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

How COVID-19 changed clinical research strategies: a global survey

Objective Clinical research has faced new challenges during the COVID-19 pandemic, leading to excessive operational demands affecting all stakeholders. We evaluated the impact of COVID-19 on clinical research strategies and compared different adaptations by regulatory bodies and academic research institutions in a global context, exploring what can be learned for possible future pandemics. Methods We conducted a cross-sectional online survey and identified and assessed different COVID-19-specific adaptation strategies used by academic research institutions and regulatory bodies. Results All 19 participating academic research institutions developed and followed similar strategies, including preventive measures, manpower recruitment, and prioritisation of COVID-19 projects. In contrast, measures for centralised management or coordination of COVID-19 projects, project preselection, and funding were handled differently amongst institutions. Regulatory bodies responded similarly to the pandemic by implementing fast-track authorisation procedures for COVID-19 projects and developing guidance documents. Quality and consistency of the information and advice provided was rated differently amongst institutions. Conclusion Both academic research institutions and regulatory bodies worldwide were able to cope with challenges during the COVID-19 pandemic by developing similar strategies. We identified some unique approaches to ensure fast and efficient responses to a pandemic. Ethical concerns should be addressed in any new decision-making process

Mild replication stress causes chromosome mis-segregation via premature centriole disengagement

Replication stress, a hallmark of cancerous and pre-cancerous lesions, is linked to structuralchromosomal aberrations. Recent studies demonstrated that it could also lead to numericalchromosomal instability (CIN). The mechanism, however, remains elusive. Here, we showthat inducing replication stress in non-cancerous cells stabilizes spindle microtubules andfavours premature centriole disengagement, causing transient multipolar spindles that lead tolagging chromosomes and micronuclei. Premature centriole disengagement depends on theG2 activity of the Cdk, Plk1 and ATR kinases, implying a DNA-damage induced deregulationof the centrosome cycle. Premature centriole disengagement also occurs spontaneously insome CIN+cancer cell lines and can be suppressed by attenuating replication stress. Finally,we show that replication stress potentiates the effect of the chemotherapeutic agent taxol, byincreasing the incidence of multipolar cell divisions. We postulate that replication stress incancer cells induces numerical CIN via transient multipolar spindles caused by prematurecentriole disengagement

How COVID-19 changed clinical research strategies: a global survey.

OBJECTIVE: Clinical research has faced new challenges during the COVID-19 pandemic, leading to excessive operational demands affecting all stakeholders. We evaluated the impact of COVID-19 on clinical research strategies and compared different adaptations by regulatory bodies and academic research institutions in a global context, exploring what can be learned for possible future pandemics. METHODS: We conducted a cross-sectional online survey and identified and assessed different COVID-19-specific adaptation strategies used by academic research institutions and regulatory bodies. RESULTS: All 19 participating academic research institutions developed and followed similar strategies, including preventive measures, manpower recruitment, and prioritisation of COVID-19 projects. In contrast, measures for centralised management or coordination of COVID-19 projects, project preselection, and funding were handled differently amongst institutions. Regulatory bodies responded similarly to the pandemic by implementing fast-track authorisation procedures for COVID-19 projects and developing guidance documents. Quality and consistency of the information and advice provided was rated differently amongst institutions. CONCLUSION: Both academic research institutions and regulatory bodies worldwide were able to cope with challenges during the COVID-19 pandemic by developing similar strategies. We identified some unique approaches to ensure fast and efficient responses to a pandemic. Ethical concerns should be addressed in any new decision-making process

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

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