Stephan Diekmann

Human kinetochore

Human mitotic checkpoint

Human kinetochore

The human centromere/kinetochore complex

We analyse the human kinetochore structure and function. We recombinantly tagged and labelled the known human kinetochore centromere proteins including the proteins of the HEC1 and hMis12 complexes. In case, these proteins are large (for example Cenp-C), we also cloned, tagged and labelled sub-fragments. We studied the interaction of these proteins in vivo by energy transfer (acceptor-bleaching FRET, FLIM, measuring associations), FCS (dynamics and concentration) and FRAP (dynamics) and the influence on the phenotype by RNAi. In our energy transfer studies, we included tagged histone proteins. Protein interactions are also studied by Yeast-Two-Hybrid analyses (collaboration with Prof. Wanker, MDC, Berlin and Dr. Hänel, HKI, Jena).

While most kinetochore proteins have been identified by now, little is known about the assembly dynamics of this chromosomal substructure. Using quantitative bleaching and fluorescence correlation spectroscopic imaging, we have assessed the incorporation and exchange rates of six inner kinetochore proteins during all phases of the cell cycle in living human cells. CENP-A, CENP-I, CENP-B, CENP-C, CENP-H and hMis12 exhibit distinct and cell cycle-specific chromatin binding stabilities with residence times ranging between seconds to hours. At mitosis, all inner kinetochore proteins become completely immobilized at centromeres. Outside centromeres, inner kinetochore proteins are highly mobile. This behaviour is consistent with a model in which these kinetochore proteins scan the nucleus by a binding-diffusion mechanism in search for their high-affinity binding sites at centromeres. These data provide a kinetic framework for inner kinetochore assembly. We are now expanding this type of analyses to all other known kinetochore constituents.

We tagged the inner kinetochore proteins CENP-A, -B, -C, -H and -I as well as the histones H2A, H3, H4 and H1 by Cerulean and EYFP, many of them at both termini, and analysed the energy transfer between them in human living cells. We found CENP-A and CENP-B in close vicinity to one another in vivo. These studies are currently extended to further kinetochore proteins.