RESUMEN
Dying cell clearance is critical for myriad biological processes such as tissue homeostasis. We herein report an enzyme-activated fluorescence cell labeling approach and its use for multicolor imaging of dying cell clearance. Diacetylated 4-hydroxymandelic acid (DHA)-conjugated dyes give rise to reactive quinone methides upon deacetylation in live cells, which in turn covalently labels cellular proteins. With partner cells tagged with distinct fluorescence, apoptotic cell clearance by Raw 264.7 macrophages and epithelial HeLa cells was captured by confocal microscopy, showing the potential of DHA-based cell labeling for investigating cell-cell interactions.
Asunto(s)
Apoptosis , Colorantes Fluorescentes/química , Ácidos Mandélicos/química , Necrosis , Animales , Bovinos , Línea Celular Tumoral , Cumarinas/síntesis química , Cumarinas/química , Cumarinas/toxicidad , Esterasas/química , Fluoresceínas/síntesis química , Fluoresceínas/química , Fluoresceínas/toxicidad , Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Humanos , Ácidos Mandélicos/síntesis química , Ácidos Mandélicos/toxicidad , Ratones , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Prueba de Estudio Conceptual , Células RAW 264.7 , Rodaminas/síntesis química , Rodaminas/química , Rodaminas/toxicidad , Coloración y Etiquetado/métodos , PorcinosRESUMEN
Sialic acid (Sia) residues on cell surface are critical for myriad cellular events such as immunity and inflammation. We herein reported the use of abiotic Sia to raise the thresholds of inflammatory cell responses. Identified from a panel of structurally diversified Sia analogs via a cell inflammation assay, Sia-2, with N-butyryl moiety at C-5, markedly lowered LPS-stimulated NF-κB activity in macrophages. Further analysis shows that Sia-2 attenuates phosphorylation of IκB and Erk1/2/p38/JNK, critical for NF-κB signaling and MAPK signaling, and lowers gene transcription of proinflammatory interleukin-6. These results support the use of abiotic Sia as promising agents to modulate cell surface Sia-pertinent cell signaling.
Asunto(s)
Antiinflamatorios/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/prevención & control , Macrófagos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Ácidos Siálicos/farmacología , Animales , Inflamación/metabolismo , Inflamación/patología , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Fosforilación , Células RAW 264.7 , Transducción de Señal , Estrés FisiológicoRESUMEN
Cell fate is largely shaped by combined activity of different types of organelles, which often feature functionally critical parameters that succumb to pathological inducers. We herein report the analysis of cell bioenergetic profiles with a dual organelle-oriented chemosensor (RC-AMI), partitioning in mitochondria to give blue fluorescence and in lysosomes to give red fluorescence. Responsive to lysosomal pH and mitochondrial transmembrane potential (ΔΨm), two parameters crucial to cell bioenergetics, RC-AMI enables dual colored reporting of lysosomal acidity and ΔΨm, revealing upregulated ΔΨm and imbalance dramatically shifted favoring ΔΨm over lysosomal acidity in cancer cells whereas the tendency is reversed in starved cells. Complementing classical homo-organelle-specific sensors, this dual organelle-oriented and fluorescently responsive probe offers a new tool to detect imbalance between lysosomal acidity and mitochondrial ΔΨm, an index critical for cancer bioenergetics.