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Abstract: Frizzled are seven-transmembrane domain G-protein coupled receptors involved in cell polarity and Wnt signaling. The mechanisms regulating their turnover at the plasma membrane remain unclear. We have identified a regulated C-terminus cleavage of Frizzled-7 in endothelial cells using ectopic expression of N- and C-termini-tagged Frizzled-7 proteins. This specific cleavage produced a 10 kDa C-terminus fragment that remained associated with intracellular vesicles and was localized within the 3rd intracytoplasmic loop using N-terminal sequencing and targeted mutagenesis. Frizzled-7 mutated forms displaying reduced C-terminus cleavage were also defective for dvl2 translocation at the plasma membrane. PMA, an activator of PKC and endocytosis, but not Wnt13A and Wnt5A, increased the appearance of Frizzled-7 C-terminus-containing vesicles and Frizzled-7 cleavage. Concanavalin-A, an inhibitor of receptor internalization decreased both constitutive and PMA-induced Frizzled-7 cleavage, while inhibition of the endocytic pathway with Δ95–295-Eps15 dominant-negative prevented only PMA-induced Frizzled-7 cleavage. Frizzled-7 C-terminus cleavage was increased with cell density and by the Ca2+ ionophore ionomycin and was decreased by specific calpain inhibitors, by the expression of DN-calpain-1 and the down-regulation of calpain-1 levels by siRNAs. Altogether, our findings pinpoint calpain-1 as a regulator of Frizzled-7 turnover at the plasma membrane and reveal a link between Frizzled-7 cleavage and its activity. [Copyright &y& Elsevier]

Building water systems promote the regrowth and survival of opportunistic pathogens, such as Legionella pneumophila, especially within biofilms, where most drinking water microbes reside. However, compared to their planktonic form, disinfection efficacy for the biofilm-associated forms of water-based pathogens is unclear. The aim of this study was to determine the effectiveness of free chlorine and monochloramine in the inactivation of biofilm-associated L. pneumophila strain Philadelphia-1 serogroup 1 (Lp). Mature (1.5-2 y old) drinking water biofilms were developed on copper (Cu) and polyvinylchloride (PVC) slides within biofilm annular reactors, then colonized with Lp at approximately 4 log10 CFU cm-2 and exposed to 2 mg L-1 of free chlorine or monochloramine. Ct (disinfectant concentration x time, expressed as mg min L-1) inactivation values for 2-, 3-, and 4-log10 reduction of planktonic and biofilm Lp were determined. For planktonic Lp, free chlorine was more effective at inactivation compared to monochloramine treatment and for biofilm-associated Lp, monochloramine was more effective on Cu biofilms, while free chlorine was more effective on PVC biofilms. In contrast to monochloramine, free chlorine treatment of Cu and PVC biofilms, negatively impacted Lp 16S rRNA gene transcript levels, and may act synergistically with Cu surfaces to further reduce transcript levels. Moreover, Lp cells shed from biofilms into the bulk water, were more resistant to disinfection compared to prepared planktonic Lp cells. Results from this study indicate that biofilm association, disinfectant type, and substratum play an important role in the survival of Legionella pneumophila in building water systems. [ABSTRACT FROM AUTHOR]