Summary of the BDS and MDI CLIC08 Working Group

This note summarizes the presentations held within the Beam Delivery System and Machine Detector Interface working group of the CLIC08 workshop. The written contributions have been provided by the presenters on a voluntary basis

Microtubule sliding activity of a kinesin-8 promotes spindle assembly and spindle length control

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by cross-linking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report for the first time on an anti-parallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule destabilizing activity. In conjunction with kinesin-5/Cin8, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a “slide-disassemble” model where Kip3’s sliding and destabilizing activity balance during pre-anaphase. This facilitates normal spindle assembly. However, Kip3’s destabilizing activity dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly

Quantification of MAP and molecular motor activities on geometrically controlled microtubule networks.

International audienceThe spatial organization of the microtubule (MT) network directs cell polarity and mitosis. It is finely regulated by hundreds of different types of microtubule-associated proteins and molecular motors whose specific functions are difficult to investigate directly in cells. Here, we have investigated their functions using geometrically controlled MT networks in vitro in cell-free assay. This was achieved by developing a new method to spatially define MT nucleation using MT microseeds adsorbed on a micropatterned glass substrate. This method could be used to control MT growth and the induction of complex MT networks. We selected the interaction of two radial arrays of dynamic and polarized MTs to analyze the formation of the central antiparallel MT bundle. We investigated the effects of the MT cross-linker anaphase spindle elongation 1 (Ase1) and the kinesin motor Klp2, which are known to regulate MT organization in the spindle midzone. We thus identified the respective roles of each protein and revealed their synergy on the establishment of stable antiparallel MT bundles by quantifying MT interactions over hundreds of comparable MT networks

Emission of greenhouse gases from French temperate hydropower reservoirs

International audienceThe emission of CO2 and CH4 by diffusion, bubbling and downstream was measured in ten reservoirs representative of the diversity of French hydropower reservoirs in 2016. In all reservoirs, higher fluxes were measured in summer than in spring and winter. Low fluxes were measured in alpine reservoirs as compared to run-of-the-river and storage reservoirs. The low temperatures as well as the low organic matter input from the watershed explained this observation. Bubbling was higher in run-of-the-river reservoirs, as compared to storage reservoirs. This was related to a higher ratio between the length of wooded river network in the watershed, and the reservoir surface area. This ratio was considered as a proxy for allochthonous particulate organic matter input per reservoir surface unit and its accumulation in the sediments. In the larger storage reservoirs, this preferential sedimentation area was limited to the river-reservoir transition zone, the extent of which is primarily a function of reservoir hydrodynamic and morphological parameters. Conversely, the long water residence time in deep storage reservoirs favoured greenhouse gas (GHG) accumulation in the bottom water and diffusion and downstream pathways as compared to bubbling. Classical drivers of GHG emissions in large reservoirs partly failed to explain our measurements, especially for bubbling which seemed to be primarily controlled by allochthonous particulate organic matter input per reservoir surface area. This may results from the small size and the large diversity of the studied reservoirs as compared to the larger systems classically used for global estimates

Solving the Challenging Synthesis of Highly Cytotoxic Silver Complexes Bearing Sterically Hindered NHC Ligands with Mechanochemistry

The use of ball-mills enabled the straightforward synthesis of a variety of silver(I) complexes featuring challenging NHC ligands. Sterically hindered including electron-poor or with very low solubility imidazolium salts were grinded with silver(I) oxide to furnish heteroleptic or homoleptic complexes in high yield and short reaction times. The synthesis of heteroleptic bis-NHC silver(I) complexes was also performed for the first time in a ball-mill. The efficiency and rapidity of the mechanochemical approach enabled the generation of a library of unprecedented NHC silver complexes, which cytotoxicity on HCT116 colorectal cancer cell line was evaluated providing a rare example of medicinal mechanochemistry. The cationic silver complexes were found more potent than the neutral analogues, with IC50 values down to 21 nM, 256 times more potent than cisplatin. </div

CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin Incorporation

Microtubules play a key role in cell division, motility, and intracellular trafficking. Microtubule lattices are generally regarded as stable structures that undergo turnover through dynamic instability of their ends [1]. However, recent evidence suggests that microtubules also exchange tubulin dimers at the sites of lattice defects, which can be induced by mechanical stress, severing enzymes, or occur spontaneously during polymerization [2-6]. Tubulin incorporation can restore microtubule integrity; moreover, "islands" of freshly incorporated GTP-tubulin can inhibit microtubule disassembly and promote rescues [3, 4, 6-8]. Microtubule repair occurs in vitro in the presence of tubulin alone [2-6, 9]. However, in cells, it is likely to be regulated by specific factors, the nature of which is currently unknown. CLASPs are interesting candidates for microtubule repair because they induce microtubule nucleation, stimulate rescue, and suppress catastrophes by stabilizing incomplete growing plus ends with lagging protofilaments and promoting their conversion into complete ones [10-17]. Here, we used in vitro reconstitution assays combined with laser microsurgery and microfluidics to show that CLASP2α indeed stimulates microtubule lattice repair. CLASP2α promoted tubulin incorporation into damaged lattice sites, thereby restoring microtubule integrity. Furthermore, it induced the formation of complete tubes from partial protofilament assemblies and inhibited microtubule softening caused by hydrodynamic-flow-induced bending. The catastrophe-suppressing domain of CLASP2α, TOG2, combined with a microtubule-tethering region, was sufficient to stimulate microtubule repair, suggesting that catastrophe suppression and lattice repair are mechanistically similar. Our results suggest that the cellular machinery controlling microtubule nucleation and growth can also help to maintain microtubule integrity

Actin filaments regulate microtubule growth at the centrosome

International audienceThe centrosome is the main microtubule-organizing centre. It also organizes a local network of actin filaments. However, the precise function of the actin network at the centrosome is not well understood. Here, we show that increasing densities of actin filaments at the centrosome of lymphocytes are correlated with reduced amounts of microtubules. Furthermore, lymphocyte activation resulted in disassembly of centrosomal actin and an increase in microtubule number. To further investigate the direct crosstalk between actin and microtubules at the centrosome, we performed in vitro reconstitution assays based on (i) purified centrosomes and (ii) on the co-micropatterning of microtubule seeds and actin filaments. These two assays demonstrated that actin filaments constitute a physical barrier blocking elongation of nascent microtubules. Finally, we showed that cell adhesion and cell spreading lead to lower densities of centrosomal actin, thus resulting in higher microtubule growth. We therefore propose a novel mechanism, by which the number of centrosomal microtubules is regulated by cell adhesion and actin-network architecture

Solving the Challenging Synthesis of Highly Cytotoxic Silver Complexes Bearing Sterically Hindered NHC Ligands with Mechanochemistry

The use of ball-mills enabled the straightforward synthesis of a variety of silver(I) complexes featuring challenging NHC ligands. Sterically hindered including electron-poor or with very low solubility imidazolium salts were grinded with silver(I) oxide to furnish heteroleptic or homoleptic complexes in high yield and short reaction times. The synthesis of heteroleptic bis-NHC silver(I) complexes was also performed for the first time in a ball-mill. The efficiency and rapidity of the mechanochemical approach enabled the generation of a library of unprecedented NHC silver complexes, which cytotoxicity on HCT116 colorectal cancer cell line was evaluated providing a rare example of medicinal mechanochemistry. The cationic silver complexes were found more potent than the neutral analogues, with IC50 values down to 21 nM, 256 times more potent than cisplatin.The use of ball-mills enabled the straightforward synthesis of a variety of silver(I) complexes featuring challenging NHC ligands. Sterically hindered including electron-poor or with very low solubility imidazolium salts were grinded with silver(I) oxide to furnish heteroleptic or homoleptic complexes in high yield and short reaction times. The synthesis of heteroleptic bis-NHC silver(I) complexes was also performed for the first time in a ball-mill. The efficiency and rapidity of the mechanochemical approach enabled the generation of a library of unprecedented NHC silver complexes, which cytotoxicity on HCT116 colorectal cancer cell line was evaluated providing a rare example of medicinal mechanochemistry. The cationic silver complexes were found more potent than the neutral analogues, with IC50 values down to 21 nM, 256 times more potent than cisplatin

Evolution des sols ennoyés sous les retenues de barrage : Influence sur l'écologie des plans d'eau et la dynamique des gaz à effet de serre

Reservoir establishment results in the submersion of soils and vegetation. Their ecological evolution follows 3 main phases.- Initial: carbon and nutrients stored in living and dead vegetation biomass supply for a rise of the trophic level and productivity of the lake. During this phase, named « trophic upsurge », which duration and intensity are variable, water anoxia, CO2 and CH4 emissions also rise especially when temperature is high.- Mean Term: Water level fluctuates according to seasonal energy needs. Waves erode submerged soils along the shoreline. If the slope is steeper than 5%, soils are redistributed downslope and this flux contributes to the trophic upsurge and to sediment accumulation. These phenomena have been poorly studied and quantified. Our measurements at Sarrans reservoir suggest that soil redistribution is a major contributor to sediment accumulation. The ecological influence of carbon and nutrient inputs to the lake may be significant in oligotrophic environments. The evolution of permanently submerged soils is poorly known. Preliminary measurements at Sarrans reservoirs show that grassland cambisols (almost) permanently submerged have lost 30% of their original carbon store since impoundment.- On the long term, both catchment soils, through erosion and nutrient supply, and soils of the littoral zone, which support vegetation communities, influence reservoir ecology. The accumulation of sedimentary carbon in reservoirs appears much higher than in natural lakes, even eutrophic, and reasons for this are many. Greenhouse gas emission is also higher, because of higher sedimentation rate and increased water level fluctuations that favour methane bubbling and water degassing at the turbine outlet.Les réservoirs résultant de la construction de barrages noient de la végétation et des sols. Leur évolution suit trois phases.- Dynamique initiale : les nouvelles niches écologiques ouvertes ainsi que les stocks de carbone et de nutriments contenus dans la végétation vivante et morte contribuent à une hausse brutale du niveau trophique et de la productivité des réservoirs. Ce phénomène, appelé « trophic upsurge », est d’importance et de durée variables. Il s’accompagne d’une désoxygénation et d’émissions de CO2 et de CH4, d’autant plus fortes que la température est élevée.- Dynamique à moyen terme : le marnage induit par l’exploitant exonde temporairement les sols noyés, qui sont alors soumis à l’érosion par le batillage des vagues. Dès que la pente dépasse 5 %, les matériaux érodés sont redistribués dans la zone toujours en eau, contribuant au « trophic upsurge » et à la sédimentation. Ces phénomènes ont été très peu quantifiés. Des résultats récents obtenus sur la retenue de Sarrans (Aveyron) suggèrent que ce phénomène est à l’origine d’une proportion importante de l’accumulation sédimentaire. L’influence écologique de l’apport de nutriments associé à la redistribution des sols pourrait être significative dans les environnements oligotrophes. On connaît très mal l’évolution des sols ennoyés au fond des retenues. Des observations préliminaires effectuées sur la retenue de Sarrans montrent que les sols conservent leur structure d’origine. Elles suggèrent aussi que des sols bruns de prairie, ennoyés en permanence depuis 80 ans, ou exondés occasionnellement, ont perdu 30 % de leur carbone, par minéralisation.- Dynamique à long terme : A long terme, ce sont les sols du bassin versant et de la zone de marnage en pente faible qui influencent l’écologie des retenues. Les sols du bassin versant dans la mesure où le contexte écologique influence l’érosion et l’apport de nutriments. Les sols de la zone littorale dans la mesure où ils soutiennent une production végétale, qui peut contribuer à filtrer l’eau, mais aussi à l’alimenter en carbone organique minéralisable. Il semble que l’émission de gaz à effet de serre par les réservoirs est, toutes choses égales par ailleurs, plus importante que celle des lacs naturels, probablement en raison de la forte sédimentation, et des variations de pression qui favorisent l’émission de CH4, en particulier lors du passage en turbine