Pathogenic LRRK2 compromises the subcellular distribution of lysosomes in a Rab12-RILPL1-dependent manner.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). Recent studies have shown that LRRK2 physiologically phosphorylates several Rab family proteins including Rab12 and that this phosphorylation is accelerated by the pathogenic mutations in LRRK2, although the significance in the PD pathogenesis remains unknown. Here we examined the effect of the overexpression of LRRK2 on the distribution of organelles in cultured cells and found that lysosomes become clustered in a perinuclear region upon the overexpression of pathogenic mutant LRRK2 in a manner dependent on its kinase activity. The perinuclear clustering of lysosomes was abolished by knocking out RAB12 as well as its effector protein RILPL1. Re-expression of Rab12 in RAB12 knockout cells suggested that the phosphorylation at Ser106 of Rab12 is required for the perinuclear clustering of lysosomes. Moreover, phosphorylated Rab12 was also accumulated on the clustered lysosomes, and the phosphorylation of Rab12 increased its interaction with RILPL1, leading us to conclude that the increase in the phosphorylation of Rab12 by pathogenic LRRK2 compromised intracellular lysosomal transport via the enhanced interaction of Rab12 with RILPL1. These data suggest the involvement of abnormal regulation of lysosomal transport in the LRRK2-mediated pathogenesis of PD.

Facile synthesis of single-crystalline MnO2 nanowire arrays with high photothermal catalytic activity.

A simple process has been developed to form single-crystalline ß-MnO2 nanowire arrays (NWAs) with a large surface area of 125 m2 g-1 on a glass plate working as a highly active three dimensional (3D) photothermal catalyst under the illumination of near infrared light due to the efficient light harvesting and heat confinement near the reaction field in addition to the large surface area.

Pixel-Level Clustering of Hematoxylin-Eosin-Stained Sections of Mouse and Human Biliary Tract Cancer.

We previously established mouse models of biliary tract cancer (BTC) based on the injection of cells with biliary epithelial stem cell properties derived from KRAS(G12V)-expressing organoids into syngeneic mice. The resulting mouse tumors appeared to recapitulate the pathological features of human BTC. Here we analyzed images of hematoxylin and eosin (H&E) staining for both the mouse tumor tissue and human cholangiocarcinoma tissue by pixel-level clustering with machine learning. A pixel-clustering model that was established via training with mouse images revealed homologies of tissue structure between the mouse and human tumors, suggesting similarities in tumor characteristics independent of animal species. Analysis of the human cholangiocarcinoma tissue samples with the model also revealed that the entropy distribution of cancer regions was higher than that of noncancer regions, with the entropy of pixels thus allowing discrimination between these two types of regions. Histograms of entropy tended to be broader for noncancer regions of late-stage human cholangiocarcinoma. These analyses indicate that our mouse BTC models are appropriate for investigation of BTC carcinogenesis and may support the development of new therapeutic strategies. In addition, our pixel-level clustering model is highly versatile and may contribute to the development of a new BTC diagnostic tool.

Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells.

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition.

Photothermal Oxidation of Cinnamyl Alcohol with Hydrogen Peroxide Catalyzed by Gold Nanoparticle/Antimony-Doped Tin Oxide Nanocrystals.

Gold nanoparticles with different mean sizes were formed on antimony-doped tin oxide nanocrystals by the temperature-varied deposition-precipitation method (Au/ATO NCs). Au/ATO NCs possess strong absorption in the near-infrared region due to Drude excitation in addition to the localized surface plasmon resonance (LSPR) of AuNPs around 530 nm. Au/ATO NCs show thermally activated catalytic activity for the oxidation of cinnamyl alcohol to cinnamaldehyde by hydrogen peroxide. The catalytic activity increases with a decrease in the mean Au particle size (dAu ) at 5.3 nm≤dAu ≤8.2 nm. Light irradiation (λex >660 nm, ∼0.5 sun) of Au/ATO NCs increases the rate of reaction by more than twice with ∼95 % selectivity. Kinetic analyses indicated that the striking enhancement of the reaction stems from the rise in the temperature near the catalyst surface of ∼30 K due to the photothermal effect of the ATO NCs.

Solid-State Structures and Photoluminescence of Lamellar Architectures of Cu(I) and Ag(I) Paddlewheel Clusters with Hydrogen-Bonded Polar Guests.

Two hexanuclear paddlewheel-like clusters appending six carboxylic-acid pendants have been isolated with the inclusion of polar solvent guests: [Cu6(Hmna)6]·7DMF (1·7DMF) and [Ag6(Hmna)6]·8DMSO (2·8DMSO), where H2mna = 2-mercaptonicotininc acid, DMF = N,N'-dimethylformamide, and DMSO = dimethyl sulfoxide. The solvated clusters, together with their fully desolvated forms 1 and 2, have been characterized by FTIR, UV-Vis diffuse reflectance spectroscopy, TG-DTA analysis, and DFT calculations. Crystal structures of two solvated clusters 1·7DMF and 2·8DMSO have been unambiguously determined by single-crystal X-ray diffraction analysis. Six carboxylic groups appended on the clusters trap solvent guests, DMF or DMSO, through H-bonds. As a result, alternately stacked lamellar architectures comprising of a paddlewheel cluster layer and H-bonded solvent layer are formed. Upon UV illumination (λex = 365 nm), the solvated hexasilver(I) cluster 2·8DMSO gives intense greenish-yellow photoluminescence in the solid state (λPL = 545 nm, ΦPL = 0.17 at 298 K), whereas the solvated hexacopper(I) cluster 1·7DMF displays PL in the near-IR region (λPL = 765 nm, ΦPL = 0.38 at 298 K). Upon complete desolvation, a substantial bleach in the PL intensity (ΦPL < 0.01) is observed. The desorption-sorption response was studied by the solid-state PL spectroscopy. Non-covalent interactions in the crystal including intermolecular H-bonds, CH⋯π interactions, and π⋯π stack were found to play decisive roles in the creation of the lamellar architectures, small-molecule trap-and-release behavior, and guest-induced luminescence enhancement.

Single-Cell Information Analysis Reveals That Skeletal Muscles Incorporate Cell-to-Cell Variability as Information Not Noise.

Cell-to-cell variability in signal transduction in biological systems is often considered noise. However, intercellular variation (i.e., cell-to-cell variability) has the potential to enable individual cells to encode different information. Here, we show that intercellular variation increases information transmission of skeletal muscle. We analyze the responses of multiple cultured myotubes or isolated skeletal muscle fibers as a multiple-cell channel composed of single-cell channels. We find that the multiple-cell channel, which incorporates intercellular variation as information, not noise, transmitted more information in the presence of intercellular variation than in the absence according to the "response diversity effect," increasing in the gradualness of dose response by summing the cell-to-cell variable dose responses. We quantify the information transmission of human facial muscle contraction during intraoperative neurophysiological monitoring and find that information transmission of muscle contraction is comparable to that of a multiple-cell channel. Thus, our data indicate that intercellular variation can increase the information capacity of tissues.

Automatic Quantitative Segmentation of Myotubes Reveals Single-cell Dynamics of S6 Kinase Activation.

Automatic cell segmentation is a powerful method for quantifying signaling dynamics at single-cell resolution in live cell fluorescence imaging. Segmentation methods for mononuclear and round shape cells have been developed extensively. However, a segmentation method for elongated polynuclear cells, such as differentiated C2C12 myotubes, has yet to be developed. In addition, myotubes are surrounded by undifferentiated reserve cells, making it difficult to identify background regions and subsequent quantification. Here we developed an automatic quantitative segmentation method for myotubes using watershed segmentation of summed binary images and a two-component Gaussian mixture model. We used time-lapse fluorescence images of differentiated C2C12 cells stably expressing Eevee-S6K, a fluorescence resonance energy transfer (FRET) biosensor of S6 kinase (S6K). Summation of binary images enhanced the contrast between myotubes and reserve cells, permitting detection of a myotube and a myotube center. Using a myotube center instead of a nucleus, individual myotubes could be detected automatically by watershed segmentation. In addition, a background correction using the two-component Gaussian mixture model permitted automatic signal intensity quantification in individual myotubes. Thus, we provide an automatic quantitative segmentation method by combining automatic myotube detection and background correction. Furthermore, this method allowed us to quantify S6K activity in individual myotubes, demonstrating that some of the temporal properties of S6K activity such as peak time and half-life of adaptation show different dose-dependent changes of insulin between cell population and individuals.Key words: time lapse images, cell segmentation, fluorescence resonance energy transfer, C2C12, myotube.