The Mitotic Spindle and Kinetochore Tension
Microtubule Dynamic Instability, Aurora B Tension Sensing, and the Mad2 Wait Signal
Tim Mitchison and Marc Kirschner discovered in 1984 that microtubules do not polymerize to a quiet equilibrium length. Instead, individual filaments switch stochastically between steady growth and rapid catastrophic shrinkage. A protective cap of GTP-tubulin stabilizes the straight growing tip, but if GTP hydrolysis catches up to the rim, curved GDP-tubulin protofilaments peel violently apart.
Mitchison and Kirschner realized in 1986 that dynamic instability turns the mitotic spindle into a high-speed search-and-capture engine. Microtubule plus ends shoot out from the centrosomes in random directions and rapidly demolish any filament that misses a chromosome. That relentless turnover captures all 46 human chromosomes in minutes rather than the hours required by diffusion.
What physical engine pulls captured chromosomes toward the spindle poles during anaphase? J. Richard McIntosh and Carlos Bustamante showed that the primary pulling force comes not from ATP-burning motors, but from the stored conformational energy of GTP hydrolysis inside the shrinking microtubule itself. A single peeling microtubule generates up to $50\text{ pN}$ of pulling force as its protofilaments curl.
To hold onto a track that is disintegrating underneath it, the kinetochore—an 80-protein machine built on centromeric CENP-A nucleosomes—grasps 15 to 20 microtubule ends simultaneously. Fibrous 80-nanometer Ndc80 rods and sliding couplers (the Dam1 ring in yeast and the SKA complex in humans) form a load-bearing sleeve. They surf the curling protofilaments without letting go.
How does a dividing cell know when sister chromatids are properly attached to opposite spindle poles (bi-orientation) rather than both hooked to the same pole? Bruce Nicklas proved the mechanism in 1967 by snaring a misattached praying-mantis chromosome with a calibrated glass microneedle. Pulling backward with $70\text{ pN}$ of tension immediately stabilized the attachment and satisfied the checkpoint.
Michael Lampson and Tarun Kapoor revealed the molecular ruler behind Nicklas's tension sensor in 2009. The kinase Aurora B is anchored to the inner centromere between sister chromatids. When sisters lack opposing tension, outer kinetochore Ndc80 tails sag inward within reach of Aurora B, which phosphorylates nine positively charged residues to electrostatically repel the negatively charged microtubule.
Once sister kinetochores hook onto microtubules pulling toward opposite spindle poles, the tug-of-war stretches the elastic centromeric chromatin between them by 100 nanometers. That physical stretch yanks the outer Ndc80 microtubule-binding domains out of the diffusive reach of inner-centromere Aurora B. Constitutive PP1 and PP2A phosphatases then strip the phosphate charges and lock the grip tight.
Conly Rieder and colleagues proved in 1995 how ruthlessly the Spindle Assembly Checkpoint guards against chromosome loss. Using a laser microbeam, they destroyed the single last unattached kinetochore in a dividing vertebrate cell. That solitary 200-nanometer plaque had been holding the entire cell in metaphase, and ablating it triggered anaphase sister separation 20 minutes later.
How can one tiny unattached kinetochore inhibit millions of Anaphase-Promoting Complex (APC/C) molecules across the cytoplasm? Andrea Musacchio showed that a kinetochore-bound Mad1-C-Mad2 core acts as a catalytic template. Soluble open Mad2 docks onto the template, flips its C-terminal safety belt into the closed conformation around Cdc20, and diffuses away to assemble the Mitotic Checkpoint Complex.
Even after every chromosome bi-orients and the Mad2 wait signal shuts off, sister chromatids remain physically locked together by protein rings called cohesin. Kim Nasmyth and Frank Uhlmann discovered in 1999 how the lock opens. Active $\text{APC/C}^{\text{Cdc20}}$ ubiquitylates an inhibitory chaperone called securin for proteasomal destruction, unleashing the giant cysteine protease separase.
Once freed from securin, active separase acts as molecular bolt cutters: it cleaves the Scc1/Rad21 kleisin subunit of the topological cohesin rings that have encircled sister DNA duplexes since S-phase. With the rings snapped open, all 46 chromosome pairs split synchronously. Peeling kinetochore microtubules then drag the separated sister chromatids to opposite poles at one micrometer per minute.
Isolated from Pacific yew bark (Taxus brevifolia) by Monroe Wall and Mansukh Wani in 1971, the chemotherapy drug paclitaxel (Taxol) works by the opposite mechanism of colchicine. Susan Horwitz proved in 1979 that paclitaxel binds inside the $\beta$-tubulin lumen to freeze microtubules against depolymerization. That stabilization robs kinetochores of dynamic tension and traps cancer cells in mitotic arrest.