Generating kinetic environments to study dynamic cellular processes in single cells

Sci Rep. 2019 Jul 12;9(1):10129. doi: 10.1038/s41598-019-46438-8.

Abstract

Cells of any organism are consistently exposed to changes over time in their environment. The kinetics by which these changes occur are critical for the cellular response and fate decision. It is therefore important to control the temporal changes of extracellular stimuli precisely to understand biological mechanisms in a quantitative manner. Most current cell culture and biochemical studies focus on instant changes in the environment and therefore neglect the importance of kinetic environments. To address these shortcomings, we developed two experimental methodologies to precisely control the environment of single cells. These methodologies are compatible with standard biochemistry, molecular, cell and quantitative biology assays. We demonstrate applicability by obtaining time series and time point measurements in both live and fixed cells. We demonstrate the feasibility of the methodology in yeast and mammalian cell culture in combination with widely used assays such as flow cytometry, time-lapse microscopy and single-molecule RNA Fluorescent in-situ Hybridization (smFISH). Our experimental methodologies are easy to implement in most laboratory settings and allows the study of kinetic environments in a wide range of assays and different cell culture conditions.

Publication types

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

MeSH terms

  • Algorithms
  • Cell Line
  • Cell Shape
  • Equipment Design
  • Gene Expression Regulation
  • Humans
  • In Situ Hybridization, Fluorescence
  • Interrupted Time Series Analysis
  • Kinetics
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism
  • Mitogen-Activated Protein Kinases / metabolism
  • Saccharomyces cerevisiae / cytology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Signal Transduction
  • Single Molecule Imaging / methods
  • Single-Cell Analysis / instrumentation
  • Single-Cell Analysis / methods*
  • Time-Lapse Imaging

Substances

  • Membrane Transport Proteins
  • STL1 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • HOG1 protein, S cerevisiae
  • Mitogen-Activated Protein Kinases