Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.

The outermost layer of centrosomes, called pericentriolar material (PCM), organizes microtubules for mitotic spindle assembly. The molecular interactions that enable PCM to assemble and resist external forces are poorly understood. Here, we use crosslinking mass spectrometry (XL-MS) to analyze PLK-1-potentiated multimerization of SPD-5, the main PCM scaffold protein in C. elegans. In the unassembled state, SPD-5 exhibits numerous intramolecular crosslinks that are eliminated after phosphorylation by PLK-1. Thus, phosphorylation induces a structural opening of SPD-5 that primes it for assembly. Multimerization of SPD-5 is driven by interactions between multiple dispersed coiled-coil domains. Structural analyses of a phosphorylated region (PReM) in SPD-5 revealed a helical hairpin that dimerizes to form a tetrameric coiled-coil. Mutations within this structure and other interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces, revealing that PCM assembly and strength are interdependent. We propose that PCM size and strength emerge from specific, multivalent coiled-coil interactions between SPD-5 proteins.

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.

During mitotic spindle assembly, microtubules generate tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. The molecular interactions that enable PCM to assemble rapidly and resist external forces are unknown. Here we use cross-linking mass spectrometry to identify interactions underlying supramolecular assembly of SPD-5, the main PCM scaffold protein in C. elegans . Crosslinks map primarily to alpha helices within the phospho-regulated region (PReM), a long C-terminal coiled-coil, and a series of four N-terminal coiled-coils. PLK-1 phosphorylation of SPD-5 creates new homotypic contacts, including two between PReM and the CM2-like domain, and eliminates numerous contacts in disordered linker regions, thus favoring coiled-coil-specific interactions. Mutations within these interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces. Thus, PCM assembly and strength are interdependent. In vitro , self-assembly of SPD-5 scales with coiled-coil content, although there is a defined hierarchy of association. We propose that multivalent interactions among coiled-coil regions of SPD-5 build the PCM scaffold and contribute sufficient strength to resist microtubule-mediated forces.

Forecasting leachate generation from pilot woodchip stockpiles using a three-dimensional transient flow model.

Leachate generation from open stockpiles of recycled woodchip materials is potentially harmful to aquatic ecosystems. There is growing interest in using numerical models to simulate leachate generation from outdoor piles, but this requires information about the hydraulic properties of the materials. The objectives of this study were to simulate leachate from woodchip piles with the numerical model HYDRUS-3D and to optimize subsets of parameters for single (SPM) and dual (DPM) pore flow models with the Bayesian Markov Chain Monte Carlo algorithm DREAMZS. Three experimental piles, each approximately 30 m3, were setup with mixtures of either once (coarse) or twice (fine) ground woodchips. Leachate continuously collected over a period of six months was similar across piles. As a result, subsets of optimized flow parameters for the coarse and fine woodchips were not different. Leachate predictions by the two pore flow models were similar and agreed reasonably with the field measurements, as indicated by Nash-Sutcliffe efficiency values greater than 0.6. This result suggests the simpler SPM is adequate for field predictions of leachate. However, leachate was consistently under-predicted by both pore models by 13-27% during rainfall events with more than 1 cm in 6 h. The optimized flow models can be used as a tool for studying pile management strategies.

Freshwater Crayfish: A Potential Benthic-Zone Indicator of Nanosilver and Ionic Silver Pollution.

Nowadays, silver nanoparticles (AgNPs) are utilized in numerous applications, raising justified concerns about their release into the environment. This study demonstrates the potential to use freshwater crayfish as a benthic-zone indicator of nanosilver and ionic silver pollution. Crayfish were acclimated to 20 L aquaria filled with Hudson River water (HRW) and exposed for 14 days to widely used Creighton AgNPs and Ag(+) at doses of up to 360 µg L(-1) to surpass regulated water concentrations. The uptake and distribution of Ag in over 650 exoskeletons, gills, hepatopancreas and muscles samples were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) in conjunction with two complementary U.S. EPA-endorsed methods: the external calibration and the standard additions. Reflecting the environmental plasticity of the two investigated species, Orconectes virilis accumulated in a dose-dependent manner more Ag than Procambarus clarkii (on average 31% more Ag). Both species showed DNA damage and severe histological changes in the presence of Ag. However, Ag(+) generally led to higher Ag accumulations (28%) and was more toxic. By the harvest day, about 14 ± 9% of the 360 µg L(-1) of AgNP exposure in the HRW oxidized to Ag(+) and may have contributed to the observed toxicities and bioaccumulations. The hepatopancreas (1.5-17.4 µg of Ag g(-1) of tissue) was identified as the best tissue-indicator of AgNP pollution, while the gills (4.5-22.0 µg g(-1)) and hepatopancreas (2.5-16.7 µg g(-1)) complementarily monitored the presence of Ag(+).