Cell-Cycle Analysis of Fission Yeast Cells by Flow Cytometry

Abstract

The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G1 and G2 phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G1 phase. We have devised a flow cytometric method exploiting the fact that cells in G1 phase contain two nuclei, whereas cells in G2 are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G1 and in G2 phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.

Spd2 assists Spd1 in modulation of RNR architecture but does not regulate deoxynucleotide pools

“…Spd1 and Spd2 levels were monitored by western blotting. The fraction of S phase cells (Knutsen et al, 2011) peaked at 50% between 80 and 120 minutes. Unlike Spd1, Spd2 was not strongly downregulated at S phase.…”

“…interpretation of FACS data in fission yeast is complicated by the fact that G1 and S phase cells are still attached to their sisters, thus giving rise to a 2C signal. However, by measuring the DNA signal width, it is possible to discriminate G1 and S cells from G2 cells (Knutsen et al, 2011), thus giving a more precise estimate of the S phase population. By using this method, we found that overexpression of Spd2 caused a strong increase in the number of cells undergoing S phase, suggesting that the protein -similarly to Spd1 -delays DNA replication ( Fig.…”

“…(B) Quantification of FACS analysis of cells overexpressing Spd1 or Spd2. Cell-cycle distribution was analyzed as reported previously (Knutsen et al, 2011). S1, the fraction of S phase cells with one nucleus; S2, binucleate cells (as defined by the width of the DNA signal) undergoing replication.…”

“…Quantification of S phase cells with the aid of the DNA signal width was performed as described previously (Knutsen et al, 2011). For each sample, a total of 30,000 cells were counted, and after gating at least 75% were still present.…”

An Imaging Flow Cytometry-based approach to analyse the fission yeast cell cycle in fixed cells

“…1D). It has been elegantly shown that other DNA-derived pulse parameters such as width can be utilised to distinguish G1 and G2 cells based on the assumption that cells with 2Â 1C nuclei should orientate through the long axis and as such have an increased time of flight through the laser beam that translates into an increased pulse width [4]. While this approach has merit, we feel that a truly accurate analysis of the fission yeast cell cycle requires an image-based approach due to the fact that not all cells will orientate correctly through the laser interrogation point and may rotate around the x, y and z axes (Fig.…”