Colchicine, Metaphase Arrest and a Temperature-Sensitive Mutant

The mitotic indices are 36/500 = 7.2%, 58/500 = 11.6%, 92/500 = 18.4% and 114/500 = 22.8%. The rise does not mean that colchicine makes cells divide faster; it does not stimulate division at all. Colchicine binds tubulin and prevents microtubule polymerisation, so no spindle forms. Chromosomes condense and align, but the sister chromatids cannot be pulled to opposite poles, the spindle assembly checkpoint is never satisfied, and the cell never enters anaphase. Cells keep arriving in mitosis from G2 while none leave it, so the proportion of cells visibly in mitosis accumulates. The stage distribution confirms this: 78% of dividing cells in the 6-hour sample are in metaphase, against 15% in the control, which is exactly what a metaphase block looks like. The reasoning point for the record is that the index by itself does not distinguish faster entry into mitosis from slower exit; the stage distribution is what identifies the mechanism, and the control value of 7.2% shows that the block, not the staining, produced the change.

The index must plateau: the root meristem has a finite population of cycling cells, prolonged colchicine reduces entry into mitosis and is eventually toxic, and cells held in metaphase for many hours may leave the cycle or die. So the curve flattens rather than climbing without limit. If the tips are washed free of colchicine after 6 hours and grown on, the arrested cells do not complete a normal division. No spindle means no segregation of chromatids, so the chromosomes are enclosed in a single restitution nucleus containing double the original chromosome number, and the cell becomes tetraploid rather than completing two diploid daughters. The mitotic index then falls back towards the control value after roughly one cycle. This is not a defect in the practical; it is exactly the principle used in plant breeding to raise autopolyploids with thicker leaves and larger fruits. The judgement to record is that a high mitotic index is evidence of an arrest, not evidence of healthy or vigorous growth.

At 39 °C the cells show condensed chromosomes aligned at the equator with no separation, so the block is at the metaphase-to-anaphase transition, not in chromosome condensation or in alignment. Because the mutated protein is required for sister-chromatid separation, the reasonable interpretation is that it must act after alignment - for example a separase or a cohesin-cleaving activity that cuts the link holding sister chromatids together. At the restrictive temperature it is inactive, so separation fails and the checkpoint holds the cell in metaphase. The permissive temperature of 32 °C is the essential control: the same genotype divides normally there, showing that the arrest is caused by the mutation and not by heat in general. What the experiment does not prove on its own is that this protein is the direct cause, because a wild-type cell line has not been tested at 39 °C; if wild-type cells also arrested, the conclusion would collapse. The design is strong because it compares one genotype at two temperatures rather than two different cell lines.