Abstract
Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment.
Highlights
During mitosis, accurate chromosome segregation is essential for the correct transmission of the genetic material to the daughter cells
For CENP-Q-EGFP and mCherry-CENP-P and for CENP-P-EGFP and mCherryCENP-O, we confirmed these results by measuring fluorescence resonance energy transfer (FRET) at kinetochores in the lifetime domain (FLIM) by time-correlated single photon counting (TCSPC) using the same fluorescent protein FRET pair EGFP-mCherry
We conclude that to some extend CENP-N, but more efficiently CENP-L and even more so CENP-K mainly recruit CENP-O, -U and -R to kinetochores but much less so CENP-Q, and not CENP-P. This finding agrees with results of Okada et al [6] who observed in human and DT40 cells that the localization of CENP-O, -P, -Q and -H was disrupted in CENP-K and CENP-L depleted cells. Our results extend their observations by identifying the pairwise interactions responsible for the observed data: Potentially CENP-P and CENP-Q are disrupted from CENP-K and -L depleted cells due to being members of the CENP-PORQU complex and not due to specific protein-protein interactions
Summary
Accurate chromosome segregation is essential for the correct transmission of the genetic material to the daughter cells. The CCAN is required for the efficient recruitment of CENP-A into centromeric nucleosomes at the end of mitosis [6,14,30,31] and the maintenance of centromeric chromatin, but is involved in chromosome alignment, kinetochore fiber stability and bipolar spindle assembly [1,2,5,6,8,32,33,34]. The CCAN was suggested to establish, in interphase, an inner kinetochore structure which functions as an assembly platform for KMN network proteins in mitosis, and only the KMN proteins connect the inner kinetochore to microtubules [3]. Ectopical CENP-T and -C alone are able to establish a functional outer kinetochore [16,35] indicating that instead of being only a structural platform, the CCAN seems to be a regulator of the mitotic kinetochore-microtubule attachment [36]
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