Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle

TY - JOUR

T1 - Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle

AU - Sakaue-Sawano, Asako

AU - Yo, Masahiro

AU - Komatsu, Naoki

AU - Hiratsuka, Toru

AU - Kogure, Takako

AU - Hoshida, Tetsushi

AU - Goshima, Naoki

AU - Matsuda, Michiyuki

AU - Miyoshi, Hiroyuki

AU - Miyawaki, Atsushi

PY - 2017/10/26

Y1 - 2017/10/26

N2 - Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitylation alone or in combination with SCFSkp2-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.

AB - Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitylation alone or in combination with SCFSkp2-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.

KW - cell cycle

KW - ubiquitin ligase

KW - Fucci

KW - embryonic stem cell

KW - interphase

KW - DNA damage

KW - fluorescent protein

KW - time-lapse imaging

KW - flow cytometry

U2 - 10.1016/j.molcel.2017.10.001

DO - 10.1016/j.molcel.2017.10.001

M3 - Article

SN - 1097-2765

JO - MOLECULAR CELL

JF - MOLECULAR CELL

ER -