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International audience; The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo-EM structures of membrane-coated CHMP2A-CHMP3 filaments from Homo sapiens of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence toward the tube interior. Inter-filament interactions are electrostatic, which may facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high-speed atomic force microscopy imaging corroborate that VPS4 can constrict and cleave CHMP2A-CHMP3 membrane tubes. We therefore conclude that CHMP2A-CHMP3-VPS4 act as a minimal membrane fission machinery.

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo electron microscopy structures of membrane-coated CHMP2A-CHMP3 filaments of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence towards the tube interior. Inter-filament interactions are electrostatic, which facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high speed atomic force microscopy imaging corroborate that CHMP2A-CHMP3 polymers and VPS4 can constrict and cleave narrow membrane tubes, thus acting as a minimal membrane fission machinery.

While memory impairment in multiple sclerosis (MS) is known to be associated with hippocampal alterations, whether hippocampal networks could dynamically reorganize as a compensation mechanism is still a matter of debate. In this context, our aim was to identify the patterns of structural and functional connectivity between the hippocampus and the rest of the brain and their possible relevance to memory performances in early MS. Thirty-two patients with a first episode suggestive of MS together with 10 matched healthy controls were prospectively explored at baseline, 1 and 5 years follow up. They were scanned with MRI and underwent a neuropsychological battery of tests that included the Selective Reminding Test and the Brief Visual Memory Test Revised to assess verbal and visuo-spatial memory, respectively. Hippocampal volume was computed together with four graph theory metrics to study the structural and functional connectivity of both hippocampi with the rest of the brain. Associations between network parameters and memory performances were assessed using linear mixed-effects (LME) models. Considering cognitive abilities, verbal memory performances of patients decreased over time while visuo-spatial memory performances were maintained. In parallel, hippocampal volumes decreased significantly while structural and functional connectivity metrics were modified, with an increase in hippocampal connections over time. More precisely, these modifications were indicating a reinforcement of hippocampal short-distance connections. LME models revealed that the drop in verbal memory performances was associated with hippocampal volume loss, while the preservation of visuo-spatial memory performances was linked to decreased hippocampal functional shortest path length. In conclusion, we demonstrated a differential impairment in memory performances in the early stages of MS and an important interplay between hippocampal-related structural and functional networks and those performances. As the structural damage increases, functional reorganization seems to be able to maintain visuo-spatial memory performances with strengthened short-distance connections.

Malformations of cortical development (MCD) of the human brain are a likely consequence of defective neuronal migration, and/or proliferation of neuronal progenitor cells, both of which are dictated in part by microtubule-dependent transport of various cargoes, including the mitotic spindle. Throughout the evolutionary spectrum, proper spindle positioning depends on cortically anchored dynein motors that exert forces on astral microtubules emanating from spindle poles. A single heterozygous amino acid change, G436R, in the conserved TUBA1A α-tubulin gene was reported to account for MCD in patients. The mechanism by which this mutation disrupts microtubule function in the developing cerebral cortex is not understood. Studying the consequence of tubulin mutations in mammalian cells is challenging partly because of the large number of α-tubulin isotypes expressed. To overcome this challenge, we have generated a budding yeast strain expressing the mutated tubulin (Tub1G437R in yeast) as one of the main sources of α-tubulin (in addition to Tub3, another α-tubulin isotype in this organism). Although viability of the yeast was unimpaired by this mutation, they became reliant on Tub3, as was apparent by the synthetic lethality of this mutant in combination with tub3Δ. We find that Tub1G437R assembles into microtubules that support normal G1 activity, but lead to enhanced dynein-dependent nuclear migration phenotypes during G2/M, and a consequential disruption of spindle positioning. We find that this mutation impairs the interaction between She1 – a negative regulator of dynein – and microtubules, as was apparent from a yeast two-hybrid assay, a co-sedimentation assay, and from live cell imaging. We conclude that a weaker interaction between She1 and Tub1G437R-containing microtubules results in enhanced dynein activity, ultimately leading to the spindle positioning defect. Our results provide the first evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation, and sheds light on a mechanism that may be causative of neurodevelopmental diseases.

International audience; Malformations of Cortical Development (MCDs) are a group of severe brain malformations associated with intellectual disability and refractory childhood epilepsy. Human missense heterozygous mutations in the 9 α-tubulin and 10 β-tubulin isoforms forming the heterodimers that assemble into microtubules (MTs) were found to cause MCDs. However, how a single mutated residue in a given tubulin isoform can perturb the entire microtubule population in a neuronal cell remains a crucial question. Here, we examined 85 MCD-associated tubulin mutations occurring in TUBA1A, TUBB2 and TUBB3 and their location in a three-dimensional (3D) microtubule cylinder. Mutations hitting residues exposed on the outer microtubule surface are likely to alter microtubule association with partners, while alteration of intra-dimer contacts may impair dimer stability and straightness. Other types of mutations are predicted to alter inter-dimer and lateral contacts, which are responsible for microtubule cohesion, rigidity and dynamics. MCD-associated tubulin mutations surprisingly fall into all categories, thus providing unexpected insights into how a single mutation may impair microtubule function and elicit dominant effects in neurons.

Malformations of the human cerebral cortex can be caused by mutations in tubulins that associate to compose microtubules. Cerebral cortical folding relies on neuronal migration and on progenitor proliferation partly dictated by microtubule-dependent mitotic spindle positioning. A single amino acid change, F265L, in the conserved TUBB2B β-tubulin gene has been identified in patients with abnormal cortex formation. A caveat for studying this mutation in mammalian cells is that nine genes encode β-tubulin in human. Here, we generate a yeast strain expressing F265L tubulin mutant as the sole source of β-tubulin. The F265L mutation does not preclude expression of a stable β-tubulin protein which is incorporated into microtubules. However, impaired cell growth was observed at high temperatures along with altered microtubule dynamics and stability. In addition, F265L mutation produces a highly specific mitotic spindle positioning defect related to Bim1 (yeast EB1) dysfunction. Indeed, F265L cells display an abnormal Bim1 recruitment profile at microtubule plus-ends. These results indicate that the F265L β-tubulin mutation affects microtubule plus-end complexes known to be important for microtubule dynamics and for microtubule function during mitotic spindle positioning., Summary: Patients with intellectual disabilities carry a F265L mutation in TUBB2B β-tubulin gene. Yeast used as a cellular model reveals spindle mis-positioning associated with reduced yeast EB1 affinity for microtubule plus-ends.

L'aïkido est un art martial japonais fondé par Morihei Ueshiba au début de l'ère Meiji (1868-1912). Il se compose de techniques à mains nues et avec des armes. En utilisant la force de l'adversaire, cet art martial vise à réduire sa tentative de nuire à néant. On pourrait considérer l'aïkido comme la matérialisation du concept de légitime défense. Cette vidéo présente l'histoire de l'aïkido et sa pratique à travers un entraïnement et des entretiens avec les membres du club GAMA. Vous découvrirez différentes facettes de l'aikïdo à travers les expériences personnelles des pratiquants et une réflexion approfondie sur ses bienfaits.Remerciements au club d'arts martiaux et d'aïkido GAMA.

SUMMARY In mammals, the C-terminal tyrosine residue of α-tubulin is subjected to removal/re-addition cycles resulting in tyrosinated microtubules and detyrosinated Glu-microtubules. CLIP170 and its yeast ortholog (Bik1) interact weakly with Glu-microtubules. Recently, we described a Microtubule- Rho1- and Bik1-dependent mechanism involved in Snc1 routing. Here, we further show a contribution of the yeast p150Glued ortholog (Nip100) in Snc1 trafficking. Both CLIP170 and p150Glued are CAP-Gly-containing proteins that belong to the microtubule +end-tracking protein family (known as +Tips). We discuss the +Tips-dependent role of microtubules in trafficking, the role of CAP-Gly proteins as possible molecular links between microtubules and vesicles, as well as the contribution of the Rho1-GTPase to the regulation of the +Tips repertoire and the partners associated with microtubules.

Nurses draw on their creativity to improve patients' quality of life. A private-practice nurse created, with the support of a local network of associations, an original sewing workshop. Patients and volunteers come together to create attractive clothes adapted to the constraints of cancer treatments.

The diversity of microtubule functions is dependent on the status of tubulin C-termini. To address the physiological role of the C-terminal aromatic residue of α-tubulin, a tub1-Glu yeast strain expressing an α-tubulin devoid of its C-terminal amino acid was used to perform a genome-wide-lethality screen. The identified synthetic lethal genes suggested links with endocytosis and related processes. In the tub1-Glu strain, the routing of the v-SNARE Snc1 was strongly impaired, with a loss of its polarized distribution in the bud, and Abp1, an actin patch or endocytic marker, developed comet-tail structures. Snc1 trafficking required dynamic microtubules but not dynein and kinesin motors. Interestingly, deletion of the microtubule plus-end-tracking protein Bik1 (a CLIP170 ortholog), which is preferentially recruited to the C-terminal residue of α-tubulin, similarly resulted in Snc1 trafficking defects. Finally, constitutively active Rho1 rescued both Bik1 localization at the microtubule plus-ends in tub1-Glu strain and a correct Snc1 trafficking in a Bik1-dependent manner. Our results provide the first evidence for a role of microtubule plus-ends in membrane cargo trafficking in yeast, through Rho1- and Bik1-dependent mechanisms, and highlight the importance of the C-terminal α-tubulin amino acid in this process.

The diversity of microtubule functions is dependent on the status of tubulin C-termini. To address the physiological role of the C-terminal aromatic residue of α-tubulin, a tub1-Glu yeast strain expressing an α-tubulin devoid of its C-terminal amino acid was used to perform a genome-wide-lethality screen. The identified synthetic lethal genes suggested links with endocytosis and related processes. In the tub1-Glu strain, the routing of the v-SNARE Snc1 was strongly impaired, with a loss of its polarized distribution in the bud, and Abp1, an actin patch or endocytic marker, developed comet-tail structures. Snc1 trafficking required dynamic microtubules but not dynein and kinesin motors. Interestingly, deletion of the microtubule plus-end-tracking protein Bik1 (a CLIP170 ortholog), which is preferentially recruited to the C-terminal residue of α-tubulin, similarly resulted in Snc1 trafficking defects. Finally, constitutively active Rho1 rescued both Bik1 localization at the microtubule plus-ends in tub1-Glu strain and a correct Snc1 trafficking in a Bik1-dependent manner. Our results provide the first evidence for a role of microtubule plus-ends in membrane cargo trafficking in yeast, through Rho1- and Bik1-dependent mechanisms, and highlight the importance of the C-terminal α-tubulin amino acid in this process., Summary: In the yeast Saccharomyces cerevisiae, cargo trafficking involves microtubule plus-ends and a Bik1-dependent mechanism that requires the presence of the C-terminal aromatic residue of β-tubulin.

International audience; Bik1p is the budding yeast counterpart of the CLIP-170 family of microtubule plus-end tracking proteins, which are required for dynein localization at plus ends and dynein-dependent spindle positioning. CLIP-170 proteins make up a CAP-Gly microtubule-binding domain, which sustains their microtubule plus-end tracking behaviour. However, in yeast, Bik1p travels towards plus ends as a cargo of the plus-end-directed kinesin Kip2p. Additionally, Kip2p behaves as a plus-end-tracking protein; hence, it has been proposed that Bik1p might track plus ends principally as a cargo of Kip2p. Here, we examined Bik1p localization in yeast strains expressing mutant tubulin lacking the C-terminal amino acid (Glu tubulin; lacking Phe), the interaction of which with Bik1p is severely impaired compared with wild type. In Glu-tubulin strains, despite the presence of robust Kip2p comets at microtubule plus ends, Bik1p failed to track plus ends. Despite Bik1p depletion at plus ends, dynein positioning at the same plus ends was unperturbed. Video microscopy and genetic evidence indicated that dynein was transported at plus ends in a Kip2p-Bik1p-dependent manner, and was then capable of tracking Bik1p-depleted plus ends. These results indicate that Bik1p interactions with tubulin are important for Bik1p plus-end tracking, and suggest alternative pathways for Bik1p-Kip2p-dependent dynein localization at plus ends.

Nurses draw on their creativity to improve patients' quality of life. A private-practice nurse created, with the support of a local network of associations, an original sewing workshop. Patients and volunteers come together to create attractive clothes adapted to the constraints of cancer treatments.

Chromatin insulators are defined as transcriptionally neutral elements that prevent negative or positive influence from extending across chromatin to a promoter. Here we show that yeast subtelomeric anti-silencing regions behave as boundaries to telomere-driven silencing and also allow discontinuous propagation of silent chromatin. These two facets of insulator activity, boundary and silencing discontinuity, can be recapitulated by tethering various transcription activation domains to tandem sites on DNA. Importantly, we show that these insulator activities do not involve direct transcriptional activation of the reporter promoter. These findings predict that certain promoters behave as insulators and partition genomes in functionally independent domains.

Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.

The requirements for target ignition with megajoule-class lasers (Nakatsuka et al., Second Annual International Conference on Solid-State Lasers for Application to ICF, Paris, October 22–25, 1995 [1] ) create new constraints on the laser technology namely due to the propagation, frequency conversion and focusing of ultrahigh energy pulses. This paper emphasizes some advanced laser topics that arise with megajoule-class lasers such as frequency conversion with narrow and broadband pulses, energy losses due to mechanical sources of beam birefringence and due to a too long length of propagation in air. Wavefront distortion is measured and corrected and a new focusing scheme with high efficiency gratings is presented. The third beamline of the Phebus laser (Fleurot et al., Infrared Technology XVI, 1990, pp. 280–291 [2] ) is dedicated to these laser physics investigations at an energy density level of 3–4 GW/cm2 that is close to the megajoule laser (LMJ) beam density value. This beamline delivers up to 1.2 kJ, 1ω light in ns square shaped pulses, i.e. typically 1 TW pulses. The output beam aperture is reduced from 23 to 18 cm to get the required energy density level. The bandwidth of the laser pulse is varied up to a few angstroms. The beam characteristics are recorded and analysed by means of the output sensor 1ω energy, time profile and spectrum diagnostics and by means of a specific set of diagnostics for the 1ω, 2ω and 3ω beam parameters, located in the switchyard on a workstation called BACF.

a pulsed radiation power of the order of 1W in the submillimeter range (102 -110 μm) was reached for the first time, with the aid of a ZnGeP 2 crystal, at the difference frequency of CO 2 laser radiations.

International audience; PtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells.

The anisotropy of the hot-electron velocity distribution in ultra-high-intensity laser produced plasma was studied with x-ray polarization spectroscopy using multilayer planar targets including x-ray emission tracer in the middle layer. This measurement serves as a diagnostic for hot-electron transport from the laser-plasma interaction region to the overdense region where drastic changes in the isotropy of the electron velocity distribution are observed. These polarization degrees are consistent with analysis of a three-dimensional polarization spectroscopy model coupled with particle-in-cell simulations. Electron velocity distribution in the underdense region is affected by the electric field of the laser and that in the overdense region becomes wider with increase in the tracer depth. A full-angular spread in the overdense region of 22.4 deg.{sub -2.4}{sup +5.4} was obtained from the measured polarization degree.

We studied the induced plasma heating in three different kind of targets: mass limited, foam targets and large mass targets. The experiment was performed at Alise laser facility of CEA/CESTA. The laser system emitted a ∼1‐ps pulse with ∼10 J energy at a wavelength of ∼1 μm. Mass limited targets had three layers with thickness 10 μm C8H8, 1 μm C8H7Cl, 10 μm C8H8 with size 100 μm×100 μm. Detailed spectroscopic analysis of X‐rays emitted from the Cl tracer showed that it was possible to heat up the plasma mass limited targets to a temperature ∼250 eV with density ∼1021 cm−3. The plasma heating is only produced by fast electron transport in the target, being the 10 μm C8H8 overcoating thick enough to prevent any possible direct irradiation of the tracer layer even taking into account mass‐ablation due to the pre‐pulse. These results demonstrate that with mass limited targets is possible to generate a plasma heated up to several hundreds eV. It is also very important for research concerning high energy density...

Results of an experimental study of multi-MeV bremsstrahlung x-ray sources created by picosecond laser pulses are presented. The x-ray source is created by focusing the short pulse in an expanding plasma obtained by heating a solid target with a time-delayed nanosecond laser beam. The high-energy part of the x-ray spectrum and emission lobe are inferred from photonuclear activation techniques. The x-ray dose is measured with silicon diodes. Two-dimensional images of the source are reconstructed from a penumbral imaging technique. These results indicate the creation of a relatively small source, below 200 µm diameter, delivering doses up to 12 mrad in air at 1 m with x-ray temperature up to 2.8 MeV. The diagnostics used give access to a whole set of coherent experimental results on the x-ray source properties which are compared to extensive numerical simulations. X-ray intensity and temperature are found to increase with the size of the preplasma. ©2009 American Institute of Physics

X‐ray polarization spectroscopy was studied to derive directly the velocity distribution function (VDF) of hot electrons propagating in plasma created with a high intensity laser pulse. Polarization measurement was made at around 1018 W/cm2 using a laser pulse (∼10 J in ∼1 ps) from Alise facility at CEA/CESTA. Chlorinated triple‐layer targets were irradiated, and Cl Heα line was observed with an x‐ray polarization spectrometer. Polarization degrees were measured as a function of the target overcoat thickness, corresponding to the depth along pre‐formed plasma. It is found that the polarization is weakly negative for thin coating, but becomes positive, and finally zero for thick coating. This result is consistent with predictions made with a time‐dependent atomic kinetics code developed to gain an insight into the generation of polarized Cl Heα radiation. The de‐polarization on the surface is attributed to excessive bulk electron temperature and that in the deep region to elastic‐scattering processes by th...

In fast ignition research, the transport dynamics of fast electrons is one of the critical issues. Fast electrons generated by an intense laser pulse show a highly anisotropic velocity distribution. To gain insight into the anisotropy of the velocity distribution of fast electrons, polarized x‐ray spectroscopy has been proposed. The polarization spectroscopy of Cl Heα radiation was experimentally demonstrated at 1017 W/cm2 (∼100 mJ in 130 fs), and a new time‐dependent atomic population kinetics code was also developed. It predicts that the high polarization arises only in a low‐density region of the target plasma. Additional x‐ray polarization measurements were done at 101717–18 W/cm2 (∼10 J in ∼1 ps). Polarization was measured as a function of the overcoat thickness of a target. The polarization is negative in the shallow region near the target surface, and becomes near zero at the laser intensity of ∼1018 W/cm2. At ∼1017 W/cm2, the polarization varies from negative to positive, and finally zero along wi...

International audience; Previous experimental and numerical results have shown that in multi-MeV Bremsstrahlung x-ray sources created by picosecond laser pulses, the delivered on-axis dose increases if the laser interacts with under-critical plasmas. The effect of the laser-plasma interaction as a function of the plasma density gradient is thus first studied here, by focusing the short pulse of the Alisé laser on a solid tantalum target coated with plastic. Interaction conditions are modified by irradiating the target front side with a secondary nanosecond heating beam, prior to the main pulse. The length of the expanding plasma is modified by adjusting the delay between the interaction and heating beams. Various diagnostics give access to a whole set of consistent experimental results on the x-ray source properties which are compared successfully to self-consistent numerical simulations obtained with coupled PIC and Monte Carlo codes. All source parameters increase as the plasma length increases, in reason of the increasing number of high-energy electrons near the axis. In a second part of this talk, we use our simulation tools to predict the x-ray and neutron (produced by photonuclear interactions in the target) flash characteristics that can be expected on future PW lasers. Favourable scaling laws for the production of an intense, directional and short duration x-ray source versus intensity are observed. However, these findings also stress that radioprotection will become an important issue for future petawatt lasers operating at the kJ level.