Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3

International audienceThe regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor. INTRODUCTION Microtubules (MTs) are polarized structures that continuously switch between periods of polymerization and depolymerization at their growing (plus) ends. This process, termed MT dynamic instability, allows rapid reorganization of the MT cytoskeleton during essential cell functions such as cell polarity and migration, mitosi

PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein over-expressing tumors

PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.This work was supported by grants BIO2010-16351 (JFD), CAM S2010/BMD-2457 (JFD), CAM S2010/BMD-2353 (JMA), BFU2011-23416 (JMA) and PharmaMar-CSIC contracts. BP had a contract from Comunidad de Madrid

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

The immunosuppressor mycophenolic acid kills activated lymphocytes by inducing a nonclassical actin-dependent necrotic signal

International audienc

Plocabulin, a novel tubulin-binding agent, inhibits angiogenesis by modulation of microtubule dynamics in endothelial cells

BACKGROUND: Vascular supply of tumors is one of the main targets for cancer therapy. Here, we investigated if plocabulin (PM060184), a novel marine-derived microtubule-binding agent, presents antiangiogenic and vascular-disrupting activities. METHODS: The effects of plocabulin on microtubule network and dynamics were studied on HUVEC endothelial cells. We have also studied its effects on capillary tube structures formation or destabilization in three-dimensional collagen matrices. In vivo experiments were performed on different tumor cell lines. RESULTS: In vitro studies show that, at picomolar concentrations, plocabulin inhibits microtubule dynamics in endothelial cells. This subsequently disturbs the microtubule network inducing changes in endothelial cell morphology and causing the collapse of angiogenic vessels, or the suppression of the angiogenic process by inhibiting the migration and invasion abilities of endothelial cells. This rapid collapse of the endothelial tubular network in vitro occurs in a concentration-dependent manner and is observed at concentrations lower than that affecting cell survival. The in vitro findings were confirmed in tumor xenografts where plocabulin treatment induced a large reduction in vascular volume and induction of extensive necrosis in tumors, consistent with antivascular effects. CONCLUSIONS: Altogether, these data suggest that an antivascular mechanism is contributing to the antitumor activities of plocabulin

Plocabulin, a novel tubulin-binding agent, inhibits angiogenesis by modulation of microtubule dynamics in endothelial cells

BACKGROUND: Vascular supply of tumors is one of the main targets for cancer therapy. Here, we investigated if plocabulin (PM060184), a novel marine-derived microtubule-binding agent, presents antiangiogenic and vascular-disrupting activities. METHODS: The effects of plocabulin on microtubule network and dynamics were studied on HUVEC endothelial cells. We have also studied its effects on capillary tube structures formation or destabilization in three-dimensional collagen matrices. In vivo experiments were performed on different tumor cell lines. RESULTS: In vitro studies show that, at picomolar concentrations, plocabulin inhibits microtubule dynamics in endothelial cells. This subsequently disturbs the microtubule network inducing changes in endothelial cell morphology and causing the collapse of angiogenic vessels, or the suppression of the angiogenic process by inhibiting the migration and invasion abilities of endothelial cells. This rapid collapse of the endothelial tubular network in vitro occurs in a concentration-dependent manner and is observed at concentrations lower than that affecting cell survival. The in vitro findings were confirmed in tumor xenografts where plocabulin treatment induced a large reduction in vascular volume and induction of extensive necrosis in tumors, consistent with antivascular effects. CONCLUSIONS: Altogether, these data suggest that an antivascular mechanism is contributing to the antitumor activities of plocabulin

Microbeam Radiation Therapy Opens a Several Days’ Vessel Permeability Window for Small Molecules in Brain Tumor Vessels

International audiencePurposeSynchrotron microbeam radiation therapy (MRT), based on an inhomogeneous geometric and microscopic irradiation pattern of the tissues with high-dose and high-dose-rate x-rays, enhances the permeability of brain tumor vessels. This study attempted to determine the time and size range of the permeability window induced by MRT in the blood-brain (tumor) barrier.Methods and MaterialsRats-bearing 9L gliomas were exposed to MRT, either unidirectional (tumor dose, 406 Gy) or bidirectional (crossfired) (2 × 203 Gy). We measured vessel permeability to molecules of 3 sizes (Gd-DOTA, Dotarem, 0.56 kDa; gadolinium-labeled albumin, ∼74 kDa; and gadolinium-labeled IgG, 160 kDa) by daily in vivo magnetic resonance imaging, from 1 day before to 10 days after irradiation.ResultsAn equivalent tumor dose of bidirectional MRT delivered from 2 orthogonal directions increased tumor vessel permeability for the smallest molecule tested more effectively than unidirectional MRT. Bidirectional MRT also affected the permeability of normal contralateral vessels to a different extent than unidirectional MRT. Conversely, bidirectional MRT did not modify the permeability of normal or tumor vessels for both larger molecules (74 and 160 kDa).ConclusionsHigh-dose bidirectional (cross-fired) MRT induced a significant increase in tumor vessel permeability for small molecules between the first and the seventh day after irradiation, whereas permeability of vessels in normal brain tissue remained stable. Such a permeability window could facilitate an efficient and safe delivery of intravenous small molecules (≤0.56 kDa) to tumoral tissues. A permeability window was not achieved by molecules larger than gado-grafted albumin (74 kDa). Vascular permeability for molecules between these 2 sizes has not been determined

Microbeam Radiation Therapy opens a several days vessel permeability window for small molecules in brain tumor vessels.

PURPOSE Synchrotron microbeam radiation therapy (MRT), based on an inhomogeneous geometric and microscopic irradiation pattern of the tissues with high dose and high dose rate X-rays, enhances the permeability of brain tumor vessels. This study attempts to determine time and size range of the permeability window induced by MRT in the blood brain (tumor) barrier. METHODS AND MATERIALS Rats-bearing 9L gliomas were exposed to MRT, either unidirectional (tumor dose 406 Gy), or bidirectional (crossfired) (2 × 203 Gy). We measured vessel permeability to molecules of 3 sizes (Gd-DOTA, Dotarem®, 0.56 kDa; gadolinium-labeled albumin, ∼74 kDa; gadolinium-labeled IgG, 160 kDa) by daily in vivo magnetic resonance imaging (MRI), from 1 day before to 10 days post-irradiation. RESULTS An equivalent tumor dose of bidirectional MRT delivered from two orthogonal directions increased tumor vessel permeability for the smallest molecule tested more effectively than unidirectional MRT. Bidirectional MRT also affected the permeability of normal contralateral vessels to a different extent than unidirectional MRT. Conversely, bidirectional MRT did not modify the permeability of normal or tumor vessels for both larger molecules (74 and 160 kDa). CONCLUSIONS High-dose bidirectional (cross-fired) MRT induced a significant increase in tumor vessel permeability for small molecules between the first and the seventh day after irradiation, while permeability of vessels in normal brain tissue remained stable. Such a permeability window could facilitate an efficient and safe delivery of intravenous small molecules (≤0.56 kDa) to tumoral tissues. A permeability window was not achieved by molecules larger than gado-grafted albumin (74 kDa). Vascular permeability for molecules between these two sizes has not been determined