There are interesting parallels between centrosome duplication and chromosome duplication. Both use a semiconservative mechanism of duplication, in which the two halves separate and serve as templates for construction of a new half. Centrosomes, like chromosomes, must replicate once and only once per cell cycle, to ensure that the cell enters mitosis with only two copies: an incorrect number of centrosomes could lead to defects in spindle assembly and thus errors in chromosome segregation.
The mechanisms that limit centrosome duplication to once per cell cycle are uncertain. In many cell types, experimental inhibition of DNA synthesis blocks centrosome duplication, providing one mechanism by which centrosome number is kept in check. Other cell types, however, including those in the early embryos of flies, sea urchins, and frogs, do not have such a mechanism and centrosome duplication continues if chromosome duplication is blocked. It is not known how such cells limit centrosome duplication to once per cell cycle.
Spindle assembly begins in early mitosis, when the two centrosomes move apart along the nuclear envelope, pulled by dynein motor proteins that link astral micro-tubules to the cell cortex (see Figure 17–25). The plus ends of the microtubules between the centrosomes interdigitate to form the interpolar microtubules, and kinesin-5 motor proteins associate with these microtubules and push the centrosomes apart (see Figure 17–25). Also in early mitosis, the number of γ-tubulin ring complexes in each centrosome increases greatly, increasing the ability of the centrosomes to nucleate new microtubules, a process called centrosome maturation.
The balance of opposing forces generated by different types of motor proteins determines the final length of the spindle. Dynein and kinesin-5 motors generally promote centrosome separation and increase spindle length. Kinesin-14 proteins do the opposite: they tend to pull the poles together (see Figure 17–25). It is not clear how the cell regulates the balance of opposing forces to generate the appropriate spindle length.
M-Cdk and other mitotic protein kinases are required for centrosome separation and maturation. M-Cdk and Aurora-A phosphorylate kinesin-5 motors and stimulate them to drive centrosome separation. Aurora-A and Plk also phosphorylate components of the centrosome and thereby promote its maturation.
The Completion of spindle assembly in animal Cells Requires nuclear-Envelope Breakdown
The centrosomes and microtubules of animal cells are located in the cytoplasm, separated from the chromosomes by the double-membrane barrier of the nuclear envelope (discussed in Chapter 12). Clearly, the attachment of sister-chromatid pairs to the spindle requires the removal of this barrier. In addition, many of the motor proteins and microtubule regulators that promote spindle assembly are associated with the chromosomes inside the nucleus, and they require nuclear-envelope breakdown to carry out their functions.
Nuclear-envelope breakdown is a complex, multistep process, which is thought to begin when M-Cdk phosphorylates several subunits of the nuclear pore complexes in the nuclear envelope. This phosphorylation initiates the disassembly of nuclear pore complexes and their dissociation from the envelope. M-Cdk
Figure 17–26 Centriole replication. The centrosome consists of a centriole pair and associated pericentriolar matrix (green). at a certain point in g1, the two centrioles of the pair separate by a few micrometers. during s phase, a daughter centriole begins to grow near the base of each mother centriole and at a right angle to it. The elongation of the daughter centriole is usually completed in g2. The two centriole pairs remain close together in a single centrosomal complex until the beginning of m phase, when the complex splits in two and the two daughter centrosomes begin to separate. Each centrosome now nucleates its own radial array of microtubules (called an aster), mainly from the mother centriole.