No abstract

Vesicular assembly from a thermo-responsive amphiphilic random copolymer is reported. Vesicle-to-micelle transition above a critical morphology transition temperature (CMTT) resulted in selective triggered release of encapsulated hydrophilic guests over hydrophobic ones. The aggregation pattern of a control polymer indicated a defined role of the methacrylamide groups in the polymer backbone for such unprecedented self-assembly from a simple polymer.

Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules. Such reporters provide information corresponding to wherever inside cells the enzyme is maximally active and preclude minor populations present in sub-cellular compartments. Here we describe a chemical imaging strategy to selectively interrogate minor, sub-cellular pools of enzymatic activity. This new technology confines the detection chemistry to a designated organelle, enabling imaging of enzymatic cleavage exclusively within the organelle. We have thus quantitatively mapped disulfide reduction exclusively in endosomes in C. elegans and identified that exchange is mediated by minor populations of enzymes PDI-3 and TRX-1 resident in endosomes. Impeding intra-endosomal disulfide reduction by knocking down TRX-1 protects nematodes from infection by Corynebacterium diphtheriae, revealing the importance of this minor pool of endosomal TRX-1. TRX-1 also mediates endosomal disulfide reduction in human cells. A range of enzymatic cleavage reactions in organelles are amenable to analysis by this new reporter strategy.Minor populations of proteins and protein complexes perform critical functions for the cell. For example, a minor population of the epidermal growth factor receptor present on exosomes mediates intercellular communication 1 . A small fraction of mammalian target of rapamycin, present on lysosomes is responsible for nutrient sensing by the cell 2 . A minor population of the KDEL receptor present in the Golgi apparatus performs the critical function of retrieving ER-resident proteins from the Golgi apparatus 3 . Small molecules that function as fluorescent reporters of enzymatic activity use highly specific and rapid Reprints and permission information is available online at www.nature.com/reprints.

Nucleophilic thiol-acrylate Michael reaction between a hydrophobic thiol and hydrophilic acrylate derivative generated a nonionic surfactant with acid-labile β-thiopropionate linker. Micellization of the surfactant, its ability to encapsulate hydrophobic dye, acid-induced disruption of the aggregate and pH-selective dye release profile have been revealed in this report. The micellar aggregates were found to be stable under neutral conditions, but they could be disrupted in acidic pH (5.3), and thus the encapsulated hydrophobic dye molecules could be selectively released. Appropriate control experiments revealed that the sulfur atom in the β-position is essential for acidic hydrolysis of the ester functionality of the surfactant.

Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome where they are bombarded with reactive oxygen (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate which simultaneously images HOCl and pH quantitatively. Using cHOClate targeted to phagosomes in live cells we successfully map phagosomal production of a specific ROS i.e. hypochlorous acid (HOCl), as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome-lysosome fusion was essential not only for phagosome acidification, but also to provide the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.