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Fluorescent indicators for simultaneous reporting of all four cell cycle phases
Bryce T Bajar1,2,10,11, Amy J Lam1,2,11, Ryan K Badiee3, Young-Hee Oh1,2,4, Jun Chu1,2,10, Xin X Zhou1,2,4, Namdoo Kim1,2,4, Benjamin B Kim1,2,4, Mingyu Chung5, Arielle L Yablonovitch6, Barney F Cruz7,
Kanokwan Kulalert8, Jacqueline J Tao1, Tobias Meyer5, Xiao-Dong Su9 & Michael Z Lin1,2,4
A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red uorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal uorescent indicators that together resolve all cell cycle phases.
Proper regulation of the cell division cycle is essential for normal development1,2, stem cell renewal3, and tissue maintenance4. In addition, cell cycle abnormalities in cancer contribute to tumor growth and further genetic damage5. As any cell cycle phase can be regulated or perturbed, methods to report all four phases in living cells would be useful. In the widely used Fucci reporter system, G1-phase cells are labeled with one fluorescent protein (FP) fused to the protein Cdt130120; while cells in S, G2, or M phases are labeled with another FP fused to the protein Geminin1110
(refs. 6 and 7). Fucci has also been modified to add a G0G1 transition reporter8. However, while Fucci and its variants reveal whether cells are within one of the proliferative phases of the cell cycle (S, G2, or M), they do not report which of these three phases cells are in.
Additional channels for live-cell imaging would be desirable for reporting multiple events such as cell cycle phases. For three-color time-lapse imaging, cyan, yellow, and red fluorescent proteins (CFPs, YFPs, and RFPs) are often used8,9, thus avoiding the phototoxic excitation wavelengths required for blue fluorescent proteins (BFPs)10. Some large-Stokes shift RFPs, those excited
exclusively by blue light and not by the yelloworange excitation wavelengths for standard RFPs, could constitute a fourth channel, but their photobleaching rates are not well suited for time-lapse imaging11.
We instead explored the possibility of creating four channels by further red shifting of far-red fluorescent proteins (far-RFPs) to enable orthogonal...