[show abstract][hide abstract] ABSTRACT: Aggresomes and related inclusion bodies appear to serve as storage depots for misfolded and aggregated proteins within cells, which can potentially be degraded by the autophagy pathway. A homogenous fluorescence-based assay was devised to detect aggregated proteins inside aggresomes and inclusion bodies within an authentic cellular context. The assay employs a novel red fluorescent molecular rotor dye, which is essentially nonfluorescent until it binds to structural features associated with the aggregated protein cargo. Aggresomes and related structures were generated within cultured cells using various potent, cell permeable, proteasome inhibitors: MG-132, lactacystin, epoxomicin and bortezomib, and then selectively detected with the fluorescent probe. Employing the probe in combination with various fluorescein-labeled primary antibodies facilitated co-localization of key components of the autophagy system (ubiquitin, p62, and LC3) with aggregated protein cargo by fluorescence microscopy. Furthermore, cytoplasmic aggregates were highlighted in SK-N-SH human neuroblastoma cells incubated with exogenously supplied amyloid beta peptide 1-42. SMER28, a small molecule modulator of autophagy acting via an mTOR-independent mechanism, prevented the accumulation of amyloid beta peptide within these cells. The described assay allows assessment of the effects of protein aggregation directly in cells, without resorting to the use of non-physiological protein mutations or genetically engineered cell lines. With minor modification, the assay was also adapted to the analysis of frozen or formalin-fixed, paraffin-embedded tissue sections, with demonstration of co-localization of aggregated cargo with β-amyloid and tau proteins in brain tissue sections from Alzheimer's disease patients.
Cell biochemistry and biophysics 12/2010; 60(3):173-85. · 3.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lysosomes are membrane-bound subcellular organelles involved in the degradation of macromolecules and pathogens in diverse processes, including endocytosis, phagocytosis, and autophagy. A red fluorescent probe was developed that is selectively sequestered in acidic organelles. U20S cells pretreated with 64 microM chloroquine for as little as 5 h show a dramatic increase in lysosome-like vesicle number and volume. The probe can be employed for highlighting lysosome-like organelles under conditions wherein cells produce vacuoles that contain most of the degradative enzymes of the lysosome but are not as acidic as the parent organelle. Using a conventional fluorescence microplate reader, the half-maximal effective concentration (EC(50)) of chloroquine was estimated. The high Z' score obtained using the assay demonstrated excellent signal-to-noise ratios. The fluorescence microplate assay was successfully employed to screen a small-molecule compound library for agents that increase lysosomal volume and number. One potential application of the new assay is in the toxicology portion of preclinical drug safety assessment (ADME-Tox) workflows, using in vitro cell culture models to aid in the drug development process.
Journal of Biomolecular Screening 03/2010; 15(4):398-405. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Enzo Life Sciences Nuclear-ID™ Red DNA stain is useful for staging cells on the basis of their DNA content. The stain can be applied to live, detergent-permeabilized or fixed cells for quantification of their distribution among the three main phases of the cell cycle (G0/G1, S and G2/M). The far-red emission of the dye facilitates mul
tiplexing with GFP and other fluorescent probes. With the dye, cells may be analyzed by fluorescence microscopy, flow cytometry and laser-scanning cytometry.