CENP-C is a blueprint for constitutive centromere-associated network assembly within human kinetochores.
Bottom Line: A 16-subunit complex named the constitutive centromere-associated network (CCAN) creates the centromere-kinetochore interface.We identified crucial determinants of this interaction whose mutation prevented kinetochore localization of CENP-HIKM and of CENP-TW, another CCAN subcomplex.When considered together with previous observations, our data point to CENP-C as a blueprint for kinetochore assembly.
Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.Show MeSH
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Mentions: We asked whether the CENP-HIKM binding interface of CENP-C is important for kinetochore localization of CCAN subunits in HeLa cells. For this, we generated inducible stable cell lines that expressed full-length wild-type GFP–CENP-C, or the 3A, W317A, or 4A mutants (Fig. S3, F and G). After depletion of endogenous CENP-C by RNAi and induction of transgene expression, the levels of CENP-HK at kinetochores were quantified (Fig. 3 A). Expression of GFP–CENP-Cwt largely rescued the kinetochore levels of CENP-HK observed in control cells. GFP–CENP-C3A and GFP–CENP-CW317A, on the other hand, only produced a partial rescue of the kinetochore levels of CENP-HK (Fig. 3 A), in agreement with their partial retention of the interaction with CENP-HIKM observed in SEC experiments (Fig. S1, C–F). Conversely, GFP–CENP-C4A was unable to produce significant rescue of the CENP-HK levels (Fig. 3, A and B), in line with the biochemical experiments.
Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.