Fluorescent Protein Sensor Research Articles

Time-Lapse Live-Cell Imaging Using Fluorescent Protein Sensors in Outflow Pathway Cells Under Fluid Flow Conditions.

The role of shear stress in regulating aqueous humor (AH) outflow and intraocular pressure (IOP) in the trabecular meshwork (TM) and Schlemm's canal (SC) of the eye is an emerging field. Shear stress has been shown to activate mechanosensitive ion channels in TM cells and induce nitric oxide production in SC cells, which can affect outflow resistance and lower IOP. Live-cell imaging using fluorescent protein sensors has provided real-time data to investigate the physiological relationship between fluid flow and shear stress in the outflow pathway cells. The successful application of time-lapse live-cell imaging in primary cultured cells has led to the identification of key cellular and molecular mechanisms involved in regulating AH outflow and IOP, including the role of autophagy and primary cilia as mechanosensors. This chapter presents a detailed protocol for conducting time-lapse live-cell imaging under fluid flow conditions in the outflow pathway cells.

A bright red fluorescent genetically encoded sensor for lactate imaging

Lactate plays a crucial role in energy metabolism and greatly impacts protein activities, exerting diverse physiological and pathological effects. Therefore, convenient lactate assays for tracking spatiotemporal dynamics in living cells are desirable. In this paper, we engineered and optimized a red fluorescent protein sensor for l-lactate named FiLa-Red. This indicator exhibited a maximal fluorescence change of 730 % and an apparent dissociation constant (Kd) of approximately 460 μM. By utilizing FiLa-Red and other sensors, we monitored energy metabolism in a multiplex manner by simultaneously tracking lactate and NAD+/NADH abundance in the cytoplasm, nucleus, and mitochondria. The FiLa-Red sensor is expected to be a useful tool for performing metabolic analysis in vitro, in living cells and in vivo.

Scaling the Functional Nanopore (FuN) Screen: Systematic Evaluation of Self-Assembling Membrane Peptides and Extension with a K+-Responsive Fluorescent Protein Sensor.

The functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanopores in vitro still constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analyzed using the functional nanopore (FuN) screen exploiting genetically encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca2+ in- and efflux patterns across the inner membrane of Escherichia coli. With a primary proof-of-concept established for the S2168 holin, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analyzed with the FuN screen and to what extent alternative fluorescent protein sensors can be adapted. Focusing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally designed nanopores. Further, the FuN screen is extended to K+-specific fluorescent protein sensors and now provides a capacity to assess the specificity of a nanopore or ion channel. Finally, a comparison to high-resolution biophysical and electrophysiological studies in planar lipid bilayers provides an experimental benchmark for future studies.

A genetically encoded fluorescent protein sensor for mitochondrial membrane damage detection

Mitochondria are essential cellular organelles; detecting mitochondrial damage is crucial in cellular biology and toxicology. Compared with existing chemical probe detection methods, genetically encoded fluorescent protein sensors can directly indicate cellular and molecular events without involving exogenous reagents. In this study, we introduced a molecular sensor system, MMD-Sensor, for monitoring mitochondrial membrane damage. The sensor consists of two molecular modules. Module I is a fusion structure of the mitochondrial localization sequence (MLS), AIF cleavage site sequence (CSS), nuclear localization sequence (NLS), N-terminus of mNeonGreen and mCherry. Module II is a fusion structure of the C-terminus of mNeonGreen, NLS sequence, and mtagBFP2. Under normal condition, Module I is constrained in the inner mitochondrial membrane anchored by MLS, while Module II is restricted to the nucleus by its NLS fusion component. If the mitochondrial membrane is damaged, CSS is cut from the inner membrane, causing Module I to shift into the nucleus guided by the NLS fusion component. After Module I enters the nucleus, the N- and C-terminus of mNeonGreen meet each other and rebuild its intact 3D structure through fragment complementation and thus generates green fluorescence in the nucleus. Dynamic migration of red fluorescence from mitochondria to the nucleus and generation of green fluorescence in the nucleus indicate mitochondrial membrane damage. Using the MMD-Sensor, mitochondrial membrane damage induced by various reagents, such as uncoupling agents, ATP synthase inhibitors, monovalent cationic carriers, and ROS, in HeLa and 293T cells are directly observed and evaluated.

How several factors, playing in concert, increase the two-photon fluorescence response of red fluorescent protein sensors

How several factors, playing in concert, increase the two-photon fluorescence response of red fluorescent protein sensors

Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH7.4, the proportionality constant for ∆F/F0vs [nicotine] (δ-slope, 2.7μM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the 'candle snuffer' mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

Engineering the ChlorON Series: Turn-On Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells.

Beyond its role as the "queen of electrolytes", chloride can also serve as an allosteric regulator or even a signaling ion. To illuminate this essential anion across such a spectrum of biological processes, researchers have relied on fluorescence imaging with genetically encoded sensors. In large part, these have been derived from the green fluorescent protein found in the jellyfish Aequorea victoria. However, a standalone sensor with a turn-on intensiometric response at physiological pH has yet to be reported. Here, we address this technology gap by building on our discovery of the anion-sensitive fluorescent protein mNeonGreen (mNG). The targeted engineering of two non-coordinating residues, namely K143 and R195, in the chloride binding pocket of mNG coupled with an anion walking screening and selection strategy resulted in the ChlorON sensors: ChlorON-1 (K143W/R195L), ChlorON-2 (K143R/R195I), and ChlorON-3 (K143R/R195L). In vitro spectroscopy revealed that all three sensors display a robust turn-on fluorescence response to chloride (20- to 45-fold) across a wide range of affinities (Kd ≈ 30-285 mM). We further showcase how this unique sensing mechanism can be exploited to directly image labile chloride transport with spatial and temporal resolution in a cell model overexpressing the cystic fibrosis transmembrane conductance regulator. Building from this initial demonstration, we anticipate that the ChlorON technology will have broad utility, accelerating the path forward for fundamental and translational aspects of chloride biology.

Oxidative and ER stress by elevated insulin biosynthesis and palmitic acid in insulin-producing cells.

The early phase of type 2 diabetes mellitus (T2DM) is characterised by insulin resistance, which can initially be compensated by elevated insulin secretion. However, as postulated by the workload hypothesis, over time harming insulin requirements contribute to β-cell dysfunction and death. The mechanisms behind this transition are complex and not fully understood but involve factors such as endoplasmic reticulum (ER) stress raised by gluco/lipotoxicity. To investigate the effect of excessive insulin folding on ER luminal H2O2 generation, ER stress and viability, insulin was expressed glucose-independently by a doxycycline-regulated Tet-On system in insulin-producing RINm5F cells. Additionally, the effect of palmitic acid (PA) as a subsidiary T2DM-associated factor was examined in this model system. Elevated insulin expression increased ER luminal H2O2 concentration quantified by the fluorescent sensor protein TriPer and reduced viability, but did not activate apoptosis. However, when combined with PA, insulin expression resulted in a significant increase in ER stress and apoptosis. Expression of ER-localised catalase verified the specificity of the applied H2O2 detection method without attenuating ER stress, caspase activation or viability loss. These findings suggest that hyperinsulinism alone can cause increased ER luminal H2O2 generation, mild ER stress and reduced viability, while hyperinsulinism in combination with PA accelerates these processes and triggers apoptosis. The inability of ER catalase to counteract these effects suggests that further damaging factors besides H2O2 are involved in cell dysfunction. Finally, reducing the high insulin demand in the initial phase of T2DM may be crucial in preventing further β-cell damage caused by gluco/lipotoxicity.

Dissecting the Permeability of the Escherichia coli Cell Envelope to a Small Molecule Using Tailored Intensiometric Fluorescent Protein Sensors.

Membranes provide a highly selective barrier that defines the boundaries of any cell while providing an interface for communication and nutrient uptake. However, despite their central physiological role, our capacity to study or even engineer the permeation of distinct solutes across biological membranes remains rudimentary. This especially applies to Gram-negative bacteria, where the outer and inner membrane impose two permeation barriers. Addressing this analytical challenge, we exemplify how the permeability of the Escherichia coli cell envelope can be dissected using a small-molecule-responsive fluorescent protein sensor. The approach is exemplified for the biotechnologically relevant macrolide rapamycin, for which we first construct an intensiometric rapamycin detector (iRapTor) while comprehensively probing key design principles in the iRapTor scaffold. Specifically, this includes the scope of minimal copolymeric linkers as a function of topology and the concomitant need for gate post residues. In a subsequent step, we apply iRapTors to assess the permeability of the E. coli cell envelope to rapamycin. Despite its lipophilic character, rapamycin does not readily diffuse across the E. coli envelope but can be enhanced by recombinantly expressing a nanopore in the outer membrane. Our study thus provides a blueprint for studying and actuating the permeation of small molecules across the prokaryotic cell envelope.

Enhanced Intrarenal pro-renin and salt sensitive hypertension in a mouse model of Type-1 diabetes

Hypertension affects up to 50% of patients with type 1 diabetes. Despite aggressive anti-hypertensive therapy, at least 30% of diabetic patients develop chronic kidney disease. Renin is the rate-limiting enzyme in the activation of the RAS and is essential for blood pressure control. Renin is expressed in Juxtaglomerular (JG) cells, as an immature pro-peptide (pro-renin), and then stored as active renin. In diabetic humans, plasma pro-renin is enhanced while renin remains unchanged or low. We hypothesize that intrarenal renin and pro-renin are enhanced in type 1 diabetes, which could potentially contribute to hypertension and diabetic nephropathy. We used the Akita mice strain, one of the few mouse models of diabetes mellitus that shows features of the human disease including hypertension and kidney damage. We found that Akita mice had higher blood pressure as measured by tailcuff (mmHg: C57=109±2.8; Aki=120.6±2.5;p<0.05, n=6), plasma glucose (in mM: C57=7.71±23.9; Aki=23.2±2.3; p<0.01; n=4) and proteinuria (mg/24hrs: C57 18.2±3.1; Aki: 29.4±3.3; p<0.05; n=5) when compared to C57 (control). In addition, plasma renin was not changed in Akita mice (n=6; p= NS). Histological staining of kidney cuts with a renin antibody (recognizes both renin and pro-renin) showed overall decreased total renin expression in Akita mice (O.D C57: 46.6±1.5; Akita=30.7±3.5). Surprisingly, Western Blots of renal cortical lysates and JG cells showed that pro-renin (43-45 kDa) expression was 79±21% higher in male Akita diabetic mice (p<0.05), demonstrating increased intra-renal pro-renin in Akita. The mechanism driving the increase in pro-renin is unknown. We previously showed that ROS potently stimulate renin release from JG cells. We found that Akita mice have increased urinary isoprostanes, supporting higher kidney ROS (ng/24hrs: C57:3.5±0.6 Aki=6.0±0.8 p<0.05; n=5). We measured H202 levels by transducing primary JG cells in culture with the fluorescent H202 protein sensor HyPer and found that JG cells isolated from Akita mice had higher baseline levels of H202 (C57= 100, Akita=117±7%, p<0.05). An activated intrarenal RAS contributes to salt sensitive hypertension, and at least one third of diabetic patients are salt-sensitive. It is not known whether diabetic Akita mice are salt sensitive. Therefore, we tested the effect of a high salt diet on blood pressure in Akita and C57 mice measured by radiotelemetry. High salt increased systolic blood pressure (SBP) in Akita (from 109±5 to 138±6 mmHg, p<0.001) but not in control C57 (from 104±2 to 117±6 mmHg, NS). We conclude that intrarenal renin and pro-renin is enhanced in Akita mice which also exhibit salt sensitive hypertension. The mechanism may involve elevated superoxide and H202 levels within JG cells stimulating pro-renin expression and release. AHA-SDG; NIH-NIDDK-RO3 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Visualization of the functional neural circuit breakdown process in a mouse model of Alzheimer's disease and application to the development of new therapies

Alzheimer's disease (AD) patients cause cognitive impairment in time and space as early symptoms, in which the hippocampus plays an essential role. In the hippocampus, place cells play an important role in processing spatial information. The hippocampus recognizes space and guides creatures to their destinations using these place cells. In AD patients, the hippocampal neuronal network that place cells form could malfunction with age. However, it has remained unknown how the disruption of the neural circuits progresses at a single-cell resolution in AD. Conventional methods such as electrophysiology experiments could only record the same cells in the hippocampal neural circuit of awake mice for a few days. To clear the detail of neuronal circuit disruption in AD, it is necessary to observe brains of the same individuals over several months. Therefore, we performed chronic two-photon calcium imaging using AD model mice expressing the fluorescent calcium sensor protein G-CaMP7 to monitor spatiotemporal representation in a virtual reality environment. The activity of hundreds of hippocampal CA1 pyramidal neurons was detected over months from the same neuronal populations. In AD mice, amyloid (Aβ) plaque-like aggregates in the stratum oriens layer of hippocampal CA1 start to develop at 2.5 months of age, increase in both number and size with age, and neurons with hyperactivity increase near Aβ plaque-like aggregates. In addition, place cells observed during VR navigation showed abnormalities in the stability of activity within the place field at 4 months old, and then the stability was further deteriorated and the number of place cells decreased at 7 months old. Thus, we demonstrated that the activity patterns of various neurons, including place cells, in the neural circuitry of the hippocampal CA1 region in AD have different failure modes, and this finding is expected to be applied to the effective drug evaluation for AD.

Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein Sensors.

The pyridine nucleotides NAD(H) and NADP(H) are key molecules in cellular metabolism, and measuring their levels and oxidation states with spatiotemporal precision is of great value in biomedical research. Traditional methods to assess the redox state of these metabolites are intrusive and prohibit live-cell quantifications. This obstacle was surpassed by the development of genetically encoded fluorescent biosensors enabling dynamic measurements with subcellular resolution in living cells. Here, we provide step-by-step protocols to monitor the intraperoxisomal NADPH levels and NAD+/NADH redox state in cellulo by using targeted variants of iNAP1 and SoNar, respectively.

Genetically encoded protein sensors for metal ion detection in biological systems: a review and bibliometric analysis.

Metal ions are indispensable elements in living organisms and are associated with regulating various biological processes. An imbalance in metal ion content can lead to disorders in normal physiological functions of the human body and cause various diseases. Genetically encoded fluorescent protein sensors have the advantages of low biotoxicity, high specificity, and a long imaging time in vivo and have become a powerful tool to visualize or quantify the concentration level of biomolecules in vivo and in vitro, temporal and spatial distribution, and life activity process. This review analyzes the development status and current research hotspots in the field of genetically encoded fluorescent protein sensors by bibliometric analysis. Based on the results of bibliometric analysis, the research progress of genetically encoded fluorescent protein sensors for metal ion detection is reviewed, and the construction strategies, physicochemical properties, and applications of such sensors in biological imaging are summarized.

Directed evolution of the genetically encoded zinc(II) FRET sensor ZapCY1

Zinc(II) ions (Zn2+) play an essential role in living systems, with their delicate concentration balance differing among the various intracellular organelles. The spatiotemporal distribution and homeostasis of Zn2+ can be monitored through photoluminescence imaging using zinc sensors. Among such biosensors, genetically encoded fluorescent sensor proteins are attractive tools owing to their subcellular localization advantage and high biocompatibility. However, the limited fluorescent properties of these proteins, such as their insufficient quantum yield and dynamic range, restrict their practical use. In this study, we developed an expression–screening–directed evolution system and used it to improve ZapCY1, a genetically encoded fluorescence resonance energy transfer (FRET) sensor. After four rounds of directed evolution, the FRET dynamic range of the modified sensor (designated ZapTV-EH) was increased by 1.5–1.7-fold. With its enhanced signal-to-noise ratio and ability to detect a wide Zn2+ concentration range, ZapTV-EH proves to be a better visualization tool for monitoring Zn2+ at the subcellular level. Combined with the simplified subcloning and expression steps and sufficient mutant libraries, this directed evolution system may provide a more simple and efficient way to develop and optimize genetically encoded FRET sensors through high-throughput screening.

Real-time fiber-optic recording of acute-ischemic-stroke signatures.

We present an experimental framework and methodology for in vivo studies on rat stroke models that enable a real-time fiber-optic recording of stroke-induced hydrogen peroxide and pH transients in ischemia-affected brain areas. Arrays of reconnectable implantable fiber probes combined with advanced optogenetic fluorescent protein sensors are shown to enable a quantitative multisite time-resolved study of oxidative-stress and acidosis buildup dynamics as the key markers, correlates and possible drivers of ischemic stroke. The fiber probes designed for this work provide a wavelength-multiplex forward-propagation channel for a spatially localized, dual-pathway excitation of genetically encoded fluorescence-protein sensors along with a back-propagation channel for the fluorescence return from optically driven fluorescence sensors. We show that the spectral analysis of the fiber-probe-collected fluorescence return provides means for a high-fidelity autofluorescence background subtraction, thus enhancing the sensitivity of real-time detection of stroke-induced transients and significantly reducing measurement uncertainties in in vivo acute-stroke studies as inherently statistical experiments operating with outcomes of multiply repeated measurements on large populations of individually variable animal stroke models.

Functional Nanopore Screen: A Versatile High-Throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli.

Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the functional nanopore (FuN) screen now provides a quantitative and dynamic readout of nanopore assembly and function in the context of the inner membrane of Escherichia coli. The assay is based on genetically encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta-, and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries, while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology, and synthetic biology.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

High-throughput and high-content bioassay enables tuning of polyester nanoparticles for cellular uptake, endosomal escape, and systemic in vivo delivery of mRNA.

Nanoparticle-based mRNA therapeutics hold great promise, but cellular internalization and endosomal escape remain key barriers for cytosolic delivery. We developed a dual nanoparticle uptake and endosomal disruption assay using high-throughput and high-content image-based screening. Using a genetically encoded Galectin 8 fluorescent fusion protein sensor, endosomal disruption could be detected via sensor clustering on damaged endosomal membranes. Simultaneously, nucleic acid endocytosis was quantified using fluorescently tagged mRNA. We used an array of biodegradable poly(beta-amino ester)s as well as Lipofectamine and PEI to demonstrate that this assay has higher predictive capacity for mRNA delivery compared to conventional polymer and nanoparticle physiochemical characteristics. Top nanoparticle formulations enabled safe and efficacious mRNA expression in multiple tissues following intravenous injection, demonstrating that the in vitro screening method is also predictive of in vivo performance. Efficacious nonviral systemic delivery of mRNA with biodegradable particles opens up new avenues for genetic medicine and human health.

Fluorescent Binding Protein Sensors for Detection and Quantification of Biochemicals, Metabolites, and Natural Products.

Membrane transporters and soluble binding proteins recognize particular nutrients, metabolites, vitamins, or ligands. By modifying genetically engineered single cysteine residues near the active sites of such proteins with extrinsic maleimide fluorophores,the approaches that we report create sensitive fluorescent sensors that detect, quantify, and monitor molecules that are relevant to the biochemistry, physiology, microbiology, and clinical properties of pro- and eukaryotic organisms. This protocol was validated in: J Biol Chem (2022), DOI: 10.1016/j.jbc.2022.101651 Graphical abstract.

Discovery of a monomeric green fluorescent protein sensor for chloride by structure-guided bioinformatics.

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over the last two decades, this understanding has been advanced by chloride-sensitive fluorescent proteins for imaging applications in living cells. To our surprise, these sensors have primarily been engineered from the green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. However, the GFP family has a rich sequence space that could already encode for new sensors with desired properties, thereby minimizing protein engineering efforts and accelerating biological applications. To efficiently sample this space, we present and validate a stepwise bioinformatics strategy focused first on the chloride binding pocket and second on a monomeric oligomerization state. Using this, we identified GFPxm163 from GFPxm found in the jellyfish Aequorea macrodactyla. In vitro characterization shows that the binding of chloride as well as bromide, iodide, and nitrate rapidly tunes the ground state chromophore equilibrium from the phenolate to the phenol state generating a pH-dependent, turn-off fluorescence response. Furthermore, live-cell fluorescence microscopy reveals that GFPxm163 provides a reversible, yet indirect readout of chloride transport via iodide exchange. With this demonstration, we anticipate that the pairing of bioinformatics with protein engineering methods will provide an efficient methodology to discover and design new chloride-sensitive fluorescent proteins for cellular applications.