The first lesson of this course opened with Virchow's claim that every cell comes from a cell, and this one is how that happens.
Division is the hardest thing a cell does, because it has to be done in the right order and exactly once. The genome must be copied completely, copied only once, and then separated into two equal sets without losing or breaking anything. A human cell doing this is handling 2.2 metres of DNA in a compartment a few micrometres across, and it succeeds nearly every time.
Four phases, and why the order is not negotiable
The cell cycle is conventionally divided into four stages. S phase is DNA synthesis. M phase is mitosis and the physical division that follows it. Between them sit two gaps, G1 before S and G2 after it, which are not idle: they are where the cell grows, and where it decides whether to proceed.
For a human cell in culture dividing every 24 hours, S takes about 8 hours, G2 about 4, M about 1, and G1 the remaining 11. G1 is by far the most variable, and a cell that stops dividing does so from G1, entering a state called G0 that may last for the rest of its life. Most cells in an adult body are in G0, and a liver hepatocyte can sit there for years and re-enter the cycle after injury.
The order matters absolutely. Dividing before replication finishes gives two incomplete genomes. Replicating twice before dividing gives a tetraploid cell. Separating chromosomes that have not all attached to the spindle loses one. Each of those failures is fatal or worse, so the cycle needs both a driver, which pushes it forward, and a supervisor, which stops it when something is not ready.
Finding the driver
Three lines of work, in three organisms, produced the answer, and they were rewarded with a shared Nobel Prize in 2001.
Leland Hartwell, working on budding yeast in the early 1970s, isolated temperature-sensitive mutants that grew normally at one temperature and arrested at a specific point in the cycle at another. That each mutant arrested at its own defined point is the key observation: it means the cycle is not a smooth continuum but a series of discrete steps, each requiring a particular gene product. He called the genes cdc, for cell division cycle, and identified a control point in G1 he named start, at which a cell commits to a division.
Paul Nurse did the same in fission yeast and found cdc2, whose product is required at more than one point in the cycle, and whose loss arrests division while certain other alleles cause cells to divide too soon at too small a size. A gene whose mutation can either block division or advance it is a controller rather than a component. Then in 1987 Nurse's laboratory did something that ought to be startling: they took a library of human complementary DNA, put it into a fission yeast lacking cdc2, and found a human gene that rescued it completely. The human protein, now called CDK1, does the job of a yeast protein across roughly a billion years of divergence.
Tim Hunt, working on sea urchin eggs in 1982, found the other half. Fertilised urchin eggs divide synchronously, so proteins can be labelled and followed across a cycle in a whole population. One protein accumulated steadily through each cycle and then disappeared abruptly at each division, over and over. He called it cyclin.
A kinase and a clock
Put the two together. CDK1 is a protein kinase, present at a roughly constant level throughout the cycle and inactive on its own. Cyclin is its activating partner, and cyclin concentration oscillates: synthesised steadily, then destroyed suddenly. The active kinase phosphorylates a large set of substrates that carry out the events of mitosis, so the cycle's timing is set by the availability of cyclin rather than by the kinase.
Different cyclins partner different kinases at different points, so the cell has several such switches in series: D-type cyclins in G1, cyclin E at the G1 to S transition, cyclin A through S phase, and cyclin B driving mitosis. Each activates the machinery for its own stage.
The destruction step is the part worth thinking about. Cyclin is not inhibited at the end of mitosis, it is ubiquitinated by a large ligase called the anaphase promoting complex and destroyed by the proteasome, using exactly the machinery described two lessons ago. Why destroy rather than inhibit? Because inhibition is reversible and destruction is not. A cell emerging from mitosis must not slip back into it, and the only way to make a molecular transition one-way is to consume something. This is the same argument that appeared in the lesson on ATP, where a biosynthetic step was made irreversible by hydrolysing pyrophosphate: irreversibility always costs a molecule.
Example. Bacteria have no cyclins and no cyclin-dependent kinases, and they still divide, coordinate replication with division, and get it right. What does that tell you about how much of what this lesson describes is a general requirement of living things?
Very little of it is general, and that is worth being clear about. The requirements that are general are logical: replicate once, segregate accurately, divide the cytoplasm, and do not start one step before the previous one is finished. The cyclin machinery is one particular implementation of those requirements, invented in the eukaryotic lineage, and bacteria implement them differently. A bacterium ties the initiation of replication to cell size and to the state of the initiator protein DnaA, segregates its chromosome partly by the physical action of replication itself pushing the daughter copies apart, and divides using a ring of FtsZ, which is a distant relative of tubulin, at a site positioned by an oscillating inhibitor system. The comparison also explains an asymmetry in difficulty. A bacterium has one small circular chromosome and can begin segregating it while still copying it, while a eukaryote has dozens of long linear ones that must be fully copied, condensed and captured before any can move, so the eukaryotic problem genuinely needs a control system that the bacterial one does not. When a piece of machinery looks universal, the useful check is whether the requirement is universal or only the solution you happen to have studied.
Now you. A cell is engineered to express a form of cyclin B that cannot be ubiquitinated and therefore cannot be destroyed. Predict what happens, and be specific about which step fails.
Answer
The cell enters mitosis normally and cannot leave it. Chromosomes condense, the nuclear envelope breaks down, the spindle forms and chromosomes align, but the cell arrests, typically in anaphase or before completing it, and never returns to interphase. The specific failures are that the substrates phosphorylated by cyclin B and CDK1 stay phosphorylated as long as the kinase is active, and dephosphorylating them is what drives nuclear envelope reformation, chromosome decondensation and spindle disassembly. There is a second, subtler failure. The same ligase that destroys cyclin B also destroys securin, whose destruction releases the protease that cuts the cohesin holding sister chromatids together, so a cell in which that ligase is prevented from acting on one substrate is often prevented from separating sisters at all. The experiment was done with a truncated cyclin B lacking its destruction box, and it is the direct demonstration that exit from mitosis requires cyclin destruction rather than merely following it in time. It also illustrates a general experimental strategy: to show that a scheduled destruction is causal rather than incidental, make the target indestructible and see whether the schedule breaks.
Supervision
The driver would run whether or not the cell was ready, so the cycle is watched by checkpoints. Each is a surveillance system that detects a problem and holds the cycle until it is fixed.
The G1 checkpoint, called the restriction point in animal cells, asks whether the cell is large enough, whether growth factors are present, and whether the DNA is damaged. Its central mechanism is the retinoblastoma protein, Rb, which binds and inhibits the E2F transcription factors that switch on S phase genes; G1 cyclin-CDK activity phosphorylates Rb, releasing E2F, and the cell commits. Damage acts through p53, which halts the cycle and can trigger apoptosis if the damage is severe.
The G2 checkpoint asks whether replication is complete and whether the DNA is intact, and blocks entry into mitosis if not.
The spindle assembly checkpoint asks whether every chromosome is properly attached, and it is the most striking of the three because of its sensitivity. A single unattached kinetochore, one out of ninety-two in a human cell, generates a diffusible inhibitory signal that prevents the anaphase promoting complex from acting anywhere in the cell. One unsatisfied attachment out of ninety-two holds the entire division.
The evidence that these are surveillance systems rather than parts of the machinery is that they can be removed. Yeast checkpoint mutants grow perfectly well under good conditions and die when the conditions are made difficult, which is exactly what one expects of a monitor and not of a component. Hartwell and Ted Weinert made that argument in 1989, and it is what gave the word checkpoint its meaning.
A related point is that attaching correctly is not simply detected but actively corrected. A chromosome whose two kinetochores attach to the same pole is under no tension, and the kinase Aurora B destabilises attachments that lack tension, so wrong attachments are released and the search is retried. Detection would only stall the cycle; correction is what lets it finish.
Example. The spindle assembly checkpoint works by generating an inhibitory "wait" signal from unattached kinetochores rather than a permissive "go" signal from attached ones. Why must it be built that way round?
Because of the arithmetic of vetoes. A human cell has ninety-two kinetochores, and the requirement is that anaphase waits if even one is unattached. An inhibitory signal satisfies this naturally: one source of inhibitor is enough to hold the cell, and the signal only ceases when the last kinetochore is attached, so the condition being detected is exactly the condition that matters. A permissive scheme would have to detect the absence of one contribution out of ninety-two, a change of about one per cent against a large background, in a system with substantial molecular noise, and it would fail silently in the dangerous direction whenever the noise obscured the shortfall. Building the signal so that the unsafe state is the one that shouts, and the safe state is silence, means that any failure of the sensor tends to delay division rather than to permit a wrong one. Engineers call this a fail-safe interlock and cells found it first. The same logic explains why the machinery detects unattached kinetochores directly, at the kinetochore, rather than inferring completeness from some global measure.
Now you. Predict what happens to a cell line in which the spindle checkpoint is weakened but not abolished, and say why complete loss is rarely seen in tumours.
Answer
A weakened checkpoint lets anaphase begin before every chromosome is correctly attached, so chromosomes are occasionally mis-segregated and the population accumulates cells with abnormal chromosome numbers. That is chromosomal instability, and it is a defining feature of many solid tumours: it generates the variation on which selection for further malignancy acts, including loss of the remaining copy of a tumour suppressor, which the two-hit argument below shows is otherwise a rare event. Complete loss is rarely seen because it is lethal. A cell with no checkpoint at all mis-segregates so severely and so often that the resulting daughters are usually inviable, so the mutations that survive are those that degrade the checkpoint rather than remove it. The practical consequence is a therapeutic idea that is being tested: if tumour cells are already close to the tolerable limit of mis-segregation and normal cells are not, then a drug that pushes segregation error rates up slightly should kill the tumour selectively, which is the reasoning behind inhibitors of the checkpoint kinases and of the spindle motors.
The mechanics
Mitosis itself is a physical problem. Each of the 46 chromosomes has been replicated into two sister chromatids held together by cohesin rings placed there during S phase, which is what guarantees that the sisters can be recognised as a pair much later.
In prophase, condensin compacts the chromosomes enormously. Chromosome 1 alone is 249 million base pairs, which is 8.5 cm of DNA, and it ends up as a metaphase chromosome about 10 µm long, a linear compaction of roughly 8,500-fold. Nothing else in the cell handles a comparable change of scale.
In prometaphase the nuclear envelope breaks down, its lamin meshwork disassembled by CDK1 phosphorylation, and the spindle microtubules gain access. They find the chromosomes by the search and capture mechanism of the previous lesson: dynamic instability sends filaments probing in all directions, and one that encounters a kinetochore is captured and stabilised.
In metaphase the chromosomes are aligned at the equator, each attached to both poles and under tension. In anaphase the anaphase promoting complex destroys securin, freeing the protease separase, which cuts cohesin. The sisters are released simultaneously and drawn to opposite poles, both by shortening of the attached microtubules and by the spindle poles moving apart.
Telophase reverses prophase: envelopes reform, chromosomes decondense. Cytokinesis is done by a contractile ring of actin and myosin that tightens around the equator and pinches the cell in two, using the same two proteins as muscle.
The accuracy is high. Chromosome mis-segregation in a normal human somatic cell is estimated at around one per hundred thousand chromosomes per division, so an error somewhere in the set occurs roughly once in two thousand divisions. In many cancer cells the rate is a hundred times higher or more, and the resulting chromosomal instability is one of their defining features.
Meiosis, in one paragraph and one calculation
Meiosis makes gametes and differs in three ways: one round of replication is followed by two divisions rather than one, homologous chromosomes pair and exchange segments by crossing over, and in the first division it is the homologues rather than the sisters that separate. The result is four cells with half the chromosome number and none of them genetically identical.
The variety generated is worth quantifying. With 23 pairs, independent assortment alone gives possible combinations per gamete, and combining two parents gives possible zygotes before crossing over is counted at all. Crossing over, at one to three exchanges per chromosome pair, makes the number effectively unbounded, since no two chromosomes produced are alike.
The first meiotic division is also where human reproduction is least reliable. Human oocytes enter meiosis before birth and arrest partway through the first division for years or decades, and the cohesion holding their chromosomes together degrades over that time. Failure to separate correctly produces a gamete with an extra or missing chromosome, and most such conceptions do not survive. Trisomy 21 is the commonest to be compatible with life, and its incidence rises steeply with maternal age, which is the direct clinical consequence of a cell cycle held in arrest for forty years.
When the supervision fails
Cancer is a disease of the cell cycle. The mutations that cause it fall into two classes distinguished by exactly the genetic logic introduced in the lesson on regulation.
Proto-oncogenes encode components that drive the cycle forward, and the cancer-causing versions are gain-of-function: a growth factor receptor permanently switched on, a signalling kinase locked active, a cyclin overexpressed. One altered copy is enough, so these mutations are dominant.
Tumour suppressors encode the brakes, and the cancer-causing versions are loss-of-function, so both copies must be lost. Alfred Knudson inferred this in 1971 from statistics alone, before either gene was cloned. Retinoblastoma occurs in a hereditary form, which appears early and usually in both eyes, and a sporadic form, which appears later and in one eye. Knudson argued that two hits are needed in the same cell, that hereditary cases inherit one and need only one more, which will happen somewhere in the retina almost certainly, and that sporadic cases need two independent hits in the same cell, which is rare and slow. The gene, RB1, is the same retinoblastoma protein that holds E2F in G1.
TP53, encoding p53, is mutated in roughly half of all human cancers, which makes it the most frequently altered gene in the disease. It sits at the junction of damage detection, cycle arrest and apoptosis, so losing it removes several safeguards at once.
The requirement for multiple independent changes explains the epidemiology. Peter Armitage and Richard Doll showed in 1954 that the incidence of most adult cancers rises roughly as the fifth or sixth power of age, which is what a model requiring five to seven rate-limiting events predicts, and which is why doubling age from 35 to 70 raises incidence by something like -fold rather than twofold. Cancer is common in old age not because ageing causes it directly but because a sequence of improbable events takes a long time to complete.
Example. A tumour is found to have lost p53 function. Predict the consequences for how it responds to radiotherapy and to DNA-damaging chemotherapy, and say why the prediction is uncomfortable.
The prediction is that it responds worse. Radiation and most classical chemotherapy kill cells by damaging DNA, and much of the killing is not direct destruction but the cell's own response: damage is detected, p53 is stabilised, and the cell either arrests or commits suicide by apoptosis. A cell without p53 does not make that decision, so it sustains the same damage and keeps dividing, and this is one well-supported reason p53 status predicts poorer response to these treatments across several cancers. The uncomfortable part is that the same treatment is a strong selective pressure. Killing the p53-competent cells in a heterogeneous tumour leaves the p53-deficient ones to repopulate it, so a treatment that shrinks a tumour can enrich it for the cells hardest to treat next time, which is a large part of why recurrence is often more aggressive than the original disease. The reasoning generalises beyond p53 and beyond cancer, since it is the same logic as antibiotic resistance: any therapy that kills most of a genetically variable population is a selection experiment.
Now you. HeLa cells, taken from Henrietta Lacks in 1951 without her knowledge or consent, are still dividing in laboratories worldwide and have never stopped. Using this course, list what must be true of them, and then say what the case raises beyond the biology.
Answer
Several things must be true and each was covered earlier. They must have active telomerase, or their telomeres would have shortened to the point of arrest long ago, and HeLa cells do. They must have lost the G1 restriction point, so they do not require growth factor signals or wait for permission to enter S phase, and in HeLa the mechanism is known: human papillomavirus 18 is integrated into the genome, and its E6 and E7 proteins respectively target p53 for degradation and inactivate Rb, taking out both brakes at once. They must be able to divide without anchorage or a normal tissue context. And they must have accumulated substantial chromosomal instability, which they have: HeLa cells carry a heavily rearranged genome with a chromosome number far from 46 and varying between sublines. Beyond the biology, the case is the standard example in research ethics. The cells were taken during a biopsy without consent, at a time when this was legal and normal; they became the most widely used human cell line in history and the basis of an industry; her family learned of it only in the 1970s, were not compensated, and had her genome published without being consulted until an agreement in 2013 gave them a say over access to it. It is worth stating plainly that nothing in the science required the ethics to be handled that way, and that a course which teaches the cell cycle using HeLa cells and does not mention where they came from is teaching an incomplete fact.
Where this leaves you
The subject began with a claim from 1855 that every cell comes from a cell, and it can now be cashed out mechanically. A cell holds itself apart from the world with a self-assembling bilayer two molecules thick, and drills it with channels, carriers and pumps that maintain a composition nothing outside shares. It pays for that with ATP, kept a hundred million times from its own equilibrium by a pathway that ferments and a machinery that breathes, the second of which works by pumping protons across a membrane and selling the gradient back through a rotary motor. The specification for every one of those proteins is written in DNA, copied semiconservatively with an error rate near one in a billion, transcribed into a disposable messenger, and translated through a code whose redundancy is arranged so that mistakes do least harm. Which parts of the specification are read is decided by regulators that were worked out from a bacterium's preference for glucose. What is made is addressed, folded, delivered and eventually destroyed on schedule. And when the cell divides, a clock built from a kinase and a protein that is manufactured and then deliberately destroyed drives the process, three checkpoints supervise it, and a spindle assembled from the same filaments that carried cargo the day before pulls two metres of DNA into two equal sets.
Everything in that paragraph is shared, in outline, by a bacterium in your gut and by the cells reading this sentence. That is the sense in which there is such a thing as the cell, and it is the strongest single piece of evidence that everything alive on Earth is related.
What has been left out is most of biology. Nothing here covers how cells signal to one another, how tissues are organised, how an embryo becomes an animal, or how immune cells recognise anything. Those are subjects of their own and they all assume this one. What a reader who has finished should be able to do is different and more useful: given a claim about a cell, work out what would have to be true for it to hold, and roughly what number to expect.