Essential role of PACSIN2/syndapin-II in caveolae membrane sculpting.

Caveolae are flask-shaped invaginations of the plasma membrane that are associated with tumor formation, pathogen entry and muscular dystrophy, through the regulation of lipids, signal transduction and endocytosis. Caveolae are generated by the fusion of caveolin-1-containing vesicles with the plasma membrane, which then participate in endocytosis via dynamin. Proteins containing membrane-sculpting F-BAR (or EFC) domains organize the membrane in clathrin-mediated endocytosis. Here, we show that the F-BAR protein PACSIN2 sculpts the plasma membrane of the caveola. The PACSIN2 F-BAR domain interacts directly with caveolin-1 by unmasking autoinhibition of PACSIN2. Furthermore, the membrane invaginations induced by the PACSIN2 F-BAR domain contained caveolin-1. Knockdown of PACSIN2 resulted in abnormal morphology of caveolin-1-associated plasma membranes, presumably as a result of decreased recruitment of dynamin-2 to caveolin-1. These results indicate that PACSIN2 mediates membrane sculpting by caveolin-1 in caveola morphology and recruits dynamin-2 for caveola fission.

Phosphoinositide binding by par-3 involved in par-3 localization.

Electrostatic interactions between lipids and proteins control many cellular events. We found that phospholipids, including phosphatidylinositol 3-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidylinositol 3,4,5-triphosphate, bound to the C-terminal coiled-coil region of par-3 at conserved, basic residues. We identified K1013 and K1014 as the phosphoinositide binding site, because the K1013E/K1014E mutation of rat par-3 abolished its lipid binding. Importantly, the K1013E/K1014E par-3 mutant exhibited significantly weaker localization at the cell-cell junctions than the wild-type par-3. Fluorescence recovery after photo-bleaching analyses confirmed the faster turnover of mutant par-3 at cell-cell junctions. The treatment of cells with an inhibitor of phosphatidylinositol 3-kinases partially increased the turnover of par-3. These data suggested that the putative phospholipid binding by par-3 is important for its localization at cell-cell junctions.

Characterization of Anti-Ice Nucleation Activity of the Extract from Coffee Refuse.

　The supercooling-facilitating (SCF) activities, that is, the anti-ice nucleation activity of the hot water extracts from five types of processed food refuse was examined. The extract with the highest activity among five hot water extracts was coffee refuse, showing 1.50℃ of SCF activity at a final concentration of 0.1 mg/ml. From the hot water extract of coffee refuse, the coffee refuse extract containing various polyphenols was prepared by the ultrafiltration (less than MWCO 10,000), a solvent fractionation of ethyl acetate. The yield of coffee refuse extract was 0.9% (w/w) from dried coffee refuse. The SCF activity of the coffee refuse extract at a final concentration of 1.0 mg/ml was 4.2℃. HPLC analysis of the coffee refuse extract showed that caffeine and chlorogenic acid, which are major components of coffee, could be found at 173 and 62.3 µg/ml, respectively. However, the SCF activities of both compounds (0.70 and 1.06℃) at a final concentration of 0.1 mg/ml were lower than those of ferulic acid and coumaric acid, respectively at 3.40 and 2.35℃. This is the first report to our knowledge on the SCF activity of caffeine. The SCF activity of caffeine at a final concentration of 1.0 mg/ml was 2.3℃. The specificity of caffeine against various ice nuclei containing calcium oxalate, 9-fluorenon, and ice nucleating bacteria was examined. Caffeine at a final concentration of 1.0 mg/ml could inhibit the ice nucleation activity of calcium oxalate, and Pseudomonas fluorescens KUIN-1 at the same level that of as silver iodide. From these results, it was suggested that the extract could be able to be applied to the field to control the frost damage of the vegetables and that the harvested vegetables might be stored unfrozen even at 0℃ or less.