Cell-Cycle States and Control Logic

Fuse a cell that is copying its DNA with a cell that has not started yet, and the second nucleus begins copying early. Fuse it with a cell that has already finished, and that nucleus refuses.

Something in the cytoplasm says “copy now”, and something in the nucleus remembers whether it already has. This lesson measures those states, then follows the fusion, frog and yeast experiments to the engine behind them: a kinase whose partner rises and falls.

Phases, and how you count them

The cycle has four phases. G1 is the gap between mitosis and the start of DNA replication. In S phase the genome is copied (the mechanics belong to DNA replication). G2 is the gap after copying and before mitosis. In M phase, mitosis sorts the duplicated chromosomes into two nuclei, and cytokinesis, the split of the cell itself, usually follows. Mammalian cells can also leave G1 for G0, a resting state they enter when conditions change and can leave again.

The first measurement is DNA content. One C is the DNA of one haploid genome, so a diploid cell has 2C in G1 and 4C in G2 and M. Doubling the DNA does not double the ploidy: a G2 cell is still diploid, with each chromosome made of two sister chromatids. To read DNA content, fix the cells and stain them with propidium iodide, a dye that fluoresces when bound to nucleic acid, plus RNase, because the dye also binds RNA. A flow cytometer measures each cell’s fluorescence one at a time and builds a histogram: a 2C peak, a 4C peak, and S-phase cells smeared between.

The histogram has two blind spots. G0 cells sit in the 2C peak with G1, and mitotic cells sit in the 4C peak with G2. Two more stains fix that. EdU, a thymidine analogue, is built into DNA made during a short pulse and is then detected by click chemistry with a fluorescent tag, so it marks cells in S. An antibody to histone H3 phosphorylated on serine 10 marks condensed mitotic chromosomes. Saldivar and colleagues combined all three, read by image-based cytometry rather than flow, to separate phases in human RPE-1 and MCF10A cells (Science, 2018).

Worked problem: fractions are not durations

The numbers here are illustrative. Suppose an asynchronous culture of a diploid human cell line doubles every 24 h, every cell is cycling, and none die. Of 10,000 cells, 5,500 fall in the 2C peak, 2,000 in the 4C peak and 2,500 between. A 30-minute EdU pulse labels 25% of cells, which agrees that the cells between the peaks are in S. And 3% are phospho-H3 positive; assume the stain marks only mitotic cells.

Step 1. Fractions: G1 0.55, S 0.25, G2 plus M 0.20, of which M is 0.03.

Step 2. The tempting move is to multiply by 24 h: 13.2, 6.0 and 4.8 h. That is wrong. Each division turns one old cell into two newborn ones, so a growing culture always holds twice as many cells just born as cells about to divide. Early phases are overrepresented.

Step 3. For a culture in steady exponential growth, the time a cell takes to pass the first fraction F of the population, ordered by age, is

t = -T × log2(1 - F/2), where T is the doubling time

• End of G1 (F = 0.55): t = -24 × log2(0.725) = 24 × 0.464 = 11.13 h.
• End of S (F = 0.80): t = -24 × log2(0.600) = 17.69 h, so S lasts about 6.55 h (working from unrounded values).
• Start of M (F = 0.97): t = -24 × log2(0.515) = 22.98 h. M lasts 24 - 22.98 = 1.02 h, and G2 lasts 22.98 - 17.69 = 5.29 h.

Check: 11.13 + 6.55 + 5.29 + 1.02 = 23.99 h, 24 h within rounding. The naive estimate made G1 two hours too long and G2 plus M (6.31 h) one and a half hours too short. The model assumes identical phase lengths in every cell and no resting or dying cells, so treat these as estimates.

Asking the cell: fusion and cytoplasm transfer

In 1970 Rao and Johnson fused cultured HeLa cells taken from different phases, so that two nuclei shared one cytoplasm. As later reviews describe the results, a G1 nucleus in a cell fused with an S-phase cell started replicating early, while a G2 nucleus in the same situation did not. Two conclusions follow. S-phase cytoplasm carries an activity that can start replication. And a nucleus that has already replicated cannot respond until it has been through mitosis, which is how DNA gets copied once per cycle and no more. (The molecular basis, licensing of replication origins in G1, belongs to DNA replication.)

Fusing a mitotic cell with an interphase cell had a different outcome: the interphase nucleus condensed its chromosomes prematurely (Johnson and Rao, 1970), and how the chromosomes looked depended on the phase of that nucleus. Mitotic cytoplasm is dominant.

A frog experiment pointed the same way. A frog oocyte waits in prophase of its first meiotic division until progesterone tells it to resume. In 1971 Yoshio Masui and Clement Markert took cytoplasm from progesterone-treated oocytes of the leopard frog Rana pipiens and injected it into untreated oocytes, which then matured. Removing the donor’s nucleus did not prevent the activity. They called it maturation-promoting factor (MPF). Smith and Ecker reported a similar transfer in the same year. The activity was later found in mitotic cells too.

Both kinds of experiment reveal an activity, not a molecule.

Asking the genes: cdc mutants in two yeasts

In 1970 Leland Hartwell, Joseph Culotti and Brian Reid described temperature-sensitive mutants of budding yeast, Saccharomyces cerevisiae, that grow at a low temperature and stop at a higher one, each with a uniform shape. They called them cdc, for cell division cycle. The bud reports the stage: none in G1, small in S, large by mitosis.

Their key idea was to separate two times. A car that runs out of fuel stops where the tank ran dry, not at the station you drove past. The execution point is when a gene’s function is normally performed. The termination point, or arrest point, is where cells collect when it is not. Shift a growing culture to the restrictive temperature and follow single cells: cells already past the execution point finish this cycle and stop in the next, while cells not yet past it stop in this one. The fraction that finishes tells you where the execution point lies. In the 1970 paper, the cdc-3 mutant kept budding and dividing its nucleus while its cells failed to separate, so steps of the cycle can come apart. One warning: their budding-yeast cdc-2, which stopped at mitosis, is not the fission-yeast cdc2 below. The budding-yeast gene that matters here is CDC28, needed at Start, the commitment point in G1.

Paul Nurse used fission yeast, Schizosaccharomyces pombe, a rod that divides in the middle, so length tells you its age. In 1975 he described a mutant that divided at a smaller size than normal. The mutation changed when division happened, not whether. Most later small-cell mutants mapped to a gene named wee1; one mapped to cdc2, whose other alleles block mitosis. So cdc2 behaves as an accelerator and wee1 as a brake. By 1981 Nurse and Yvonne Bissett concluded that cdc2 acts both in G1 and in G2. In 1982 David Beach, Barbara Durkacz and Nurse found that budding-yeast DNA carrying CDC28 rescued a fission-yeast cdc2 mutant: two distant yeasts, one job.

Cyclins meet Cdc2: one engine

In 1983 Tim Hunt’s group followed new proteins in dividing embryos of the sea urchin Arbacia punctulata. One built up through each cycle and was destroyed at each division; they named it cyclin and saw similar proteins in another sea urchin and a clam. Disappearing at division is a correlation. Tests came next. Clam cyclin A mRNA injected into Xenopus oocytes made them resume meiosis (Swenson, Farrell and Ruderman, 1986). In Xenopus egg extracts that cycle in a tube, destroying the messenger RNA stopped the cycles in interphase and adding back cyclin mRNA restored them, while a cyclin that could not be destroyed held the extract in mitosis (Murray and Kirschner, with Solomon, 1989).

The lines met in 1988. Lohka, Hayes and Maller purified MPF from Xenopus eggs, and antibodies to fission-yeast Cdc2 recognised its 32 kDa protein (Gautier and colleagues), and Dunphy and colleagues reached Cdc2 by a separate route; in 1990 the other component proved to be cyclin B. MPF is Cdc2, now called CDK1, bound to cyclin B.

The logic, as it is now taught:

• CDKs (cyclin-dependent kinases) are the catalytic subunits, inactive alone. Cyclins are regulatory subunits that switch them on and steer them to targets; cyclin levels rise and fall while CDK levels stay fairly even. In mammals, cyclin D with CDK4 or CDK6 acts in G1, cyclin E with CDK2 at the G1/S transition, cyclin A with CDK2 in S, and cyclins A and B with CDK1 in G2 and M. Growth-factor signals reach cyclin D (see Ras to MAPK), and the Rb brake it releases is in cancer genetics.
• Wee1 adds an inhibitory phosphate to CDK1, holding the complex off while cyclin accumulates; the phosphatase Cdc25 removes it (the chemistry is in the phosphorylation switch).
• CDK inhibitor proteins, such as p21 and p27 in mammals, bind the complex and block it.
• Two ubiquitin ligases destroy regulators: the SCF at several points, notably around G1/S, the APC/C in mitosis, where destroying cyclin B ends M phase (ubiquitin and proteolysis).

Active CDK1 also activates Cdc25 and inhibits Wee1, two positive loops that make mitotic entry a switch (signaling circuits). In Xenopus egg extracts, entry needed about 32 to 40 nM of added nondegradable cyclin B, but an extract already in mitosis stayed there down to about 16 to 24 nM, and unreplicated DNA raised the entry threshold. Read the fusion results in this light: mitotic cytoplasm is full of active CDK1-cyclin B, enough to condense any nucleus it meets.

Design problem

Using cell fusion, design experiments to ask whether S-phase cytoplasm can drive replication in another nucleus, and why a G2 nucleus might not respond.