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Exit from Quiescence Displays a Memory of Cell Growth and Division

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Exit from Quiescence Displays a Memory of Cell Growth and Division ARTICLE DOI: 10.1038/s41467-017-00367-0 OPEN Exit from quiescence displays a memory of cell growth and division Xia Wang1,2, Kotaro Fujimaki1, Geoffrey C. Mitchell1,3, Jungeun Sarah Kwon1, Kimiko Della Croce1, Chris Langsdorf4, Hao Helen Zhang5 & Guang Yao1,6 Reactivating quiescent cells to proliferate is critical to tissue repair and homoeostasis. Quiescence exit is highly noisy even for genetically identical cells under the same environmental conditions. Deregulation of quiescence exit is associated with many diseases, but cellular mechanisms underlying the noisy process of exiting quiescence are poorly understood. Here we show that the heterogeneity of quiescence exit reflects a memory of preceding cell growth at quiescence induction and immediate division history before quiescence entry, and that such a memory is reflected in cell size at a coarse scale. The deterministic memory effects of preceding cell cycle, coupled with the stochastic dynamics of an Rb-E2F bistable switch, jointly and quantitatively explain quiescence-exit heterogeneity. As such, quiescence can be defined as a distinct state outside of the cell cycle while displaying a sequential cell order reflecting preceding cell growth and division variations. 1 Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA. 2 School of Biological and Medical Engineering, Hefei University of Technology, Hefei, Anhui 230009, China. 3 Department of Biology, Wofford College, Spartanburg, SC 29303, USA. 4 Molecular Probes, Thermo Fisher Scientific, Eugene, OR 97402, USA. 5 Department of Mathematics, University of Arizona, Tucson, AZ 85721, USA. 6 Arizona Cancer Centre, University of Arizona, Tucson, AZ 85719, USA. Xia Wang, Kotaro Fujimaki and Geoffrey C. Mitchell equally contributed to this work. Correspondence and requests for materials should be addressed to G.Y. (email: [email protected]) NATURE COMMUNICATIONS | 8: 321 | DOI: 10.1038/s41467-017-00367-0 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00367-0 ut of the 1013 ~1014 cells in our body, the vast majority Particularly, is this heterogeneity caused by stochastic events, Oare non-dividing. While many non-dividing cells can no or deterministic and predictable variations, in the cell population? longer proliferate, such as cells in senescence or terminal Given that a critical size control has been observed during the – differentiation, quiescent cells (e.g., lymphocytes, hepatocytes, G1-S transition of cycling eukaryotic cells10 12, and that quiescent stem and progenitor cells) retain their proliferative potential. In hematopoietic cells were shown to need to grow in size before response to physiological signals, typically serum growth factors, restarting proliferation13,wefirst examined whether quiescence- quiescent cells can be activated to re-enter the cell cycle, exit heterogeneity was associated with cell size differences in a rat – which serves as the basis for tissue homoeostasis and repair1 3. embryonic fibroblast (REF) cell model. We found that depending Recent studies have shown that quiescence is not simply a passive on experimental conditions, cell size may or may not appear to be fall-back state lacking proliferative activities, but is rather an associated with the observed quiescence-exit heterogeneity. actively maintained state1, 2, 4 that provides protection against Further modelling and experimental analysis showed that long-term cellular stress and toxicity1, 5. quiescence-exit heterogeneity was associated with both the Quiescence exit is highly heterogeneous. In a clonal culture preceding cell growth at quiescence induction by serum induced to quiescence by the same condition (e.g., serum star- starvation and the cell division status prior to quiescence entry vation), individual quiescent cells exhibit significantly different (‘preceding cell growth and division’ for short). Meanwhile, cell – paces in restarting the cell cycle upon serum stimulation6 8. size reflected preceding cell growth and division at a coarse but Furthermore, upon non-saturating serum stimulation (at an not fine scale. Our study showed that the deterministic variations intermediate concentration or with a short pulse), some cells re- in preceding cell cycle, coupled with stochastic noise in an enter the cell cycle while others remain quiescent6, 7, 9. Con- Rb-E2F bistable switch that underlies the quiescence-to- ceivably, a heterogeneous transition from quiescence to proliferation transition9, 14, determine the heterogeneity of proliferation can be beneficial in vivo by avoiding exhausting a quiescence exit and cell cycle re-entry. Lastly, our analysis pool of quiescent cells completely with a single stimulus. also suggests that quiescence, while being a distinct state outside It meanwhile poses a therapeutic challenge since cells remaining of the cell cycle, displays a sequential cell order reflecting quiescent (e.g., certain cancer stem cells) are difficult to target. a memory of preceding cell growth and division. This new Mechanisms underlying the heterogeneity in quiescence exit are, quiescence model helps settle the long debate over whether however, poorly understood. quiescent cells are located in a distinct G0 phase or simply paused In this study, we set out to investigate what accounts for the along a G1 continuum, and reveals a previously underappreciated heterogeneous quiescence exit in a supposedly homogeneous, mechanism underlying the heterogeneous growth responses of clonal cell population under the same culture conditions. quiescent cells. ab c 1.0 0.12 Serum pulse 0.8 (20% serum, pd hrs) 0.08 0.6 0.4 0.04 Measurement 0.2 Distal tail Promixmal end 0.02% 0.0 0.00 Q E2F−ON probability density E2F−ON cumulative fraction 024681012 0 pd 24 (h) 121086420 Serum−pulse duration (h) Serum−pulse duration (h) deMeasurment Measurment 21 h 24 h 38 h Serum Serum S’ L’ S L pulse pulse 0 h 4 h 0 h 0 h 8 h 50% 40% 4 h E2F-OFF-ON 30% Large cells 20% 3.5 h Small Cell fraction cells 8 h 10% SSC CT dye Intensity d1 d0 0% E2F-dGFP CT dye Intensity S’ L’ S L Fig. 1 Quiescence-exit distribution and cell size correlation. a Experimental scheme for measuring quiescence-exit heterogeneity. Q, quiescent state (the same below). b Cumulative fraction of quiescence-exit (E2F-ON) cells over serum pulse duration. Each dot represents a unique sample. The sigmoid curve indicates a best fit of data points y ¼ a . c Quiescence-exit distribution as the derivative of the sigmoidal curve in b. d Serum-starved quiescent 1þeÀbðxÀcÞ REF/E23 cells were stained with CT violet dye at time 0, and stimulated with serum pulse at indicated durations. E2F-dGFP and CT levels were measured at the indicated time points after serum pulse initiation. e Serum-starved, CT-labelled quiescent cells as in d were stimulated with serum pulses at indicated durations. CT dye intensity and SSC were measured at the 38th hour after serum pulse initiation, as shown in the density plot (left); the percentage of cells in each labelled CT bin (S’,L’,S,L, as described in the text) is shown for each serum pulse group (right). For simplicity, CT bins were considered not to overlap. Cells of d1 and d0 labels correspond to those that divided and did not divide following a serum pulse, respectively 2 NATURE COMMUNICATIONS | 8: 321 | DOI: 10.1038/s41467-017-00367-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00367-0 ARTICLE a b Drive cells to different early-to-late G1 positions Significantly larger (P < 0.01) 26 First serum pulse Second serum pulse m) 5, 10, 15 h 0, 4, 6 h µ 24 Return to quiescence Quiescence-exit measurement 22 (no division) Q Cell diameter ( 20 0 2.5 d (First pulse) Mimosine 5 h 5 h 10 h 15 h 10 h 15 h (G1/S block) 0 1 d Time 0 Right after Right before first pulse second pulse c Second pulse d Right before second pulse 0 h 4 h 6 h 0.14 30 0.12 (First pulse) 0.10 5 h 20 0.08 15 h 0.06 E2F-ON% 10 0.04 0.02 Probability density 0 0 5 10 15 5 10 15 5 10 15 (h) 15 17 19 21 23 25 27 29 31 33 First pulse Cell diameter (µm) e f Significantly larger First serum pulse Second serum pulse 26 (P < 0.01) 15, 20 h 5 h m) µ 24 22 Quiescence-exit measurement 20 (Cell division) 18 Q Cell diameter ( 16 0 3 d 15 h 20 h 15 h 20 h (First pulse) 0 1 d Time 0 Right after Right before CT dye first pulse second pulse g h 15 h first pulse group 20 h first pulse group Right after first pulse Right before second pulse 105 105 (38.7%) (57.8%) 0.16 0.16 4 (First pulse) (First pulse) 10 104 0.12 15 h 0.12 15 h 3 3 0.08 20 h 0.08 20 h 10 10 0 0 0.04 0.04 E2F-dGFP level 0 0 Probability density Probability density 3 4 5 3 4 5 15 19 23 27 29 15 19 23 27 29 17 21 25 31 33 17 21 25 31 33 10 10 10 10 10 10 Cell diameter (µm) Cell diameter (µm) CT dye intensity Fig. 2 Quiescence-exit likelihood is correlated with cell size at quiescence induction. a Experimental scheme. E2F-dGFP activity was measured one day after the initiation of the second serum pulse. See text for details. b Cell size measured right before the first serum pulse (time 0), after the first pulse (of 5–15 h), and before the second serum pulse, following the scheme in a. Star sign indicates statistical significance P < 0.01) in one-sided (arrow pointed) t-test comparing the means of cell diameters in two cell populations (20,000–30,000 cells each).
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