Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Abstract

Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research.

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

“…However, we were unable to observe any CR-induced lifespan extension using the microfluidic dissection platform. This discrepancy between the two methods cannot be explained by differences in medium, the absence of temperature cycles or by a general inability of the microfluidic dissection platform to generate lifespan extension, because we previously demonstrated that the microfluidic platform generates identical RLS data for several longevity mutants as the classical dissection method [e.g., median RLS of 32 vs. 34 buds for fob1Δ and 13 vs. 13 buds for sir2Δ (22)]. Thus, apparently, CR-induced lifespan extension also depends on subtle, yet unknown, differences between the methods used to determine RLS.…”

“…RLS was determined using a microfluidic dissection platform (22,37) and an inverted fluorescence microscope (Eclipse Ti-E; Nikon Instruments). The microscope was placed in an incubator (Life Imaging Services) for cultivation at 30°C.…”

“…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.…”

“…We performed a series of 10 independent RLS measurements at 2% and 0.5% (wt/vol) glucose with the microfluidic dissection platform developed by Lee et al (22). Although at 2% (wt/vol) glucose the RLS measured with the microfluidic dissection platform was similar to the average RLS that we determined from the literature (26.5 ± 0.5 vs. 25.9 ± 0.1 buds; P = 0.8), we could not find any lifespan extending effect of CR with the microfluidic dissection platform.…”

“…After removing such biased data, we determined the literature-wide average RLS of BY4741 and BY4742 at 2% (wt/vol) glucose and under CR conditions. As small sample sizes are inherent to the laborious microdissection technique most commonly used to measure RLS, we performed additional aging experiments with a recently developed microfluidic dissection platform that allows acquisition of data for large numbers of cells in a fully automated manner (22). Although identical RLSs were measured at 2% (wt/vol) glucose with the microfluidic dissection platform as with the classical microdissection method, CR failed to extend lifespan in the microfluidic dissection platform.…”