18.8: Spindle Assembly

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.

During eukaryotic cell division, microtubules assemble around the duplicated chromosomes in a bipolar array, forming the spindle apparatus. Spindle assembly is aided by multiple collaborating mechanisms.

Spindle assembly begins after two centrosomes are positioned at opposite ends of the cell. In animal cells, spindle poles are located at the centrosomes.

The centrosomes mature and nucleate bipolar microtubules, where the minus-end is anchored to the spindle pole, and the plus-end radiates outwards.

Simultaneously, multifunctional enzyme complexes, called M-Cdks, phosphorylate several nuclear envelope components, triggering the nuclear envelope to breakdown and expose the condensed chromosomes to the cytoplasm.

Microtubule nucleation at the centrosomes generates three types of microtubules. Interpolar microtubules come from opposite poles whose plus ends overlap, creating an antiparallel array at the spindle midzone. Kinetochore microtubules have their plus-ends connect with the kinetochores of exposed chromosomes. Astral microtubules have their plus-ends contact and interact with the cell cortex, positioning the spindle pole.

In the absence of centrosomes, mitotic chromosomes aid in acentrosomal spindle assembly. Mitotic chromosomes activate Ran-GTP, a nuclear protein. Activated Ran-GTP induces the release of microtubule-stabilizing proteins from protein complexes in the cytosol. The local activation of these factors promotes localized microtubule nucleation and stabilization.

Several microtubule-dependent motor proteins also contribute to spindle assembly and stabilization.

Dynein links astral microtubule plus-ends with cell-cortex components and pulls the spindle poles toward the cell-cortex. In the spindle midzone, kinesin-5 associates with interpolar microtubule plus-ends, to slide them past each other and generate a force that pushes the poles apart.

Kinesin-14 cross-links the interpolar microtubules at the spindle midzone and generates tension that pulls the poles together. Kinesin-4 and kinesin-10, the chromokinesins,  connect microtubules with chromosomal arms to push chromosomes away from the poles.

The balance of these opposing forces generated by the motor proteins determines the final length and position of the assembled spindle.