Kinetochore - Kinetochore Structure in Animal Cells

Kinetochore Structure in Animal Cells

The kinetochore is composed of several layers, observed initially by conventional fixation and staining methods of electron microscopy, (reviewed by C. Rieder in 1982) and more recently by rapid freezing and substitution.

The deepest layer in the kinetochore is the inner plate, which is organized on a chromatin structure containing nucleosomes presenting a specialized histone (named CENP-A, which substitutes histone H3 in this region), auxiliary proteins and DNA. DNA organization in the centromere (satellite DNA) is one of the least known aspects in vertebrate kinetochores. The inner plate appears like a discrete heterochromatin domain throughout the cell cycle.

Outside the inner plate we find the outer plate, composed mostly by proteins. This structure is assembled in the surface of the chromosomes when the nuclear envelope breaks down. The outer plate in vertebrate kinetochores contains about 20 anchoring sites for MTs (+) ends (named kMTs, after kinetochore MTs), whereas kinetochore's outer plate in yeast (Saccharomyces cerevisiae) contains only one anchoring site.

The outermost domain in the kinetochore forms a fibrous corona, which can be visualized by conventional microscopy, yet only in absence of MTs. This corona is formed by a dynamic network of resident and temporary proteins implicated in the spindle checkpoint, in MTs anchoring and in the regulation of chromosome behavior.

During mitosis, each sister chromatid forming the complete chromosome has its own kinetochore. Distinct sister kinetochores can be observed at first at the end of G2 phase in cultured mammalian cells. These early kinetochores show a mature laminar structure before the nuclear envelope breaks down (reviewed by Pluta et al. in 1995). The molecular pathway for kinetochore assembly in high eukaryotes has been studied using gene knockouts in mice and in cultured chicken cells, as well as using RNA interference (RNAi) in C. elegans, Drosophila and human cells. Yet no simple linear route can describe the data obtained so far.

The first protein to be assembled on the kinetochore is CENP-A (Cse4 in Saccharomyces cerevisiae). This protein is a specialized isoform of histone H3. CENP-A is required for incorporation of the inner kinetochore proteins CENP-C, CENP-H and CENP-I/MIS6. The relative position of these proteins in the CENP-A dependent pathway are not completely defined. For instance, CENP-C localization requires CENP-H in chicken cells, but it is independent of CENP-I/MIS6 in human cells. In C. elegans and metazoa, the incorporation of many proteins in the outer kinetochore depends ultimately on CENP-A.

Kinetochore proteins can be grouped according to their concentration at kinetochores during mitosis: some proteins remain bound throughout cell division, whereas some others change in concentration; furthermore, they can be recycled in their binding site on kinetochores either slowly (they are rather stable) or rapidly (dynamic).

• Proteins whose levels remain stable from prophase until late anaphase include constitutive components of the inner plate and the stable components of the outer kinetocore, such as the Ndc80 complex, KNL/KBP proteins (kinetochore-null/KNL-binding protein), MIS proteins and CENP-F. Together with the constitutive components, these proteins seem to organize the nuclear core of the inner and outer structures in the kinetochore.
• The dynamic components that vary in concentration on kinetochores during mitosis include the molecular motors CENP-E and dynein (as well as their target components ZW10 and ROD), and the spindle checkpoint proteins (such as Mad1, Mad2, BubR1 and Cdc20). These proteins assemble on the kinetochore in high concentration in absence of microtubules; however, the higher the number of MTs anchored to the kinetochore, the lower the concentration of these proteins. At metaphase, CENP-E, Bub3 and Bub1 levels disminish about 3 to 4x as compared to free kinetochores, whereas dynein/dynactin, Mad1, Mad2 and BubR1 levels are reduced >10-100x.
• Whereas the spindle checkpoint protein levels present in the outer plate diminish as MTs anchor, other components such as EB1, APC and proteins in the Ran pathway (RanGap1 and RanBP2) associate to kinetochores only when MTs are anchored. This may belong to a mechanism in the kinetochore to recognize the MTs plus-end (+), ensuring their proper anchoring and regulating their dynamic behavior as they remain anchored.

A 2010 study uses a complex method (termed multiclassifier combinatorial proteomics or MCCP) to analize the proteomic composition of vertebrate chromosomes, including kinetochores. Although this study does not include a biochemical enrichment for kinetochores, obtained data include all the centromeric subcomplexes, with peptides from all 125 known centromeric proteins. According to this study, there are still about one hundred unknown kinetochore proteins, doubling the known structure during mitosis, which confirms the kinetochore as one of the most complex cellular substructures.