STIM1 functions as a proton sensor to coordinate cytosolic pH with store-operated calcium entry.

The meticulous regulation of intracellular pH (pHi) is crucial for maintaining cellular function and homeostasis, impacting physiological processes such as heart rhythm, cell migration, proliferation, and differentiation. Dysregulation of pHi is implicated in various pathologies such as arrhythmias, cancer, and neurodegenerative diseases. Here, we explore the role of STIM1, an ER calcium (Ca2+) sensor mediating Store Operated Ca2+ Entry (SOCE), in sensing pHi changes. Our study reveals that STIM1 functions as a sensor for pHi changes, independent of its Ca2+-binding state. Through comprehensive experimental approaches including confocal microscopy, FRET-based sensors, and mutagenesis, we demonstrate that changes in pHi induce conformational alterations in STIM1, thereby modifying its subcellular localization and activity. We identify two conserved histidine within STIM1 essential for sensing pHi shifts. Moreover, intracellular alkalization induced by agents such as Angiotensin II or NH4Cl enhances STIM1-mediated SOCE, promoting cardiac hypertrophy. These findings reveal a novel facet of STIM1 as a multi-modal stress sensor that coordinates cellular responses to both Ca2+ and pH fluctuations. This dual functionality underscores its potential as a therapeutic target for diseases associated with pH and Ca2+ dysregulation.

A cancer-targeted glutathione-gated probe for self-sufficient ST/CDT combination therapy and FRET-based miRNA imaging.

A cancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. The CGT probe is constructed using MnO2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin. After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn2+-mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy.

Intracellular and extracellular moesins differentially regulate Src activity and ß-catenin translocation to the nucleus in breast cancer cells.

It is increasingly recognized that a single protein can have multiple, sometimes paradoxical, roles in cell functions as well as pathological conditions depending on its cellular locations. Here we report that moesins (MSNs) in the intracellular and extracellular domains present opposing roles in pro-tumorigenic signaling in breast cancer cells. Using live cell imaging with fluorescence resonance energy transfer (FRET)- and green fluorescent protein (GFP)-based biosensors, we investigated the molecular mechanism underlying the cellular location-dependent effect of MSN on Src and ß-catenin signaling in MDA-MB-231 breast cancer cells. Inhibition of intracellular MSN decreased the activities of Src and FAK, whereas overexpression of intracellular MSN increased them. By contrast, extracellular MSN decreased the activities of Src, FAK, and RhoA, as well as ß-catenin translocation to the nucleus. Consistently, Western blotting and MTT-based analysis showed that overexpression of intracellular MSN elevated the expression of oncogenic genes, such as p-Src, ß-catenin, Lrp5, MMP9, Runx2, and Snail, as well as cell viability, whereas extracellular MSN suppressed them. Conditioned medium derived from MSN-overexpressing mesenchymal stem cells or osteocytes showed the anti-tumor effects by inhibiting the Src activity and ß-catenin translocation to the nucleus as well as the activities of FAK and RhoA and MTT-based cell viability. Conditioned medium derived from MSN-inhibited cells increased the Src activity, but it did not affect the activities of FAK and RhoA. Silencing CD44 and/or FN1 in MDA-MB-231 cells blocked the suppression of Src activity and ß-catenin accumulation in the nucleus by extracellular MSN. Collectively, the results suggest that cellular location-specific MSN is a strong regulator of Src and ß-catenin signaling in breast cancer cells, and that extracellular MSN exerts tumor-suppressive effects via its interaction with CD44 and FN1.

Real-time visualization and quantitation of cell death and cell cycle progression in 2D and 3D cultures utilizing genetically encoded probes.

Cancer cells grown as 3D-structures are better models for mimicking in vivo conditions than the 2D-culture systems employable in drug discovery applications. Cell cycle and cell death are important determinants for preclinical drug screening and tumor growth studies in laboratory conditions. Though several 3D-models and live-cell compatible approaches are available, a method for simultaneous real-time detection of cell cycle and cell death is required. Here we demonstrate a high-throughput adaptable method using genetically encoded fluorescent probes for the real-time quantitative detection of cell death and cell cycle. The cell-cycle indicator cdt1-Kusabira orange (KO) is stably integrated into cancer cells and further transfected with the Fluorescence Resonance Energy Transfer-based ECFP-DEVD-EYFP caspase activation sensor. The nuclear cdt1-KO expression serves as the readout for cell-cycle, and caspase activation is visualized by ECFP/EYFP ratiometric imaging. The image-based platform allowed imaging of growing spheres for prolonged periods in 3D-culture with excellent single-cell resolution through confocal microscopy. High-throughput screening (HTS) adaptation was achieved by targeting the caspase-sensor at the nucleus, which enabled the quantitation of cell death in 3D-models. The HTS using limited compound libraries, identified two lead compounds that induced caspase-activation both in 2D and 3D-cultures. This is the first report of an approach for noninvasive stain-free quantitative imaging of cell death and cell cycle with potential drug discovery applications.

Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment.

cAMP signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live-cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in vivo in mice of both sexes following optic nerve crush injury. These findings define a novel neuronal compartment that confers cAMP regulation of neuroprotection and axon growth and that may be therapeutically targeted in disease.SIGNIFICANCE STATEMENT cAMP is a second messenger responsible for the regulation of diverse cellular processes including neuronal neurite extension and survival following injury. Signal transduction by cAMP is highly compartmentalized in large part because of the formation of discrete, localized multimolecular signaling complexes by A-kinase anchoring proteins. Although the concept of cAMP compartmentation is well established, the function and identity of these compartments remain poorly understood in neurons. In this study, we provide evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein mAKAPα that is necessary and sufficient for the induction of neurite outgrowth in vitro and for the survival of retinal ganglion cells in vivo following optic nerve injury.

Monitoring the Heavy Metal Lead Inside Living Drosophila with a FRET-Based Biosensor.

The harmful impact of the heavy metal lead on human health has been known for years. However, materials that contain lead remain in the environment. Measuring the blood lead level (BLL) is the only way to officially evaluate the degree of exposure to lead. The so-called "safe value" of the BLL seems to unreliably represent the secure threshold for children. In general, lead's underlying toxicological mechanism remains unclear and needs to be elucidated. Therefore, we developed a novel genetically encoded fluorescence resonance energy transfer (FRET)-based lead biosensor, Met-lead, and applied it to transgenic Drosophila to perform further investigations. We combined Met-lead with the UAS-GAL4 system to the sensor protein specifically expressed within certain regions of fly brains. Using a suitable imaging platform, including a fast epifluorescent or confocal laser-scanning/two-photon microscope with high resolution, we recorded the changes in lead content inside fly brains ex vivo and in vivo and at different life stages. The blood-brain barrier was found to play an important role in the protection of neurons in the brain against damage due to the heavy metal lead, either through food or microinjection into the abdomen. Met-lead has the potential to be a powerful tool for the sensing of lead within living organisms by employing either a fast epi-FRET microscope or high-resolution brain imaging.

FRET-Based Ca2+ Biosensor Single Cell Imaging Interrogated by High-Frequency Ultrasound.

Fluorescence resonance energy transfer (FRET)-based biosensors have advanced live cell imaging by dynamically visualizing molecular events with high temporal resolution. FRET-based biosensors with spectrally distinct fluorophore pairs provide clear contrast between cells during dual FRET live cell imaging. Here, we have developed a new FRET-based Ca2+ biosensor using EGFP and FusionRed fluorophores (FRET-GFPRed). Using different filter settings, the developed biosensor can be differentiated from a typical FRET-based Ca2+ biosensor with ECFP and YPet (YC3.6 FRET Ca2+ biosensor, FRET-CFPYPet). A high-frequency ultrasound (HFU) with a carrier frequency of 150 MHz can target a subcellular region due to its tight focus smaller than 10 µm. Therefore, HFU offers a new single cell stimulations approach for FRET live cell imaging with precise spatial resolution and repeated stimulation for longitudinal studies. Furthermore, the single cell level intracellular delivery of a desired FRET-based biosensor into target cells using HFU enables us to perform dual FRET imaging of a cell pair. We show that a cell pair is defined by sequential intracellular delivery of the developed FRET-GFPRed and FRET-CFPYPet into two target cells using HFU. We demonstrate that a FRET-GFPRed exhibits consistent 10-15% FRET response under typical ionomycin stimulation as well as under a new stimulation strategy with HFU.

Quantification of Trastuzumab-HER2 Engagement In Vitro and In Vivo.

Human EGF Receptor 2 (HER2) is an important oncogene driving aggressive metastatic growth in up to 20% of breast cancer tumors. At the same time, it presents a target for passive immunotherapy such as trastuzumab (TZM). Although TZM has been widely used clinically since 1998, not all eligible patients benefit from this therapy due to primary and acquired drug resistance as well as potentially lack of drug exposure. Hence, it is critical to directly quantify TZM-HER2 binding dynamics, also known as cellular target engagement, in undisturbed tumor environments in live, intact tumor xenograft models. Herein, we report the direct measurement of TZM-HER2 binding in HER2-positive human breast cancer cells and tumor xenografts using fluorescence lifetime Forster Resonance Energy Transfer (FLI-FRET) via near-infrared (NIR) microscopy (FLIM-FRET) as well as macroscopy (MFLI-FRET) approaches. By sensing the reduction of fluorescence lifetime of donor-labeled TZM in the presence of acceptor-labeled TZM, we successfully quantified the fraction of HER2-bound and internalized TZM immunoconjugate both in cell culture and tumor xenografts in live animals. Ex vivo immunohistological analysis of tumors confirmed the binding and internalization of TZM-HER2 complex in breast cancer cells. Thus, FLI-FRET imaging presents a powerful analytical tool to monitor and quantify cellular target engagement and subsequent intracellular drug delivery in live HER2-positive tumor xenografts.

Gaussian FRET two-hybrid assays for determining the stoichiometry of hetero-oligomeric complexes in single living cells.

Here we integrate multiple Gaussian-functions analysis into fluorescence resonance energy transfer (FRET) two-hybrid assays (Gaussian FRET two-hybrid assay) to determine the stoichiometric ratios of intracellular hetero-oligomers in single living cells. This method adopts in multiple Gaussian-functions to fit the E-count histograms of both donor- and acceptor-centric FRET efficiency (ED and EA) images of a single cell for obtaining the peak values (EDi and EAi), thus yielding the corresponding stoichiometric ratios (EDi/EAi) of intracellular hetero-oligomers. We performed Gaussian FRET two-hybrid assay for living Hela cells coexpressing different FRET tandem plasmids, and obtained consistent results with the expected values. Gaussian FRET two-hybrid assay for cells coexpressing Bad-CFP and Bcl-XL-YFP reveals that Bcl-XL binds with Bad to form a hetero-oligomeric complex with a stoichiometry of 2:1 on mitochondria.

Time-lapse FRET analysis reveals the ability of Bax dimer to trigger mitochondrial outer membrane permeabilization.

Bax oligomerization is essential for triggering mitochondrial outer membrane permeabilization (MOMP) in many apoptotic programs. However, it is controversial whether Bax dimer is sufficient to trigger MOMP. In this report, multiple Gaussian function-based FRET analysis (Multi-Gaussian FRET analysis) was used to dissect the dimerization and then tetramerization of Bax in relation to MOMP. Multi-Gaussian FRET analysis on the time-lapse FRET images of single living cells co-expressing CFP-Bax and YFP-Bax revealed that formation of mitochondrial Bax homodimers preceded MOMP within 3 min and Bax dimer transformed into tetramer within 6 min concomitantly with complete MOMP within 10 min, providing direct evidence in support of the sufficient ability of Bax dimers to trigger MOMP at least in natural cells.

Imaging cAMP nanodomains in the heart.

3'-5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that modulates multiple cellular functions. It is now well established that cAMP can mediate a plethora of functional effects via a complex system of local regulatory mechanisms that result in compartmentalized signalling. The use of fluorescent probes to monitor cAMP in intact, living cells have been instrumental in furthering our appreciation of this ancestral and ubiquitous pathway and unexpected details of the nano-architecture of the cAMP signalling network are starting to emerge. Recent evidence shows that sympathetic control of cardiac contraction and relaxation is achieved via generation of multiple, distinct pools of cAMP that lead to differential phosphorylation of target proteins localized only tens of nanometres apart. The specific local control at these nanodomains is enabled by a distinct signalosome where effectors, targets, and regulators of the cAMP signal are clustered. In this review, we focus on recent advances using targeted fluorescent reporters for cAMP and how they have contributed to our current understanding of nanodomain cAMP signalling in the heart. We briefly discuss how this information can be exploited to design novel therapies and we highlight some of the questions that remain unanswered.

Multichannel wide-field microscopic FRET imaging based on simultaneous spectral unmixing of excitation and emission spectra.

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has inherent ability resolving spectral crosstalks, two key issues of quantitative fluorescence resonance energy transfer (FRET) measurement, of both the excitation and emission spectra between donor and acceptor without additional corrections. We here set up a filter-based multichannel wide-field microscope for ExEm unmixing-based FRET imaging (m-ExEm-spFRET) containing a constant system correction factor (fsc ) for a stable system. We performed m-ExEm-spFRET with four- and two-wavelength excitation respectively on our system to quantitatively image single living cells expressing FRET tandem constructs, and obtained accurate FRET efficiency (E) and concentration ratio of acceptor to donor (RC ). We also performed m-ExEm-spFRET imaging for single living cells coexpressing CFP-Bax and YFP-Bax, and found that the E values were about 0 for control cells and about 28% for staurosporin-treated cells when RC were larger than 1, indicating that staurosporin induced significant oligomerisation.

Circuit-dependent striatal PKA and ERK signaling underlies rapid behavioral shift in mating reaction of male mice.

The selection of reward-seeking and aversive behaviors is controlled by two distinct D1 and D2 receptor-expressing striatal medium spiny neurons, namely the direct pathway MSNs (dMSNs) and the indirect pathway MSNs (iMSNs), but the dynamic modulation of signaling cascades of dMSNs and iMSNs in behaving animals remains largely elusive. We developed an in vivo methodology to monitor Förster resonance energy transfer (FRET) of the activities of PKA and ERK in either dMSNs or iMSNs by microendoscopy in freely moving mice. PKA and ERK were coordinately but oppositely regulated between dMSNs and iMSNs by rewarding cocaine administration and aversive electric shocks. Notably, the activities of PKA and ERK rapidly shifted when male mice became active or indifferent toward female mice during mating behavior. Importantly, manipulation of PKA cascades by the Designer Receptor recapitulated active and indifferent mating behaviors, indicating a causal linkage of a dynamic activity shift of PKA and ERK between dMSNs and iMSNs in action selection.

Role of PKA signaling in D2 receptor-expressing neurons in the core of the nucleus accumbens in aversive learning.

The nucleus accumbens (NAc) serves as a key neural substrate for aversive learning and consists of two distinct subpopulations of medium-sized spiny neurons (MSNs). The MSNs of the direct pathway (dMSNs) and the indirect pathway (iMSNs) predominantly express dopamine (DA) D1 and D2 receptors, respectively, and are positively and negatively modulated by DA transmitters via Gs- and Gi-coupled cAMP-dependent protein kinase A (PKA) signaling cascades, respectively. In this investigation, we addressed how intracellular PKA signaling is involved in aversive learning in a cell type-specific manner. When the transmission of either dMSNs or iMSNs was unilaterally blocked by pathway-specific expression of transmission-blocking tetanus toxin, infusion of PKA inhibitors into the intact side of the NAc core abolished passive avoidance learning toward an electric shock in the indirect pathway-blocked mice, but not in the direct pathway-blocked mice. We then examined temporal changes in PKA activity in dMSNs and iMSNs in behaving mice by monitoring Förster resonance energy transfer responses of the PKA biosensor with the aid of microendoscopy. PKA activity was increased in iMSNs and decreased in dMSNs in both aversive memory formation and retrieval. Importantly, the increased PKA activity in iMSNs disappeared when aversive memory was prevented by keeping mice in the conditioning apparatus. Furthermore, the increase in PKA activity in iMSNs by aversive stimuli reflected facilitation of aversive memory retention. These results indicate that PKA signaling in iMSNs plays a critical role in both aversive memory formation and retention.

Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca2+ Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons.

Dysfunctions of NO-cGMP signaling have been implicated in various neurological disorders. We have studied the potential crosstalk of cGMP and Ca2+ signaling in cerebellar granule neurons (CGNs) by simultaneous real-time imaging of these second messengers in living cells. The NO donor DEA/NO evoked cGMP signals in the granule cell layer of acute cerebellar slices from transgenic mice expressing a cGMP sensor protein. cGMP and Ca2+ dynamics were visualized in individual CGNs in primary cultures prepared from 7-day-old cGMP sensor mice. DEA/NO increased the intracellular cGMP concentration and augmented glutamate-induced Ca2+ transients. These effects of DEA/NO were absent in CGNs isolated from knockout mice lacking NO-sensitive guanylyl cyclase. Furthermore, application of the cGMP analogues 8-Br-cGMP and 8-pCPT-cGMP, which activate cGMP effector proteins such as cyclic nucleotide-gated cation channels and cGMP-dependent protein kinases (cGKs), also potentiated glutamate-induced Ca2+ transients. Western blot analysis failed to detect cGK type I or II in our primary CGNs. The addition of phosphodiesterase (PDE) inhibitors during cGMP imaging showed that CGNs degrade cGMP mainly via Zaprinast-sensitive PDEs, most likely PDE5 and/or PDE10, but not via PDE1, 2, or 3. In sum, these data delineate a cGK-independent NO-cGMP signaling cascade that increases glutamate-induced Ca2+ signaling in CGNs. This cGMP⁻Ca2+ crosstalk likely affects neurotransmitter-stimulated functions of CGNs.

cGMP Imaging in Brain Slices Reveals Brain Region-Specific Activity of NO-Sensitive Guanylyl Cyclases (NO-GCs) and NO-GC Stimulators.

Impaired NO-cGMP signaling has been linked to several neurological disorders. NO-sensitive guanylyl cyclase (NO-GC), of which two isoforms-NO-GC1 and NO-GC2-are known, represents a promising drug target to increase cGMP in the brain. Drug-like small molecules have been discovered that work synergistically with NO to stimulate NO-GC activity. However, the effects of NO-GC stimulators in the brain are not well understood. In the present study, we used Förster/fluorescence resonance energy transfer (FRET)-based real-time imaging of cGMP in acute brain slices and primary neurons of cGMP sensor mice to comparatively assess the activity of two structurally different NO-GC stimulators, IWP-051 and BAY 41-2272, in the cerebellum, striatum and hippocampus. BAY 41-2272 potentiated an elevation of cGMP induced by the NO donor DEA/NO in all tested brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP increases in the cerebellum and striatum, but not in the hippocampal CA1 area or primary hippocampal neurons. The brain-region-selective activity of IWP-051 suggested that it might act in a NO-GC isoform-selective manner. Results of mRNA in situ hybridization indicated that the cerebellum and striatum express NO-GC1 and NO-GC2, while the hippocampal CA1 area expresses mainly NO-GC2. IWP-051-potentiated DEA/NO-induced cGMP signals in the striatum of NO-GC2 knockout mice but was ineffective in the striatum of NO-GC1 knockout mice. These results indicate that IWP-051 preferentially stimulates NO-GC1 signaling in brain slices. Interestingly, no evidence for an isoform-specific effect of IWP-051 was observed when the cGMP-forming activity of whole brain homogenates was measured. This apparent discrepancy suggests that the method and conditions of cGMP measurement can influence results with NO-GC stimulators. Nevertheless, it is clear that NO-GC stimulators enhance cGMP signaling in the brain and should be further developed for the treatment of neurological diseases.

Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells.

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.

Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.

"Store-operated" cAMP signaling contributes to Ca2+-activated Cl- secretion in T84 colonic cells.

Apical cAMP-dependent CFTR Cl(-) channels are essential for efficient vectorial movement of ions and fluid into the lumen of the colon. It is well known that Ca(2+)-mobilizing agonists also stimulate colonic anion secretion. However, CFTR is apparently not activated directly by Ca(2+), and the existence of apical Ca(2+)-dependent Cl(-) channels in the native colonic epithelium is controversial, leaving the identity of the Ca(2+)-activated component unresolved. We recently showed that decreasing free Ca(2+) concentration ([Ca(2+)]) within the endoplasmic reticulum (ER) lumen elicits a rise in intracellular cAMP. This process, which we termed "store-operated cAMP signaling" (SOcAMPS), requires the luminal ER Ca(2+) sensor STIM1 and does not depend on changes in cytosolic Ca(2+). Here we assessed the degree to which SOcAMPS participates in Ca(2+)-activated Cl(-) transport as measured by transepithelial short-circuit current (Isc) in polarized T84 monolayers in parallel with imaging of cAMP and PKA activity using fluorescence resonance energy transfer (FRET)-based reporters in single cells. In Ca(2+)-free conditions, the Ca(2+)-releasing agonist carbachol and Ca(2+) ionophore increased Isc, cAMP, and PKA activity. These responses persisted in cells loaded with the Ca(2+) chelator BAPTA-AM. The effect on Isc was enhanced in the presence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), inhibited by the CFTR inhibitor CFTRinh-172 and the PKA inhibitor H-89, and unaffected by Ba(2+) or flufenamic acid. We propose that a discrete component of the "Ca(2+)-dependent" secretory activity in the colon derives from cAMP generated through SOcAMPS. This alternative mode of cAMP production could contribute to the actions of diverse xenobiotic agents that disrupt ER Ca(2+) homeostasis, leading to diarrhea.

Development of a Compensated Förster Resonance Energy Transfer Imaging for Improved Assessment of the Intrapulmonary Distribution of Polymeric Nanoparticles.

Inhalation-based drug delivery systems have gained attention as potential therapeutic options for various respiratory diseases. Among these systems, nanoparticles are being explored as drug carriers because of their ability to deliver therapeutic agents directly to the lungs. It is essential to accurately evaluate the intrapulmonary behavior of nanoparticles to optimize drug delivery and achieve selective targeting of lung lesions. Prior research used the Förster resonance energy transfer (FRET) phenomenon to study the in vivo behavior of nanoparticles as drug carriers. In this study, image reconstruction involving bleed-through compensation was used to quantitatively assess the behavior of FRET nanoparticles in the lungs. When the nanoparticles for FRET fluorescence imaging, which employed 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) as the donor and as 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine iodide (DiR) the acceptor, were administered to mouse lungs, whole-body in vivo imaging could not compensate for the influence of respiration and heartbeat. However, ex vivo imaging of excised lungs enabled the quantitative evaluation of the time-concentration profiles and distribution of nanoparticles within the lungs. This imaging technique is particularly useful for the development of inhalable nanoparticles that specifically target the lesions and exhibit controlled-release capabilities within the lungs.