Organelle-Localizable Fluorescent Chemosensors for Site-Specific Multicolor Imaging of Nucleoside Polyphosphate Dynamics in Living Cells

Abstract

ATP and its derivatives (nucleoside polyphosphates (NPPs)) are implicated in many biological events, so their rapid and convenient detection is important. In particular, live cell detection of NPPs at specific local regions of cells could greatly contribute understanding of the complicated roles of NPPs. We report herein the design of two new fluorescent chemosensors that detect the dynamics of NPPs in specific regions of living cells. To achieve imaging of NPPs on plasma membrane surfaces (2-2Zn(II)), a lipid anchor was introduced into xanthene-based Zn(II) complex 1-2Zn(II), which was previously developed as a turn-on type fluorescent chemosensor for NPPs. Meanwhile, for subcellular imaging of ATP in mitochondria, we designed rhodamine-type Zn(II) complex 3-2Zn(II), which possesses a cationic pyronin ring instead of xanthene. Detailed spectroscopic studies revealed that 2-2Zn(II) and 3-2Zn(II) can sense NPPs with a several-fold increase of their fluorescence intensities through a sensing mechanism similar to 1-2Zn(II), involving binding-induced recovery of the conjugated form of the xanthene or pyronin ring. In live cell imaging, 2-2Zn(II) containing a lipid anchor selectively localized on the plasma membrane surface and detected the extracellular release of NPPs during cell necrosis induced by streptolysin O. On the other hand, rhodamine-type complex 3-2Zn(II) spontaneously localized at mitochondria inside cells, and sensed the local increase of ATP concentration during apoptosis. Multicolor images were obtained through simultaneous use of 2-2Zn(II) and 3-2Zn(II), allowing detection of the dynamics of ATP in different cellular compartments at the same time.

Mitochondria Regulate Neutrophil Activation by Generating ATP for Autocrine Purinergic Signaling

“…Although this method was highly sensitive, it had major drawbacks including the need for an excitation wavelength in the ultraviolet range, which resulted in significant phototoxicity (23). The small molecular probe we used in the current study was developed in the laboratory of Dr. Hamachi (11,24). This ATP probe features a membrane anchor that has allowed us to attach the probe to the cell surface of living PMNs and to assess ATP concentration changes at the cell surface for prolonged periods of time.…”

“…After attachment of cells to the fibronectin-coated glass, PMNs were washed, and the supernatants were replaced with fresh HBSS containing 500 nM of the cell surface-targeting ATP probe, 2-2Zn(II), which was kindly provided by Drs. Kurishita and Hamachi from Kyoto University (11). Fluorescence imaging was done with a Leica DMI 6000B microscope (Leica, Wetzlar, Germany) equipped with a Spot Boost EMCCD camera (Diagnostic Instruments Inc., Sterling Heights, MI).…”

“…For that purpose, we used a novel fluorescent ATP probe, termed 2-2Zn(II), that had been generated and kindly provided to us by Drs. Kurishita and Hamachi from Kyoto University (11). This probe has a membrane-anchoring structure that allows it to bind to the cell surface on PMNs.…”

Mitochondria Are Gate-keepers of T Cell Function by Producing the ATP That Drives Purinergic Signaling

“…To study this possibility, we developed a new imaging technique that has allowed us to monitor the spatiotemporal dynamics of ATP release during IS formation. For that purpose, we used a novel fluorescent small molecular ATP probe designed by Dr. Kurishita in the laboratory of Professor Hamachi at Kyoto University (17). This probe, named 2-2Zn(II) features a lipid anchor residue which allows it to bind to the outer membrane of living cells This ATP probe made it possible to monitor submicromolar ATP concentration changes at the T cell surface in real-time using live-cell fluorescence imaging techniques (supplemental Fig.…”

CR1-mediated ATP Release by Human Red Blood Cells Promotes CR1 Clustering and Modulates the Immune Transfer Process

“…Visualization of CR1-mediated ATP Release Using a Fluorescent ATP-sensing Probe-To directly image ATP release promoted by RBC CR1 ligation, we employed a novel fluorescent nucleotide-sensing probe, 2-2Zn(II), which binds the extracellular side of the plasma membrane, and upon interaction with ATP, fluoresces in an ATP concentration-dependent manner (13,19,20). First, to test the specificity of the probe for ATP, 2-2Zn(II)-treated RBC were incubated at room temperature (5 min) in the presence or absence of ATP diphosphohydrolase (apyrase) and subsequently analyzed by flow cytometry.…”