Figure 1.
Acceptor-bleaching FRET of the protein pairs EGFP-CENP-U/CENP-R-mCherry (FRET signal) and EGFP-CENP-Q/CENP-P-mCherry (no FRET signal).
Typical HEp-2 cell nuclei are displayed in (A) and (D) showing centromere location of all four CEN proteins. Two of these locations, spot 1 and spot 2 in each of the two graphs, were selected for fluorescence intensity analysis before and after acceptor bleaching (see enlargements below). Spot 1 served as control and showed no detectable intensity change. At spot 2, the acceptor fluorophore mCherry was bleached (compare pre-bleach and post-bleach in (A) and (D)). In (B) and (E), the time course of the fluorescence intensity of the donor and the acceptor of both FRET pairs are shown. The acceptor intensities in the ROI (“region of interest”; open squares) were averaged and normalized to the mean intensity measured at the three time points before bleaching. The donor intensities in the ROI were averaged and normalized to the intensity measured at the time point after bleaching. Bleaching of the acceptor resulted in a fluorescence intensity increase of the donor (black dots) for EGFP-CENP-U (B) indicating the presence of FRET (see arrow), but no fluorescence intensity increase for EGFP-CENP-Q (E) indicating the absence of FRET. (C) and (F): Donor fluorescence intensity variation observed during acceptor bleaching normalized to the intensity measured at the first time point after bleaching. Control: spot 1 (acceptor not bleached) yielding Evar (grey bars), FRET measurement: spot 2 (acceptor bleached) yielding EFRET (black bars). For protein pairs indicated, number of observed single cases (grouped into Evar or EFRET value ranges of 4%) displayed versus values of Evar or EFRET. (C) EGFP-CENP-U (donor) and CENP-R-mCherry (acceptor): distribution of EFRET (40 bleached kinetochores) is clearly distinct from the distribution of Evar (39 non-bleached kinetochores) indicating FRET. (F): EGFP-CENP-Q (donor) and CENP-P-mCherry (acceptor): distribution of EFRET (52 bleached kinetochores) superimposes the distribution of Evar (51 non-bleached kinetochores) indicating no FRET.
Table 1.
FRET interactions between CENP-O class proteins.
Figure 2.
Cell cycle-dependent FRET between CENP-Q C- (grey bars) and N-termini (white bars).
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).
Figure 3.
Centromere protein interactions analyzed by F3H assay.
(A) EGFP tagged centromere proteins (bait, green) were recruited to the lac operator repeat array by the GFP-binding protein fused to the Lac repressor (LacI-GBP). Co-expressed mCherry tagged centromere proteins (prey, red) may either interact with the GFP-tagged protein (yellow in the overlay) or may not interact resulting in a disperse distribution. Upper two rows: interaction between EGFP-CENP-O and mCherry-CENP-P, EGFP-CENP-P and mCherry-CENP-O. Lower two rows: EGFP-CENP-Q did not interact with and recruit mCherry-CENP-O to the lac spot, but shows a weak interaction in the reverse combination. For all results see Table 1. Bar: 5 µm. (B): Strong F3H interactions are displayed (++: thick lines, +: thin lines). Black bars: interactions between CENP-PORQU proteins, red arrows: recruitment of CENP-PORQU proteins by CENP-K, -L, and -N. CENP-C is not able to recruit any of the CENP-PORQU proteins (data see Table 2).
Table 2.
F3H analysis of CENP-O class protein interactions.
Figure 4.
FCCS measurements displaying G versus lag time.
Red: mCherry (A, B) or mRFP (C), green: EGFP, black: auto-correlation. Count rates are displayed over 10 sec (inserts a1) indicating the absence of photobleaching, and 1 sec (inserts a2) indicating the absence of larger protein aggregates. The cross-correlation analyses are amplified in inserts b. (A) EGFP-CENP-O and mCherry-CENP-P indicate complex formation in the nucleoplasm (amplitude of cross-correlation/amplitude of mCherry signal: 29%). The amplitude of the cross-correlation curve A(CC), relative to the diffusion-related amplitude of one of the autocorrelation curves A(AC) of EGFP or mCherry, is a measure of binding or dynamic colocalization [49], [50]. According to this ratio of amplitudes A(CC)/A(AC), up to 20–30% of nucleoplasmic CENP-O and -P are hetero-dimers. Count rates were recorded simultaneously for both fluorophores. The count rate detected in a 10 sec measurement (insert a1) demonstrates the absence of photobleaching, while the count rate in a 1 sec resolution time scale (insert a2) indicates the absence of larger protein aggregates. The autocorrelations yielded 1.069 and 1.073 for EGFP-CENP-O and mCherry-CENP-P, respectively. The cross-correlation analysis (with a magnified scale of G(τ); insert b) resulted in a correlation of 1.02 indicating that 29% of the molecules are co-migrating in the nucleoplasm. (B) EGFP and mCherry expressed as single non-fused proteins (negative control) do not show any cross-correlation (A(CC)/A(EGFP) = 0%). The count rates (inserts a1 and a2) indicate the absence of photobleaching and larger proteins. The autocorrelations yielded 1.316 and 1.116 for EGFP and mRFP, respectively. The cross-correlation curve (with a magnified scale of G(τ), insert b) resulted in a value of 1.001 indicating the absence of any complexation between EGFP and mRFP. (C) mRFP-EGFP fusion protein (positive control) shows cross-correlation (A(CC)/A(mRFP) = 49%). The count rates indicate that photobleaching and the presence of larger protein aggregates can be excluded (inserts a1, a2) and that the autocorrelations of EGFP (1.06) and mRFP (1.09) were comparable to the values obtained for EGFP-CENP-O and mCherry-CENP-P. Cross-correlating the two channels against each other, we obtained a value of 1.029 indicating that about 50% of the molecules are detected as a complex (with a magnified scale of G(τ) in insert b).
Figure 5.
CENP-O loading to kinetochores measured by the SNAP-tag technology.
(A) Top: schematic representation of the performed experiment. Below: representative images of cells showing TMR-star fluorescence for SNAP-CENP-O in G2, M-phase and the following G1. Cell cycle phases G2 (CENP-F staining of the whole nucleus) and mitosis (specific kinetochore binding of CENP-F) are clearly identified. (B) The same experiment as in (A) was performed with U2OS cells stably expressing PCNA-GFP. SNAP-CENP-O fluorescence appears at kinetochores in late S-phase as judged from cellular PCNA distributions. (C) Top: schematic representation of the performed experiment. Below: representative images of cells expressing SNAP-CENP-O showing no fluorescence at kinetochores during G1. CENP-O is thus loaded to kinetochores in S-phase.
Figure 6.
Fluorescence recovery after photobleaching of EGFP-CENP-P in mid S-phase.
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.
Table 3.
Long term FRAP results for the CENP-PORQU proteins.
Figure 7.
Levels of CENP-O/P/Q total protein during the cell cycle.
(A) Quantitative immunoblot of CENP-O relative to α-Tubulin. Protein amounts are measured at G1/S (0 h), 2, 4, 6, 8 and 10 hrs after release from the double thymidine block in synchronised human HEp-2 cells. CENP-F and PCNA staining identify the time points 2, 4, and 6 hrs as S-phase, time point 8 hrs as G2 and 10 hrs as M-phase. The cellular amount of CENP-O reduces in G2 and further in M-phase. (B, C) Quantitative immuno-blots of CENP-P and CENP-Q protein levels relative to α-Tubulin at 0 (G1/S), 2 (early S), 4 (middle S), 6 (late S-phase), 8 (G2) hrs after release from double thymidine block in synchronized HeLa cells. Cycle stages were attributed from FACs analysis, PCNA staining and phase contrast microscopy (data not shown). (D) Representative immunoblots showing CENP-P, CENP-Q, Cyclin-B1 and α-Tubulin at the 0 (G1/S), 2 (early S), 4 (middle S) hrs time points and cells arrested in mitosis with nocodazole (16 hrs). (E) Quantitative four-colour immuno-flourence using anti-CENP-Q (red), CREST (green), DAPI (blue) and anti-PCNA (far red) antibodies in the same cells used in panel B. Pixel intensities of CENP-Q (signal – background) at kinetochores (n = 50 from 5 cells) are shown for each time point after release from double thymidine block (E) and representative images (F). CENP-Q loads onto kinetochores during S-phase reaching maximal binding in late S-phase (6 h). Scale bar = 5 µm.