Distinct organization and regulation of the outer kinetochore KMN network downstream of CENP-C and CENP-T

Abstract

The kinetochore provides a vital connection between chromosomes and spindle microtubules [1, 2]. Defining the molecular architecture of the core kinetochore components is critical for understanding the mechanisms by which the kinetochore directs chromosome segregation. The KNL1/Mis12 complex/Ndc80 complex (KMN) network acts as the primary microtubule-binding interface at kinetochores [3] and provides a platform to recruit regulatory proteins [4]. Recent work found that the inner kinetochore components CENP-C and CENP-T act in parallel to recruit the KMN network to kinetochores [5-8]. However, due to the presence of these dual pathways, it has not been possible to distinguish differences in the nature of kinetochore assembly downstream of CENP-C or CENP-T. Here, we separated these pathways by targeting CENP-C and CENP-T independently to an ectopic chromosomal locus in human cells. Our work reveals that the organization of the KMN network components downstream of CENP-C and CENP-T is distinct. CENP-C recruits the Ndc80 complex through its interactions with KNL1 and the Mis12 complex. In contrast, CENP-T directly interacts with Ndc80, which in turn promotes KNL1/Mis12 complex recruitment through a separate region on CENP-T, resulting in functional relationships for KMN network localization that are inverted relative to the CENP-C pathway. We also find that distinct regulatory paradigms control the assembly of these pathways, with Aurora B kinase promoting KMN network recruitment to CENP-C and cyclin-dependent kinase (CDK) regulating KMN network recruitment to CENP-T. This work reveals unexpected complexity for the architecture and regulation of the core components of the kinetochore-microtubule interface.

Figure 1. KMN network components display separable recruitment to CENP-T

A) Immunofluorescence images showing positive (GFP-CENP-C (1-100)-LacI) or negative (GFP-CENP-C (101-234)-LacI) co-localization with anti-Ndc80 in nocodazole-treated cells. Chosen cells lacked overlap between the GFP focus and endogenous kinetochores marked by anti-CENP-A. Images were scaled independently to show the full range of data. Arrowheads indicate position of the GFP focus. B) Summary of immunofluorescence experiments assessing co-localization of KMN components with the indicated CENP-C-LacI fusions. >90% of cells display indicated behavior (N = 50 cells/condition). C) Representative immunofluorescence images showing localization of GFP-CENP-T-LacI foci and KMN components in nocodazole-arrested mitotic cells. Images were scaled independently to show full range of data. Numbers in lower right indicate number of mitotic cells showing co-localization. D) Summary of co-localization of KMN components with CENP-T-LacI fusions. >90% of cells observed display indicated behavior (N = 50 cells/condition). Scale bars, 5 μm. See also Figures S1 and S2.

Figure 2. The KMN network displays distinct dependency relationships for recruitment downstream of CENP-C and CENP-T

A and B) Top: Representative immunofluorescence images of GFP-CENP-C (1-100)-LacI (A) and GFP-CENP-T (1-250)-LacI (B) foci following depletion of KMN components. Bottom: Quantification of antibody/GFP intensity ratio at the focus, normalized to control RNAi (+/− SEM). N = 20. Right: Schematic of interdependency of KMN components for the CENP-C (A) and CENP-T (B) pathways determined by RNAi (Fig. 2) and truncation analyses (Fig. 1). For CENP-T, KNL1/Mis12 complex recruitment requires the Ndc80 complex and a second region on CENP-T. C) Representative immunofluorescence images showing anti-CENP-A and anti-Mis12 complex levels in HeLa cells. Quantification of Mis12 is shown in bottom right as fraction of control RNAi +/− standard deviation. N = 10. Student’s t-test (for panels A-C) - NS: not significant, *: p<0.05, **: p<0.01; ***: p<0.001. Scale bars, 5 μm.

Figure 3. Recruitment of KMN network to CENP-T is dependent on CDK phosphorylation

A) CENP-T schematic indicating its KMN network recruitment [6] and histone fold [25] domains. Indicated residues correspond to CDK phosphorylation sites [5, 24] with those analyzed in this study in red. Sequences corresponding to hatched regions are shown below. Alignment was performed using EMBOSS Water [26]. B) Representative immunofluorescence images showing GFP-CENP-T-LacI foci co-stained for Ndc80 or Mis12 complexes in nocodazole-treated cells. Images were scaled independently to show the full range of data. Numbers in lower right indicate the number of mitotic cells with co-localization between GFP and the indicated KMN component. Wild type CENP-T (1-250) images are duplicated from Fig. S1. Scale bar, 5 μm. C) Quantification of antibody/GFP fluorescence ratio (+/− SEM) at the indicated foci normalized to wild type CENP-T. N = 10 cells/condition. Student’s t-test, NS: not significant, *: p<0.05, **: p<0.01; ***: p<0.001. D) Immunofluorescence images of cells following 72 hr IPTG washout. Cells with >1 GFP focus are marked with arrowheads. Centromeres stained with anti-centromere antibodies (ACA). Scale bar, 15 μm. E) Table showing the frequency of cells with multiple GFP foci following 72 hr IPTG washout. N = 100 cells/condition. See also Figure S3.

Figure 4. Aurora B kinase regulates KMN network recruitment downstream of CENP-C

A and B) Left: Quantification of antibody/GFP intensity normalized to DMSO treatment for the indicated conditions (+/− SEM). N=20. Right: Representative immunofluorescence images of GFP-CENP-C (1-100)-LacI (A) and GFP-CENP-T (1-250)-LacI (B) foci after treatment. C) Representative immunofluorescence images showing CENP-A and Mis12 levels in HeLa cells. Quantification of Mis12 at mitotic kinetochores is shown in bottom right as a fraction of control RNAi + DMSO +/− standard deviation. N = 10. All images were scaled relative to their DMSO and RNAi control. D) Representative immunofluorescence images of GFP-Dsn1 wild type and Aurora B phospho-mimetic (S28D S78D S100D S109D) mutant expressing HeLa cells after RNAi depletion of endogenous Dsn1. GFP fluorescence is shown in bottom right as a fraction of mitotic kinetochore fluorescence for each construct +/− standard deviation. N = 90-300 kinetochores/condition from multiple cells. Images were scaled equivalently within each cell line. E) Model for KMN network recruitment at CENP-T or CENP-C foci (left) or endogenous kinetochores (right). It remains unknown whether KNL1/Mis12 interact directly with CENP-T, whether two Ndc80 complexes bind simultaneously to CENP-T, and whether a single KNL1/Mis12 complex can bridge CENP-T and CENP-C at endogenous kinetochores. Stars indicate phosphorylation by the indicated kinase. Student’s t-test (for panels A-D) - NS: not significant, *: p < 0.05, ***: p < 0.001. Scale bars, 5 μm. See also Figure S4.