Visualization of prekinetochore locus on the centromeric region of highly extended chromatin fibers: does kinetochore autoantigen CENP-C constitute a kinetochore organizing center?

Kinetochore is morphologically defined as a trilaminated, highly differentiated structure at the primary constriction of mitotic chromosomes. This subcellular organella is assumed to be composed of DNA and proteins. Immunoelectron microscopy has shown that centromere autoantigens CENP-C and CENP-B localize to the kinetochore inner plate and the underlying centromeric region respectively. We previously indicated that both are DNA-binding proteins that constitute centromeric heterochromatin throughout the cell cycle. Here, we tried to elucidate how these molecules are involved in the kinetochore/centromere organization in vivo by analyzing their morphological behavior in nuclei. Using immunofluorescence microscopy, we found that CENP-C remained as round discrete dots, whereas CENP-B displayed larger surrounding materials. To examine the CENP-C-binding locus on the genome, we prepared highly extended chromatin fibers and performed simultaneous immunofluorescence and fluorescence in situ hybridization. We obsreved that centromeric alphoid DNA, targeted by CENP-B, was highly dispersed, whereas the CENP-C antigen persisted as small dots well situated on the fibers. These features reminded us of the 'ball and cup' structure that had been presented for 'prekinetochore'. We propose here that CENP-C constitutes a 'kinetochore organizing center' tightly associating with DNA, whereas CENP-B heterochromatin offers the solid support during kinetochore maturation.