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

Adaptive Radiation within Marine Anisakid Nematodes: A Zoogeographical Modeling of Cosmopolitan, Zoonotic Parasites

Parasites of the nematode genus Anisakis are associated with aquatic organisms. They can be found in a variety of marine hosts including whales, crustaceans, fish and cephalopods and are known to be the cause of the zoonotic disease anisakiasis, a painful inflammation of the gastro-intestinal tract caused by the accidental consumptions of infectious larvae raw or semi-raw fishery products. Since the demand on fish as dietary protein source and the export rates of seafood products in general is rapidly increasing worldwide, the knowledge about the distribution of potential foodborne human pathogens in seafood is of major significance for human health. Studies have provided evidence that a few Anisakis species can cause clinical symptoms in humans. The aim of our study was to interpolate the species range for every described Anisakis species on the basis of the existing occurrence data. We used sequence data of 373 Anisakis larvae from 30 different hosts worldwide and previously published molecular data (n = 584) from 53 field-specific publications to model the species range of Anisakis spp., using a interpolation method that combines aspects of the alpha hull interpolation algorithm as well as the conditional interpolation approach. The results of our approach strongly indicate the existence of species-specific distribution patterns of Anisakis spp. within different climate zones and oceans that are in principle congruent with those of their respective final hosts. Our results support preceding studies that propose anisakid nematodes as useful biological indicators for their final host distribution and abundance as they closely follow the trophic relationships among their successive hosts. The modeling might although be helpful for predicting the likelihood of infection in order to reduce the risk of anisakiasis cases in a given area

Candida albicans-induced epithelial damage mediates translocation through intestinal barriers

ABSTRACT Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans, a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans, candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin. IMPORTANCE Candida albicans, usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer

Rapid proliferation due to better metabolic adaptation results in full virulence of a filament-deficient Candida albicans strain.

The ability of the fungal pathogen Candida albicans to undergo a yeast-to-hypha transition is believed to be a key virulence factor, as filaments mediate tissue damage. Here, we show that virulence is not necessarily reduced in filament-deficient strains, and the results depend on the infection model used. We generate a filament-deficient strain by deletion or repression of EED1 (known to be required for maintenance of hyphal growth). Consistent with previous studies, the strain is attenuated in damaging epithelial cells and macrophages in vitro and in a mouse model of intraperitoneal infection. However, in a mouse model of systemic infection, the strain is as virulent as the wild type when mice are challenged with intermediate infectious doses, and even more virulent when using low infectious doses. Retained virulence is associated with rapid yeast proliferation, likely the result of metabolic adaptation and improved fitness, leading to high organ fungal loads. Analyses of cytokine responses in vitro and in vivo, as well as systemic infections in immunosuppressed mice, suggest that differences in immunopathology contribute to some extent to retained virulence of the filament-deficient mutant. Our findings challenge the long-standing hypothesis that hyphae are essential for pathogenesis of systemic candidiasis by C. albicans

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

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).

<p>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 E_var (grey bars), FRET measurement: spot 2 (acceptor bleached) yielding E_FRET (black bars). For protein pairs indicated, number of observed single cases (grouped into E_var or E_FRET value ranges of 4%) displayed versus values of E_var or E_FRET. (C) EGFP-CENP-U (donor) and CENP-R-mCherry (acceptor): distribution of E_FRET (40 bleached kinetochores) is clearly distinct from the distribution of E_var (39 non-bleached kinetochores) indicating FRET. (F): EGFP-CENP-Q (donor) and CENP-P-mCherry (acceptor): distribution of E_FRET (52 bleached kinetochores) superimposes the distribution of E_var (51 non-bleached kinetochores) indicating 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