采用基于NADPH的荧光寿命成像法选择性探测多核形细胞中N

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses
the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading
pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and
oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and
function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by
means of biexponentialNAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670±140 picoseconds
as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to
oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors,
we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity
of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of
NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular
function. 超快门宽增强型CCD相机