Single cell analysis of yeast replicative aging using a new generation of microfluidic device

PLoS One. 2012;7(11):e48275. doi: 10.1371/journal.pone.0048275. Epub 2012 Nov 8.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / metabolism
  • Calibration
  • Cell Division
  • Chitin / metabolism
  • Gene Expression
  • Gene Expression Regulation, Fungal
  • Genes, Reporter
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / genetics
  • Microfluidic Analytical Techniques / instrumentation*
  • Peptide Elongation Factor 1
  • Peptide Elongation Factors / genetics
  • Peptide Elongation Factors / metabolism
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae / physiology*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Single-Cell Analysis / instrumentation*
  • Staining and Labeling
  • Stress, Physiological

Substances

  • Biomarkers
  • Luminescent Proteins
  • Peptide Elongation Factor 1
  • Peptide Elongation Factors
  • Saccharomyces cerevisiae Proteins
  • TEF2 protein, S cerevisiae
  • red fluorescent protein
  • Chitin