The cell cycle is the process in which a growing cell duplicates all of its DNA, copying each base precisely 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 into two; and two resting gap phases separating them, G1 before S phase and G2 between S and M.
The cell cycle is tightly regulated; ensuring that cells proceed through it at the proper pace and at the proper time is essential to maintaining an organism’s healthy development. But the cycle can be broken. Cancer is the ultimate example of the cell cycle gone awry, as cancer cells divide uncontrollably, often picking up DNA damage as they go.
In a laboratory setting, it’s often useful to know which phase of the cell cycle one’s specimens are currently in. A researcher might want to know if all the cells in a dish are growing synchronously, for example, or to confirm that the cells she treated with her favorite drug are in fact stopping the cycle as expected.
At this point, you could only tell whether living cells were currently either cycling or stalled—to figure out where they were in the cell cycle required killing them. The most commonly used cycling-detection system is called Fucci, for fluorescence ubiquitination cell cycle indicator. It employs a red fluorescent protein linked to something that’s at the end of the G1 phase and a green fluorescent protein linked to something that’s only present in cells going through the S, G2, or M phase.
So Fucci cannot distinguish among these three growth phases. This is partially because it has been hard to generate four differently colored fluorescent protein labels that can be viewed simultaneously and partially because we don’t have the right things to link them to. Geminin, which the green fluorescent protein is linked to, appears at the beginning of S phase but isn’t degraded until the cycle starts again, at the start of the next G1.
Enter researchers from Stanford, who invented Fucci4. Their first innovation was mutating a red fluorescent protein to make a new color. After 26 mutations, they ended up with mMaroon1, a protein that, when excited by the appropriate wavelength of light, fluoresces darkly enough that it can be easily distinguished from orange and yellow fluorescent proteins. Along with blue and green fluorescent proteins, they now have four colors—one to mark each phase of the cell cycle. Transitions between phases can be watched by looking at how the colors appear in combination.
Their next tweak was to link one of their markers—the blue one—to a protein that is degraded immediately after S phase. So now they have the initial G1 marker, a marker linked to a yellow fluorescent protein, and as in the original system, the G1-S transition is denoted by the appearance of a green fluorescent protein. The blue protein’s disappearance now indicates the transition from S phase into G2; mMaroon1 shows up 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 entered G1 again. Since all of these colors can be tracked in living cells, you can now watch a cell cycle without terminating it.