FRET Biosensor Research Articles

A Novel Single-Color FRET Sensor for Rho-Kinase Reveals Calcium-Dependent Activation of RhoA and ROCK.

Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII.

Abstract We096: Gene therapy of MAOA fine-tunes nuclear cAMP nanodomain and improves cardiac hypertrophy in mice

Cardiac hypertrophy, a precursor to heart failure, represents a leading cause of global mortality and morbidity. Current therapeutic strategies focused on reducing the heart's workload, demonstrate limited efficacy. Nuclear-localized cAMP emerges as a promising therapeutic target, intervening in hypertrophic gene transcription and exhibiting overactivation in hypertrophic cardiomyocytes. Despite its potential, the spatiotemporal regulation and molecular targets of nuclear-localized cAMP (NLS-cAMP) remain elusive. Employing super-resolution imaging, we identified a pool of β1 adrenergic receptors (β1AR) at the nuclear envelope that transduces NLS-cAMP-PKA signaling in cardiomyocytes. Utilizing a nuclear-localized FRET biosensor, our investigations revealed that the deletion or inhibition of monoamine oxidase A (MAOA) amplifies the norepinephrine-induced NLS-β1AR activation signal. Conversely, overexpressing MAOA prevents NLS-B1AR activation, underscoring the critical role of MAOA in regulating the NLS-cAMP nanodomain. Single-cell transcriptome analysis unveiled a decline in MAOA expression in a mouse cardiac hypertrophy model. Cardiac-specific deletion of MAOA in mice led to simultaneous hypertrophy and eventual heart failure. Mechanistically, MAOA deletion increased PKA-dependent phosphorylation of HDAC1, H3K27 acetylation, and chromatin accessibility of hypertrophic genes, including Nppa and Nppb. To validate therapeutic potential, we intraventricularly injected adeno-associated virus (AAV) 9 vectors expressing mcherry or mcherry-MAOA under the control of the cTNT promoter into mice. Inducing cardiac hypertrophy through Angiotensin II infusion five weeks later, mice preinjected with AAV-MAOA exhibited attenuated cardiac hypertrophy and improved cardiac function. Our findings suggest that MAOA gene therapy holds promise for treating hypertrophy by specifically targeting the NLS-cAMP nanodomain, opening new avenues for targeted interventions in cardiac hypertrophy and heart failure.

A multiplexed high throughput screening assay using flow cytometry identifies glycolytic molecular probes in bloodstream form Trypanosoma brucei

Kinetoplastid organisms, including Trypanosoma brucei, are a significant health burden in many tropical and semitropical countries. Much of their metabolism is poorly understood. To better study kinetoplastid metabolism, chemical probes that inhibit kinetoplastid enzymes are needed. To discover chemical probes, we have developed a high-throughput flow cytometry screening assay that simultaneously measures multiple glycolysis-relevant metabolites in live T. brucei bloodstream form parasites. We transfected parasites with biosensors that measure glucose, ATP, or glycosomal pH. The glucose and ATP sensors were FRET biosensors, while the pH sensor was a GFP-based biosensor. The pH sensor exhibited a different fluorescent profile from the FRET sensors, allowing us to simultaneously measure pH and either glucose or ATP. Cell viability was measured in tandem with the biosensors using thiazole red. We pooled sensor cell lines, loaded them onto plates containing a compound library, and then analyzed them by flow cytometry. The library was analyzed twice, once with the pooled pH and glucose sensor cell lines and once with the pH and ATP sensor cell lines. Multiplexing sensors provided some internal validation of active compounds and gave potential clues for each compound's target(s). We demonstrated this using the glycolytic inhibitor 2-deoxyglucose and the alternative oxidase inhibitor salicylhydroxamic acid. Individual biosensor-based assays exhibited a Z′-factor value acceptable for high-throughput screening, including when multiplexed. We tested assay performance in a pilot screen of 14,976 compounds from the Life Chemicals Compound Library. We obtained hit rates from 0.2 to 0.4% depending on the biosensor, with many compounds impacting multiple sensors. We rescreened 44 hits, and 28 (64%) showed repeatable activity for one or more sensors. One compound exhibited EC50 values in the low micromolar range against two sensors. We expect this method will enable the discovery of glycolytic chemical probes to improve metabolic studies in kinetoplastid parasites.

A novel single-color FRET biosensor for Rho-kinase activity reveals calcium-dependent activation of RhoA and ROCK.

Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to other kinases. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity, and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII.

A sandwich FRET biosensor for lysozyme detection based on peptide-functionalized gold nanoparticles and FAM-labeled aptamer

Lysozyme (LYZ) plays a crucial role in the body's immune defense system. Monitoring LYZ levels can provide valuable insights into the diagnosis and severity assessment of various diseases. Traditionally, antibody-based sandwich assays are employed for LYZ detection, but they are often time-consuming and operationally complicated. In this research, a novel sandwich FRET biosensor was developed, which enables rapid detection of LYZ based on peptide-functionalized gold nanoparticles (pAuNPs) and FAM-labeled aptamer (Apt-FAM). Initially, a mixture of Apt-FAM and pAuNPs resulted in partial quenching of the Apt-FAM fluorescence emission through an inner filter effect (IFE), with negligible energy transfer because of the electrostatic repulsion between the negatively charged pAuNPs and Apt-FAM. The introduction of LYZ into the mixture drove the specific binding of Apt-FAM and pAuNPs to LYZ, facilitating the formation of a pAuNPs-LYZ-aptamer sandwich structure. The formation of this complex drew the pAuNPs and Apt-FAM into close enough proximity to enable FRET to occur, which in turn effectively quenched the fluorescence emission of FAM. The decrease in FAM fluorescence intensity was correlated with the increasing concentration of LYZ. Thus, a sandwich FRET biosensor was successfully developed for LYZ detection with a linear detection range of 0–1.75 μM and a detection limit of 85 nM. Additionally, the biosensor allowed visual detection of LYZ in a 96-well microplate, with a rapid response time of just 15 s. This study introduces a innovative sandwich FRET biosensor that combines aptamer and peptide recognition elements, offering a fast and antibody-free method for protein detection.

SORL1 is a receptor for tau that promotes tau seeding

Sortilin-related receptor 1 (SORL1) is an intracellular sorting receptor genetically implicated in Alzheimer's disease (AD) that impacts amyloid precursor protein trafficking. The objective of these studies was to test the hypothesis that SORL1 binds tau, modulates its cellular trafficking and impacts the aggregation of cytoplasmic tau induced by pathological forms of tau. Using surface plasmon resonance measurements, we observed high-affinity binding of tau to SORL1 and the vacuolar protein sorting 10 domain of SORL1. Interestingly, unlike LDL receptor-related protein 1, SORL1 binds tau at both pH 7.4 and pH 5.5, revealing its ability to bind tau at endosomal pH. Immunofluorescence studies confirmed that exogenously added tau colocalized with SORL1 in H4 neuroglioma cells, while overexpression of SORL1 in LDL receptor-related protein 1-deficient Chinese hamster ovary (CHO) cells resulted in a marked increase in the internalization of tau, indicating that SORL1 can bind and mediate the internalization of monomeric forms of tau. We further demonstrated that SORL1 mediates tau seeding when tau RD P301S FRET biosensor cells expressing SORL1 were incubated with high molecular weight forms of tau isolated from the brains of patients with AD. Seeding in H4 neuroglioma cells is significantly reduced when SORL1 is knocked down with siRNA. Finally, we demonstrate that the N1358S mutant of SORL1 significantly increases tau seeding when compared to WT SORL1, identifying for the first time a potential mechanism that connects this specific SORL1 mutation to Alzheimer's disease. Together, these studies identify SORL1 as a receptor that contributes to trafficking and seeding of pathogenic tau.

Characterization of PDGF-Induced Subcellular Calcium Regulation through Calcium Channels in Airway Smooth Muscle Cells by FRET Biosensors

The homeostasis of cellular calcium is fundamental for many physiological processes, while the calcium levels remain inhomogeneous within cells. During the onset of asthma, epithelial and inflammatory cells secrete platelet-derived growth factor (PDGF), inducing the proliferation and migration of airway smooth muscle (ASM) to the epidermal layer, narrowing the airway. The regulation of ASM cells by PDGF is closely related to the conduction of calcium signals. In this work, we generated subcellular-targeted FRET biosensors to investigate calcium regulation in the different compartments of ASM cells. A PDGF-induced cytoplasmic calcium [Ca2+]C increase was attributed to both extracellular calcium influx and endoplasmic reticulum (ER) calcium [Ca2+]ER release, which was partially regulated by the PLC-IP3R pathway. Interestingly, the removal of the extracellular calcium influx led to inhibited ER calcium release, likely through inhibitory effects on the calcium-dependent activation of the ER ryanodine receptor. The inhibition of the L-type calcium channel on the plasma membrane or the SERCA pump on the ER resulted in both reduced [Ca2+]C and [Ca2+]ER from PDGF stimulation, while IP3R channel inhibition led to reduced [Ca2+]C only. The inhibited SERCA pump caused an immediate [Ca2+]C increase and [Ca2+]ER decrease, indicating active calcium exchange between the cytosol and ER storage in resting cells. PDGF-induced calcium at the outer mitochondrial membrane sub-region showed a similar regulatory response to cytosolic calcium, not influenced by the inhibition of the mitochondrial calcium uniporter channel. Therefore, our work identifies calcium flow pathways among the extracellular medium, cell cytosol, and ER via regulatory calcium channels. Specifically, extracellular calcium flow has an essential function in fully activating ER calcium release.

Abstract 1563 A novel FRET biosensor for visualizing membrane-mediated dimerization of PIP5K

Abstract 1563 A novel FRET biosensor for visualizing membrane-mediated dimerization of PIP5K

Live-cell FRET biosensors for high-throughput screening targeting the SERCA2A-DWORF complex

Live-cell FRET biosensors for high-throughput screening targeting the SERCA2A-DWORF complex

SORL1 regulates tau trafficking

AbstractBackgroundWhile LRP1 is a cellular receptor that mediates tau uptake and potentiates tau seeding, we observed LRP1‐independent uptake of tau in LRP1‐genetically deficient cells and hypothesized that the sortilin‐related receptor (SORL1) could also be involved in tau processing.MethodSurface plasmon resonance was used to assess tau binding to SORL1. 125I‐labelled tau uptake assays were used to investigate the role of SORL1 in endocytosis. The P301S FRET biosensor was used to assess SORL1’s role in cytosolic tau seeding, using both WT and AD‐associated mutations in the SORL1 gene (G511R and N1358S). Confocal microscopy was used to investigate colocalization and intracellular trafficking of tau and SORL1. Finally, we investigated the impact of SORL1 on tau processing in vivo by injecting human pathogenic tau isolated from AD patients into the hippocampus of SORL1 knockout vs wildtype mice and analyzing via immunohistochemistry.ResultWe confirmed high‐affinity binding of tau to SORL1 and found that fluorescently labeled tau colocalizes with overexpressed SORL1 in H4 neuroglioma cells. When we knocked down SORL1 expression using siRNA in H4 cells, we observed a significant increase in the LRP1‐mediated degradation of 125I‐labeled tau, revealing that SORL1 directs tau away from lysosomal degradative pathways. To determine if this occurs in vivo, we injected pathogenic forms of human tau into the hippocampal region of WT and SORL1‐deficient mice. Two days following injection, preliminary results suggest less human tau retention in the brains of SORL1‐deficient mice when compared to WT controls. Together, these data suggest that SORL1 may have a dual function in tau metabolism: impacting both uptake of tau, and trafficking of pathogenic tau to lysosomes. We also found that SORL1 promotes cytosolic tau seeding induced by pathogenic forms of tau. The AD‐associated N1358S mutation SORL1 gene demonstrated a significant increase in tau seeding when compared with WT SORL1.ConclusionThese studies identify SORL1 as a sorting receptor for tau that contributes to seeding of pathogenic tau. Furthermore, they indicated that SORL1 directs tau away from lysosomal degradation pathways, and identify promotion of tau seeding as a potential mechanism by which the SORL1 N1358S mutation contributes to AD.

Hsp40 molecular chaperones are potent regulators of tau seeding and accumulation

AbstractBackgroundThe misfolding of the microtubule associated protein tau is associated with Alzheimer’s disease and other tauopathies. In tauopathies, aberrant tau spreads through synaptically connected regions of the brain by templating the misfold of endogenous tau. Work from our group and others has identified molecular chaperones as regulators of tau accumulation and toxicity, but many tau regulators have yet to be identified.MethodWe performed a screen of members from seven major chaperone families using Tau RD P301S FRET Biosensor cells. Hits were assessed by secondary knockdown studies in cultured cells and validated in tau expressing primary neurons. Additional in vitro experiments to dissect the mechanism of activity are ongoing, which will be followed by in vivo studies.ResultOur data support the use of Tau RD P301S FRET Biosensor cells as a robust screening tool to identify known and novel tau regulators. The Hsp40 family is enriched with tau regulators, including some not previously known to affect tau. Studies are ongoing to determine how these proteins alter tau levels.ConclusionMultiple members of the Hsp40 family regulate tau accumulation.

DnaJs are enriched in tau regulators

The aberrant accumulation of tau protein is implicated as a pathogenic factor in many neurodegenerative diseases. Tau seeding may underlie its predictable spread in these diseases. Molecular chaperones can modulate tau pathology, but their effects have mainly been studied in isolation. This study employed a semi-high throughput assay to identify molecular chaperones influencing tau seeding using Tau RD P301S FRET Biosensor cells, which express a portion of tau containing the frontotemporal dementia-related P301S tau mutation fused to a FRET biosensor. Approximately fifty chaperones from five major families were screened using live cell imaging to monitor FRET-positive tau seeding. Among the tested chaperones, five exhibited significant effects on tau in the primary screen. Notably, three of these were from the DnaJ family. In subsequent studies, overexpression of DnaJA2, DnaJB1, and DnaJB6b resulted in significant reductions in tau levels. Knockdown experiments by shRNA revealed an inverse correlation between DnaJB1 and DnaJB6b with tau levels. DnaJB6b overexpression, specifically, reduced total tau levels in a cellular model with a pre-existing pool of tau, partially through enhanced proteasomal degradation. Further, DnaJB6b interacted with tau complexes. These findings highlight the potent chaperone activity within the DnaJ family, particularly DnaJB6b, towards tau.

Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State.

Ca2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide (DPc10) binding promotes leaky RyR2 in cardiomyocytes and reports on that endogenous state. Conversely, calmodulin (CaM) binding inhibits RyR2 leak and low CaM affinity is diagnostic of leaky RyR2. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. We used FRET to clarify the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca2+, DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca2+, DPc10 decreased FRETmax without affecting CaM/RyR2 binding affinity. This correlates with the analysis of fluorescence-lifetime-detected FRET, indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP/A-CaM distances independent of [Ca2+]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, with this effect being larger in micromolar versus nanomolar Ca2+. Moreover, A-DPc10/RyR2 binding is cooperative in a CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by the analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

Abstract P3128: Synapse-associated Protein 97 Is Necessary For Enhancing Cardiac Contractility Via Beta1 Adrenoceptor-neuronal Nitric Oxide Synthase Pathway And Guanosine3',5'-cyclic Monophosphate Signaling

Objective: Synapse-associated protein 97 (SAP97) is a scaffold protein widely expressed in the brain, heart, and kidney. SAP97 has been suggested function in the regulation of cardiac contractility. SAP97 directly binds to cardiac beta1 adrenceptor. Our recent studies have shown that increased cAMP and cGMP levels are involved in enhancing excitation-contraction (E-C) coupling after sympathetic nervous system activation. We aim to study the role of SAP97 in beta1 adrenoceptor-induced increases in cyclic nucleotides and cardiac E-C coupling. Results and Methods: Cardiac function in mice with cardiomyocyte-specific deletion of the SAP97 (SAP97-cKO) was comparable to that in flox/flox control littermates. Ligand-induced cGMP signal in freshly isolated adult ventricular myocytes (AVMs) using FRET biosensor Gi500. The cGMP responsiveness caused by β1AR agonist as dobutamine was reduced in SAP97-cKO versus f/f cells. No significant difference between the groups using nonselective beta-adrenergic stimulation isoproterenol (ISO). In comparison, the dobutamine-induced cAMP levels were not reduced in SAP97-cKO when compared to f/f controls. The dobutamine-induced ejection fraction in hearts and sarcomere shortening in AVMs were significantly attenuated in SAP97-cKO hearts and AVMs when compared to f/f controls, respectively. On the other hand, no difference was observed between f/f and cKO AVMs and hearts after stimulation by ISO. To further examine the LTCC activity, whole-cell currents (ICa) were recorded from freshly isolated AVMs. The results showed that the LTCC current was attenuated in SAP97-cKO AVMs than cKO controls when exposed to dobutamine. These results show SAP97 is necessary for increasing intracellular cGMP level, LTCC current, and cardiac E-C coupling and contractility after β1AR stimulation. Conclusion: SAP97 plays a crucial role in β 1 AR signaling, which is necessary for triggering E-C coupling through cAMP and cGMP pathways.

Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

Nuclear lamina strain states revealed by intermolecular force biosensor

Nuclear lamins have been considered an important structural element of the nucleus. The nuclear lamina is thought both to shield DNA from excessive mechanical forces and to transmit mechanical forces onto the DNA. However, to date there is not yet a technical approach to directly measure mechanical forces on nuclear lamins at the protein level. To overcome this limitation, we developed a nanobody-based intermolecular tension FRET biosensor capable of measuring the mechanical strain of lamin filaments. Using this sensor, we were able to show that the nuclear lamina is subjected to significant force. These forces are dependent on nuclear volume, actomyosin contractility, functional LINC complex, chromatin condensation state, cell cycle, and EMT. Interestingly, large forces were also present on nucleoplasmic lamins, indicating that these lamins may also have an important mechanical role in the nucleus. Overall, we demonstrate that the nanobody-based approach allows construction of biosensors for complex protein structures for mechanobiology studies.

Vangl-dependent Wnt/planar cell polarity signaling mediates collective breast carcinoma motility and distant metastasis

BackgroundIn light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis.MethodsVangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model.ResultsWe observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics.ConclusionsWe conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation.

Flagellar brake protein YcgR interacts with motor proteins MotA and FliG to regulate the flagellar rotation speed and direction.

In E. coli and related species, flagellar brake protein YcgR responds to the elevated intracellular c-di-GMP, decreases the flagellar rotation speed, causes a CCW rotation bias, and regulates bacterial swimming. Boehm et al. suggested that c-di-GMP-activated YcgR directly interacted with the motor protein MotA to curb flagellar motor output. Paul et al. proposed that YcgR disrupted the organization of the FliG C-terminal domain to bias the flagellar rotation. The target proteins are controversial, and the role of motor proteins remains unclear in flagellar rotation speed and direction regulation by YcgR. Here we assayed the motor proteins' affinity via a modified FRET biosensor and accessed the role of those key residue via bead assays. We found that YcgR could interact with both MotA and FliG, and the affinities could be enhanced upon c-di-GMP binding. Furthermore, residue D54 of YcgR-N was needed for FliG binding. The mutation of the FliG binding residue D54 or the MotA binding ones, F117 and E232, restored flagellar rotation speed in wild-type cells and cells lacking chemotaxis response regulator CheY that switched the flagellar rotation direction and decreased the CCW ratio in wild-type cells. We propose that c-di-GMP-activated YcgR regulated the flagellar rotation speed and direction via its interaction with motor proteins MotA and FliG. Our work suggest the role of YcgR-motor proteins interaction in bacterial swimming regulation.

Endosome and Lysosome Membrane Properties Functionally Link to γ-Secretase in Live/Intact Cells.

Our unique multiplexed imaging assays employing FRET biosensors have previously detected that γ-secretase processes APP C99 primarily in late endosomes and lysosomes in live/intact neurons. Moreover we have shown that Aβ peptides are enriched in the same subcellular loci. Given that γ-secretase is integrated into the membrane bilayer and functionally links to lipid membrane properties in vitro, it is presumable that γ-secretase function correlates with endosome and lysosome membrane properties in live/intact cells. In the present study, we show using unique live-cell imaging and biochemical assays that the endo-lysosomal membrane in primary neurons is more disordered and, as a result, more permeable than in CHO cells. Interestingly, γ-secretase processivity is decreased in primary neurons, resulting in the predominant production of long Aβ42 instead of short Aβ38. In contrast, CHO cells favor Aβ38 over the Aβ42 generation. Our findings are consistent with the previous in vitro studies, demonstrating the functional interaction between lipid membrane properties and γ-secretase and provide further evidence that γ-secretase acts in late endosomes and lysosomes in live/intact cells.

Fluorescent Biosensor Imaging of Nitrate in Arabidopsis thaliana.

Nitrate (NO3-) is an essential element and nutrient for plants and animals. Despite extensive studies on the regulation of nitrate uptake and downstream responses in various cells, our knowledge of the distribution of nitrogen forms in different root cell types and their cellular compartments is still limited. Previous physiological models have relied on in vitro biochemistry and metabolite level analysis, which limits the ability to differentiate between cell types and compartments. Here, to address this, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named nlsNitraMeter3.0, for the quantitative visualization of nitrate concentration and distribution at the cellular level in Arabidopsis thaliana. This biosensor was specifically designed for nitrate measurements, not nitrite. Through genetic engineering to create and select sensors using yeast, Xenopus oocyte, and Arabidopsis expression systems, we developed a reversible and highly specific nitrate sensor. This method, combined with fluorescence imaging systems such as confocal microscopy, allows for the understanding and monitoring of nitrate transporter activity in plant root cells in a minimally invasive manner. Furthermore, this approach enables the functional analysis of nitrate transporters and the measurement of nitrate distribution in plants, providing a valuable tool for plant biology research. In summary, we provide a protocol for sensor development and a biosensor that can be used to monitor nitrate levels in plants. Key features This protocol builds upon the concept of FRET biosensors for in vivo visualization of spatiotemporal nitrate levels at a cellular resolution. Nitrate levels can be quantified utilizing the biosensor in conjunction with either a plate reader or a fluorescence microscope.