Spindle assembly occurs through three, often coexisting, pathways – the centrosome-mediated pathway, the chromatin-mediated pathway, and the microtubule-mediated pathway – collectively contributing to form a robust spindle apparatus.

In most cells, centrosomes are the primary microtubule nucleation centers. In the centrosome-mediated pathway, the G2-prophase transition triggers centrosome maturation and increased microtubule nucleation. Progressive nucleation results in a microtubule array emanating from both centrosomes. The plus-ends of these microtubules seek out and capture the chromosomes via their kinetochores.

Chromatin-mediated microtubule nucleation occurs near the chromosomes, driven by a nuclear protein, Ran-GTP, which exists at high concentrations close to the chromosomes. Ran-GTP binds to importin-beta, causing the release of its cargo, the spindle assembly factors (SAFs). SAFs promote localized microtubule nucleation in the vicinity of the chromosomes.

Existing microtubules also support further microtubule formation through the microtubule-mediated microtubule nucleation pathway. A protein complex, augmin, associates with existing microtubules and mediates the recruitment of gamma-tubulin ring complex (gammaTuRC) to initiate nucleation. The microtubule-mediated nucleation contributes to an increase in microtubule density within the spindle, adding to its robustness.

Spindle assembly results in a bipolar microtubule array containing three categories of microtubules. The kinetochore microtubules (K-MTs) tether the chromosomes to the spindle poles. The astral microtubules (A-MTs) radiate towards the cell cortex and aid in spindle positioning. The non-kinetochore microtubules (nK-MTs) fail to connect with kinetochores but serve to separate the poles and provide stability to the spindle.