Theresa C. Swayne

Mitochondrial quality control during inheritance is associated with lifespan and mother-daughter age asymmetry in budding yeast

Fluorescence loss in photobleaching experiments and analysis of mitochondrial function using superoxide and redox potential biosensors revealed that mitochondria within individual yeast cells are physically and functionally distinct. Mitochondria that are retained in mother cells during yeast cell division have a significantly more oxidizing redox potential and higher superoxide levels compared to mitochondria in buds. Retention of mitochondria with more oxidizing redox potential in mother cells occurs to the same extent in young and older cells and can account for the age‐associated decline in total cellular mitochondrial redox potential in yeast as they age from 0 to 5 generations. Deletion of Mmr1p, a member of the DSL1 family of tethering proteins that localizes to mitochondria at the bud tip and is required for normal mitochondrial inheritance, produces defects in mitochondrial quality control and heterogeneity in replicative lifespan (RLS). Long‐lived mmr1Δ cells exhibit prolonged RLS, reduced mean generation times, more reducing mitochondrial redox potential and lower mitochondrial superoxide levels compared to wild‐type cells. Short‐lived mmr1Δ cells exhibit the opposite phenotypes. Moreover, short‐lived cells give rise exclusively to short‐lived cells, while the majority of daughters of long‐lived cells are long lived. These findings support the model that the mitochondrial inheritance machinery promotes retention of lower‐functioning mitochondria in mother cells and that this process contributes to both mother–daughter age asymmetry and age‐associated declines in cellular fitness.

Role for cER and Mmr1p in Anchorage of Mitochondria at Sites of Polarized Surface Growth in Budding Yeast

Mitochondria accumulate at neuronal and immunological synapses and yeast bud tips and associate with the ER during phospholipid biosynthesis, calcium homeostasis, and mitochondrial fission. Here we show that mitochondria are associated with cortical ER (cER) sheets underlying the plasma membrane in the bud tip and confirm that a deletion in YPT11, which inhibits cER accumulation in the bud tip, also inhibits bud tip anchorage of mitochondria. Time-lapse imaging reveals that mitochondria are anchored at specific sites in the bud tip. Mmr1p, a member of the DSL1 family of tethering proteins, localizes to punctate structures on opposing surfaces of mitochondria and cER sheets underlying the bud tip and is recovered with isolated mitochondria and ER. Deletion of MMR1 impairs bud tip anchorage of mitochondria without affecting mitochondrial velocity or cER distribution. Deletion of the phosphatase PTC1 results in increased Mmr1p phosphorylation, mislocalization of Mmr1p, defects in association of Mmr1p with mitochondria and ER, and defects in bud tip anchorage of mitochondria. These findings indicate that Mmr1p contributes to mitochondrial inheritance as a mediator of anchorage of mitochondria to cER sheets in the yeast bud tip and that Ptc1p regulates Mmr1p phosphorylation, localization, and function.

Inheritance of the fittest mitochondria in yeast

Eukaryotic cells compartmentalize their biochemical processes within organelles, which have specific functions that must be maintained for overall cellular health. As the site of aerobic energy mobilization and essential biosynthetic activities, mitochondria are critical for cell survival and proliferation. Here, we describe mechanisms to control the quality and quantity of mitochondria within cells with an emphasis on findings from the budding yeast Saccharomyces cerevisiae. We also describe how mitochondrial quality and quantity control systems that operate during cell division affect lifespan and cell cycle progression.

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae

Fzo1p contributes to mitochondrial inheritance by fusion of mitochondria that enter the bud to mitochondria that are anchored in the bud tip. This promotes retention of mitochondria in the bud tip. However, it also promotes anchorage of lower-functioning mitochondria in the bud tip, which inhibits clearance of those organelles from buds.

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

Blocking farnesylation of the prelamin A variant in Hutchinson-Gilford progeria syndrome alters the distribution of A-type lamins.

Mutations in the lamin A/C gene that cause Hutchinson-Gilford progeria syndrome lead to expression of a truncated, permanently farnesylated prelamin A variant called progerin. Blocking farnesylation leads to an improvement in the abnormal nuclear morphology observed in cells expressing progerin, which is associated with a re-localization of the variant protein from the nuclear envelope to the nuclear interior. We now show that a progerin construct that cannot be farnesylated is localized primarily in intranuclear foci and that its diffusional mobility is significantly greater than that of farnesylated progerin localized predominantly at the nuclear envelope. Expression of non-farnesylated progerin in transfected cells leads to a redistribution of lamin A and lamin C away from the nuclear envelope into intranuclear foci but does not significantly affect the localization of endogenous lamin B1 at nuclear envelope. There is a similar redistribution of lamin A and lamin C into intranuclear foci in transfected cells expressing progerin in which protein farnesylation is blocked by treatment with a protein farnesyltransferase inhibitor. Blocking farnesylation of progerin can lead to a redistribution of normal A-type lamins away from the inner nuclear envelope. This may have implications for using drugs that block protein prenylation to treat children with Hutchinson-Gilford progeria syndrome. These findings also provide additional evidence that A-type and B-type lamins can form separate microdomains within the nucleus.

Chapter 19 Visualization of mitochondrial movement in yeast

Publisher Summary This chapter describes methods for using mitochondria-specific vital dyes and optical microscopy to study mitochondrial movement and morphology in living cells. The budding yeast, Saccharomyces cerevisiae, is used as a model system. However, the techniques described are readily applicable to the study of mitochondrial dynamics in other eukaryotes. Many potential-sensing mitochondrial dyes have been developed. The chapter focuses on several that are known to work well in yeast: DiOC 6 (3), DASPMI, rhodamine 123, rhodamine B hexyl ester, and the MitoTracker family. The selection of a suitable dye for a given application should be based on several factors. The chapter explores that, if double labeling is desired (with a fluorescent protein or another vital dye), the mitochondrial dye chosen must have nonoverlapping excitation and emission spectra. A dye should be tested on the strain of interest to find a concentration that provides sufficient sensitivity and specificity. Finally, the fluorescence must be stable enough to persist for the required observation time without cytotoxicity.

Visualization of mitochondria in budding yeast.

Publisher Summary This chapter describes the labeling methods and optical approaches for visualizing yeast mitochondria, using fluorescence microscopy. It is now feasible for laboratories to acquire and analyze high-resolution multidimensional images of yeast and structures within yeast, including mitochondria. As mitochondria cannot be detected in yeast using transmitted-light microscopy (phase-contrast or differential interference contrast), methods have been developed to visualize the organelle with vital fluorescent dyes, immunofluorescence, or targeted fluorescent proteins (FPs). For studies of morphology and dynamics of mitochondrial membranes, the laboratory uses FPs targeted to the matrix or the inner surface of the inner mitochondrial membrane. FPs can be targeted to mitochondria by two methods both of which employ fusion proteins. One approach relies on ectopic expression of fusion proteins consisting of mitochondrial signal sequences fused to FPs. The other method is the fusion of FPs to endogenous mitochondrial proteins. Signal sequences contain all the information that is required for import of proteins from the cytosol into mitochondria. They may reside at the N-terminus, C-terminus, or within nuclear-encoded mitochondrial proteins. Mitochondrial signal sequences have been used to target FPs to mitochondria and specific compartments within mitochondria. The chapter discusses several plasmid-borne targeted FPs to label yeast mitochondria. All of the targeted FPs used produces a robust fluorescent signal that is specific for mitochondria and have no deleterious effect on cell growth or on mitochondrial morphology, motility, or respiratory activity. Applications and outcomes for each of the commonly used approaches are also summarized in the chapter.

Imaging of the Actin Cytoskeleton and Mitochondria in Fixed Budding Yeast Cells

The budding yeast Saccharomyces cerevisiae is widely used as a model system to study the organization and function of the cytoskeleton. In the past, its small size, rounded shape, and rigid cell wall created obstacles to explore the cell biology of this model eukaryote. It is now possible to acquire and analyze high-resolution and super-resolution multidimensional images of the yeast cell. As a result, imaging of yeast has emerged as an important tool in eukaryotic cell biology. This chapter describes labeling methods and optical approaches for visualizing the cytoskeleton and interactions of the actin cytoskeleton with mitochondria in fixed yeast cells using wide-field and super-resolution fluorescence microscopy.

Fluorescence imaging of mitochondria in yeast.

The budding yeast Saccharomyces cerevisiae has many advantages as a model system, but until recently high-resolution microscopy was not often attempted in this organism. Its small size, rounded shape, and rigid cell wall were obstacles to exploring the cell biology of this model eukaryote. However, it is now feasible for laboratories to acquire and analyze high-resolution, multidimensional images of yeast cell biology, including the mitochondria. As a result, imaging of yeast has emerged as an important tool in eukaryotic cell biology. This chapter describes labeling methods and optical approaches for visualizing yeast mitochondria using fluorescence microscopy.