CR1-mediated ATP release by human red blood cells promotes CR1 clustering and modulates the immune transfer process.

Humans and other higher primates are unique among mammals in using complement receptor 1 (CR1, CD35) on red blood cells (RBC) to ligate complement-tagged inflammatory particles (immune complexes, apoptotic/necrotic debris, and microbes) in the circulation for quiet transport to the sinusoids of spleen and liver where resident macrophages remove the particles, but allow the RBC to return unharmed to the circulation. This process is called immune-adherence clearance. In this study we found using luminometric- and fluorescence-based methods that ligation of CR1 on human RBC promotes ATP release. Our data show that CR1-mediated ATP release does not depend on Ca(2+) or enzymes previously shown to mediate an increase in membrane deformability promoted by CR1 ligation. Furthermore, ATP release following CR1 ligation increases the mobility of the lipid fraction of RBC membranes, which in turn facilitates CR1 clustering, and thereby enhances the binding avidity of complement-opsonized particles to the RBC CR1. Finally, we have found that RBC-derived ATP has a stimulatory effect on phagocytosis of immune-adherent immune complexes.

Synthetic self-localizing ligands that control the spatial location of proteins in living cells.

Small-molecule ligands that control the spatial location of proteins in living cells would be valuable tools for regulating biological systems. However, the creation of such molecules remains almost unexplored because of the lack of a design methodology. Here we introduce a conceptually new type of synthetic ligands, self-localizing ligands (SLLs), which spontaneously localize to specific subcellular regions in mammalian cells. We show that SLLs bind their target proteins and relocate (tether) them rapidly from the cytoplasm to their targeting sites, thus serving as synthetic protein translocators. SLL-induced protein translocation enables us to manipulate diverse synthetic/endogenous signaling pathways. The method is also applicable to reversible protein translocation and allows control of multiple proteins at different times and locations in the same cell. These results demonstrate the usefulness of SLLs in the spatial (and temporal) control of intracellular protein distribution and biological processes, opening a new direction in the design of small-molecule tools or drugs for cell regulation.

Organelle-localizable fluorescent chemosensors for site-specific multicolor imaging of nucleoside polyphosphate dynamics in living cells.

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.

Rational design of FRET-based ratiometric chemosensors for in vitro and in cell fluorescence analyses of nucleoside polyphosphates.

Ratiometric fluorescence sensing is a useful technique for the precise and quantitative analysis of biological events occurring under complex conditions, such as those inside cells. We report herein the design of new ratiometric chemosensors for nucleoside polyphosphates such as ATP that are based on binding-induced modulation of fluorescence resonance energy transfer (FRET) coupled with a turn-on fluorescence-sensing mechanism. We designed these new FRET-based ratiometric chemosensors by utilizing spectral overlap changes to modulate the FRET efficiency. Introduction of coumarin fluorophores as the FRET donors into a binuclear zinc complex as the FRET acceptor provided the ratiometric chemosensors. These chemosensors exhibited a clear dual-mission signal change upon binding with strong affinity (K(app) ≈ 10(6)-10(7) M(-1)) to nucleoside polyphosphates in aqueous solution, whereas no detectable emission change was observed with monophosphates and phosphodiester species or various other anions. These chemosensors were used for real-time fluorescence monitoring of enzyme reactions such as saccharide synthesis by glycosyltransferase and phosphorylation by protein kinase, both of which involve nucleoside polyphosphates as substrates. The utility of ratiometric sensing by chemosensors was further demonstrated in a fluorescence-imaging study of the nucleoside polyphosphates inside living cells, wherein we ratiometrically visualized the stimulus-responsive concentration change of ATP, an indicator of the cellular energy level.

Fluorescence imaging of ATP in neutrophils from patients with sepsis using organelle-localizable fluorescent chemosensors.

BACKGROUND: The activation of polymorphonuclear neutrophils (PMNs) plays an important role in sepsis. Previously, we showed that ATP release and feedback via ATP receptors are essential for PMN activation; however, the dynamics remain poorly understood. Two new fluorescent chemosensors, PMAP-1 and MitoAP-1, were developed to detect ATP in the plasma membrane and mitochondria of living cells, respectively. In this study, we aimed to evaluate ATP localization using these chemosensors in PMNs of sepsis patients. METHODS: Live PMNs isolated from 16 sepsis patients and healthy controls (HCs) were stained with these chemosensors and observed by confocal microscopy, and their mean fluorescence intensities (MFIs) were evaluated using flow cytometry. CD11b expression in PMNs was also evaluated. RESULTS: The MFIs of PMAP-1 and MitoAP-1 and CD11b expression in PMNs from sepsis patients on days 0-1 were significantly higher than those of HCs. The MFI of PMAP-1 and CD11b expression on days 3-4 decreased significantly compared to those observed at days 0-1, whereas MitoAP-1 MFI was maintained at a high level. The PMAP-1 MFI was significantly positively correlated with CD11b expression, white blood cell counts, neutrophil counts, and C-reactive protein levels in patients. CONCLUSIONS: The higher MFIs of PMAP-1 and MitoAP-1 in sepsis patients suggest a pivotal role of ATP for PMN activation. The temporal difference in ATP levels suggests that ATP plays different roles in the mitochondria and on the cell surface. These data should contribute to the understanding of the dynamics of ATP in PMNs and help to develop a novel therapy for sepsis.

Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function.

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.

Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging.

We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.