Fluorescent indicators for simultaneous reporting of all four cell cycle phases

Abstract

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 fluorescent 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 fluorescent indicators that together resolve all cell cycle phases.

Figure 1

Development of a four-color imaging method and a S/G2 transition reporter. (a) mTurquoise2, Clover, mKO2, and mMaroon1 can be imaged orthogonally with appropriate excitation (left) and emission (right) filters. Excitation filters are: 440/10-nm for CFP, 490/10-nm for GFP, 545/10-nm for OFP, and 610/10-nm for far-RFP. Emission filters are: 472/30-nm for CFP, 525/30-nm for GFP, 575/25-nm for OFP, and 665/65-nm for far-RFP. The spectrum above the emission chart indicates definitions for blue (B), green (G), yellow (Y), orange (O), and red (R) in the CRC Handbook of Fundamental Spectroscopic Correlation Charts. (b) A mKO2-SLBP fusion is degraded after the end of S phase, as marked by disappearance of PCNA foci in HeLa cells. Scale bar, 10 μm. (c) FP fusions to SLBP fragments tested as candidate S/G2 transition reporters. (d) mTurquoise2-SLBP(18-126) is degraded after the end of S phase, as marked by disappearance of EYFP-PCNA foci. Arrows point to a doubly transfected cell and its daughter cells. Scale bar, 10μm. (e) Time-courses of mTurquoise2-SLBP(18-126) fluorescence during and after S phase. For each cell, fluorescence intensity was normalized to the intensity at PCNA foci disappearance. Time of nuclear envelope (NE) formation is indicated for each trace. (f) mTurquoise2-SLBP(18-126) fluorescence is cyclical through multiple cell divisions.

Figure 2

Fucci4, a reporter system for visualizing all four cell cycle stages. (a) Diagram of indicators mTurquoise2-SLBP(18-126), Clover-Geminin(1-110), mKO2-Cdt1(30–120), and H1.0-mMaroon1. (b,c) Tracking of cell cycle stages in HeLa cells via time-lapse imaging of Fucci4, including a mother cell dividing into two daughter cells (b) and daughter cells dividing into granddaughter cells (c). Deduced cell cycle phase is indicated to the right. Scale bar, 10 μm. (d) With Fucci4, S phase can be identified by high SLBP expression and low Cdt1 expression, revealing S-phase arrest by double-thymidine block. n = 125 and 226 cells for control and double thymidine treatment, respectively. (e) Representative field of cells treated by double-thymidine block. Scale bar, 10 μm.