig), and mitochondrial morphology was not affected by D-alanine in strains lacking the Su9-DAO construct (S2C and S2D Fig). Thus, production of H 2 O 2 in the mitochondrial matrix induces mitochondrial fragmentation under nonlethal conditions, similar to exogenously added H 2 O 2 . The respiratory chain is thought to constitute the major source of mitochondrial ROS [44]. To test whether it is also required for H 2 O 2 -induced mitochondrial fragmentation, we examined growth and mitochondrial morphology in Su9-DAO expressing Δmip1 cells lacking the mitochondrial DNA polymerase. This mutant is respiratory deficient as it lacks the mtDNA-encoded respiratory chain subunits. Growth impairment and mitochondrial fragmentation in Δmip1 cells were as severe as in wild-type cells (Fig 2E–2G), indicating that respiratory chain activity is not required. Fragmentation of mitochondria under ROS stress suggests that mitochondrial fusion might be compromised. To test this, we assayed mitochondrial fusion in zygotes. Haploid cells of opposite mating types containing mitochondria labeled with either GFP or ERFP were grown in L-alanine or D-alanine-containing media, mixed to allow mating and zygote formation, and analyzed by fluorescence microscopy. Zygotes obtained from cells grown in L-alanine-containing media showed almost complete mixing of mitochondrial labels, indicating efficient mitochondrial fusion. In contrast, upon incubation in D-alanine-containing media only about 20% of zygotes showed fused mitochondria when both mating partners contained Su9-DAO, and 40% when a Su9-DAO expressing strain was mated with a wild type lacking this construct (Figs 2H, 2I, and S2E). We conclude that mitochondrial ROS stress severely impairs mitochondrial fusion.

Mitochondrial dynamics or retrograde actin cable flow are not critical for partitioning of ROS-stressed mitochondria We have previously reported that transport of a critical mitochondrial quantity to the bud depends on a fine-tuned balance of anterograde movement powered by Myo2 and mitochondrial fusion and fission. Δfzo1 mutants lacking the major mitochondrial fusion protein, Fzo1, contain fragmented mitochondria and show a significant inheritance defect suggesting that fragmented mitochondria are less efficiently transported into the bud than fused and tubular mitochondria [61]. Therefore, we asked whether the impairment of mitochondrial inheritance under ROS stress is caused by mitochondrial fragmentation. To test this, we quantified mitochondrial inheritance in strains that are unable to undergo mitochondrial fragmentation. Δdnm1 mutants lack the major mitochondrial fission protein, Dnm1, and contain fused, interconnected mitochondrial networks due to a block of mitochondrial fission with ongoing mitochondrial fusion. Δdnm1 Δfzo1 cells have tubular mitochondria, similar to the wild type, which are no longer dynamic due to simultaneous blocks of fusion and fission [62,63]. Mitochondria were efficiently transported into medium-sized buds in Su9-DAO-expressing cells in the presence of L-alanine. Induction of mitochondrial ROS stress by the addition of D-alanine resulted in mitochondrial inheritance defects in fission-defective mutants, very similar to the wild type, while mitochondria maintained an interconnected morphology (Fig 4A and 4B). Also, Su9-DAO-dependent growth defects were similar in Δdnm1 Δfzo1 double and Δdnm1 single mutant and wild-type strains (Fig 4C). We conclude that impaired inheritance of ROS-stressed mitochondria is not caused by mitochondrial fragmentation. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Retention of ROS-stressed mitochondria is independent of mitochondrial dynamics and actin cable dynamics. (A and B) Cells expressing Su9-DAO and mtGFP were incubated for 3 h in the presence of L-alanine or D-alanine. Fluorescence images are z stacks subjected to deconvolution. Mitochondrial inheritance was quantified in 100 cells per sample (triplicate experiments, error bars indicate SD calculated from the sum of cells showing reduced or efficient inheritance). In this and the following figures, the percentage of cells lacking mitochondria in their bud corresponds to the difference between the sum of the categories “efficient” plus “reduced” and 100%. (C) Ten-fold serial dilutions of cells were spotted on agar plates containing minimal complete medium with glucose and incubated at 30°C. (D and E) Cells were analyzed as in (A and B). Bars, 5 μm. Data pooling and statistics are detailed in S2 Table. The data underlying this figure can be found in S1 Datasheet. DAO, D-amino acid oxidase; mtGFP, mitochondria-targeted GFP; ROI, region of interest; SD, standard deviation. https://doi.org/10.1371/journal.pbio.3002310.g004 Bundles of actin filaments (cables) serve as tracks for Myo2-dependent organelle transport towards the bud. Actin polymerization in the bud or bud neck and pulling forces produced by myosins generate a constant flow of actin cables towards the pole of the mother cell that is distal to the bud. This retrograde actin cable flow (RACF) constitutes an opposing force that has to be overcome by mitochondria to reach the bud [24,27]. RACF is promoted by the class II myosin, Myo1, and limited by the tropomyosin, Tpm2 [64]. It has been reported that deletion of the TPM2 gene enhances the asymmetry of mitochondrial redox state in the mother cell and bud, whereas deletion of MYO1 abrogates it. Based on these observations, it was suggested that increased RACF in Δtpm2 selects more reduced mitochondria for inheritance, whereas reduced RACF in Δmyo1 allows the entry of more oxidized mitochondria into the bud. Thus, RACF might serve as a filter to prevent low-functioning (i.e., more oxidized) mitochondria from moving into the bud [24]. To test whether RACF affects the inheritance of mitochondria containing active Su9-DAO, we quantified mitochondria in buds of Su9-DAO-expressing Δtpm2 cells. If RACF plays a major role in preventing these mitochondria from entering the bud, it can be expected that Δtpm2 buds contain fewer mitochondria than the wild type. However, we observed that their mitochondrial content was even slightly increased (Fig 4D and 4E), suggesting that increased RACF is not sufficient to prevent dysfunctional mitochondria from moving into the bud. Deletion of the MYO1 gene is lethal in some genetic backgrounds [65,66] and produces severe cytokinesis defects in others [67–69]. We generated viable haploid Δmyo1 mutants by tetrad dissection of a heterozygous diploid deletion mutant and observed that cells were misshapen and/or multibudded, and buds were very heterogeneous in size (S3 Fig). Thus, it was not possible to analyze the role of Myo1 in mitochondrial inheritance in the genetic background used in this study.

Intracellular distribution of Ypt11 and Mmr1 is affected by mitochondrial ROS stress As recruitment of Myo2 appears to be critical for asymmetric inheritance of mitochondria under ROS stress, we further examined the roles of Ypt11 and Mmr1. GFP-Ypt11 expressed from plasmids at slightly elevated levels rescues mitochondrial inheritance defects in Δypt11 and localizes to bud tips, thereby mirroring the localization of Myo2 [32]. Consistently, GFP-Ypt11 colocalized with mitochondria in tips of small or medium-sized buds (Fig 6A). Mitochondrial inheritance was still ROS stress-sensitive in GFP-Ypt11 expressing cells (Fig 6B), and mitochondrial ROS stress led to the disappearance of GFP-Ypt11 from buds (Fig 6A and 6C), likely reflecting the mitochondrial inheritance defect under these conditions. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. Localization of Ypt11 and Mmr1 is altered upon mitochondrial ROS stress. (A and B) Δypt11 cells expressing mtERFP and Su9-DAO were transformed with plasmid p416 (MET25)-GFP-YPT11, grown in selective medium, and analyzed by fluorescence microscopy using identical camera settings. Fluorescence images are maximum intensity projections of z stacks. Mitochondrial inheritance was quantified in 100 cells per sample (triplicate experiments, error bars indicate SD calculated from the sum of cells showing reduced or efficient inheritance). (C) Cells were grown as in (A) and medium-sized buds lacking a GFP-Ypt11 signal were quantified in 100 cells per sample (triplicate experiments ± SD). (D and E) Cells carrying a genome-inserted MMR1-yEFGP allele and expressing mtERFP and Su9-DAO were analyzed by fluorescence microscopy. Fluorescence images are z stacks subjected to deconvolution. Cells were grouped into four categories: (i) diffuse Mmr1-GFP signal, (ii) signal enriched in the mother cell, (iii) at the bud neck, or (iv) in the bud. Representative cells for each category are shown in the bottom panel. Mmr1-GFP was quantified in 100 cells per sample (triplicate experiments ± SD). Bars, 5 μm. Data pooling and statistics are detailed in S2 Table. The data underlying this figure can be found in S1 Datasheet. (F) MMR1 (WT) and MMR1-yEFGP (Mmr1-GFP) cells expressing Su9-DAO were grown in the presence of L-alanine or D-alanine and cell extracts were analyzed by western blotting using anti-GFP antibodies. Hexokinase (Hxk1) served as a loading control. DAO, D-amino acid oxidase; mtERFP, mitochondria-targeted enhanced red fluorescent protein; ROS, reactive oxygen species; SD, standard deviation. https://doi.org/10.1371/journal.pbio.3002310.g006 Next, we tagged the MMR1 gene with yEGFP in the chromosome. The MMR1-yEGFP allele is functional as cells containing this allele together with the Δypt11 allele are viable (S4 Fig). Mmr1-GFP colocalized with mitochondria in tips of small and medium-sized buds or at the bud neck of large budded cells, again mirroring the localization of Myo2. This polarized distribution of Mmr1-GFP was largely lost upon induction of H 2 O 2 production in mitochondria (Fig 6D and 6E). The loss of focal Mmr1-GFP signals in the majority of stressed cells suggests that foci containing active Mmr1/Myo2 complexes disappear from the surface of H 2 O 2 producing mitochondria. Recently, it was shown that Mmr1 is subject to ubiquitination and degradation when newly inherited mitochondria reach the bud tip [70]. We asked whether enhanced turnover of Mmr1 can be triggered by mitochondrially produced H 2 O 2 . To test this, we determined Mmr1-GFP levels in western blots of total cell extracts of Su9-DAO expressing cells. We observed that Mmr1-GFP levels were very similar in cells grown in L-alanine and D-alanine-containing media (Fig 6F). This suggests that Mmr1 is released from stressed and immobilized mitochondria, but not subsequently degraded.

Mmr1 is critical for retention of ROS-stressed mitochondria in mother cells If Ypt11 and/or Mmr1 play a critical role in retention of mitochondria upon ROS stress, mitochondrial inheritance should be rendered ROS-insensitive when the YPT11 or MMR1 gene is deleted. Therefore, we examined mitochondrial inheritance in Δypt11 or Δmmr1 mutant cells in the absence or presence of Su9-DAO activity. Consistent with published observations [29,34], Δypt11 and Δmmr1 single mutants showed a moderate mitochondrial inheritance defect under non-stressed conditions (Fig 7A and 7B). Production of H 2 O 2 in mitochondria resulted in a severe reduction of mitochondrial inheritance in wild-type and Δypt11 cells. Strikingly, we found that deletion of MMR1 rendered mitochondrial inheritance insensitive to mitochondrial ROS stress, i.e., mitochondrial inheritance in Su9-DAO expressing Δmmr1 cells was very similar in L-alanine and D-alanine-containing media (Fig 7B). This suggests that Mmr1 is critical for the retention of ROS-stressed mitochondria in yeast mother cells. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. Mmr1 is required for retention of damaged mitochondria in mother cells. (A and B) Cells expressing Su9-DAO and mtGFP were incubated for 3 h in the presence of L-alanine or D-alanine. Fluorescence images are z stacks subjected to deconvolution. Mitochondrial inheritance was quantified in at least 100 cells per sample (triplicate experiments, error bars indicate SD calculated from the sum of cells showing reduced or efficient inheritance). (C) Δmmr1 Δypt11 cells expressing Tom20-Inp2-GFP and mtERFP were grown to logarithmic growth phase and analyzed by fluorescence microscopy. Fluorescence images are z stacks subjected to deconvolution. (D) Wild-type, single, and double mutant strains containing a URA3-based plasmid encoding Tom20-Inp2-GFP were allowed to lose the plasmid in medium containing uracil. Then, 10-fold serial dilutions of cells were spotted on agar plates containing minimal complete medium either without uracil or supplemented with uracil and 5-FOA, which counterselects against the URA3 marker. Lack of growth on 5-FOA medium indicates inability to lose the plasmid; i.e., the gene encoded by the plasmid is essential in this background. (E and F) Wild-type cells expressing Su9-DAO and mtERFP and containing plasmid pRS416 (empty vector control) and Δmmr1 Δypt11 cells expressing Su9-DAO, Tom20-Inp2-GFP, and mtERFP were incubated for 3 h in the presence of L-alanine or D-alanine. Fluorescence images are z stacks subjected to deconvolution. Mitochondrial inheritance was quantified in 100 cells per sample (triplicate experiments, error bars indicate SD calculated from the sum of cells showing reduced or efficient inheritance). Bars, 5 μm. Data pooling and statistics are detailed in S2 Table. The data underlying this figure can be found in S1 Datasheet. 5-FOA, 5-fluoroorotic acid; DAO, D-amino acid oxidase; mtERFP, mitochondria-targeted enhanced red fluorescent protein; mtGFP, mitochondria-targeted GFP; SD, standard deviation. https://doi.org/10.1371/journal.pbio.3002310.g007 To further test this idea, we constructed a yeast strain that is able to recruit Myo2 to mitochondria independent of Mmr1 and Ypt11. Inp2 is the Myo2 receptor on peroxisomes and interacts with the subdomain of the Myo2 cargo-binding domain distal of the Mmr1 binding site [59,71,72]. We fused the large, cytosol-exposed part of Inp2 to the membrane anchor of the mitochondrial outer membrane protein Tom20 and GFP. Tom20-Inp2-GFP formed punctate structures that largely colocalized with mitochondria in yeast cells (Fig 7C). Importantly, expression of this construct rescued lethality of the Δmmr1 Δypt11 double deletion, indicating that it replaces the function of the mitochondrial Myo2 adaptor proteins (Fig 7D). Mitochondria were inherited as efficiently as in wild-type cells under non-stressed conditions. Remarkably, their inheritance was not affected by mitochondrially generated H 2 O 2 (Fig 7E and 7F). Thus, replacement of Mmr1/Ypt11 by Tom20-Inp2-GFP renders mitochondrial inheritance insensitive to ROS stress. We conclude that the presence of Mmr1 is critical for retention of damaged mitochondria in mother cells.

Retention of ROS-stressed mitochondria in mother cells is a highly selective process So far, our results suggest that oxidative damage results in immobilization of mitochondria triggered by the release or inactivation of Myo2 and Mmr1. However, it is unclear whether this is a general stress response that affects all mitochondria in the cell. Alternatively, the cellular transport machinery might be able to differentiate between stressed and non-stressed mitochondria to selectively transport healthy mitochondria and thereby retain damaged organelles in mother cells. To test this, we developed an assay that allowed us to simultaneously observe the behavior of individual mitochondria that share the same cytosol in the same cell. To be able to track the behavior of individual mitochondria over a long period of time, we wanted to avoid fusion and matrix content mixing. Therefore, we employed the Δdnm1 Δfzo1 double mutant, whose mitochondria cannot fuse but have a wild type-like morphology [62,63] (see also Fig 4A). We constructed a Δdnm1 Δfzo1 strain that is MATa and expresses Su9-DAO and mtGFP, and another Δdnm1 Δfzo1 strain that is MATα and expresses mtERFP but lacks the Su9-DAO construct. Cells were pre-cultured in medium containing galactose to induce expression of the fluorescent mitochondrial marker proteins and either L-alanine (i.e., non-stressed control) or D-alanine (i.e., induction of mitochondrial ROS stress). Then, glucose was added to shut off the synthesis of mtGFP and mtERFP, and cultures were mixed to allow mating and zygote formation. Cells were then further incubated to allow bud formation at the zygote, and inheritance of differently labeled mitochondria was quantified (Fig 8A). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 8. Healthy mitochondria are selectively inherited in buds formed at zygotes. (A) Schematic outline of the experiment: Δdnm1 Δfzo1 cells expressing Su9-DAO and mtGFP (green) or only mtERFP (magenta) were grown in L-alanine (control) or D-alanine (ROS stress)-containing media, expression of fluorescent proteins was stopped by addition of glucose to the media, cells were mated and allowed to form buds for 3 h. (B) Fluorescence images are z stacks subjected to deconvolution. Asterisks indicate medial buds of zygotes. Bar, 5 μm. (C) Relative mitochondrial inheritance was quantified in 50 zygotes per sample by calculating the GFP and ERFP intensity ratios as shown in the equation. Arrows point to the zygote that is closest to the value of 1 for each experiment (gray, L-alanine; black, D-alanine). Inset, mean values of relative mitochondrial inheritance (arbitrary units; triplicate experiments). Data pooling and statistics are detailed in S2 Table. The data underlying this figure can be found in S1 Datasheet. DAO, D-amino acid oxidase; mtERFP, mitochondria-targeted enhanced red fluorescent protein; mtGFP, mitochondria-targeted GFP; ROS, reactive oxygen species. https://doi.org/10.1371/journal.pbio.3002310.g008 We observed that both mating partners passed on their mitochondria equally efficiently to the newly formed bud in the presence of L-alanine. When mitochondrial ROS stress was selectively induced in one mating partner by the addition of D-alanine, we observed a strong preference for transmission of mitochondria from the non-stressed counterpart. Intriguingly, more than 80% of the buds received mitochondria mainly or exclusively from the mating partner that lacked the Su9-DAO construct (Fig 8B and 8C). To exclude that this was due to the mating type or other strain-specific effects, we constructed a new set of strains with switched mating types and obtained the same result (S5 Fig). Analysis of zygotes by live cell microscopy showed that mitochondria contributed by the stressed parental cell were less mobile in the zygote, whereas mitochondria contributed by the non-stressed parental cell migrated efficiently into the emerging bud (S6 and S7 Videos). These observations demonstrate that mitochondria that are present in the same cell behave remarkably differently depending on their physiological conditions. DAO activity in the matrix renders them almost immotile and prevents their entry into the newly formed bud, whereas mitochondria without DAO activity continue to show efficient movements directed towards the bud. Obviously, these strikingly different behaviors are not due to a general deterioration of cell physiology or inactivation of cytosolic factors or cytoskeletal tracks. Rather, it appears that mitochondrial motility is regulated directly at the organellar level.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002310

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.