Plk1-Dependent Recruitment of γ-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components

The nucleation of microtubules requires protein complexes containing γ-tubulin, which are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. We have previously shown that these interactions require the γ-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional γ-tubulin to the centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. However, the molecular details of this important pathway are unknown and a Plk1 substrate that controls γ-tubulin recruitment has not been identified. Here we show that Plk1 associates with GCP-WD in mitosis and Plk1 activity contributes to phosphorylation of GCP-WD. Plk1 depletion or inhibition prevents accumulation of GCP-WD at mitotic centrosomes, but GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit γ-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal γ-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of γ-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates

The centrosome protein NEDD1 as a potential pharmacological target to induce cell cycle arrest

<p>Abstract</p> <p>Background</p> <p>NEDD1 is a protein that binds to the gamma-tubulin ring complex, a multiprotein complex at the centrosome and at the mitotic spindle that mediates the nucleation of microtubules.</p> <p>Results</p> <p>We show that NEDD1 is expressed at comparable levels in a variety of tumor-derived cell lines and untransformed cells. We demonstrate that silencing of NEDD1 expression by treatment with siRNA has differential effects on cells, depending on their status of p53 expression: p53-positive cells arrest in G1, whereas p53-negative cells arrest in mitosis with predominantly aberrant monopolar spindles. However, both p53-positive and -negative cells arrest in mitosis if treated with low doses of siRNA against NEDD1 combined with low doses of the inhibitor BI2536 against the mitotic kinase Plk1. Simultaneous reduction of NEDD1 levels and inhibition of Plk1 act in a synergistic manner, by potentiating the anti-mitotic activity of each treatment.</p> <p>Conclusion</p> <p>We propose that NEDD1 may be a promising target for controlling cell proliferation, in particular if targeted in combination with Plk1 inhibitors.</p

A lateral belt of cortical LGN and NuMA guides mitotic spindle movements and planar division in neuroepithelial cells

Knockdown or mislocalization of LGN complex components disrupts the stereotypic biphasic spindle movements regulating planar cell division and neuroepithelial structure in chick embryos

Dependence of atmospheric muon flux on seawater depth measured with the first KM3NeT detection units: The KM3NeT Collaboration

KM3NeT is a research infrastructure located in the Mediterranean Sea, that will consist of two deep-sea Cherenkov neutrino detectors. With one detector (ARCA), the KM3NeT Collaboration aims at identifying and studying TeV–PeV astrophysical neutrino sources. With the other detector (ORCA), the neutrino mass ordering will be determined by studying GeV-scale atmospheric neutrino oscillations. The first KM3NeT detection units were deployed at the Italian and French sites between 2015 and 2017. In this paper, a description of the detector is presented, together with a summary of the procedures used to calibrate the detector in-situ. Finally, the measurement of the atmospheric muon flux between 2232–3386 m seawater depth is obtained

Deep-sea deployment of the KM3NeT neutrino telescope detection units by self-unrolling

KM3NeT is a research infrastructure being installed in the deep Mediterranean Sea. It will house a neutrino telescope comprising hundreds of networked moorings — detection units or strings — equipped with optical instrumentation to detect the Cherenkov radiation generated by charged particles from neutrino-induced collisions in its vicinity. In comparison to moorings typically used for oceanography, several key features of the KM3NeT string are different: the instrumentation is contained in transparent and thus unprotected glass spheres; two thin Dyneema® ropes are used as strength members; and a thin delicate backbone tube with fibre-optics and copper wires for data and power transmission, respectively, runs along the full length of the mooring. Also, compared to other neutrino telescopes such as ANTARES in the Mediterranean Sea and GVD in Lake Baikal, the KM3NeT strings are more slender to minimise the amount of material used for support of the optical sensors. Moreover, the rate of deploying a large number of strings in a period of a few years is unprecedented. For all these reasons, for the installation of the KM3NeT strings, a custom-made, fast deployment method was designed. Despite the length of several hundreds of metres, the slim design of the string allows it to be compacted into a small, re-usable spherical launching vehicle instead of deploying the mooring weight down from a surface vessel. After being lowered to the seafloor, the string unfurls to its full length with the buoyant launching vehicle rolling along the two ropes. The design of the vehicle, the loading with a string, and its underwater self-unrolling are detailed in this paper.French National Research Agency (ANR) ANR-15-CE31-0020Centre National de la Recherche Scientifique (CNRS)European Union (EU)Institut Universitaire de France (IUF)LabEx UnivEarthS ANR-10-LABX-0023 ANR-18-IDEX-0001Paris Ile-de-France Region, FranceShota Rustaveli National Science Foundation of Georgia (SRNSFG), Georgia FR-18-1268German Research Foundation (DFG)Greek Ministry of Development-GSRTIstituto Nazionale di Fisica Nucleare (INFN), Ministero dell'Universita e della Ricerca (MUR), PRIN Italy NAT-NET 2017W4HA7SMinistry of Higher Education, Scientific Research and Professional Training, MoroccoNetherlands Organization for Scientific Research (NWO) Netherlands GovernmentNational Science Center, Poland National Science Centre, Poland 2015/18/E/ST2/00758National Authority for Scientific Research (ANCS), RomaniaMinisterio de Ciencia, Innovación, Investigación y Universidades (MCIU): Programa Estatal de Generación de Conocimiento (MCIU/FEDER) PGC2018-096663-B-C41 PGC2018-096663-B-A-C42 PGC2018-096663-B-BC43 PGC2018-096663-B-B-C44Severo Ochoa Centre of Excellence and MultiDark Consolider (MCIU), Junta de Andalucía SOMM17/6104/UGRGeneralitat Valenciana GRISOLIA/2018/119 CIDEGENT/2018/034La Caixa Foundation LCF/BQ/IN17/11620019EU: MSC program, Spain 71367

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Stability of the small ?-tubulin complex requiresHCA66, a protein of the centrosome and the nucleolus.

To investigate changes at the centrosome during the cell cycle,we analyzed the composition of the pericentriolar materialfrom unsynchronized and S-phase-arrested cells by gelelectrophoresis and mass spectrometry. We identified HCA66,a protein that localizes to the centrosome from S-phase to mitosisand to the nucleolus throughout interphase. Silencing of HCA66expression resulted in failure of centrosome duplicationand in the formation of monopolar spindles, reminiscentof the phenotype observed after ?-tubulin silencing.Immunofluorescence microscopy showed that proteins of the?-tubulin ring complex were absent from the centrosome inthese monopolar spindles. Immunoblotting revealed reducedprotein levels of all components of the ?-tubulin small complex(?-tubulin, GCP2, and GCP3) in HCA66-depleted cells. Bycontrast, the levels of ?-tubulin ring complex proteins such asGCP4 and GCP-WD/NEDD1 were unaffected. We propose thatHCA66 is a novel regulator of ?-tubulin function that plays arole in stabilizing components of the ?-tubulin small complex,which is in turn essential for assembling the larger ?-tubulinring complex

NuMA is required for proper spindle assembly and chromosome alignment in prometaphase

NuMA is a protein that has been previously shown to play a role in focusing microtubules at the mitotic spindle poles. However, most previous work relies on experimental methods that might cause dominant side effects on spindle formation, such as microinjection of antibodies, overexpression of mutant protein, or immunodepletion of NuMA-containing protein complexes

Assembly and regulation of γ-tubulin complexes

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