Cell Based Assays in Cell Cycle II

GFP-HDHB(CT)/RFP-HDHB(CT)
The C-terminal region of human DNA helicase B (HDHB) is a 99 amino acids polypeptide that contains both a putative nuclear localization signal (NLS) and a nuclear export signal (NES). It undergoes cell cycle-dependent translocation. GFP-HDHBCT is a fusion of GFP and the C-terminal region of HDHB. It remains inside the nucleus during G1 phase, then translocates into and remains in the cytoplasm during S and G2 phases. After nuclear envelope breaks down in M phase, this biosensor is localized throughout the cell. After nuclear envelope reformation at the end of M phase, the G1 phase biosensor is imported into the nucleus.

Hahn AT, Jones JT, Meyer T. Quantitative analysis of cell cycle phase durations and PC12 differentiation using fluorescent biosensors. Cell Cycle. 2009 Apr 1;8(7):1044-52. Epub 2009 Apr 2.

PM-YFP-NLS
This biosensor is a fusion protein comprising three components, a reversible plasma membrane–targeting domain fused to the N terminus of enhanced yellow fluorescent protein (EYFP), which is in turn fused to the N terminus of a nuclear localization signal (NLS). The nuclear localization signal would anchor the reporter’s localization to the nucleus during the G1, S and G2 phases (interphase) of the cell cycle. Upon the nuclear envelope breakdown at the onset of prometaphase, the plasma membrane–binding domain would cause reversible translocation of the biosensor to the plasma membrane where the fluorescence could be monitored by microscopy.

Jones JT, Myers JW, Ferrell JE, Meyer T. Probing the precision of the mitotic clock with a live-cell fluorescent biosensor. Nat Biotechnol. 2004 Mar;22(3):306-12.

E2F-mClaverGR TRE Reporter
The E2F family of transcription factors is a key regulator of cell-cycle checkpoints in mammalian cells. The E2F protein is a major target of the retinoblastoma gene product (Rb) and the activity of E2F/pRb is intimately connected with the G1-S transition of the cell cycle. The E2F protein forms a heterodimer complex with DP1, which binds to E2F response elements and initiate transcription.
The E2F-responsive mClaverGR construct encodes a photoconvertible green-to-red fluorescent protein reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the E2F transcriptional response element (TRE). We have experimentally optimized the number of response elements as well as the intervening sequence between response elements to maximize the signal to noise ratio.

Ki67p-GFP
Ki67, encoded by MKI67 gene, is expressed in all phases of the active cell cycle (G1, S, G2 and M phase), while is absent in the resting phase (G0). Therefore, it is routinely used as a marker of cell cycling and proliferation. Recently, Zambon AC[1] cloned and characterized the 1.5 kb proximal promoter (Ki67p) of the human Ki67 gene. A reporter, Ki67p-GFP, was further constructed to express GFP under Ki67p. Their data verified that GFP driven by Ki67p is co-expressed in cells with endogenous Ki67 and is correlated with cells transitioning through G1/S/G2/M phases of the cell cycle. Mitomycin C induced G1/S/G2/M blocking or cell-density induced cell cycle arrest both attenuate Ki67p activity.

Zambon AC. Use of the Ki67 promoter to label cell cycle entry in living cells.Cytometry A. 2010 Jun;77(6):564-70.

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Tags: cell assays, cell cycle, G1 phase G2 phase, interphase, M phase, nuclear envelop, nuclear localization, S Phase

Cell Cycle Assays-Part I

This is the first part of a series of blogs about using fluorescent proteins in cell based assays with established examples, a common theme here at the AlleleBlog.

FUCCI Cell Cycle Sensor

The FUCCI Cell Cycle Sensor is composed of a red (RFP) and a green (GFP) fluorescent protein fused to different regulators of the cell cycle: cdt1 and geminin.

During the cell cycle, these two proteins are ubiquitinated at different time points by specific ubiquitin E3 ligases, which tag them for degradation in the proteasome. The E3 ligases’ activities are regulated temporally and result in the biphasic cycling of GERMINI and CDT1 levels during the cell cycle. In the G1 phase of the cell cycle, GERMINI is degraded; therefore, only CDT1 tagged with RFP is present and appears as red fluorescence within the nuclei. In the S, G2, and M phases, CDT1 is degraded; only GERMINI tagged with GFP is present, resulting in cells with green fluorescent nuclei.

During the G1/S transition, when CDT1 levels are decreasing and GERMINI levels increasing, both proteins are present, so are the tagged fluorescent proteins. When the green and red images are overlaid, nuclei fluoresce yellow. This dynamic color change, from red-to-yellow-to-green, represents the entire cell cycle. This representation can be used to study the effects of elements that may influence cell cycles.

Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, Imamura T, Ogawa M, Masai H, Miyawaki A.Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008 Feb 8;132(3):487-98.

CCNB1-CyclinB(NT)-GFP

In late S phage, CCNB1 promoter will be switched on to drive the expression of Cyclin B N-terminus-GFP expression; thereafter the fluorescent signal will be switched off at the destruction box in Cyclin B N-terminus at the end of Mitosis phase. During the intervening phase the fusion reporter protein will translocate from cytoplasm to nucleus by the cytoplasmic retention signal in the Cyclin B N-terminus.

Thomas N. Lighting the circle of life: fluorescent sensors for covert surveillance of the cell cycle. Cell Cycle. 2003 Nov-Dec;2(6):545-9.

GFP-PCNA/YFP-PCNA
GFP-PCNA, a fusion of GFP and PCNA, has been widely used as a convenient tool to monitor the progress of S phase. At the onset of S phase, GFP-PCNA translocates into the nucleus; at mitosis the nuclear envelope breaks down and the nuclear accumulation of PCNA-GFP dissipates.

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Tags: cell based assays, cell cycle, cell cycle arrest, DNA-repair, Fluorescent proteins, M phase, mitosis, S Phase