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Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programmed cell death. The biochemical milieu is known to affect mitochondrial function and contribute to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of ageing. However, the physical characteristics of the extracellular matrix are also altered in cancer and in aging tissues. We demonstrate that cells sense the physical properties of the extracellular matrix and activate a mitochondrial stress response that adaptively tunes mitochondrial function via SLC9A1-dependent ion exchange and HSF1-dependent transcription. Overall, our data indicate that adhesion-mediated mechanosignaling may play an unappreciated role in the altered mitochondrial functions observed in aging and cancer.Graphical Abstract

In yeast, actin cables are F-actin bundles that are essential for cell division through their function as tracks for cargo movement from mother to daughter cell. Actin cables also affect yeast lifespan by promoting transport and inheritance of higher-functioning mitochondria to daughter cells. Here, we report that actin cable stability declines with age. Our genome-wide screen for genes that affect actin cable stability identified the open reading frame YKL075C. Deletion of YKL075C results in increases in actin cable stability and abundance, mitochondrial fitness, and replicative lifespan. Transcriptome analysis revealed a role for YKL075C in regulating branched-chain amino acid (BCAA) metabolism. Consistent with this, modulation of BCAA metabolism or decreasing leucine levels promotes actin cable stability and function in mitochondrial quality control. Our studies support a role for actin stability in yeast lifespan, and demonstrate that this process is controlled by BCAA and a previously uncharacterized ORF YKL075C, which we refer to as actin, aging and nutrient modulator protein 1 (AAN1).

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPR(ER)), the unfolded protein response of the mitochondria (UPR(MT)), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters are described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g. RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Mitochondria are highly dynamic organelles that undergo directed movement and anchorage, which in turn are critical for calcium buffering and energy mobilization at specific regions within cells or at sites of contact with other organelles. Physical and functional interactions between mitochondria and other organelles also impact processes, including phospholipid biogenesis and calcium homeostasis. Indeed, mitochondrial motility, localization, and interaction with other organelles are compromised in many neurodegenerative diseases. Here, we describe methods to visualize and carry out quantitative analysis of mitochondrial movement in two genetically-manipulatable, widely-used model systems: Drosophila neurons and the budding yeast, Saccharomyces cerevisiae. We also describe approaches for multi-color imaging in living yeast cells that may be used to visualize colocalization of proteins within mitochondria, as well as interactions of mitochondria with other organelles.

There exists a phenomenon in aging research whereby early life stress can have positive impacts on longevity. The mechanisms underlying these observations suggest a robust, long-lasting induction of cellular defense mechanisms. These include the various unfolded protein responses of the endoplasmic reticulum (ER), cytosol, and mitochondria. Indeed, ectopic induction of these pathways, in the absence of stress, is sufficient to increase lifespan in organisms as diverse as yeast, worms, and flies. Here, we provide an overview of the protein quality control mechanisms that operate in the cytosol, mitochondria, and ER and discuss how they affect cellular health and viability during stress and aging.

Loss of mitochondrial DNA (mtDNA) results in loss of mitochondrial respiratory activity, checkpoint-regulated inhibition of cell cycle progression, defects in growth, and nuclear genome instability. However, after several generations, yeast cells can adapt to the loss of mtDNA. During this adaptation, rho0cells, which have no mtDNA, exhibit increased growth rates and nuclear genome stabilization. Here, we report that an immediate response to loss of mtDNA is a decrease in replicative lifespan (RLS). Moreover, we find that adapted rho0cells bypass the mtDNA inheritance checkpoint, exhibit increased mitochondrial function, and undergo an increase in RLS as they adapt to the loss of mtDNA. Transcriptome analysis reveals that metabolic reprogramming to compensate for defects in mitochondrial function is an early event during adaptation and that up-regulation of stress response genes occurs later in the adaptation process. We also find that specific subtelomeric genes are silenced during adaptation to loss of mtDNA. Moreover, we find that deletion of SIR3, a subtelomeric gene silencing protein, inhibits silencing of subtelomeric genes associated with adaptation to loss of mtDNA, as well as adaptation-associated increases in mitochondrial function and RLS extension.

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., Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud.

Maintenance and regulation of proper mitochondrial dynamics and functions are necessary for cellular homeostasis. Numerous diseases, including neurodegeneration and muscle myopathies, and overall cellular aging are marked by declining mitochondrial function and subsequent loss of multiple other cellular functions. For these reasons, optimized protocols are needed for visualization and quantification of mitochondria and their function and fitness. In budding yeast, mitochondria are intimately associated with the actin cytoskeleton and utilize actin for their movement and inheritance. This chapter describes optimal approaches for labeling mitochondria and the actin cytoskeleton in living budding yeast cells, for imaging the labeled cells, and for analyzing the resulting images.

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.