Following the assembly of a bipolar microtubule array, the second major step in spindle formation is the attachment of the array to the sister-chromatid pairs. Spindle microtubules become attached to each chromatid at its kinetochore, a giant, multilayered protein structure that is built at the centromeric region of the chromatid (Figure 17–30; also see Chapter 4). In metaphase, the plus ends of kinetochore microtubules are embedded head-on in specialized microtubuleattachment sites within the outer region of the kinetochore, furthest from the DNA. The kinetochore of an animal cell can bind 10–40 microtubules, whereas a budding yeast kinetochore can bind only one. Attachment of each microtubule depends on multiple copies of a rod-shaped protein complex called the Ndc80 complex, which is anchored in the kinetochore at one end and interacts with the sides of the microtubule at the other, thereby linking the microtubule to the kinetochore while still allowing the addition or removal of tubulin subunits at this end (Figure 17–31). Regulation of plus-end polymerization and depolymerization at the kinetochore is critical for the control of chromosome movement on the spindle, as we discuss later.
Kinetochore attachment to the spindle occurs by a complex sequence of events. At the end of prophase in animal cells, the centrosomes of the growing spindle generally lie on opposite sides of the nuclear envelope. Thus, when the envelope breaks down, the sister-chromatid pairs are bombarded by microtubule
Figure 17–29 bipolar spindle assembly without centrosomes in parthenogenetic embryos of the insect Sciara (or fungus gnat). The microtubules are stained green, the chromosomes red. The top fluorescence micrograph shows a normal spindle formed with centrosomes in a normally fertilized Sciara embryo. The bottom micrograph shows a spindle formed without centrosomes in an embryo that initiated development without fertilization. note that the spindle with centrosomes has an aster at each pole of the spindle, whereas the spindle formed without centrosomes does not. Both types of spindles are able to segregate the replicated chromosomes. (From B. de saint phalle and W. sullivan, J. Cell Biol. 141:1383–1391, 1998. With permission from The Rockefeller University press.) plus ends coming from two directions. However, the kinetochores do not instantly achieve the correct ‘end-on’ microtubule attachment to both spindle poles. Instead, detailed studies with light and electron microscopy show that most initial attachments are unstable lateral attachments, in which a kinetochore attaches to the side of a passing microtubule, with assistance from kinesin motor proteins in the outer kinetochore. Soon, however, the dynamic microtubule plus ends capture the kinetochores in the correct end-on orientation (Figure 17–32).
Another attachment mechanism also plays a part, particularly in the absence of centrosomes. Careful microscopic analysis suggests that short microtubules in the vicinity of the chromosomes become embedded in the plus-end-binding sites of the kinetochore. Polymerization at these plus ends then results in growth of the microtubules away from the kinetochore. The minus ends of these kinetochore microtubules are eventually cross-linked to other minus ends and focused by motor proteins at the spindle pole (see Figure 17–28).
The success of mitosis demands that sister chromatids in a pair attach to opposite poles of the mitotic spindle, so that they move to opposite ends of the cell when direction of chromatid movement
Figure 17–30 The kinetochore.
a fluorescence micrograph of a metaphase chromosome stained with a dna-binding fluorescent dye and with human autoantibodies that react with specific kinetochore proteins. The two kinetochores, one associated with each sister chromatid, are stained red.