Xiangxing Kong

A Highly Selective Mitochondria-Targeting Fluorescent K+ Sensor

Regulation of intracellular potassium (K(+) ) concentration plays a key role in metabolic processes. So far, only a few intracellular K(+) sensors have been developed. The highly selective fluorescent K(+) sensor KS6 for monitoring K(+) ion dynamics in mitochondria was produced by coupling triphenylphosphonium, borondipyrromethene (BODIPY), and triazacryptand (TAC). KS6 shows a good response to K(+) in the range 30-500 mM, a large dynamic range (Fmax /F0 ≈130), high brightness (ϕf =14.4 % at 150 mM of K(+) ), and insensitivity to both pH in the range 5.5-9.0 and other metal ions under physiological conditions. Colocalization tests of KS6 with MitoTracker Green confirmed its predominant localization in the mitochondria of HeLa and U87MG cells. K(+) efflux/influx in the mitochondria was observed upon stimulation with ionophores, nigericin, or ionomycin. KS6 is thus a highly selective semiquantitative K(+) sensor suitable for the study of mitochondrial potassium flux in live cells.

K(+) regulates Ca(2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells.

P2X7 purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real time. The use of KS6, a novel sensor for selective and sensitive dynamic visualization of intracellular potassium flux in live cells, multiplexed with the intracellular calcium sensor Fluo-4, revealed a coordinated relationship between potassium and calcium. Interestingly, the mitochondrial potassium pool was mobilized in a P2X7 signaling, and ATP dose-dependent manner, suggesting a role for mitochondrial sensing of cytosolic ion perturbation. Through treatment with extracellular potassium we found that potassium efflux was necessary to permit sustained calcium entry, but not transient calcium flux from intracellular stores. Further, intracellular calcium chelation with BAPTA-AM indicated that P2X7-induced potassium depletion was independent of calcium mobilization. This evidence suggests that both potassium efflux and calcium influx are necessary for mitochondrial reactive oxygen generation upstream of NLRP3 inflammasome assembly and pyroptotic cell death. We propose a model wherein potassium efflux is necessary for calcium influx, resulting in mitochondrial reactive oxygen generation to trigger the NLRP3 inflammasome.

A polymer-based ratiometric intracellular glucose sensor

A new polymeric ratiometric glucose sensor was synthesized and used for dynamically monitoring intracellular glucose concentrations in HeLa cells.

Ratiometric fluorescent pH-sensitive polymers for high-throughput monitoring of extracellular pH

Extracellular pH has a strong effect on cell metabolism and growth. Precisely detecting extracellular pH with high throughput is critical for cell metabolism research and fermentation applications. In this research, a series of ratiometric fluorescent pH sensitive polymers are developed and the ps-pH-neutral is characterized as the best one for exculsive detection of extracellular pH. Poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) is used as the host polymer to increase the water solubility of the pH sensitive polymer without introducing cell toxicity. The fluorescent emission spectra from the polymeric sensor under excitation at the isosbestic point 455 nm possess two fluorescence peaks at 475 nm and 505 nm, which have different responding trends to pH. This enables the polymer to detect pH using fluorescent maxima at 475 nm and 505 nm (I475nm /I505nm ) ratiometrically. The cell impermeability ensures the sensor can solely detect the environmental pH. The sensor is tested to detect the extracellular pH of bacteria or eukaryotic cells in high throughput assays using a microplate reader. Results showed that the pH sensor can be used for high throughput detection of extracellular pH with high repeatability and low photobleaching effect.

1,8-Naphthalimide Derivative Dyes with Large Stokes Shifts for Targeting Live-Cell Mitochondria.

An ideal fluorescent dye for staining cell organelles should have multiple properties including specificity, stability, biocompatibility, and a large Stokes shift. Tunable photophysical properties enable 1,8‐naphthalimide to serve as an excellent fluorophore in biomedical applications. Many naphthalimide derivatives have been developed into drugs, sensors, and other dyes. In this study, a series of 1,8‐naphthalimide derivatives targeting live cell mitochondria were synthesized. Among these probes, Mt‐4 was characterized as the best one, with highly specific mitochondrial localization, low cytotoxicity, and a large Stokes shift. More importantly, Mt‐4 stood out as a potential mitochondrial dye for living‐cell experiments involving induced mitochondrial stress arising from the treatments because Mt‐4 shows enhanced fluorescence in mitochondrial stress situations.

The oxindole Syk inhibitor OXSI-2 blocks nigericin-induced inflammasome signaling and pyroptosis independent of potassium efflux.

The inflammasome is a caspase-1-activating complex that is implicated in a growing number of acute and chronic pathologies. Interest has increased in identifying small molecular inhibitors of inflammasome signaling because of its role in clinically relevant diseases. It was recently reported that the protein tyrosine kinase, Syk, regulates pathogen-induced inflammasome signaling by phosphorylating a molecular switch on the adapter protein ASC. However, several aspects of the role of Syk in inflammasome signaling and the effects of its inhibition remain unclear. The aim of the present study is to explore in detail the effects of the oxindole Syk inhibitor OXSI-2 on various aspects of nigericin-induced inflammasome signaling. Our results indicate that OXSI-2 inhibits inflammasome assembly, caspase-1 activation, IL-1β processing and release, mitochondrial ROS generation, and pyroptotic cell death. Using a novel live cell potassium sensor we show that Syk inhibition with OXSI-2 has no effect on potassium efflux kinetics and that blockade of potassium efflux with extracellular potassium alters Syk phosphorylation. The effects of OXSI-2 identified in this study provide context for the role of Syk in inflammasome signaling and demonstrate its importance in oxidative signaling upstream of inflammasome activation and downstream of ion flux.

Multifunctional PHPMA-Derived Polymer for Ratiometric pH Sensing, Fluorescence Imaging, and Magnetic Resonance Imaging

In this paper, we report synthesis and characterization of a novel multimodality (MRI/fluorescence) probe for pH sensing and imaging. A multifunctional polymer was derived from poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) and integrated with a naphthalimide-based-ratiometric fluorescence probe and a gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex (Gd-DOTA complex). The polymer was characterized using UV-vis absorption spectrophotometry, fluorescence spectrofluorophotometry, magnetic resonance imaging (MRI), and confocal microscopy for optical and MRI-based pH sensing and cellular imaging. In vitro labeling of macrophage J774 and esophageal CP-A cell lines shows the polymers ability to be internalized in the cells. The transverse relaxation time (T2) of the polymer was observed to be pH-dependent, whereas the spin-lattice relaxation time (T1) was not. The pH probe in the polymer shows a strong fluorescence-based ratiometric pH response with emission window changes, exhibiting blue emission under acidic conditions and green emission under basic conditions, respectively. This study provides new materials with multimodalities for pH sensing and imaging.