FUCCI sensors: powerful new tools for analysis of cell proliferation

The FUCCI concept. (a) The original FUCCI sensors mark cells residing in G1 phase with red fluorescence, while cells in S/G2/M are labeled in green. During a short period at the G1/S transition, both probes are present and hence the cells appear yellow. (b) Domain structure of the human Geminin‐based S/G2/M sensors. DB, destruction box; NLS, nuclear localization signal; CC, coiled‐coil domain. (c) Domain structure of the human Cdt1‐based G1 sensor. PIP, PCNA interaction motif; Cy, Cy motif; CC, coiled‐coil domain. (d) Time plot illustrating the sequential degradation of the FUCCI probes. Nuclear mAG‐hGem_1–110 or pan‐localized mAG‐hGem_1–60 accumulates during S and G2 phase, but is targeted for degraded during late mitotis and G1 phase by the E3 ligase, APC/C. The nuclear mKO‐hCdt1_30–120 probe accumulates during G1 phase and is degraded during S and G2 phase by the SCF^Skp2 complex. (e) Overview of the fluorescent proteins that produce functional FUCCI sensors.

Timeline illustrating the invention of the different FUCCI variants and the key discoveries that have been made with them.

FUCCI in flies. (a) Fly‐FUCCI marks cells in G1 phase in green, cells in S phase are labeled in red, and cells in G2 phase express both markers and therefore appear yellow. (b) Domain structure of Drosophila E2F1. PIP, PCNA interaction motif; DNA, DNA binding motif; MB, marked box; TA, transactivation domain. (c) Schematic of Drosophila cyclin B. DB, destruction box; CB, cyclin box. (d) Time plot illustrating the sequential destruction of the Fly‐FUCCI probes. GFP‐E2F1_1–230 accumulates during G1 phase, but is rapidly destroyed during S phase by CRL4^Cdt2‐mediated degradation. Levels of GFP‐E2F1_1–230 recover during G2 phase. mRFP1‐CycB_1–266 degradation is mediated by APC/C and lasts from mid‐mitosis throughout G1 phase. Levels of mRFP1‐CycB_1–266 increase in S phase and reach their maximum at the end of G2 phase. (e) Table describing the promoter/fluorochrome combinations of the currently available Fly‐FUCCI transgenes. (f) Schematic of the multicistronic Fly‐FUCCI construct that has been optimized for the use in Drosophila cell lines. T2A autocleavage sites separate both, the Fly‐FUCCI probes and the neomycin resistance gene, thereby allowing rapid selection of stable cell lines.

(a) The zebrafish FUCCI systems labels cells residing in G1 phase by red fluorescence, whereas green fluorescence indicates cells in S or G2 phase. (b) Domain structure of zebrafish Cdt1 compared to human Cdt1. PIP, PCNA interaction motif; Cy, Cy motif; CC, coiled‐coil domain. (c) Comparison of the functional domains of zebrafish and human Geminin. DB, destruction box; NLS, nuclear localization signal; CC, coiled‐coil domain. (d) Time plot illustrating the sequential degradation of the zFUCCI probes. Nuclear mAG‐zGem_1–100 or pan‐localized mAG‐zGem_1–60 accumulates during S and G2 phase, but is targeted for proteolysis during late mitotic stages and G1 phase by APC/C. The nuclear mKO‐hCdt1_30–120 probe is rapidly destroyed upon initiation of DNA replication by the S‐phase‐specific E3 ligase CRL4^Cdt2. (e) Schematic of the multicistronic dual FUCCI construct. Cerulian‐3x‐FLAG‐zGem_1–100 and Cherry‐zCdt_1–190 are expressed as a single polypeptide under the control of the zebrafish ubiquitin promoter. Both FUCCI probes are separated by a 2A sequence, which self‐cleaves upon expression.

FUCCI‐expressing mice. (a) In R26P‐FUCCI 2 mice, G1 cells are constitutively labeled by red fluorescence, whereas cells in S and G2 phases are marked in yellow. (b) Diagram of the R26P‐FUCCI 2 expression construct. mCherry‐hCdt1_30–120 and mVenus‐hGem_1–110 are bidirectionally expressed from the ubiquitous rosa26 promoter (R26p). Two copies of the cHSC4 insulator separate the individual components of the R26P‐FUCCI 2 construct. (c) R26R‐mCherry‐hCdt1_30–120 mice conditionally mark cells in G1 phase by red fluorescence. (d) R26R‐mVenus‐hGem_1–110 mice conditionally label cells in S/G2 phase by yellow fluorescence. (e) Either mCherry‐hCdt1_30–120 or mVenus‐hGem_1–110 was targeted to the rosa26 locus. To control the expressions of mCherry‐hCdt1_30–120 or mVenus‐hGem_1–110 in time and space, a neo cassette (neomycin‐resistant gene expressed under the control of the PGK1 promoter) flanked by loxP sequences was placed in front of each probe. The neo cassette can be excised by Cre‐mediated loxP recombination, resulting in expression of the FUCCI probes in all Cre‐expressing cells. SA, adenovirus splice acceptor. (g) R26R‐FUCCI2aR mice conditionally mark cells in G1 phase by red fluorescence and cells S/G2 phase by yellow fluorescence. (f) The multicistron FUCCIa2R construct was inserted in reverse orientation into the Rosa26 locus. The targeting construct contains the CAG promotor, a stop cassette consisting of the neomycin resistance gene flanked by loxP sites, mCherry‐hCdt1_30–120 and mVenus‐hGem_1–110. Both FUCCI probes are separated by a 2A auto‐cleavages site, which produces iso‐stoichiometric quantities of both FUCCI probes.