Aging Yeast Cells Undergo a Sharp Entry into Senescence Unrelated to the Loss of Mitochondrial Membrane Potential

103

Cellular aging plays an important role in many diseases, such as cancers, metabolic syndromes, and neurodegenerative disorders. There has been steady progress in identifying aging-related factors such as reactive oxygen species and genomic instability, yet an emerging challenge is to reconcile the contributions of these factors with the fact that genetically identical cells can age at significantly different rates. Such complexity requires single-cell analyses designed to unravel the interplay of aging dynamics and cell-to-cell variability. Here we use microfluidic technologies to track the replicative aging of single yeast cells and reveal that the temporal patterns of heterochromatin silencing loss regulate cellular life span. We found that cells show sporadic waves of silencing loss in the heterochromatic ribosomal DNA during the early phases of aging, followed by sustained loss of silencing preceding cell death. Isogenic cells have different lengths of the early intermittent silencing phase that largely determine their final life spans. Combining computational modeling and experimental approaches, we found that the intermittent silencing dynamics is important for longevity and is dependent on the conserved Sir2 deacetylase, whereas either sustained silencing or sustained loss of silencing shortens life span. These findings reveal that the temporal patterns of a key molecular process can directly influence cellular aging, and thus could provide guidance for the design of temporally controlled strategies to extend life span.replicative aging | single-cell analysis | microfluidics | chromatin silencing | computational modeling C ellular aging is generally driven by the accumulation of genetic and cellular damage (1, 2). Although much progress has been made in identifying molecular factors that influence life span, what remains sorely missing is an understanding of how these factors interact and change dynamically during the aging process. This is in part because aging is a complex process wherein isogenic cells have various intrinsic causes of aging and widely different rates of aging. As a result, static population-based approaches could be insufficient to fully reveal sophisticated dynamic changes during aging. Recent developments in single-cell analyses to unravel the interplay of cellular dynamics and variability hold the promise to answer that challenge (3-5). Here we chose the replicative aging of yeast S. cerevisiae as a model and exploited quantitative biology technologies to study the dynamics of molecular processes that control aging at the single-cell level.

Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Multigenerational silencing dynamics control cell aging

Jin

O’Laughlin

et al. 2017

103

“…As the classical microdissection method is a labor intensive technique to determine RLS, especially when larger sample sizes are required, we questioned whether the recently developed microfluidic dissection methods (22,27,28) could generate similar lifespan data. The advantage of using a microfluidic dissection method is that RLS can be measured in a semiautomated manner with a large number of cells under constant environmental conditions, i.e., without incubation at low temperature during the night and with constant glucose concentration.…”

Section: Cr Does Not Extend Replicative Lifespan In a Microfluidic DImentioning

confidence: 99%

Calorie restriction does not elicit a robust extension of replicative lifespan inSaccharomyces cerevisiae

Huberts

González

Lee

et al. 2014

Calorie restriction (CR) is often described as the most robust manner to extend lifespan in a large variety of organisms. Hence, considerable research effort is directed toward understanding the mechanisms underlying CR, especially in the yeast Saccharomyces cerevisiae. However, the effect of CR on lifespan has never been systematically reviewed in this organism. Here, we performed a meta-analysis of replicative lifespan (RLS) data published in more than 40 different papers. Our analysis revealed that there is significant variation in the reported RLS data, which appears to be mainly due to the low number of cells analyzed per experiment. Furthermore, we found that the RLS measured at 2% (wt/vol) glucose in CR experiments is partly biased toward shorter lifespans compared with identical lifespan measurements from other studies. Excluding the 2% (wt/vol) glucose experiments from CR experiments, we determined that the average RLS of the yeast strains BY4741 and BY4742 is 25.9 buds at 2% (wt/vol) glucose and 30.2 buds under CR conditions. RLS measurements with a microfluidic dissection platform produced identical RLS data at 2% (wt/vol) glucose. However, CR conditions did not induce lifespan extension. As we excluded obvious methodological differences, such as temperature and medium, as causes, we conclude that subtle methodspecific factors are crucial to induce lifespan extension under CR conditions in S. cerevisiae.I n the last decades, a nutritious diet low in calories, commonly referred to as calorie restriction (CR), has been reported to extend the lifespan of a broad range of organisms, such as yeast, worms, fruit flies, mice, rats, and monkeys (1-6). This seemingly evolutionary conserved effect of CR on aging has sparked intense investigation into its underlying molecular mechanisms, especially in the budding yeast Saccharomyces cerevisiae (7-10). Some studies suggest that nutrient-responsive pathways, such as target of rapamycin (TOR), protein kinase A (PKA), and Sch9 (11), mediate CR-induced lifespan extension, whereas others hypothesize that CR increases the activity of sirtuin deacetylases (e.g., SIR2) extending lifespan by suppressing ribosomal DNA recombination (9, 12, 13).In S. cerevisiae, lifespan can either be defined by the time cells remain viable in absence of nutrients, called chronological lifespan, or by the number of daughter cells a yeast cell produces during its life, which is referred to as replicative lifespan (RLS) (14). Although RLS can vary greatly between individual yeast cells, the average RLS (i.e., across a population of cells) is assumed to be a genotypic trait (15-19). However, besides CR, other environmental conditions, such as pH and carbon source, are known to have a strong impact on RLS (20, 21).Here, through a systematic analysis of RLS data of S. cerevisiae using data from more than 27,000 cells, we obtained indication that small sample sizes might be the cause for the often observed significant variation between RLS data. Further, we identified that partly biased data exis...

“…Microfluidic devices have been developed to capture yeast cells for high-resolution imaging analysis during vegetative growth (16)(17)(18)(19)(20). Recently, such devices have been designed that enable the tracking of yeast cells throughout their lifespan, making it possible to record and study cellular phenotypic changes during aging (21)(22)(23).…”

Section: Saccharomyces Cerevisiaementioning

confidence: 99%

High-throughput analysis of yeast replicative aging using a microfluidic system

Liu

et al. 2015

150

174

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a highthroughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction.microfluidics | high-throughput | replicative aging | calorie restriction | Saccharomyces cerevisiae