Mitochondria must grow with the growing cell to ensure proper cellular physiology and inheritance upon division. We measured the physical size of mitochondrial networks in budding yeast and found that mitochondrial network size increased with increasing cell size and that this scaling relation occurred primarily in the bud. The mitochondria to cell size ratio continually decreased in aging mothers over successive generations. However, regardless of mother age or mitochondrial content, all buds attained the same average ratio. Thus, yeast populations achieve a stable scaling relation between mitochondrial content and cell size despite asymmetry in inheritance.

A major limitation to yeast aging study has been the inability to track mother cells and observe molecular markers during the aging process. The traditional lifespan assay relies on manual micro-manipulation to remove daughter cells from the mother, which is laborious, time consuming, and does not allow long term tracking with high resolution microscopy. Recently, we have developed a microfluidic system capable of retaining mother cells in the microfluidic chambers while removing daughter cells automatically, making it possible to observe fluorescent reporters in single cells throughout their lifespan. Here we report the development of a new generation of microfluidic device that overcomes several limitations of the previous system, making it easier to fabricate and operate, and allowing functions not possible with the previous design. The basic unit of the device consists of microfluidic channels with pensile columns that can physically trap the mother cells while allowing the removal of daughter cells automatically by the flow of the fresh media. The whole microfluidic device contains multiple independent units operating in parallel, allowing simultaneous analysis of multiple strains. Using this system, we have reproduced the lifespan curves for the known long and short-lived mutants, demonstrating the power of the device for automated lifespan measurement. Following fluorescent reporters in single mother cells throughout their lifespan, we discovered a surprising change of expression of the translation elongation factor TEF2 during aging, suggesting altered translational control in aged mother cells. Utilizing the capability of the new device to trap mother-daughter pairs, we analyzed mother-daughter inheritance and found age dependent asymmetric partitioning of a general stress response reporter between mother and daughter cells.

SummaryBudding yeast has served as an important model organism for aging research, and previous genetic studies have led to the discovery of conserved genes/pathways that regulate lifespan across species. However, the molecular causes of aging and death remain elusive, because it is very difficult to directly observe the cellular and molecular events accompanying aging in single yeast cells by the traditional approach based on micromanipulation. We have developed a microfluidic system to track individual mother cells throughout their lifespan, allowing automated lifespan measurement and direct observation of cell cycle dynamics, cell/organelle morphologies, and various molecular markers. We found that aging of the wild‐type cells is characterized by an increased general stress and a progressive lengthening of the cell cycle for the last few cell divisions; these features are much less apparent in the long‐lived FOB1 deletion mutant. Following the fate of individual cells revealed that there are different forms of cell death that are characterized by different terminal cell morphologies, and associated with different levels of stress and lifespan. We have identified a molecular marker – the level of the expression of Hsp104, as a good predictor for the lifespan of individual cells. Our approach allows detailed molecular phenotyping of single cells in the process of aging and thus provides new insight into its mechanism.

SUMMRAY Telomerase is required for long-term telomere maintenance and protection. Using single budding yeast mother cell analyses we found that, even Early after Telomerase Inactivation (ETI), yeast mother cells show transient DNA Damage Response (DDR) episodes, stochastically altered cell cycle dynamics, and accelerated mother cell aging. The acceleration of ETI mother cell aging was not explained by increased reactive oxygen species (ROS), Sir protein perturbation, or deprotected telomeres. ETI occurred well before the population senescence and caused late after telomerase inactivation (LTI). ETI phenotypes were morphologically distinct from LTI senescence, genetically uncoupled from telomere length, and were rescued by elevating dNTP pools. Our combined genetic and single-cell analyses show that well before critical telomere shortening, telomerase is continuously required to respond to transient DNA replication stress in mother cells, and that a lack of telomerase accelerates otherwise normal aging.

Single-cell transcriptome analysis through next-generation sequencing (single-cell RNA-seq) has been used broadly to address important biological questions. It has proved to be very powerful, and many exciting new biological discoveries have been achieved in the last decade. Its application has greatly improved our understanding of diverse biological processes and the underlying molecular mechanisms, an understanding that would not have been achievable by conventional analysis based on bulk populations. However, so far, single-cell RNA-seq analysis has been used mostly for higher organisms. For microorganisms, single-cell RNA-seq has not been widely used, mainly because the stiff cell wall prevents effective lysis, much less starting RNA material is obtained, and the RNA lacks polyadenylated tails for universal priming of mRNA molecules. In general, the detection efficiency of current single-cell RNA-seq technologies is very low, and further development or improvement of these technologies is required for exploring the microbial world at single-cell resolution. Here, we briefly review recent developments in single-cell RNA-seq of microorganisms and discuss current challenges and future directions.

The budding yeast Saccharomyces cerevisiae (S. cerevisiae) has relatively short lifespan and is genetically tractable, making it a widely used model organism in aging research. Here, we carried out a systematic and quantitative investigation of yeast aging with single‐cell resolution through transcriptomic sequencing. We optimized a single‐cell RNA sequencing (scRNA‐seq) protocol to quantitatively study the whole transcriptome profiles of single yeast cells at different ages, finding increased cell‐to‐cell transcriptional variability during aging. The single‐cell transcriptome analysis also highlighted key biological processes or cellular components, including oxidation–reduction process, oxidative stress response (OSR), translation, ribosome biogenesis and mitochondrion that underlie aging in yeast. We uncovered a molecular marker of FIT3, indicating the early heterogeneity during aging in yeast. We also analyzed the regulation of transcription factors and further characterized the distinctive temporal regulation of the OSR by YAP1 and proteasome activity by RPN4 during aging in yeast. Overall, our data profoundly reveal early heterogeneity during aging in yeast and shed light on the aging dynamics at the single cell level.

Triazole derivativesTriazole derivatives R 0280 Fundamental Structure-Activity Relationships Associated with a New Structural Class of Respiratory Syncytial Virus Inhibitor. -A series of benzotriazolederived inhibitors of respiratory syncytial virus fusion is prepared and their structure-activity relationships are examined. The results indicate that the topology of the side chain is important but the terminus element offers considerable latitude to modulate physical properties. -(YU, K.-L.; ZHANG, Y.; CIVIELLO, R. L.; KADOW, K. F.; CIANCI, C.; KRYSTAL, M.; MEANWELL*, N. A.; Bioorg. Med. Chem. Lett. 13 (2003) 13, 2141-2144; Dep. Chem., Bristol-Myers Squibb Pharm. Res. Inst., Wallingford, CT 06492, USA; Eng.) -F. Santoso 42-129

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