The cell cycle and mitosis

Three phases before anything divides

A cell that is going to divide works through the same sequence every time. In G1 it grows and makes more of the organelles it will have to share out. In S phase it replicates its DNA, semi-conservatively, so that every chromosome ends up as two identical sister chromatids held together at a centromere. In G2 it keeps growing and checks the copy for errors. Only then does mitosis begin.

That ring is drawn for a cell cycling in about 24 hours, and the proportions are a typical case rather than a constant of nature. G1 stretches and shrinks: a root-tip meristem cell runs the whole cycle in less than a day, and mitosis then takes a larger share of a smaller total. Keep the ordering, which never changes, and treat the exact tenth as a figure attached to a 24-hour cycle.

Some cells leave the cycle. A neurone or a mature muscle fibre stops at the end of G1 and enters G0, which is less a phase than the exit from one. Neither of those cells will replicate its DNA again, but other cells in G0, such as liver cells and resting lymphocytes, can be signalled back into the cycle. Most of your cells are in G0 now.

The chromosome count does not change during S phase, which trips people up. A human cell in G1 has 46 chromosomes; after S phase it still has 46 chromosomes, but each one is now two chromatids, so there is twice as much DNA. Count centromeres, not strands, and the arithmetic behaves.

Chromatid

One of the two identical copies of a chromosome, joined to its sister at the centromere after DNA replication.

Homologous pair

Two chromosomes of the same size carrying the same genes at the same loci, one inherited from each parent. Not the same thing as a pair of chromatids.

Spindle

The framework of protein microtubules built across the cell before the chromosomes are moved, running from pole to pole and attaching to every centromere; shortening the fibres is what separates the chromatids.

Mitotic index

The proportion of cells in a sample that are visibly in mitosis.

Telomeres and the end-replication problem

S phase copies almost the whole chromosome. The exception is at the two ends, and it follows from the mechanism of replication rather than from a fault in any one enzyme.

Two constraints on DNA polymerase apply. It builds a new strand only in the 5 prime to 3 prime direction, and it can only add nucleotides to the 3 prime end of something that is already there, which is why a short RNA primer has to be laid down first. On the leading strand that is no trouble: one primer at the start, then the polymerase runs continuously to the far end of the template. The lagging strand is built backwards in fragments, each with its own primer, and the primers are afterwards removed and the gaps filled by extending the fragment next door. The last primer at the very end of the chromosome has no fragment beyond it. When it is removed there is no 3 prime end for the polymerase to work from, so that gap stays open and a short stretch of the end is never copied.

Describe the drawing

Two panels. In the upper one the leading strand at one end of a chromosome is drawn as a single primer followed by an unbroken run of new DNA reaching the tip, so every base of the template is copied. The lagging strand below it is drawn as four Okazaki fragments, each with its own primer at the end nearer the tip. When the primers are taken out, three of the gaps are filled by the fragment beyond them extending into the space, and the fourth, at the very tip, has no fragment beyond it to be extended from and stays open, so the tip goes uncopied and no better enzyme could reach it. The lower panel plots telomere length against division number from ten thousand base pairs of non-coding repeat: a body cell loses seventy-five base pairs a division and so has a hundred and thirty-three divisions of repeats to spend before the erosion reaches a gene, while a germ line cell, in which telomerase adds the repeats back, holds a flat line and can divide without limit, which is what most cancers switch back on.

A chromosome therefore becomes slightly shorter at every division. In a human cell the loss is of the order of fifty to a hundred base pairs per division, and it is cumulative: it continues at every division and is inherited by every descendant cell.

This is what the ends of chromosomes are for. Each one is capped by a telomere: a long run of a short sequence repeated over and over, TTAGGG in humans and something similar in most other eukaryote, thousands of copies of it, and coding for nothing. Because the repeats are non-coding, the shortening erodes repeats rather than genes, and a cell can divide many dozens of times before the erosion reaches a transcribed sequence.

The telomere has a second function. An uncapped double-stranded end resembles a chromosome broken in half. Repair enzymes join a free end to any other free end available, and the damage checkpoints halt the cycle until they have. Two chromosomes fused end to end are then torn apart at the next anaphase and the damage compounds. A telomere, with the proteins bound along it, is folded back and capped so that the repair enzymes do not act on it as though it were a break. A telomere therefore protects the coding sequence from erosion and protects the end itself from inappropriate repair.

Cells that divide indefinitely require their telomeres to be maintained. Telomerase is an enzyme that carries a short RNA molecule of its own and uses it as a template to add telomere repeats back onto the end, restoring what replication takes. It is active in the cells of the germ line, which make gametes and must hand a full-length chromosome to the next generation, and in stem cells and some cells of the immune system. In most ordinary body cells it is switched off, telomeres shorten with every division, and a cell whose telomeres have grown too short stops dividing altogether or dies. Most cancer cells express telomerase again, which is one factor allowing a tumour to continue dividing beyond the usual replicative limit.

Telomere

A cap of short non-coding sequences repeated many times at each end of a chromosome, which is eroded instead of the genes when replication fails to copy the end, and which stops the end being treated as a break in the DNA.

Telomerase

An enzyme carrying its own RNA template, which adds telomere repeats back onto the end of a chromosome; it is active in germ line cells, stem cells and most cancer cells.

The three checkpoints

Between the phases lie checkpoints. Learn each by what it checks as well as by name. The cycle does not proceed past a checkpoint until the relevant condition is met, and the checking is carried out by the cell's own regulatory proteins, which is why a mutation in one of those proteins has such large consequences.

The G1 checkpoint is the point of no return. A cell that passes it is committed to copying its entire genome whether conditions stay favourable or not, so cell size, nutrients, signals from neighbours and DNA damage all get their say here. Failing it has more than one outcome: most cells pause, repair the fault and try again, some withdraw into G0, and a cell that cannot be mended may stop dividing permanently or be removed by apoptosis.

The G2 checkpoint is a proofreading stop. S phase copies about six thousand million base pairs in a human cell and does not do it flawlessly, so this checkpoint halts a cell with unrepaired breaks. Damage that gets past is handed to both daughters.

The spindle checkpoint sits inside mitosis rather than between two phases, and it is why anaphase is so abrupt: one chromosome not yet attached to both poles holds the whole cell at metaphase, and when the last attachment is made every centromere divides at once.

Distinguish two outcomes. A checkpoint delays the cycle, and most cells held at one repair the fault and proceed. Apoptosis ends the cell and occurs when repair fails.

The four stages of mitosis

Mitosis is conventionally divided into four stages. The division is a convention, since the process is continuous, but each stage is defined by features visible under a microscope, which is why a stage can be identified from a photograph.

Describe the drawing

Mitosis animated through its four stages in order. Prophase: the chromosomes condense inside the nucleus, each already two chromatids joined at a centromere, and the nuclear envelope begins to break down. Metaphase: the chromosomes line up along the equator with spindle fibres running to them from poles on each side. Anaphase: the centromeres divide and the chromatids are pulled to opposite poles. Telophase: a nuclear envelope re-forms around each group and the cytoplasm divides, giving two genetically identical daughter cells.

Two of the four stages are defined by the behaviour of the spindle. It is a framework of protein microtubules, assembled during prophase and running from one pole of the cell to the other. In an animal cell the poles are organised by a pair of centrioles; a plant cell assembles the same spindle without them. Fibres attach to every centromere from both sides, and anaphase consists of those fibres shortening.

Cytokinesis follows, and it is not part of mitosis proper: mitosis divides the nucleus, cytokinesis divides the cytoplasm. In animal cells a ring of protein filaments under the membrane contracts and pinches it inwards. In plant cells it cannot, because of the cell wall, so vesicles line up along the middle, fuse into a cell plate, and a new wall is built outwards from it. Organelles are distributed approximately rather than in equal numbers.

The mitotic index

The mitotic index is the calculation this topic requires, and it is obtained from a root-tip squash. The tip of a root is a meristem: a few millimetres in which every cell is still dividing. Tissue cut more than 5 mm back contains cells that have left the cycle and begun elongating.

The preparation determines whether the count is reliable. The tip is warmed in dilute hydrochloric acid, which breaks down the middle lamella holding the cells together, then stained with toluidine blue or acetic orcein, both of which bind to DNA, and squashed under a coverslip to a single layer. Overlapping layers cannot be counted reliably, because a chromosome in one plane of focus is not readily distinguished from one in another.

Then count. Score every cell in the field rather than a selection, because a sample chosen by eye is biased towards cells in mitosis. Count several fields and add the totals, and state the total number of cells counted alongside the index.

Describe the drawing

The same field of two hundred and forty cells drawn twice, with thirty-six of them shown in a visibly mitotic state. On the left every cell in the field is counted, giving thirty-six in mitosis out of two hundred and forty and an index of nought point one five. On the right the twenty most conspicuous cells are ringed, and because a cell with condensed chromosomes is what stands out down a microscope, all twenty of them are in mitosis and the index from that sample is one point nought nought, with sixteen mitotic cells left unringed and two hundred and twenty cells never counted at all. Beneath, a bar for a twenty-hour cycle is divided so that mitosis takes the honest index of it, which is about three hours, because the fraction of a population caught in a stage is the fraction of the cycle that stage occupies.

One inference the index licenses, and one it does not. A fixed slide is a snapshot of a population, so the fraction caught in a stage is the fraction of the cycle that stage occupies: cycle 20 hours, index 0.15, mitosis about 3 hours. What it cannot show alone is how fast the tissue divides, since equal indices mean different rates when the cycles differ in length.

Binary fission, and how meiosis differs

Mitosis addresses two features of eukaryotic cells: a nuclear envelope that must be disassembled and rebuilt, and several long linear chromosomes that must be separated without tangling. A prokaryote has neither, and divides by binary fission. The prokaryote's single circular chromosome is replicated, and dedicated proteins move the two copies into opposite halves of the growing cell. Older textbooks had the copies attached to the membrane and carried apart passively as the cell grew, but the segregation is now known to be an active process. Plasmids are replicated too, though not to a fixed number, and a new membrane and wall are laid down across the middle.

State three differences from mitosis: there is no spindle, there is no nuclear envelope to break down or re-form, and there is no alignment at an equator, so do not describe the chromosome as lining up. Binary fission is also rapid: Escherichia coli divides in twenty minutes in favourable conditions, against about a day for a human cell.

The other nuclear division on the specifications is meiosis, which halves the chromosome number and produces genetically different cells, and is taught in meiosis and the sources of genetic variation.

Loss of checkpoint control and tumour formation

Every daughter cell from mitosis is genetically identical to the parent, barring the rare copying error or missorted chromosome that slips through the checkpoints, which is what makes mitosis the right tool for growth, for repair, and for asexual reproduction: a yeast cell buds off a daughter cell, a Hydra grows a bud that breaks away, and a strawberry plant sends out runners, all by mitosis. The same property makes a fault in the process serious: an error is copied faithfully into everything descended from that cell.

Mutations in the genes controlling the checkpoints can leave a cell dividing when it should have stopped. Two categories matter. Proto-oncogenes normally stimulate division, and a mutation can leave one permanently switched on. Tumour suppressor genes normally halt the cycle at a checkpoint, and a mutation can prevent one being expressed. In either case the cycle is no longer restrained, division continues unchecked, and a mass of cells accumulates as a tumour.

That also explains why many chemotherapy drugs work the way they do. Some prevent DNA replication in S phase, some disrupt spindle formation so metaphase cannot complete. Neither targets cancer cells specifically. Both target dividing cells, which is why hair follicles and the gut lining, both dividing rapidly, suffer the familiar side effects.