Figure 1.

Phase-dependent biosensor localization and combinatorial usage. (A) The G₁ phase biosensor construct consists of the HDHB C-terminus and tdimer2 (a dimeric red fluorescent protein). In G₁ phase (post nuclear envelope formation in the daughter cells) the biosensor is nuclear, as shown in the schematic and transfected HeLa cell (left). As the cell progresses through the cell cycle, the G₁ phase biosensor translocates to the cytoplasm (G₁/S), becoming nuclear excluded in S and G₂ phases. Breakdown of the nuclear envelope in M phase allows for fluorescence to spread throughout the rounded cell, with exclusion only at the condensed chromatin (right). Time is in hours:minutes. (B) The nuclear localized S phase biosensor consists of an NLS (SV40 nuclear location signal), EYFP, a linker (18 hydrophilic amino acids) and PCNA. Epifluorescence time-lapse images of a HeLa cell (top) and a schematic (bottom) show S phase biosensor localization through one cell cycle. The nuclei of the schematic S phase cell have been enlarged to better illustrate puncta formation and their change in morphology, which indicates replicative progression. Time is in hours:minutes. (C) Confocal images of a HeLa cell with a punctate S phase biosensor (green) and cytoplasmic G₁ phase biosensor (red) denotes S phase DNA replication. (D) Single frame analysis of coexpressed G₁ and S phase biosensors allows for the identification of the four phases. When the G₁ phase biosensor is nuclear and the S phase biosensor is nuclear but not punctuate, the cell is in G₁ phase (arrows). A cytoplasmically localized G₁ phase biosensor and a punctate S phase biosensor identify S phase cells (*). G₂ cells have cytoplasmic G₁ phase biosensor fluorescence and non-punctate S phase biosensor fluorescence (circle). Coalignment of the G₁ and S phase fluorescence in the rounded mitotic cell body occurs post nuclear envelope breakdown (square). S phase puncta are not always easy to identify, especially in early and mid S phase (#).