Chromosome behaviour in mitosis

By the time prophase begins, S phase of interphase has already replicated all the DNA. This is critical to remember: chromosomes are not created during mitosis — they have existed throughout interphase (as chromatin) and have been copied during S phase.

After S phase, each chromosome consists of:

• Two identical sister chromatids (the two copies of the DNA produced by semi-conservative replication),
• joined at the centromere (a specific region of repetitive DNA).

The total chromosome number has not changed — a human cell still has 46 chromosomes — but the total amount of DNA has doubled (so we sometimes refer to '46 chromosomes, 92 chromatids' or '4C DNA content').

Why does this matter for mitosis? Because the role of mitosis is to separate these pre-existing sister chromatids so that each daughter nucleus receives one chromatid (= one chromosome's worth of DNA) per chromosome pair. The 'doubling' has already been done; the 'sorting' is mitosis.

During G2, the cell has also:

• Synthesised microtubule subunits ready for spindle assembly.
• Replicated centrosomes (in animal cells), so there are now two centrosomes that will move to opposite poles in prophase.
• Carried out DNA damage checks; if damage is found, p53 can pause the cycle to allow repair or trigger apoptosis.

Prophase is the first and usually longest stage of mitosis. Three things happen in parallel:

1. Chromosome condensation. The long, thin chromatin fibres coil and supercoil, becoming progressively shorter, thicker and visible under the light microscope. Each chromosome now appears as two sister chromatids joined at the centromere. (X-shaped appearance for chromosomes with a central centromere, V-shaped for those with an off-centre centromere.) Condensation makes the chromosomes mechanically robust so they can be moved without tangling or breaking.

2. Nuclear envelope breakdown. The double membrane of the nuclear envelope fragments into small vesicles and disperses into the cytoplasm. The nucleolus also disappears (it will reform during telophase). This frees the chromosomes from the nucleus and allows the spindle fibres to reach them.

3. Spindle formation. Microtubules organise into a spindle, a fan-shaped array of fibres extending across the cell:

   • Animal cells. The two centrosomes (each containing a pair of centrioles) move to opposite poles of the cell. Microtubules grow from the centrosomes (microtubule-organising centres at their core) and extend across the cell.
   • Plant cells. Plant cells lack centrioles; the spindle is organised from less structured microtubule-organising centres (MTOCs) at the cell poles. The spindle still forms in essentially the same fan shape.

By the end of prophase, each chromosome has spindle microtubules attached to specialised protein complexes — the kinetochores — assembled on its centromere. The chromosomes are now ready to be moved.

In metaphase, the chromosomes are moved by the spindle to the equator of the cell:

• The centromeres line up on the metaphase plate — an imaginary plane midway between the two poles. The sister chromatids extend either side of this plane.
• Each chromosome is attached, at its centromere, to spindle microtubules from both poles — one chromatid is linked to one pole, the other chromatid to the opposite pole.
• The spindle is now under tension. Microtubule shortening from one side is balanced by microtubule shortening from the other side, holding the chromosome stationary on the equator.

Metaphase is the stage at which chromosomes are most easily seen and identified under a microscope, because they are maximally condensed and lined up neatly. Karyotyping (the analysis of an individual's chromosome set) is normally done on cells arrested in metaphase using colchicine (a drug that prevents spindle function).

Metaphase checkpoint (spindle assembly checkpoint). Before the cell is allowed to proceed to anaphase, every chromosome must be correctly attached to spindle fibres from both poles. Any chromosome that is not properly attached generates a 'wait' signal. Only when ALL chromosomes are correctly attached does the cell move into anaphase. This prevents unequal distribution of chromosomes (aneuploidy), which would be disastrous for the daughter cells.

Anaphase is the briefest stage of mitosis (often just 2-3 minutes), but it is the most dramatic visually. Two events define it:

1. Centromeres divide. The protein 'glue' (cohesin) holding the two sister chromatids together at the centromere is cleaved by the enzyme separase. Each chromatid is now free of its sister and is, from this point on, counted as a separate chromosome. (So briefly, during anaphase, the cell contains TWICE the diploid number — but only momentarily.)

2. Spindle fibres shorten and pull chromatids apart. Microtubules attached to the kinetochores depolymerise from their ends, shortening so that each chromatid is pulled towards the pole it was attached to. The separated chromatids appear V-shaped because they are dragged centromere-first, with the chromatid arms trailing behind. Other spindle microtubules push the poles further apart, elongating the cell.

The energy for these movements comes from ATP hydrolysis. Motor proteins (e.g. dyneins, kinesins) walk along the microtubules and provide the force.

By the end of anaphase, one complete set of chromosomes has arrived at each pole. Each set is genetically identical to the parent set (the chromatids were copies of the original chromosomes) and identical to the set at the other pole.

Telophase is essentially the reverse of prophase: the cell rebuilds the nuclear structure around each set of chromosomes that has arrived at a pole.

• The separated chromatids (now called chromosomes again, since each is one DNA molecule) reach the two opposite poles. There is one complete set of chromosomes at each pole, identical to the parent set.
• The chromosomes decondense — uncoiling until they are once more long, thin and no longer individually visible under the light microscope. This makes the DNA accessible to RNA polymerase for transcription.
• A new nuclear envelope reforms around each chromosome set, using vesicles from the original nuclear envelope that broke down during prophase. The two new envelopes seal off two distinct nuclei.
• The nucleolus reappears in each new nucleus (forming around the genes encoding ribosomal RNA).
• The spindle disassembles, with the microtubule subunits returning to the cytoplasmic pool.

By the end of telophase, mitosis (= nuclear division) is complete. The cell now contains two genetically identical daughter nuclei, with chromosomes again in their decondensed chromatin form. Cytokinesis then follows to divide the cytoplasm.

Cytokinesis is the division of the cytoplasm that follows mitosis. It is not strictly part of mitosis itself (which is nuclear division), but it is part of the cell cycle. The mechanism differs sharply between animal and plant cells because plant cells have a rigid cellulose cell wall.

Cytokinesis in animal cells. A ring of actin and myosin filaments assembles just inside the cell-surface membrane around the equator (where the metaphase plate had been). The ring contracts (like the drawstring on a purse), pulling the membrane inwards to form a cleavage furrow. The furrow deepens until the cell is pinched in two. Because the membrane is flexible and there is no cell wall, this 'pinching' is straightforward.

Cytokinesis in plant cells. Plant cells cannot pinch in because of the rigid cell wall, so they build a new partition from scratch:

• Golgi-derived vesicles containing cell-wall material (polysaccharides, glycoproteins) line up across the equator.
• The vesicles fuse with one another to form a flat, disc-shaped structure: the cell plate.
• The cell plate grows outwards from the centre until it meets and joins the existing cell wall on all sides.
• New middle lamella (rich in pectins) and primary cell walls then form on each side of the cell plate, separating the two daughter cells.

In both cases, the result is two genetically identical daughter cells, each with one of the daughter nuclei. Only the mechanism of dividing the cytoplasm differs.

The mitotic index (MI) is a numerical measure of how rapidly a tissue is dividing. It is the proportion of cells in a microscope field that are visibly in mitosis (showing condensed chromosomes), expressed as a percentage:

$\text{Mitotic index} = \dfrac{\text{number of cells in mitosis}}{\text{total number of cells}} \times 100$

How to count: Examine a stained preparation under high power. A cell is counted as 'in mitosis' if its chromosomes are visible as discrete structures (in prophase, metaphase, anaphase or telophase). Cells with diffuse chromatin (no visible chromosomes) are in interphase. Count both groups in the same field of view and apply the formula.

Typical values:

• High MI (10-20%): actively dividing tissues — onion / garlic root tip (meristem), bone marrow, gut crypt epithelium, basal layer of skin, and most tumours.
• Low MI (<1%): mature, fully differentiated tissues — neurons, muscle, mature leaf parenchyma, liver hepatocytes (very low except during regeneration).

Why measure mitotic index?

• In plant biology, MI is used to study the effects of factors (temperature, growth regulators, mitotic poisons) on cell division rates in root tips.
• In medicine, a persistently high MI in a tissue that should be quiescent (e.g. a breast biopsy) is a marker of malignancy — pathologists routinely report 'mitotic count' as part of tumour grading.

Limitations:

• MI does not distinguish between healthy and abnormal mitosis.
• A high MI measured at one moment is a 'snapshot' — it does not tell you whether the cells are completing division successfully.
• Different parts of a tissue may have very different MIs (e.g. root meristem vs root cortex).

Cambridge 9700 examines a classic practical to observe the stages of mitosis: the temporary root-tip squash preparation.

Method.

1. Grow roots. Suspend an onion or garlic bulb over water (or use germinating broad bean seeds) for a few days until 1-2 cm of root has grown.
2. Cut the root tip. Cut off the terminal ~5 mm — this contains the rapidly dividing meristematic zone where mitosis is occurring.
3. Hydrolyse / soften. Place the root tip in hot 1 mol dm$^{-3}$ HCl at ~60 °C for 4-5 minutes. The acid hydrolyses the middle lamella (the pectin-rich layer between cells), allowing the cells to be separated and spread out as a single layer.
4. Stain. Transfer the softened tip to a few drops of aceto-orcein stain (or toluidine blue) on a microscope slide for 5-10 minutes. The stain is a basic dye that binds to acidic DNA, colouring chromosomes deep red / purple against the unstained cytoplasm.
5. Squash. Cover with a cover slip and press firmly (e.g. with the wooden end of a mounting needle or your thumb on filter paper) to spread the cells out into a single layer. This is essential — without squashing, the cells lie on top of each other and chromosomes cannot be seen clearly.
6. Observe. View under low power first to locate the densely-stained meristematic region, then switch to high power.

Identifying mitotic stages:

• Interphase: nucleus visible but no distinct chromosomes.
• Prophase: chromosomes visible as thread-like structures; nuclear envelope may still be present early on.
• Metaphase: chromosomes lined up across the centre of the cell.
• Anaphase: V-shaped chromatids being pulled apart to opposite ends.
• Telophase: two clusters of chromatids at opposite ends; new nuclear envelopes forming.

Calculating MI. Count the number of cells in mitosis (any of P, M, A, T) and the total number of cells in the same field of view; apply the formula.

Safety. Dilute HCl is corrosive and is heated — wear eye protection, use a heat-resistant water bath, and use forceps. The stain may be irritant — avoid skin contact.