CENP-C/H/I/K/M/T/W/N/L and hMis12 but not CENP-S/X participate in complex formation in the nucleoplasm of living human interphase cells outside centromeres

<div><p>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 (K_d) 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.</p></div

Cross-correlation by DC-FCCS.

<p>Cross-correlation by DC-FCCS.</p

Protein-protein co-migration of CCAN and Mis12 proteins.

<p>The degree of co-migration (corrected) in the nucleoplasm outside centromeres of human interphase cells is color-coded (red dashed arrows: no or hardly detectable co-migration, green arrow: 5–30%, blue arrows: 30–60%, black arrows: above 60% co-migration). Please note: these green, blue or black arrows do not necessarily indicate direct protein-protein interaction; the labeled proteins co-migrate in a complex: their interaction might be either direct or mediated by a third (or more) protein.</p

Cross-correlation by SW-FCCS.

<p>Cross-correlation by SW-FCCS.</p

DC-FCCS of EGFP-(s)-CENP-T and mCherry-(s)-CENP-S.

<p>A) Displayed are G versus lag time. Red: FCS- or autocorrelation-curve G (τ) for mCherry, green: FCS- or autocorrelation-curve G (τ) for EGFP, black: cross-correlation-curve G (τ), AC = autocorrelation. CC = cross-correlation, A(AC) = amplitude of autocorrelation curve, A(CC) = amplitude of cross-correlation curve. The cross-correlation analyses are amplified in inserts a. Count rates are displayed over 1 sec (inserts b; green = EGFP and red = mCherry). For the pair EGFP-(s)-CENP-T and mCherry-(s)-CENP-S no indication for complex formation in the nucleoplasm was detected (A(CC)/A(AC_mCherry) = 0%). The cross-correlation analysis (with a magnified scale of G (τ); insert a) resulted in a correlation of 1.001, whereas the autocorrelations yielded 1.322 for EGFP-(s)-CENP-T and 1.106 for mCherry-(s)-CENP-S. This ratio indicates that no nucleoplasmic CENP-T and -S are part of a common complex. B) Localization of cotransfected EGFP-(s)-CENP-T (EGFP) and mCherry-(s)-CENP-S (mCherry) in living human HEp-2 cells which were used for FCCS analysis. White bar = 10 μm. A cell nucleus is displayed showing co-localisation at centromeres (merge) and weak fluorescence in the nucleoplasm. Two locations of the same size and shape, a centromere (spot 1) and the centromere-free position of an FCCS measurement, as shown in Fig 4A (spot 2), in the nucleoplasm were selected for fluorescence intensity analysis. For the analyzed centromere in spot 1 the ratios of nucleoplasmic to centromeric fluorescence intensities was 1:43 for EGFP-(s)-CENP-T and 1:33 for mCherry-(s)-CENP-S. The concentrations of nucleoplasmic proteins, estimated by FCCS, was 6 nM for EGFP-(s)-CENP-T and 14 nM for mCherry-(s)-CENP-S.</p

SW-FCCS analysis of CENP-T and CENP-W.

<p>(a): ACF curves of EGFP-(s)-CENP-W (green) and mCherry-(s)-CENP-T (red), and CCF curves (blue, purple) in the nucleoplasm of interphase HEK293 cells. The data show cross-correlation between CENP-T and CENP-W, indicating interaction. (b) and (c): <i>K</i>_<i>d</i> determination using Scatter plot (b) and a histogram (c) of multiple SW-FCCS measurements to determine an effective <i>K</i>_<i>d</i> of the interaction. (d): ACF curves of EGFP-(s)-CENP-T^∆N (green) and mCherry-(s)-CENP-W (red), and CCF curves (blue, purple) in the nucleoplasm of interphase HEK293 cells. The data show reduced cross-correlation between CENP-T^∆N and CENP-W. (e) and (f): <i>K</i>_<i>d</i> determination using Scatter plot (e) and a histogram (f) of multiple SW-FCCS measurements to determine the effective <i>K</i>_<i>d</i> of this interaction between the histone-fold domain of CENP-T (CENP-T^∆N) and CENP-W. A defined interaction is detected by both, the linear fit of the scatter plot as well as the log-normal fit of the histogram.</p

FRET interactions between CENP-O class proteins.

<p>The FRET pair EGFP-mCherry is used. “F” indicates that for these fusions FRET was detected also by FLIM. ++: strong FRET, +: weak FRET, −: no FRET.</p

F3H analysis of CENP-O class protein interactions.

<p>GFP-tagged CENP-O class proteins, CENP-K, -L, -N and -C (rows) were bound to ectopic chromosomes sites. When RFP-tagged CENP-O class proteins, CENP-K, -L, -N and -C (lines) were recruited to these proteins, this was visible by a yellow dot. Signal intensity at the nuclear spot was used an indicator for interaction strength. ++, +: strong interaction; +−: weak interaction; −: no interaction.</p

Fluorescence recovery after photobleaching of EGFP-CENP-P in mid S-phase.

<p>Normalised mean fluorescence values of 55 kinetochores taken in time steps of 30 min over 4 hours. Recovery levels off, indicative of an about 40% immobile fraction.</p

Cell cycle-dependent FRET between CENP-Q C- (grey bars) and N-termini (white bars).

<p>In late S-phase and G2, significant FRET is observed (p<0.001). In G1, early and mid S-phase, no FRET is observed (p>0.005).</p