Small Plasma Membrane-Targeted Fluorescent Dye for Long-Time Imaging and Protein Degradation Analyses.

Plasma membrane (PM)-targeted fluorescent dyes have become an important tool to visualize morphological and dynamic changes in the cell membrane. However, most of these PM dyes are either too large and thus might potentially perturb the membrane and affect its functions or exhibit a short retention time on the cell membrane. The rapid internalization problem is particularly severe for PM dyes based on cationic and neutral hydrophobic fluorescent dyes, which can be easily transported into the cells by transmembrane potential and passive diffusion mechanisms. In this paper, we report a small but highly specific PM fluorescent dye, PM-1, which exhibits a very long retention time on the plasma membrane with a half-life of approximately 15 h. For biological applications, we demonstrated that PM-1 can be used in combination with protein labeling probes to study ectodomain shedding and endocytosis processes of cell surface proteins and successfully demonstrated that native transmembrane human carbonic anhydrase IX (hCAIX) is degraded via the ectodomain shedding mechanism. In contrast, hCAIX undergoes endocytic degradation in the presence of sheddase inhibitors. We believe that PM-1 can be a versatile tool to provide detailed insights into the dynamic processes of the cell surface proteins.

Protein-Labeling Fluorescent Probe for Folate Receptor α.

GPI-anchored folate receptor α (FRα) is an attractive anticancer drug target and diagnosis marker in fundamental biology and medical research due to its significant expression on many cancer cells. Currently, analyses of FRα expression levels are usually achieved using immunological methods. Due to the continual FRα synthesis and degradation, immunological methods are not suitable for studying real-time dynamic activities of FRα in living cells. In this paper, we introduce a rapid and specific FRα protein-labeling fluorescent probe, FR1, to facilitate the study of the dynamics of expression and degradation processes of endogenous FRα in living cells. With this labeling probe, insights on FRα protein lifetime and shedding from the cell surface can be obtained using fluorescence live-cell imaging and electrophoresis techniques. We revealed that FRα undergoes soluble domain release and endocytosis degradation simultaneously. Imaging results showed that most of the membrane FRα are transported to the lysosomes after 2 h of incubation. Furthermore, we also showed that the secretion of a FRα soluble domain into the environment is most likely accomplished by phospholipase. We believe that this protein-labeling approach can be an important tool for analyzing various dynamic processes involving FRα.

Affinity-Switchable Interaction of Biotin and Streptavidin for the Signal-ON Detection of Small Molecules.

Lateral flow assay (LFA) based on gold nanoparticles (AuNPs) is a widely used analytical device for the rapid analysis of environmental hazards and biomarkers. Typically, a sandwich-type format is used for macromolecule detection, in which the appearance of a red test line indicates a positive result (Signal-ON). In contrast, small molecule detection usually relies on a competitive assay, where the absence of a test line indicates positive testing (Signal-OFF). However, such a "Signal-OFF" reading is usually detected within a narrower dynamic range and tends to generate false-negative signals at a low concentration. Moreover, inconsistent readings between macromolecule and small molecule testing might lead to misinterpretation when used by nonskilled individuals. Herein, we report a "Signal-ON" small molecule competitive assay based on the sterically modulated affinity-switchable interaction of biotin and streptavidin. In the absence of a small molecule target, a large steric hindrance can be imposed on the biotin to prevent interaction with streptavidin. However, in the presence of the small molecule target, this steric effect is removed, allowing the biotin to bind to streptavidin and generate the desired test line. In this article, we demonstrate the selective detection of two small molecule drugs, sulfonamides and trimethoprim, using this simple and modular affinity-switchable lateral flow assay (ASLFA). We believe that this affinity-switchable approach can also be adapted in drug discovery and clinical diagnosis, where the competitive assay format is always used for the rapid analysis of small molecules.

Protein-Labeling Fluorescent Probe Reveals Ectodomain Shedding of Transmembrane Carbonic Anhydrases.

Ectodomain shedding is a form of limited proteolysis in which a protease cleaves a transmembrane protein, releasing the extracellular domain from the cell surface. Cells use this process to regulate a wide variety of biological events. Typically, immunological detection methods are employed for the analysis of ectodomains secreted into the cultured media. In this paper, we describe a new strategy using an affinity-based protein-labeling fluorescent probe to study ectodomain shedding. We analyzed the ectodomain shedding of cell surface carbonic anhydrases (CAIX and CAXII), which are important biomarkers for tumor hypoxia. Using both chemical and genetic approaches, we identified that the ADAM17 metalloprotease is responsible for the shedding of carbonic anhydrases. Compared to current immunological methods, this protein-labeling approach not only detects ectodomain released into the culture media but also allows real-time living cell tracking and quantitative analysis of remnant proteins on the cell surface, thereby providing a more detailed insight into the mechanism of ectodomain shedding as well as protein lifetime on the cell surface.