Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex

Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment

Fission yeast Dicer harbours a unique dsRBD that participates in the spatial organization of the RNAi pathway

The term RNAi describes a set of conserved pathways found in most eukaryotes. RNAi is involved in various cellular processes, ranging from the control of gene expression to the establishment of heterochromatic structures. Common to all RNAi pathways is the association of small RNAs with members of the Argonaute family of proteins, forming the core component of a diverse set of protein-RNA complexes. The small RNAs guide these complexes via base-pairing interactions to homologous sequences, which usually results in reduced activity of these targets. The vast majority of small RNA molecules are generated by Dicer enzymes by endonucleolytically processing double-stranded RNAs. The Schizosaccharomyces pombe RNAi pathway is required for the formation of centromeric heterochromatin, and this process has been biochemically characterized in great detail. However, our knowledge about the spatial organization of RNAi in Schizosaccharomyces pombe is very limited. The few experiments performed so far, which have mainly addressed the cellular localization of Dicer and Argonaute, have resulted in data conflicting with the biochemical observations. In my PhD thesis, I have employed yeast genetics, biochemical and proteomics approaches to untangle these conflicting data with a major focus on the investigation of Dicer localization. I was able to demonstrate for the first time that Dicer is primarily localized to the nucleus where it associates with the nuclear periphery. Furthermore, I showed that nuclear retention of Dicer is essential for the formation of centromeric heterochromatin. These findings are consistent with the existing biochemical data and further support our model proposed for the formation of centromeric heterochromatin by the RNAi pathway. My early work demonstrated that nuclear localization of Dicer depends on its C-terminus. In a subsequent collaborative effort, we have solved the solution structure of this C-terminus, which showed that it encodes for a unique type of dsRBD and revealed novel insights into the mechanisms of nuclear retention of Dicer. Importantly, I have found that binding of this domain to RNA is dispensable for RNAi. Rather, the dsRBD represents a novel regulatory module for RNAi, which can mediate nucleo-cytoplasmic shuttling of Dicer. This feature seems to be conserved in higher eukaryotes. My work does also suggest a new function for RNAi in fission yeast, which is different from the well-established RNAi-mediated formation of heterochromatin at the centromeres and is likely to function in controlling environmentally regulated genes. Future studies in our laboratory will focus on the dissection of the mechanistic details of this novel mode of gene regulation

An extended dsRBD with a novel zinc-binding motif mediates nuclear retention of fission yeast Dicer

Dicer proteins function in RNA interference (RNAi) pathways by generating small RNAs (sRNAs). Here, we report the solution structure of the C-terminal domain of Schizosaccharomyces pombe Dicer (Dcr1). The structure reveals an unusual double-stranded RNA binding domain (dsRBD) fold embedding a novel zinc-binding motif that is conserved among dicers in yeast. Although the C-terminal domain of Dcr1 still binds nucleic acids, this property is dispensable for proper functioning of Dcr1. In contrast, disruption of zinc coordination renders Dcr1 mainly cytoplasmic and leads to remarkable changes in gene expression and loss of heterochromatin assembly. In summary, our results reveal novel insights into the mechanism of nuclear retention of Dcr1 and raise the possibility that this new class of dsRBDs might generally function in nucleocytoplasmic trafficking and not substrate binding. The C-terminal domain of Dcr1 constitutes a novel regulatory module that might represent a potential target for therapeutic intervention with fungal diseases.ISSN:0261-4189ISSN:1460-207

Dynamics of CENP-N kinetochore binding during the cell cycle

Accurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated

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

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

Levels of CENP-O/P/Q total protein during the cell cycle.

<p>(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.</p

FCCS measurements displaying G versus lag time.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044717#pone.0044717-Bacia1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044717#pone.0044717-Bacia2" target="_blank">[50]</a>. 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).</p

CENP-O loading to kinetochores measured by the SNAP-tag technology.

<p>(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.</p