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. 2014 Feb 6:4:4012.
doi: 10.1038/srep04012.

A novel cell-cycle-indicator, mVenus-p27K-, identifies quiescent cells and visualizes G0-G1 transition

Toshihiko Oki  1 , Koutarou Nishimura  2 , Jiro Kitaura  2 , Katsuhiro Togami  2 , Akie Maehara  2 , Kumi Izawa  2 , Asako Sakaue-Sawano  3 , Atsushi Niida  4 , Satoru Miyano  5 , Hiroyuki Aburatani  6 , Hiroshi Kiyonari  7 , Atsushi Miyawaki  3 , Toshio Kitamura  1
Affiliations

A novel cell-cycle-indicator, mVenus-p27K-, identifies quiescent cells and visualizes G0-G1 transition

Toshihiko Oki et al. Sci Rep. .

Abstract

The quiescent (G0) phase of the cell cycle is the reversible phase from which the cells exit from the cell cycle. Due to the difficulty of defining the G0 phase, quiescent cells have not been well characterized. In this study, a fusion protein consisting of mVenus and a defective mutant of CDK inhibitor, p27 (p27K(-)) was shown to be able to identify and isolate a population of quiescent cells and to effectively visualize the G0 to G1 transition. By comparing the expression profiles of the G0 and G1 cells defined by mVenus-p27K(-), we have identified molecular features of quiescent cells. Quiescence is also an important feature of many types of stem cells, and mVenus-p27K(-)-transgenic mice enabled the detection of the quiescent cells with muscle stem cell markers in muscle in vivo. The mVenus-p27K(-) probe could be useful in investigating stem cells as well as quiescent cells.

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Figures

Figure 1
Figure 1. mVenus-p27K− probe identifies quiescent cells.
(A) Various constructs with concatenated mVenus and p27. (B) Typical images of NIH3T3 transfectants (Scale, 200 μm). (C) Representative histograms for DNA contents of NIH3T3 (right) and of the fluorescent positive cells (red or green solid line), and the percentages of the fluorescent positive cells in G1/0 cells (left). (D) Representative histograms (upper) and relative median intensity (%) (Relative F.I.) (lower) of fluorescence of NIH3T3 in cycling (black) or serum starvation (red). (E–F) Representative Ki-67 staining of NIH3T3 (E) or Ba/F3 cells (F) or Lewis lung carcinoma cells (F) expressing mVenus-p27K-. NC, isotype control.
Figure 2
Figure 2. mVenus-p27K− and mCherry-hCdt1(30/120) probes effectively visualize G0 to G1 transition.
(A) Typical images and (B) changes in normalized fluorescent intensity (Flu.Int.) of mVenus-p27K−, mCherry-hCdt1(30/120), and AmCyan-hGeminin(1/110) in NIH3T3 cells at cell cycle entry (Scale, 10 μm).
Figure 3
Figure 3. Expression profiles of G0(p27K−(+)/hCdt1(30/120)(+)) cells and G1((p27K− (−)/hCdt1(30/120)(+)) cells.
(A) FACS analysis for G0 and G1 cells. (B) Scatter plot with X and Y axis indicating the expression level of each gene in G1 and G0 cells respectively. (C–D) Catalogue of differently expressed genes: the top genes enriched (C) in G0 cells and (D) in G1 cells. (E) GSEA comparison of G0 against G1 cells. NES, normalized enrichment score; FDR, false discovery rate.
Figure 4
Figure 4. Enhanced expression of several tumor suppressors was observed in the G0 phase.
(A) The expression for the G0-associated genes and Mki67. (B–D) Changes in the levels of tumor suppressors in NIH3T3 cells after (B) serum addition following serum starvation or (C) serum starvation or (D) in Ba/F3 cells after the serum and IL-3 addition following their depletion. Data were analyzed by qPCR and represented as values normalized to (A) G1 cells (mean ± S.D, n = 4) or (B–D) the levels at 0 hr (mean ± S.D, n = 3). *, P < 0.05.
Figure 5
Figure 5. mVenus-p27K− positive population in skeletal muscle includes muscle satellite cells.
(A) Typical FACS analysis. BM, bone marrow; Sp, spleen; Thy, thymus (B) Typical fluorescent images of sections of muscles (Scale, 200 μm) (right). (C) FACS and (D) qPCR (mean ± S.D, n = 3) for marker expression of muscles. *, P < 0.05.
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References

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