The cell cycle is the process by which a growing cell duplicates all of its DNA, copies each base exactly once, and then divides into two daughter cells. It consists of four phases: S, for synthesis, when the new DNA is made; M, for mitosis, when the cell splits in two; and two resting phases that separate them, G1 before S phase and G2 between S and M.
The cell cycle is tightly regulated; ensuring that cells move through it at the right rate and at the right time is essential to maintaining an organism’s healthy development. But the circle can be broken. Cancer is the ultimate example of cell cycle failure, as cancer cells divide uncontrollably, often picking up DNA damage in the process.
In a laboratory setting, it is often helpful to know what stage of the cell cycle the specimens are currently in. For example, a researcher wants to know if all the cells in a dish grow synchronously, or to confirm that the cells she treated with her drug of choice actually stop the cycle as expected.
At this point you could only tell if living cells were cycling or stalling at the time – to find out where they were in the cell cycle they had to be killed. The most commonly used cycle detection system is called Fucci, for fluorescence ubiquitination cell cycle indicator. It uses a red fluorescent protein linked to something that is at the end of the G1 phase and a green fluorescent protein linked to something that is only present in cells that pass through the S, G2, or M go phase.
So Fucci cannot distinguish between these three growth phases. This is partly because it was difficult to generate four differently colored fluorescent protein tags that can be viewed at the same time and partly because we don’t have the right things to link them to. Geminin, to which the green fluorescent protein is linked, appears at the start of S phase, but is not broken down until the cycle starts again, at the start of the next G1.
Enter researchers at Stanford, the inventor of Fucci4. Their first innovation was to mutate a red fluorescent protein to make a new color. After 26 mutations, they ended up with mMaroon1, a protein that, when excited by the correct wavelength of light, fluoresces so darkly that it can be easily distinguished from orange and yellow fluorescent proteins. Together with blue and green fluorescent proteins, they now have four colors: one to highlight each phase of the cell cycle. Transitions between phases can be viewed by watching how the colors appear in combination.
Their next modification was to link one of their markers – the blue one – to a protein that is broken down immediately after S phase. So now they have the initial G1 marker, a marker linked to a yellow fluorescent protein, and just like in the original system, the G1-S transition is indicated by the appearance of a green fluorescent protein. The disappearance of the blue protein now indicates the transition from S phase to G2; mMaroon1 appears at the G2-M transition.
Since this is a cycle, the loss of green and the reappearance of yellow and blue indicates that we have re-entered G1. Since all these colors can be tracked in living cells, you can now view a cell cycle without terminating it.
Nature Methods2016. DOI: 10.1038/nmeth.4045 (About DOIs).