pH-sensitive Fluorescent Protein Research Articles

A genetically-encoded fluorescence-based reporter to spatiotemporally investigate mannose-6-phosphate pathway.

Maintenance of a pool of active lysosomes with acidic pH and degradative hydrolases is crucial for cell health. Abnormalities in lysosomal function are closely linked to diseases, such as lysosomal storage disorders, neurodegeneration, intracellular infections, and cancer among others. Emerging body of research suggests the malfunction of lysosomal hydrolase trafficking pathway to be a common denominator of several disease pathologies. However, available conventional tools to assess lysosomal hydrolase trafficking are insufficient and fail to provide a comprehensive picture about the trafficking flux and location of lysosomal hydrolases. To address some of the shortcomings, we designed a genetically-encoded fluorescent reporter containing a lysosomal hydrolase tandemly tagged with pH sensitive and insensitive fluorescent proteins, which can spatiotemporally trace the trafficking of lysosomal hydrolases. As a proof of principle, we demonstrate that the reporter can detect perturbations in hydrolase trafficking, that are induced by pharmacological manipulations and pathophysiological conditions like intracellular protein aggregates. This reporter can effectively serve as a probe for mapping the mechanistic intricacies of hydrolase trafficking pathway in health and disease and is a utilitarian tool to identify genetic and pharmacological modulators of this pathway, with potential therapeutic implications.

Progress in pH-Sensitive sensors: essential tools for organelle pH detection, spotlighting mitochondrion and diverse applications.

pH-sensitive fluorescent proteins have revolutionized the field of cellular imaging and physiology, offering insight into the dynamic pH changes that underlie fundamental cellular processes. This comprehensive review explores the diverse applications and recent advances in the use of pH-sensitive fluorescent proteins. These remarkable tools enable researchers to visualize and monitor pH variations within subcellular compartments, especially mitochondria, shedding light on organelle-specific pH regulation. They play pivotal roles in visualizing exocytosis and endocytosis events in synaptic transmission, monitoring cell death and apoptosis, and understanding drug effects and disease progression. Recent advancements have led to improved photostability, pH specificity, and subcellular targeting, enhancing their utility. Techniques for multiplexed imaging, three-dimensional visualization, and super-resolution microscopy are expanding the horizon of pH-sensitive protein applications. The future holds promise for their integration into optogenetics and drug discovery. With their ever-evolving capabilities, pH-sensitive fluorescent proteins remain indispensable tools for unravelling cellular dynamics and driving breakthroughs in biological research. This review serves as a comprehensive resource for researchers seeking to harness the potential of pH-sensitive fluorescent proteins.

Studying Selective Autophagy of Protein Aggregates Using Particles Induced by Multimerization (PIMs).

Selective autophagy of protein aggregates, called aggrephagy, is vital for maintaining cellular homeostasis. Classically, studying aggrephagy has been challenging due to the infrequent occurrence of autophagic events and the lack of control over the specificity and timing of protein aggregation. We previously reported two variants of a PIM (particles induced by multimerization) assay that enable the formation of chemically induced, fluorescently labeled protein aggregates in cells. PIMs are recognized by the selective autophagy machinery and are subsequently degraded in the lysosome. By making use of pH-sensitive fluorescent proteins, such as GFP or mKeima, the PIM assay allows for direct visualization of aggregate clearance in cells. Here, we describe a protocol for the use of the PIM assay to study aggrephagy in live and fixed cells.

Investigating lysosomal exocytosis as a pathway of beta‐amyloid release from neurons

AbstractBackgroundThe endosomal‐lysosomal system has long been linked to the production of beta‐amyloid (Aβ), but the specific intracellular compartments involved in Aβ secretion remain contentious. Conventionally, lysosomes are associated with Aβ degradation, yet studies have also shown that lysosomes are a site of Aβ accumulation. Lysosomal exocytosis is a major secretory pathway in non‐neuronal cells, but few studies have investigated this pathway in neurons. This project examined the role and mechanism of lysosomal exocytosis in neurons, and we hypothesized that lysosomal exocytosis is a pathway for Aβ secretion from neurons.MethodsWe have established human iPSC‐derived neurons in culture, including cells bearing the APP Swedish mutation. Cells were transduced with pH sensitive fluorescent (pHluorin/pHmScarlet) tagged compartment markers to examine neurosecretory granules (synaptophysin), early endosomes (Rab5), late endosomes (Rab9) and lysosomes (LAMP1). Cells were loaded with fluorescent exogenous Aβ or Amytracker to label endogenous Aβ. We then stimulated secretion with ionomycin and used live cell Total Internal Reflection Fluorescence (TIRF) microscopy to examine molecular events occurring within 250 nm to the plasma membrane. shRNA against proteins known to be involved with lysosomal secretion (Rab27b, Munc13‐4 and synaptotagmin‐7) were used as controls. Imaris software was used to analyze the number and timing of lysosomal secretory events and the secretion of Aβ.ResultsAs previously reported, exogenous Aβ in wild‐type cells and endogenous Aβ in APP Swedish cells accumulates in lysosomes, but not in early endosomes. TIRF microscopy captured videos of lysosomes approaching and fusing with the plasma membrane. Fusion appears as a reduction in normal fluorescent LAMP1 signal and an increase in pH sensitive fluorescent protein marker signal (reflecting the loss of luminal acidity due to fusion). Intracellular Aβ was released from neurons as lysosomes fused with the plasma membrane, seen as a simultaneous decrease in fluorescent Aβ labelling. Silencing of Rab27b significantly reduced lysosomal exocytosis and Aβ secretion, which confirmed that this is a lysosomal process.ConclusionOur results demonstrate that lysosomal exocytosis is a novel secretion pathway for Aβ in human neurons and identify potential pharmacological targets that regulate clearance of Aβ.

Live-cell imaging of endocytosed synaptophysin around individual hippocampal presynaptic active zones.

In presynaptic terminals 4 types of endocytosis, kiss-and-run, clathrin-mediated, bulk and ultrafast endocytosis have been reported to maintain repetitive exocytosis of neurotransmitter. However, detailed characteristics and relative contribution of each type of endocytosis still need to be determined. Our previous live-cell imaging study demonstrated individual exocytosis events of synaptic vesicle within an active-zone-like membrane (AZLM) formed on glass using synaptophysin tagged with a pH-sensitive fluorescent protein. On the other hand, individual endocytosis events of postsynaptic receptors were recorded with a rapid extracellular pH exchange method. Combining these methods, here we live-cell imaged endocytosed synaptophysin with total internal reflection fluorescence microscopy in rat hippocampal culture preparations. Clathrin-dependent and -independent endocytosis, which was seemingly bulk endocytosis, occurred within several seconds after electrical stimulation at multiple locations around AZLM at room temperature, with the locations varying trial to trial. The contribution of clathrin-independent endocytosis was more prominent when the number of stimulation pulses was large. The skewness of synaptophysin distribution in intracellular vesicles became smaller after addition of a clathrin inhibitor, which suggests that clathrin-dependent endocytosis concentrates synaptophysin. Ultrafast endocytosis was evident immediately after stimulation only at near physiological temperature and was the predominant endocytosis when the number of stimulation pulses was small.

POpsicle: An all-optical reporter system for synaptic vesicle recycling combining pH-sensitive fluorescent proteins with optogenetic manipulation of neuronal activity.

pH-sensitive fluorescent proteins are widely used to study synaptic vesicle (SV) fusion and recycling. When targeted to the lumen of SVs, fluorescence of these proteins is quenched by the acidic pH. Following SV fusion, they are exposed to extracellular neutral pH, resulting in a fluorescence increase. SV fusion, recycling and acidification can thus be tracked by tagging integral SV proteins with pH-sensitive proteins. Neurotransmission is generally activated by electrical stimulation, which is not feasible in small, intact animals. Previous in vivo approaches depended on distinct (sensory) stimuli, thus limiting the addressable neuron types. To overcome these limitations, we established an all-optical approach to stimulate and visualize SV fusion and recycling. We combined distinct pH-sensitive fluorescent proteins (inserted into the SV protein synaptogyrin) and light-gated channelrhodopsins (ChRs) for optical stimulation, overcoming optical crosstalk and thus enabling an all-optical approach. We generated two different variants of the pH-sensitive optogenetic reporter of vesicle recycling (pOpsicle) and tested them in cholinergic neurons of intact Caenorhabditis elegans nematodes. First, we combined the red fluorescent protein pHuji with the blue-light gated ChR2(H134R), and second, the green fluorescent pHluorin combined with the novel red-shifted ChR ChrimsonSA. In both cases, fluorescence increases were observed after optical stimulation. Increase and subsequent decline of fluorescence was affected by mutations of proteins involved in SV fusion and endocytosis. These results establish pOpsicle as a non-invasive, all-optical approach to investigate different steps of the SV cycle.

Selecting for pH sensitive flourescent proteins with library screening methods.

Intracellular pH (pHi) is known to regulate diverse cell behaviors including cell migration, proliferation, and metabolism. For most of these behaviors, an increase in pHi from ∼7.2 to ∼7.6 has been shown to be a permissive cue. Our current understanding of pHi dynamics during these cell processes is limited by a lack of bright, red-shifted, photostable pH-sensitive fluorescent protein biosensors. Most existing pH biosensors are incompatible with long-term imaging, in vivo pHi measurements, and deep tissue imaging required to understand physiological pHi dynamics during processes like wound healing, cell-cycle progression, and metabolic adaptation. Here we establish a platform for selection of new red-shifted pH-sensitive fluorescent proteins using both random mutagenesis and rational protein design in combination with library selection strategies. We are applying this platform to develop pH biosensors with improved photophysical properties for imaging 3D organoid models as well as increase the linear dynamic range of existing pH-sensitive fluorescent proteins. New red-shifted pH biosensors can be used to track pHi changes inside cells over long time-lapse experiments, while avoiding cellular autofluorescence and allowing for simultaneous imaging of pH and GFP-targeted proteins of interest. The application of these tools will expand our understanding of pHi dynamics and heterogeneity in both normal cell processes as well as models for diseases where pHi is dysregulated like neurodegeneration (low pHi) and cancer (high pHi).

Quantifying pH in Malaria Using pHluorin and Flow Cytometry.

Intracellular pH (pHi) plays a critical role in the regulation of numerous biological functions where specific pH ranges are required for optimal operation within cells. Slight pH changes can impact the regulation of diverse molecular processes, including enzymatic activities, ion channels, and transporters, which all play a role in cell functions. Methods for quantifying pHi continue to evolve and include various optical methods using fluorescent pH indicators. Here, we provide a protocol to measure pHi in the cytosol of Plasmodium falciparum blood stage parasites by means of flow cytometry and using pHluorin2, a pH-sensitive fluorescent protein that has been introduced into the genome of the parasite.

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates.

pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK′ = 6.6) and EGF-CoGFP-mCRISPRed (pK′ = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein–drug conjugates in cancer therapy.

Spontaneously Recycling Synaptic Vesicles Constitute Readily Releasable Vesicles in Intact Neuromuscular Synapses.

Emerging evidence shows that spontaneous synaptic transmission plays crucial roles on neuronal functions through presynaptic molecular mechanisms distinct from that of action potential (AP)-evoked transmission. However, whether the synaptic vesicle (SV) population undergoing the two forms of transmission is segregated remains controversial due in part to the conflicting results observed in cultured neurons. Here we address this issue in intact neuromuscular synapses using transgenic zebrafish larvae expressing two different indicators targeted in the SVs: a pH-sensitive fluorescent protein, pHluorin, and a tag protein, HaloTag. By establishing a quantitative measure of recycled SV fractions, we found that ∼85% of SVs were mobilized by high-frequency AP firings. In contrast, spontaneously recycling SVs were mobilized only from <8% of SVs with a time constant of 45 min at 25°C, although prolonged AP inhibition mobilized an additional population with a delayed onset. The mobilization of the early-onset population was less temperature-sensitive and resistant to tetanus toxin, whereas that of the late-onset population was more sensitive to temperature and was inhibited by tetanus toxin, indicating that prolonged AP inhibition activated a distinct molecular machinery for spontaneous SV fusion. Therefore, the early-onset population limited to <8% was likely the only source of spontaneous release that occurred physiologically. We further showed that this limited population was independent from those reluctant to fuse during AP firing and was used in both the hypertonic stimulation and the immediate phase of AP-evoked releases, thereby matching the characteristics of the readily releasable pool.SIGNIFICANCE STATEMENT Synaptic vesicles (SVs) are divided into functionally distinct pools depending on how they respond to action potential (AP) firing. The origin of SVs used for spontaneous fusion remains enigmatic despite intensive studies in cultured preparations. We addressed this question in intact neuromuscular synapses and provided two findings. First, prolonged AP inhibition activated a distinct population of fusion, which needs to be distinguished from genuine spontaneous fusion arising from a highly limited fraction. Second, the limited fraction observed early in the AP inhibition period exhibited the characteristics of readily releasable pool in the subsequent round of stimulation. Our study revealed that the origin of spontaneous SV fusion is restricted to the readily releasable pool among the SV pools involved in AP-evoked fusion.

Quantification of mitophagy using mKeima-mito in cultured human primary retinal pigment epithelial cells

The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of visual disorders, including age-related macular degeneration (AMD). Utilizing primary RPE cultures to investigate disease is an important step in understanding disease mechanisms. However, the use of primary RPE cultures presents a number of challenges, including the limited number of cells available and the presence of auto-fluorescent pigment that interferes with quantifying fluorescent probes. Additionally, primary RPE are difficult to transfect with exogenous nucleic acids traditionally used for fluorescent imaging. To overcome these challenges, we used an adeno-associated viral (AAV) vector to express a pH sensitive fluorescent protein, mKeima, fused to the mitochondrial targeting sequence of cytochrome oxidase subunit 8A (mKeima-mito). mKeima-mito allows for quantification of mitochondrial autophagy (mitophagy) in live-cell time-lapse imaging experiments. We also developed an image analysis pipeline to selectively quantify mKeima-mito while removing the signal of auto-fluorescent pigment from the dataset by utilizing information from the mKeima fluorescent channels. These techniques are demonstrated in primary RPE cultures expressing mKeima-mito treated with 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), an uncoupler that depolarizes the mitochondrial membrane and leads to mitochondrial fragmentation and mitophagy. The techniques outlined provide a roadmap for investigating disease mechanisms or the effect of treatments utilizing fluorescent probes in an important cell culture model.

Manipulating charge migration through a fluorescent protein

ArcLight is a genetically encoded voltage indicator (GEVI) that fuses a pH sensitive fluorescent protein (FP) to a voltage sensing domain. We have recently reported that an electrostatic interaction between two neighboring FPs in the cytoplasm is altered when the transmembrane segments respond to voltage due to a negative charge on the surface of the FP (Kang et al., 2021). The chromophore of the FP is inside a protective barrel structure consisting of 11 β-sheets. Since the FP is pH sensitive, pathways must exist for charge to migrate from the external surface to the internal chromophore.

Methods for library screening of pH sensitive fluorescent proteins

Intracellular pH (pHi) regulates diverse cell behaviors including cell migration, proliferation, and metabolism. For most of these processes, transient increases in pHi (from ∼7.2 to ∼7.6) have been shown to be a permissive cue. However, our understanding of how pHi dynamics and heterogeneity contribute to these processes is limited by the lack of bright, photostable, and red-shifted pH biosensors. Current tools to measure pHi include chemical dyes and genetically-encoded fluorescent protein sensors.

MOrange2, a Genetically Encoded, pH Sensitive Fluorescent Protein, is an Alternative to BCECF-AM to Measure Intracellular pH to Determine NHE3 and DRA Activity.

NHE3 (Na+/H+ exchanger3) and SLC26A3 (Cl-/HCO3- exchanger, DRA) are the major components of the intestinal neutral NaCl absorptive process and based on the intestinal segment, contribute to HCO3- absorption and HCO3- secretion. NHE3 and DRA are highly regulated by changes in second messengers, cAMP, cGMP and Ca2+. Precise and convenient measurement of exchanger activity is necessary to allow rapid study of physiologic and pharmacologic functions. Some epithelial cells are difficult to load with AM ester dyes and loading may not be uniform. The use of a genetically modified fluorescent protein, mOrange2 was explored as an intracellular pH sensor protein to measure exchange activity of NHE3 and DRA. The model used was FRT cells stably expressing NHE3 or DRA with intracellular pH measured by changes of mOrange2 fluorescence intensity. Intracellular pH was monitored using a) Isolated single clones of FRT/mOrange2/HA-NHE3 cells studied in a confocal microscope with time-lapse live cell imaging under basal conditions and when NHE3 was inhibited by exposure to forskolin and stimulated by dexamethasone, b) coverslip grown FRT/mOrange2 cells expressing NHE3 or DRA using a computerized fluorometer with a perfused cuvette with standardization of the mOrange2 absorption and emission signal using K+/Nigericin as an internal standard in each experiment. A similar rate of intracellular alkalization by Na+ addition in cells expressing NHE3 and by Cl- removal in cells expressing DRA was found in mOrange2 expressing cells compared to the same cells loaded with BCECF-AM, both using the same pH calibration with K+/Nigericin. Using mOrange2 as the pH sensor, NHE3 basal activity was quantitated and shown to be inhibited by forskolin and stimulated by dexamethasone, and DRA was oppositely shown to be stimulated by forskolin, responses similar to results found using BCECF-AM. This study demonstrates that mOrange2 protein can be an effective alternate to BCECF-AM in measuring intracellular pH (preferred setting Ex520nm, Em 563nm) as affected by NHE3 and DRA activity, with the advantage, compared to AM ester dyes, that genetic expression can provide uniform expression of the pH sensor.

Quantitative Analysis of Presynaptic Vesicle Luminal pH in Cultured Neurons.

Newly generated synaptic vesicles (SVs) are re-acidified by the activity of the vacuolar-type H+-ATPases. Since H+ gradient across SV membrane drives neurotransmitter uptake into SVs, precise measurements of steady-state vesicular pH and dynamics of re-acidification process will provide important information concerning the H+-driven neurotransmitter uptake. Indeed, we recently demonstrated distinct features of steady state and dynamics of vesicular pH between glutamatergic vesicles and GABAergic vesicles in cultured hippocampal neurons. In this article, we focus on an experimental protocol and setup required to determine steady-state luminal pH of SVs in living neurons. This protocol is composed of efficient expression of a pH-sensitive fluorescent protein in the lumen of SVs in cultured neurons, and recordings of its fluorescence changes under a conventional fluorescent microscope during local applications of acidic buffer and ionophores-containing solution at a given pH. The method described here can be easily applied for measuring luminal pH of different types of secretory organelles and other acidic organelles such as lysosomes and endosomes in cultured cell preparations.

Chemical Sectioning for Immunofluorescence Imaging.

Immunofluorescence (IF) is a powerful investigative tool in biological research and medical diagnosis, whereas conventional imaging methods are always conflict between speed, contrast/resolution, and specimen volume. Chemical sectioning (CS) is an effective method to overcome the conflict, which works by chemically manipulating the off/on state of fluorescent materials and turning on only the extremely superficial surface fluorescence of tissues to realize the sectioning capacity of wide-field imaging. However, the current mechanism of CS is only applicable to samples labeled with pH-sensitive fluorescent proteins and still cannot fulfill samples immunolabeled with frequently used commercial fluorescent dyes. Here, immunofluorescence chemical sectioning (IF-CS) is described to present an off/on mechanism for Alexa dyes by complexation reactions, allowing CS imaging of IF labeled tissues. IF-CS enables IF freeing from out-of-focus interference in wide-field imaging and satisfying with multicolor imaging. IF-CS demonstrates the utility of the 3D submicron-resolution imaging of large immunolabeled tissues on the wide-field block-face system. IF-CS may remarkably facilitate systematic studies of refined subcellular architectures of endogenous proteins in intact biological systems.

PHmScarlet is a pH-sensitive red fluorescent protein to monitor exocytosis docking and fusion steps

pH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. Superecliptic pHluorin (SEP), a green pH-sensitive FP, has been widely used for imaging single-vesicle exocytosis. However, the docking step cannot be visualized using this FP, since the fluorescence signal inside vesicles is too low to be observed during docking process. Among the available red pH-sensitive FPs, none is comparable to SEP for practical applications due to unoptimized pH-sensitivity and fluorescence brightness or severe photochromic behavior. In this study, we engineer a bright and photostable red pH-sensitive FP, named pHmScarlet, which compared to other red FPs has higher pH sensitivity and enables the simultaneous detection of vesicle docking and fusion. pHmScarlet can also be combined with SEP for dual-color imaging of two individual secretory events. Furthermore, although the emission wavelength of pHmScarlet is red-shifted compared to that of SEP, its spatial resolution is high enough to show the ring structure of vesicle fusion pores using Hessian structured illumination microscopy (Hessian-SIM).

Imaging endocytic vesicle formation at high spatial and temporal resolutions with the pulsed-pH protocol.

Endocytosis is a fundamental process occurring in all eukaryotic cells. Live cell imaging of endocytosis has helped to decipher many of its mechanisms and regulations. With the pulsed-pH (ppH) protocol, one can detect the formation of individual endocytic vesicles (EVs) with an unmatched temporal resolution of 2 s. The ppH protocol makes use of cargo protein (e.g., the transferrin receptor) coupled to a pH-sensitive fluorescent protein, such as superecliptic pHluorin (SEP), which is brightly fluorescent at pH 7.4 but not fluorescent at pH <6.0. If the SEP moiety is at the surface, its fluorescence will decrease when cells are exposed to a low pH (5.5) buffer. If the SEP moiety has been internalized, SEP will remain fluorescent even during application of the low pH buffer. Fast perfusion enables the complete exchange of low and high pH extracellular solutions every 2 s, defining the temporal resolution of the technique. Unlike other imaging-based endocytosis assays, the ppH protocol detects EVs without a priori hypotheses on the dynamics of vesicle formation. Here, we explain how the ppH protocol quantifies the endocytic activity of living cells and the recruitment of associated proteins in real time. We provide a step-by-step procedure for expression of the reporter proteins with transient transfection, live cell image acquisition with synchronized pH changes and automated analysis. The whole protocol can be performed in 2 d to provide quantitative information on the endocytic process being studied.

Protein expression-independent response of intensity-based pH-sensitive fluorophores in Escherichia coli.

Fluorescent proteins that modulate their emission intensities when protonated serve as excellent probes of the cytosolic pH. Since the total fluorescence output fluctuates significantly due to variations in the fluorophore levels in cells, eliminating the dependence of the signal on protein concentration is crucial. This is typically accomplished with the aid of ratiometric fluorescent proteins such as pHluorin. However, pHluorin is excited by blue light, which can complicate pH measurements by adversely impacting bacterial physiology. Here, we characterized the response of intensity-based, pH-sensitive fluorescent proteins that excite at longer wavelengths where the blue light effect is diminished. The pH-response was interpreted in terms of an analytical model that assumed two emission states for each fluorophore: a low intensity protonated state and a high intensity deprotonated state. The model suggested a scaling to eliminate the dependence of the signal on the expression levels as well as on the illumination and photon-detection settings. Experiments successfully confirmed the scaling predictions. Thus, the internal pH can be readily determined with intensity-based fluorophores with appropriate calibrations irrespective of the fluorophore concentration and the signal acquisition setup. The framework developed in this work improves the robustness of intensity-based fluorophores for internal pH measurements in E. coli, potentially extending their applications.