Kinetochore is a macro-molecular protein complex built by at least 26 different core-kinetochore proteins during metaphase and additional corona proteins, also known as SAC proteins, which are contributed during prometaphase or at unattached kinetochores. The core-kinetochore proteins are classified into two groups: inner kinetochore and outer kinetochore proteins. Inner kinetochore proteins, commonly known as CCAN (Constitutive Centromere Associated Network) proteins, localize at centromeric chromatin throughout the cell cycle. CCAN proteins include CENP-A, CENP-C, CENP-T complex, and CENP-H/I complex. Outer kinetochore proteins, also known as the highly conserved KMN (Knl1, Mis12 complex, and Ndc80 complex) network, localize at kinetochores during mitosis.
All kinetochore proteins have multiple copies; for example, a single human kinetochore at metaphase has ~250 molecules of Ndc80 complex, ~215 molecules of CENP-C, and ~72 molecule of CENP-T. Are these multiple copies of kinetochore proteins randomly mixed, like a salad bowl, at the kinetochore? The answer is No. It may be surprising, but the core-kinetochore proteins systematically build a kinetochore architecture within a diffraction limited spot (~200 nm dimensions) at centromeric chromatin on the chromosome (Figure 1). An example of the systematic building is the inner kinetochore CENP-T and outer kinetochore Ndc80 that overlap, but are not fully co-localized at a single human kinetochore (Figure 3). However, because the optical resolution limit of light microscopy is ~200 nm for x- and y-axis, and ~500 nm for z-axis, the use of super-resolution microscopy is required to study kinetochore protein architecture and elucidate kinetochore functions.
In order to resolve these spatial and temporal difficulties, we use a calibrated confocal microscope, light-sheet microscopy, and super-resolution microscopy including SIM, STED, and 2D/3D fluorescence co-localization methods we recently developed for the following projects.
(1) How kinesin contributes to proper kinetochore functions.
We recently found that a highly conserved Kinesin-5 in budding yeast, Cin8, recruits PP1 (Protein Phosphatase 1) to the kinetochore near Ndc80 microtubule binding domains, and Cin8-PP1 complex is critical for proper mitotic progression as well as proper force generation at Ndc80. We are now working to elucidate molecular mechanisms in kinetochore functions of Cin8 and other Kinetochore MAPs in budding yeast and humans.
(2) Kinetochore deformation in mitotic progression.
The kinetochore, a diffraction limited structure, is known to deform during mitotic progression. We are determining the kinetochore structural changes and elucidating its functions in faithful chromosome segregation using super-resolution microscopy.
(3) SAC protein architecture.
SAC (Spindle Assembly Checkpoint) proteins, also known as corona proteins, play a critical role in arresting cells before metaphase, until all kinetochores have properly attached to microtubules. A great majority of SAC components dissociate from kinetochores when the checkpoint is satisfied at metaphase. They are also major kinetochore structural components during G2, prophase, and prometaphase as well as at unattached kinetochores (kinetochores without microtubules). Thus, it important to answer the questions of where and how SAC proteins assemble at kinetochores. We are determining their nm-scale positions, as well as protein copy numbers, within above kinetochores, using super-resolution microscopy and quantitative light microscopy.