CAMSAP2 and CAMSAP3 localize at microtubule intersections to regulate the spatial distribution of microtubules.

Microtubule networks support many cellular processes and have a highly ordered architecture. However, due to the limited axial resolution of conventional light microscopy, the structural features of these networks cannot be resolved in three-dimensional (3D) space. Here, we use customized ultra-high resolution interferometric single-molecule localization microscopy to characterize the microtubule networks in Caco2 cells. We find that the microtubule minus-ends associated protein CAMSAPs localize at a portion of microtubule intersections. Further investigation shows that depletion of CAMSAP2 and CAMSAP3 leads to the narrowing of the inter-microtubule distance. We find that CAMSAPs recognize microtubule defects, which are often associated with microtubule intersections, and then recruit katanin to remove the damaged microtubules. Therefore, the CAMSAP-katanin complex is a regulating module for the distance between microtubules. Taken together, our results characterize the architecture of the cellular microtubule networks in high resolution and provide molecular insights into how the 3D structure of microtubule networks is controlled.

CAMSAP3 accumulates in the pericentrosomal area and accompanies microtubule release from the centrosome via katanin.

The epithelium has an apico-basal axis polarity that plays an important role in absorption, excretion and other physiological functions. In epithelial cells, a substantial number of non-centrosomal microtubules (MTs) are scattered in the cytoplasm with an apico-basal polarity and reorientate as epithelial cells perform different functions. Several previous studies have found that non-centrosomal MTs are nucleated at the centrosome, and then released and translocated elsewhere. However, the detailed process and molecular mechanism remain largely unknown. In this study, we found that Nezha, also called calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a non-centrosomal MT minus-end protein, accumulates in the pericentrosomal area and accompanies the release of MTs from the centrosome; whereas depletion of CAMSAP3 prevented MT release and instead caused focusing of MTs at centrosomes. Further studies demonstrated that CAMSAP3 precisely coordinates with dynein and katanin to regulate the MT detachment process. In conclusion, our results indicate that CAMSAP3 is a key molecule for generation of non-centrosomal MTs.

Minus end-directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions.

The adherens junction (AJ) plays a crucial role in maintaining cell-cell adhesion in epithelial tissues. Previous studies show that KIFC3, a minus end-directed kinesin motor, moves into AJs via microtubules that grow from clusters of CAMSAP3 (also known as Nezha), a protein that binds microtubule minus ends. The function of junction-associated KIFC3, however, remains to be elucidated. Here we find that KIFC3 binds the ubiquitin-specific protease USP47, a protease that removes ubiquitin chains from substrates and hence inhibits proteasome-mediated proteolysis, and recruits it to AJs. Depletion of KIFC3 or USP47 promotes cleavage of E-cadherin at a juxtamembrane region of the cytoplasmic domain, resulting in the production of a 90-kDa fragment and the internalization of E-cadherin. This cleavage depends on the E3 ubiquitin protein ligase Hakai and is inhibited by proteasome inhibitors. E-cadherin ubiquitination consistently increases after depletion of KIFC3 or USP47. These findings suggest that KIFC3 suppresses the ubiquitination and resultant degradation of E-cadherin by recruiting USP47 to AJs, a process that may be involved in maintaining stable cell-cell adhesion in epithelial sheets.

An autoinhibited structure of α-catenin and its implications for vinculin recruitment to adherens junctions.

α-Catenin is an actin- and vinculin-binding protein that regulates cell-cell adhesion by interacting with cadherin adhesion receptors through ß-catenin, but the mechanisms by which it anchors the cadherin-catenin complex to the actin cytoskeleton at adherens junctions remain unclear. Here we determined crystal structures of αE-catenin in the autoinhibited state and the actin-binding domain of αN-catenin. Together with the small-angle x-ray scattering analysis of full-length αN-catenin, we deduced an elongated multidomain assembly of monomeric α-catenin that structurally and functionally couples the vinculin- and actin-binding mechanisms. Cellular and biochemical studies of αE- and αN-catenins show that αE-catenin recruits vinculin to adherens junctions more effectively than αN-catenin, partly because of its higher affinity for actin filaments. We propose a molecular switch mechanism involving multistate conformational changes of α-catenin. This would be driven by actomyosin-generated tension to dynamically regulate the vinculin-assisted linkage between adherens junctions and the actin cytoskeleton.

Nezha/CAMSAP3 and CAMSAP2 cooperate in epithelial-specific organization of noncentrosomal microtubules.

Major microtubules in epithelial cells are not anchored to the centrosome, in contrast to the centrosomal radiation of microtubules in other cell types. It remains to be discovered how these epithelial microtubules are generated and stabilized at noncentrosomal sites. Here, we found that Nezha [also known as calmodulin-regulated spectrin-associated protein 3 (CAMSAP3)] and its related protein, CAMSAP2, cooperate in organization of noncentrosomal microtubules. These two CAMSAP molecules coclustered at the minus ends of noncentrosomal microtubules and thereby stabilized them. Depletion of CAMSAPs caused a marked reduction of microtubules with polymerizing plus ends, concomitantly inducing the growth of microtubules from the centrosome. In CAMSAP-depleted cells, early endosomes and the Golgi apparatus exhibited irregular distributions. These effects of CAMSAP depletion were maximized when both CAMSAPs were removed. These findings suggest that CAMSAP2 and -3 work together to maintain noncentrosomal microtubules, suppressing the microtubule-organizing ability of the centrosome, and that the network of CAMSAP-anchored microtubules is important for proper organelle assembly.

Analytical study of endocrine-disrupting chemicals in leachate treatment process of municipal solid waste (MSW) landfill sites.

Influent and processed water were sampled at different points in the leachate treatment facilities of five municipal solid waste (MSW) landfill sites. Then, the concentrations of endocrine-disrupting chemicals (EDCs), namely, alkylphenols (APs), bisphenol A (BPA), phthalic acid esters (PAEs) and organotin compounds (OTs), in the treated leachate samples were determined and the behavior of the EDCs in the treatment processes was discussed. The concentrations of APs were as low as those in surface waters, and no OTs were detected (detection limit: 0.01 microg/L). Meanwhile, diethylhexyl phthalate (DEHP), which was the most abundant of the four substances measured as PAEs, and BPA were found in all of the influent samples. BPA was considerably degraded by aeration, except when the water temperature was low and the total organic carbon (TOC) was high. By contrast, aeration, biological treatment, and coagulation/sedimentation removed only a small amount of DEHP.

Oxygen intrusion into waste in old landfills of low organic content.

Oxygen invasion into old landfills was studied by assuming the installation of gas venting pipes to promote stabilization of waste. In an experiment using a column pack with old incombustible waste, oxygen intrusion was observed and the oxygen consumption rate was estimated. Oxygen diffused into the waste layer very quickly in the initial stage of the experiment, but oxygen concentration increased only gradually due to reduced gradient and decreasing oxygen consumption. The maximum oxygen consumption rate in packed waste was one-third of that in loosely deposited waste in a beaker measured in a respiration test. A mathematical model was created which fitted the experimental data well and a three-dimensional simulation of a full-scale landfill and a sensitivity analysis were performed.

Batch separation of shredded bulky waste by gas-solid fluidized bed at laboratory scale.

A gas-solid fluidized bed separator using various bed materials was used to separate shredded municipal bulky waste (SBW). Using 290 microm glass beads as the bed material, the apparent density of the fluidized bed was 1.5 g/cm(3) and the SBW could be separated into combustibles such as wood, paper and plastics and incombustibles such as metals and glass. The overall efficiency (Newton's efficiency) of the separation was calculated to be 0.93. In order to obtain high efficiency, the superficial velocity must be adjusted so that the fluidized bed is agitated moderately and at the same time there is no weak fluidized region. Using a mixture of particles of nylon shot and 68 microm glass beads, the apparent density of the fluidized mixture bed could be varied between 0.63 and 0.99 g/cm(3) by changing the mixing ratio of the two materials. In the case of a mixing ratio of 20% for glass beads, an apparent density of 0.65 g/cm(3) was produced, in which wood and paper components were recovered while plastics remained in the bed to give a final overall efficiency of 0.88.

Leaching behaviour of elements and evaluation of pre-treatment methods for municipal solid waste incinerator residues in column leaching tests.

Two new pre-treatment methods (water-washing/carbonation and carbonation/phosphate stabilization) of municipal solid waste (MSW) incinerator residues were evaluated by column leaching tests under aerobic conditions and anaerobic conditions (which were changed to aerobic conditions after 10 months). A mixture of bottom ash and fly ash (5:1 ratio) was pre-treated using each method. Shredded incombustible residues (SIR) were added to each ash preparation in proportions similar to the ratios present in landfills. For comparison, landfill wastes typical of Japan, namely, a mixture of bottom ash, chelating-pre-treated fly ash, and SIR, were also examined. Leachate samples were collected periodically and analysed over a 15-month period. When compared with chelating pretreatment, both water-washing/carbonation and carbonation/ phosphate stabilization reduced the leaching of Pb, Al, and Cu by about one to two orders of magnitude. Moreover, the initial concentrations of Ca and Pb in leachates from column of water-washing/carbonation were 56-57% and 84-96% less than those from the column of carbonation/phosphate stabilization. Therefore, water-washing/carbonation was considered to be a promising approach to obtain early waste stabilization and to reduce the release of heavy metals to near-negligible levels. The leaching behaviour of elements was also discussed.

Gas permeability and tortuosity for packed layers of processed municipal solid wastes and incinerator residue.

To make a proper evaluation of gas component movement inside a landfill site, it is important to investigate the different parameters related to gas flow. In this work gas-filled porosity, intrinsic permeability, tortuosity and equivalent pore radius were determined for various packed wastes, such as incineration ash, shredded bulky waste and shredded incombustible waste. These parameters were measured/inferred for samples packed in a column and exposed to a controlled gas flow. The effect of waste conditions, especially the moisture content, on these parameters was also investigated. The intrinsic permeability of such packed wastes was generally in the order of 10(-10) to 10(-9) m2, except for some ash that was one to two orders lower. The tortuosity of waste layer was greater than that of a particulate material and ranged between 2 and 10. The equivalent pore radius was generally in the order of 10(-4) m, which means that gas diffusion is still ordinary in such packed waste layer. The obtained results will be utilized when simulating gas flow inside a landfill site for biogas extraction or site aeration.

Behavior of endocrine-disrupting chemicals in leachate from MSW landfill sites in Japan.

Endocrine-disrupting chemicals (EDCs) in landfill leachates and the effluent from leachate treatment facilities have been analyzed by many researchers. However, seasonal and yearly variations and the influence of landfill age are still not clear. In this study, leachate was sampled on four occasions each, at different seasons, from two MSW landfills which receive different waste material. Then, the quantities of alkylphenols (APs), bisphenol A (BPA), phthalic acid esters (PAEs) and organotin compounds (OTs) in leachate were determined. By sampling leachate from landfill cells of different age, the long-term behavior of EDCs was studied. Furthermore, leachate was also sampled at different points in the process of a leachate treatment system, and then the behavior of EDCs in the facility was studied. The concentrations of APs were as low as in surface waters, and OTs were not detected (detection limit was 0.01 microg/l), while BPA and DEHP, which were the most abundant of the four substances measured as PAEs, were found in all the leachates that were measured. Concentrations of BPA and DEHP were almost constant regardless of season, except for a couple of low concentrations observed for BPA. The varying composition of landfilled waste did not influence BPA and DEHP in leachate. Concentration of BPA in raw leachate tends to decrease as the years go by, but the concentration of DEHP was observed to remain at a constant level. BPA was considerably degraded by aeration for leachates from the two landfills, except when the leachate temperature was low. Aeration, coagulation/sedimentation, and biological treatment could not remove DEHP.

Evaluation of pre-treatment methods for landfill disposal of residues from municipal solid waste incineration.

This study presents results concerning leaching of Pb, Cd, Ca, and Cl with reference to one individual batch of bottom ash and fly ash (5:1) resulting from a municipal solid waste incinerator (MSWI) in Japan. This ash mixture was stabilised by the following pre-treatments: water washing, carbonation, and phosphate stabilisation. Once the optimum processing condition for each pre-treatment was determined, the performances were evaluated using both pH-stat leaching (pH 6, 9, 12) and availability tests. These performance tests were carried out with only fly ash without considering the mixture of MSWI residues, in order to accurately determine leaching differences among the pre-treatments. Water washing effectively removed the major elements from MSWI residues and also reduced the leachability of trace metals, such as Pb and Cd. A washing time of 15 minutes with a liquid/solid ratio of 5 was reasonably effective. Carbonation had a significant effect on leachability in alkaline ranges (pH 9 and 12), when the reaction occurred only on the surface of MSWI residues, moreover a moisture content of 10-16.7% was proved suitable for carbonation. On the other hand, phosphate stabilisation, even with small amounts of phosphate (0.16 mol-PO4(3-) kg(-1)), was very effective in reducing the leachability of heavy metals.

Study on composition and particle characteristics of shredded municipal waste for the improvement of separation efficiency in a municipal bulky waste processing facility.

In order to propose a new and more efficient method of separation for shredded municipal bulky waste (SBW), particle characteristics (namely shape, density and terminal settling velocity) were investigated. It was found that particles of the combustibles such as wood, paper and plastics were included in the fraction smaller than 50 mm. Consequently, separation of SBW into combustibles and non-combustibles by particle size is difficult. Since the particles of the various materials are of all shapes, it is difficult to sort them by shape discrimination. The overall separation efficiencies using air flow and density for SBW separation were estimated and the maximum values are as high as approximately 90% and 99% respectively. However, because of the overlapping ranges of the terminal settling velocities in air for glass, metals, rubber, and plastics, the overall efficiency will become smaller if those materials are major components of the feed. It is concluded that density separation is more suitable for the separation of combustibles and non-combustibles in SBW than air separation.

Determination of oxygen consumption for landfilled municipal solid wastes.

One of the serious problems of landfilling is the long time required for stabilisation. Nevertheless, site aeration proved to be effective to attenuate such problems. Oxygen consumption rate and air permeability/diffusion are the key parameters to set up the best allocation and design for vertical pipes to promote aeration and hence stabilisation of waste. This paper presents an investigation into the oxygen consumption for various landfilled wastes in Japan such as incineration ash, incombustible waste and under-sieve fraction of shredded bulky waste. Fresh waste sampled directly from processing facilities, as well as old waste sampled from landfill sites, were subjected to analytical determination and measurement of oxygen consumption. Bulky waste showed the highest oxygen consumption, however the old waste revealed a significant loss of biodegradability potential and lower oxygen consumption records. The oxygen consumption for short testing periods (10-days), tends to increase with the ignition loss (IL). The experimental data of respiratory tests (for old waste) were modelled and the fitting parameters are correlated to waste characteristics.

Solubility and adsorption characteristics of Pb in leachate from MSW incinerator bottom ash.

The solubility and adsorption characteristics of Pb in leachate from municipal solid waste incineration (MSWI) bottom ash was studied by batch experiments. The solubility of Pb in leachate was 1-2 orders of magnitude lower than that of Pb in carbonate solutions. Adsorption experiments showed bottom ash have high adsorption capacity for Pb. To evaluate a controlling component in bottom ash, quartz, Al and Fe powder as well as their ions were examined over wide ranges of pH at 25 degrees C. The results showed that only Fe powder had high adsorption capacities for Pb, indicating that iron oxides in bottom ash would be one major adsorbent for Pb. Adsorption diagram, constructed by using the data of adsorption experiments, fitted the experimental data of Pb in leachate very well. It suggests that the decrease of Pb solubility in leachate is mainly due to adsorption reaction.