Date Published: March 6, 2018
Publisher: Public Library of Science
Author(s): Christian Hoischen, Sibel Yavas, Thorsten Wohland, Stephan Diekmann, Yanchang Wang.
Kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. Here, we measured the co-migration between protein pairs of the constitutive centromere associated network (CCAN) and hMis12 complexes by fluorescence cross-correlation spectroscopy (FCCS) in the nucleoplasm outside centromeres in living human interphase cells. FCCS is a method that can tell if in living cells two differently fluorescently labelled molecules migrate independently, or co-migrate and thus are part of one and the same soluble complex. We also determined the apparent dissociation constants (Kd) of the hetero-dimers CENP-T/W and CENP-S/X. We measured co-migration between CENP-K and CENP-T as well as between CENP-M and CENP-T but not between CENP-T/W and CENP-S/X. Furthermore, CENP-C co-migrated with CENP-H, and CENP-K with CENP-N as well as with CENP-L. Thus, in the nucleoplasm outside centromeres, a large fraction of the CENP-H/I/K/M proteins interact with CENP-C, CENP-N/L and CENP-T/W but not with CENP-S/X. Our FCCS analysis of the Mis12 complex showed that hMis12, Nsl1, Dsn1 and Nnf1 also form a complex outside centromeres of which at least hMis12 associated with the CENP-C/H/I/K/M/T/W/N/L complex.
Chromosome segregation is executed by a conserved molecular machinery which contains a large number of subunits and recruits many additional regulatory proteins (recently reviewed in [1–5]). A multi-protein complex, the “kinetochore”, assembles onto centromeric chromatin [6–15]. During mitosis, the kinetochore mediates the interaction between DNA and the mitotic spindle [16–28] (reviewed in ). Kinetochores are built from an inner layer, directly contacting centromeric chromatin, and an outer layer, binding to the spindle microtubules. The inner kinetochore controls outer kinetochore assembly [30–32], influences microtubule binding [31,33], and contributes to epigenetic specification of centromeres [21–26,34–37].
16 CCAN proteins, forming the sub-complexes CENP-T/W/S/X, CENP-H/I/K/M, CENP-L/N and CENP-P/O/R/Q/U, assemble at the centromere during all phases of the cell cycle. We studied if these proteins and the four proteins of the hMis12 complex form pre-complexes in the nucleoplasm outside centromeres.
In this work, by FCCS we measured the co-migration of CCAN and hMis12 complex protein pairs in the nucleoplasm outside centromeres in living human interphase cells. Since cross-correlation values obtained from DC-FCCS experiments under-estimate the percentage of co-migrating molecules, for some protein pairs we carried out SW-FCCS experiments which correct for these effects. Furthermore, the fluorescent tags might introduce sterical hindrance, preventing the detection of protein-protein interaction. Therefore, we labeled proteins at both termini and, in cases we detected no co-migration, introduced longer protein linkers between tag and CENP. For the hetero-dimerisation of CENP-T/W and CENP-S/X, we also determined apparent dissociation constants (Kd). We detected co-migration between CENP-K and CENP-T as well as between CENP-M and CENP-T but not between CENP-T/W and CENP-S/X. Furthermore, CENP-C co-migrated with CENP-H, and CENP-K with CENP-N as well as with CENP-L. Thus, in the nucleoplasm outside centromeres, the CENP-H/I/K/M proteins interact with CENP-C, CENP-N/L and CENP-T/W but not with CENP-S/X. Probably these interactions take place in the nucleoplasm independent of modifications specifically occurring at centromeres. Nevertheless, we assume that at the centromere cell cycle and location specific protein modification of at least some of the proteins will enhance their interactions with other CCAN proteins. Furthermore, at the centromere CCAN protein-protein interactions will be further stabilized by multiple protein-protein binding . Our FCCS analysis of the hMis12 complex showed that also these four proteins form a diffusing complex outside centromeres with at least hMis12 interacting with members of the CENP-C/H/I/K/M/T/W/N/L complex. Overall we show here that human CCAN and hMis12 kinetochore proteins assemble in the nucleoplasm outside centromeres, potentially forming dynamic multimeric complexes.