FRET-based Sensor Research Articles

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami–based cargo scaffolds to probe the individual and ensemble properties of GIPC–myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor–motor linkage, and that increased flexibility correlates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR–myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR–myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI dimers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.

Turn –on FRET-based cysteine sensor by sulfur-doped carbon dots and Au nanoparticles decorated WS2 nanosheet

Cysteine is an essential biothiol that plays various functions in the human body. Decrease or exceeding of this excellent antioxidant from the expected range will lead to so many problems. Thus, appropriate sensing of it would be of great importance. Sulfur-doped carbon Dots(S-CDs) owe excellent fluorescence emission. Therefore, designing a Fluorescence resonance energy transfer (FRET) system between S-CDs as donor and Au nanoparticles (AuNPs) decorated tungsten disulfide nanosheet (WS2 NSs) would be a perfect strategy for cysteine detection. Excitation at 340 nm will give the maximum quantum yield of S-CDs (21%) and fluorescence emission peak at 460 nm. In the presence of cysteine, the FRET mechanism inhibited through the affinity of cysteine's functional groups (-SH and -NH2) toward AuNPs and S-CDs fluorescence emission was recovered. To find the best efficiency of the system, optimization of pH, temperature, and time was investigated. Here the linear range of 3–275 µM and limit of detection of 0.01 µM was obtained. Finally, the fluorescence method was applied to the analysis of cysteine in human blood serum, which poses the potential of rapid and sensitive sensing. It can detect both lower and higher amounts of serum cysteine.

Time-Domain Fluorescence Lifetime Imaging of cAMP Levels with EPAC-Based FRET Sensors.

Second messenger molecules in eukaryotic cells relay the signals from activated cell surface receptors to intracellular effector proteins. FRET-based sensors are ideal to visualize and measure the often rapid changes of second messenger concentrations in time and place. Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative technique for measuring FRET. Given the recent development of commercially available, sensitive and photon-efficient FLIM instrumentation, it is becoming the method of choice for FRET detection in signaling studies. Here, we describe a detailed protocol for time domain FLIM, using the EPAC-based FRET sensor to measure changes in cellular cAMP levels with high spatiotemporal resolution as an example.

Assaying Protein Kinase A Activity Using a FRET-Based Sensor Purified from Mammalian Cells.

Protein Kinase A (PKA) is the major intracellular receptor for cAMP. Research into this prototype kinase is supported by kinase assays that are typically performed in vitro using radio-labeled ATP. For in vivo studies, genetically encoded FRET-based sensors have become popular for monitoring PKA activity. Here, we show that it is also possible to apply such reporters in vitro. We describe how to express and purify milligram quantities of a FRET-based PKA activity reporter using cultured human embryonic kidney cells. We demonstrate how to utilize the purified reporter in a plate reader to determine the IC50 for the widely utilized PKA inhibitor H89 in the presence of a physiologically relevant concentration of ATP. The protocol takes advantage of the economical transfection reagent polyethylenimine and can be performed in a standard cell culture facility. Whereas assays based on radiolabelling are more sensitive, the approach presented here has several advantages: It enables continuous measurement of changes in substrate phosphorylation; a single preparation produces enough reporter for thousands of recordings; the reporter has a long shelf life; and it avoids the safety considerations that arise when working with radioactive material.

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus.

GLT-1, the major glutamate transporter in the mammalian central nervous system, is expressed in presynaptic terminals that use glutamate as a neurotransmitter, in addition to astrocytes. It is widely assumed that glutamate homeostasis is regulated primarily by glutamate transporters expressed in astrocytes, leaving the function of GLT-1 in neurons relatively unexplored. We generated conditional GLT-1 knockout (KO) mouse lines to understand the cell-specific functions of GLT-1. We found that stimulus-evoked field extracellular postsynaptic potentials (fEPSPs) recorded in the CA1 region of the hippocampus were normal in the astrocytic GLT-1 KO but were reduced and often absent in the neuronal GLT-1 KO at 40 weeks. The failure of fEPSP generation in the neuronal GLT-1 KO was also observed in slices from 20 weeks old mice but not consistently from 10 weeks old mice. Using an extracellular FRET-based glutamate sensor, we found no difference in stimulus-evoked glutamate accumulation in the neuronal GLT-1 KO, suggesting a postsynaptic cause of the transmission failure. We hypothesized that excitotoxicity underlies the failure of functional recovery of slices from the neuronal GLT-1 KO. Consistent with this hypothesis, the non-competitive NMDA receptor antagonist MK801, when present in the ACSF during the recovery period following cutting of slices, promoted full restoration of fEPSP generation. The inclusion of an enzymatic glutamate scavenging system in the ACSF conferred partial protection. Excitotoxicity might be due to excess release or accumulation of excitatory amino acids, or to metabolic perturbation resulting in increased vulnerability to NMDA receptor activation. Previous studies have demonstrated a defect in the utilization of glutamate by synaptic mitochondria and aspartate production in the synGLT-1 KO in vivo, and we found evidence for similar metabolic perturbations in the slice preparation. In addition, mitochondrial cristae density was higher in synaptic mitochondria in the CA1 region in 20–25 weeks old synGLT-1 KO mice in the CA1 region, suggesting compensation for loss of axon terminal GLT-1 by increased mitochondrial efficiency. These data suggest that GLT-1 expressed in presynaptic terminals serves an important role in the regulation of vulnerability to excitotoxicity, and this regulation may be related to the metabolic role of GLT-1 expressed in glutamatergic axon terminals.

GPCR signaling is highly compartmentalized in human cardiomyocytes and severely remodeled in atrial fibrillation

Atrial fibrillation (AF) has been linked to the remodeling of membrane receptors and alterations in downstream cAMP-dependent regulation. However, to date, no study has elucidated how the increase on cAMP upon different G-protein-coupled receptors (GPCRs) can lead to different physiological compartmentalized responses. The aim of this study was to investigate the compartmentally specific effects of GPCRs on cAMP levels in human atrial myocytes (HAMs) from patients with AF and control patients without AF (Ctl), and how these compartmentalized effects are altered in AF. HAMs were isolated from 60 AF and 76 Ctl patient tissues. Cells were transduced with adenoviruses (Epac1-camps, pm-Epac1-camps and Epac1-JNC) and cultured for 48 hours to express the FRET-based cAMP sensor inthe cytosolic, membrane, and RYR2 nanodomains. Förster-resonance energy transfer (FRET) was used to measure cAMP levels in 525 HAMs stimulated with isoprenaline (100 µM), serotonin (100 µM), or the A2AR agonist CGS (200 nM). A desensitization to β-adrenergic receptor stimulation was exclusively found in the cytosol of AF myocytes, while no difference was seen in the RYR2 or LTCC compartment. Similar effects were observed upon serotonin stimulation with a significant desensitization in the cytosol, and no difference in the RYR2 compartment. In response to A2ARs stimulation AF myocytes displayed a significantly higher cytosolic increase in cAMP levels. However, no response was seen in the LTCC compartment in response to serotonin or A2AR stimulation. Collectively, our data show that cAMP levels are highly compartmentalized and differentially regulated by GPCRs. Furthermore, these results provide a mechanistic insight for the previously reported functional effects seen upon stimulation of these three receptors.

Fluorescent Sensor Array for Quantitative Determination of Saccharides.

Accurate monitoring of sugar levels is essential for many fields from food industry to human health. Here, we developed FRET-based dual chromophore sensors for saccharides that form oxazolidine boronate and may be employed as a noninvasive method for monitoring of sugar levels in biological fluids, namely, urine. The saccharide-binding properties of the sensors were studied using fluorescence spectroscopy and utilized in the determination of saccharides in a high-throughput manner. Here, two fluorescent sensors were successful in the classification of nine different monosaccharides and disaccharides with 100% correct classification. Furthermore, the dual chromophore self-assembled sensors were successfully utilized for the quantitative determination of important carbohydrates such as glucose in the presence of competitive saccharides (fructose) and in complex media (urine) without sample pretreatment. The present fluorescent sensors allow for quantification of glucose in a concentration range of 0-60 mM, which matches the concentration range of frequently used urinalysis test strips.

Actin-generated force applied during endocytosis measured by Sla2-based FRET tension sensors

Mechanical forces are integral to many cellular processes, including clathrin-mediated endocytosis, a principal membrane trafficking route into the cell. During endocytosis, forces provided by endocytic proteins and the polymerizing actin cytoskeleton reshape the plasma membrane into a vesicle. Assessing force requirements of endocytic membrane remodeling is essential for understanding endocytosis. Here, we determined actin-generated force applied during endocytosis using FRET-based tension sensors inserted into the major force-transmitting protein Sla2 in yeast. We measured at least 8 pN force transmitted over Sla2 molecule, hence possibly more than 300-880 pN applied during endocytic vesicle formation. Importantly, decreasing cell turgor pressure and plasma membrane tension reduced force transmitted over the Sla2. The measurements in hypotonic conditions and mutants lacking BAR-domain membrane scaffolds then showed the limits of the endocytic force-transmitting machinery. Our study provides force values and force profiles critical for understanding the mechanics of endocytosis and potentially other key cellular membrane-remodeling processes.

Modular and Single-Cell Sensors of Bacterial Ser/Thr Kinase Activity.

At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation. We demonstrate the utility of these sensors in measuring the activity of PrkC, a conserved bacterial Ser/Thr kinase, in different growth conditions from single cells to colonies. We also show that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). These sensors have a modular design that should generalize to other bacterial signaling systems in the future.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy.

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700µδ6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700µδ6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700µδ6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Abstract 91: FLT3 is a major driver of glucose consumption in squamous cell lung cancer

Abstract Squamous cell lung cancer (SqCLC) represents 30% of all cases of non-small cell lung cancer. Despite the prevalence of SqCLC, there are few molecularly targeted therapies for SqCLC patients. There is an urgent need for new approaches in this area. SqCLC cells consume elevated levels of glucose and are highly dependent on glucose consumption for their survival. Targeting glucose consumption may be a valuable therapeutic strategy in SqCLC but is challenged by the fact that glucose consumption is critical for vital organs. Our ability to selectively target glucose consumption in SqCLC is limited by an incomplete understanding of the shared and distinct mechanisms through which SqCLC and healthy cells drive glucose consumption. We recently developed and validated a high-throughput glucose consumption assay that serves as a platform technology for identifying new proteins and pathways that drive glucose consumption in cancerous and healthy cells. In this project, we screened H520, SK-MES-1, and H596 SqCLC cell lines against 3555 bioactive small molecules including a library of kinase inhibitors and FDA-approved drugs, and measured glucose consumption 24 hours after drug treatment. We discovered and validated 62 compounds that decreased glucose consumption per cell by >50% in at least one cell line. From this list, we searched for compounds that decreased glucose consumption in all three cell lines, by >70% in at least one cell line, and with an EC50 value <~1 µM; that have been used in mice and people; that have not been previously linked to glucose consumption; and that do not significantly decrease cell growth at the time point measured (24 hours post-treatment). This led us to further investigate Pacritinib, a small molecule kinase inhibitor in clinical trials for the treatment of myelofibrosis. Pacritinib reduces glucose consumption in all three SqCLC cell lines with an EC50 value of 1.2 µM in cell culture and blocks glucose consumption in cell line xenografts in vivo without affecting glucose consumption in healthy tissues. In the SqCLC cell lines, Pacritinib decreases protein levels of Hexokinase 1 and Hexokinase 2 as well as hexokinase activity as measured by a FRET-based glucose sensor. Hexokinase 1 overexpression in the SqCLC cell lines blocks Pacritinib from inhibiting glucose consumption. Pacritinib targets JAK2, TYK2, FLT3, and additional kinases at low nanomolar concentrations in vitro. Additional small molecule inhibitors of JAK2 and TYK2 had no effect on SqCLC glucose consumption. However additional inhibitors of FLT3 blocked glucose consumption in all three SqCLC cell lines. FLT3 overexpression increased SqCLC glucose consumption and blocked Pacritinib from inhibiting glucose consumption. In conclusion, using our high-throughput technology, we discovered that FLT3 is a selective and targetable driver of glucose consumption in SqCLC. Our data suggest that FLT3 activates glucose consumption by increasing Hexokinase 1 levels. Citation Format: Chiara Ghezzi, Robert Damoiseaux, Peter M. Clark. FLT3 is a major driver of glucose consumption in squamous cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 91.

289-OR: Cleavage of Protein Kinase C d by Caspase-3 Mediates Cytokine-Induced ß-Cell Death

In type 1 diabetes (T1D) autoreactive T-cells secrete cytokines such as tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and interferon-γ (IFNγ) that can initiate β-cell apoptosis. Protein kinase C delta (PKCδ) plays a role in mediating cytokine-induced β-cell apoptosis. The exact mechanisms of PKCδ regulation in β-cell death and role in T1D are unknown. In cancer cells PKCδ mediates apoptosis by translocating to the nucleus and being cleaved by caspase 3. We hypothesize that PKCδ mediates cytokine-induced β-cell apoptosis by translocating to the nucleus and cleavage by caspase 3. Islets from WT or mice with a β-cell specific PKCδ knockout were cultured for 24hr with cytokines (10ng/ml TNFα, 5ng/ml IL-1β, 100ng/ml IFNγ), caspase 3 inhibitor (Z-DEVD-FMK, 33µg/ml), or adenovirus to produce GFP-PKCδ, GFP with a constitutively active nuclear localization sequence, and a PKCδ mutant that cannot be cleaved by caspase 3 (CM-GFP-PKCδ). Human islets were treated with 10μM PKCδ inhibitor (δV1-1) with cytokines. Apoptosis was determined by fluorescent live/dead staining. PKCδ translocation was determined by GFP colocalization with a nuclear stain and activity was determined with a FRET-based sensor. Decreasing PKCδ activity protected mouse (p<0.01) and human (p=0.02) islets against cytokine-induced apoptosis. Cytokines induced PKCδ translocation to the nucleus at 3 (p=0.05) and 24hr (p<0.01) and increased nuclear PKCδ activity at 3hr (p=0.04). WT islets with Z-DEVD-FMK and CM-GFP-PKCδ islets were protected from cytokine-induced death. Production of PKCδ with an active nuclear localization sequence did not impact apoptosis. Inhibiting PKCδ protected islets from cytokine-induced β-cell death. Pro-inflammatory cytokines induce nuclear translocation and activity of PKCδ. Nuclear localization of PKCδ alone is insufficient for apoptosis but caspase-3 cleavage is required. Our data suggests that targeted inhibition of PKCδ has the potential to prevent or delay β-cell death in T1D onset. Disclosure J. Collins: None. R. K. Benninger: None. N. L. Farnsworth: None. Funding JDRF (D1671-FAC-2020-891-A-N)

Temporal analysis of T-cell receptor-imposed forces via quantitative single molecule FRET measurements

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.

A modification of cAMP compartmentation is involved in the doxorubicin-induced cardiotoxicity

Doxorubicin (Dox) is one of the most commonly used chemotherapy but also one of the most cardiotoxic. As of today, only the Iron chelator Dexrazoxane has been FDA and EMA approved as a cardioprotective agent for Dox but its usage is still controversial. Thus, there is an urgent need to identify new targets to develop an effective protection against Dox-induced cardiotoxicity (DIC) without compromising its anti-tumor action. Well-known and studied DIC mechanisms include Topoisomerase II inhibition, DNA damages, induction of ROS and bioenergetic distress that lead to the death of the cardiomyocyte. Alternative mechanisms are emerging such as the alteration of the ß adrenergic system. cAMP is one of the most important second messengers in the heart and is found compartmentalized in specific subcellular domain modulating downstream effectors and thus many physiological processes (contraction/relaxation). Our objective is to study DIC mechanisms with a specific focus on Dox effect on the ß adrenergic system and especially on the cAMP compartmentation. We used FRET-based sensors for cAMP targeted to different compartment of the cell (cytosol and nucleus) in order to study cAMP production in adult rat ventricular myocyte under Isoprenalin stimulation after Dox treatment. Involvement of the main molecular actors of the cAMP compartmentation system has been studied. Under β adrenergic stimulation, cAMP production decreases in the Dox treated cells compare to the control, in the cytosol as well as in the nucleus. Furthermore, the maximal level of cAMP production reached by the Dox treated myocytes is also lower than the control. Based on this data we looked at the expression of AC5 and 6, the two most expressed in myocytes. AC5 but not AC6 mRNA expression is decreased by Dox treatment indicating a potential implication of AC5 in the DIC. Moreover, PDEs involvement cannot be exclude yet. Here, we show for the first time the involvement of cAMP compartmentation in the DIC.

Probing mechanotransduction in living cells by optical tweezers and FRET-based molecular force microscopy

Cells sense mechanical signals and forces to probe the external environment and adapt to tissue morphogenesis, external mechanical stresses and a wide range of diverse mechanical cues. Here, we propose a combination of optical tools to manipulate single cells and measure the propagation of mechanical and biochemical signals inside them. Optical tweezers are used to trap microbeads that are used as handles to manipulate the cell plasma membrane; genetically encoded FRET-based force sensors inserted in F-actin and alpha-actinin are used to measure the propagation of mechanical signals to the cell cytoskeleton, while fluorescence microscopy with single-molecule sensitivity can be used with a huge array of biochemical and genetic sensors. We describe the details of the setup implementation, the calibration of the basic components and preliminary characterization of actin and alpha-actinin FRET-based force sensors.

Temporal Analysis of T-Cell Receptor-Imposed Forces Via Quantitative Single Molecule Fret Measurements

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and dynamical behavior are still poorly defined. We here report a FRET-based sensor equipped with a TCR-reactive single chain antibody fragment, which was tethered to planar supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5pN and moderate force loading rates on the TCR of 1.5pN per second. When facing fluid SLBs instead, T-cells still exerted tensile forces yet with threefold reduced magnitude and only prior to but not upon activation. Our findings not only provide first truly molecular information on TCR-imposed forces within the immunological synapse, they also recalibrate their significance in antigen recognition.

Lab-on-Microsphere-FRET-Based Multiplex Sensor Platform.

Here we report on the development and investigation of a novel multiplex assay model based on polymer microspheres (PMS) encoded with ternary AIS/ZnS quantum dots (QDs). The system was prepared via layer-by-layer deposition technique. Our studies of Förster resonance energy transfer (FRET) between the QD-encoded microspheres and two different cyanine dyes have demonstrated that the QD photoluminescence (PL) quenching steadily increases with a decrease in the QD-dye distance. We have found that the sensitized dye PL intensity demonstrates a clear maximum at two double layers of polyelectrolytes between QDs and Dye molecules on the polymer microspheres. Time resolved PL measurements have shown that the PL lifetime decreases for the QDs and increases for the dyes due to FRET. The designed system makes it possible to record spectrally different bands of FRET-induced dye luminescence with different decay times and thereby allows for the multiplexing by wavelength and photoluminescence lifetimes of the dyes. We believe that PMS encoded with AIS/ZnS QDs have great potential for the development of new highly selective and sensitive sensor systems for multiplex analysis to detect cell lysates and body fluids’ representative biomarkers.

Pathological activation of CaMKII induces arrhythmogenicity through TRPM4 overactivation.

TRPM4 is a Ca2+-activated nonselective cation channel involved in cardiovascular physiology and pathophysiology. Based on cellular experiments and numerical simulations, the present study aimed to explore the potential arrhythmogenicity of CaMKII-mediated TRPM4 channel overactivation linked to Ca2+ dysregulation in the heart. The confocal immunofluorescence microscopy, western blot, and proximity ligation assay (PLA) in HL-1 atrial cardiomyocytes and/or TRPM4-expressing TSA201 cells suggested that TRPM4 and CaMKII proteins are closely localized. Co-expression of TRPM4 and CaMKIIδ or a FRET-based sensor Camui in HEK293 cells showed that the extent of TRPM4 channel activation was correlated with that of CaMKII activity, suggesting their functional interaction. Both expressions and interaction of the two proteins were greatly enhanced by angiotensin II treatment, which induced early afterdepolarizations (EADs) at the repolarization phase of action potentials (APs) recorded from HL-1 cells by the current clamp mode of patch clamp technique. This arrhythmic change disappeared after treatment with the TRPM4 channel blocker 9-phenanthrol or CaMKII inhibitor KN-62. In order to quantitatively assess how CaMKII modulates the gating behavior of TRPM4 channel, the ionomycin-permeabilized cell-attached recording was employed to obtain the voltage-dependent parameters such as steady-state open probability and time constants for activation/deactivation at different [Ca2+]i. Numerical simulations incorporating these kinetic data into a modified HL-1 model indicated that > 3-fold increase in TRPM4 current density induces EADs at the late repolarization phase and CaMKII inhibition (by KN-62) completely eliminates them. These results collectively suggest a novel arrhythmogenic mechanism involving excessive CaMKII activity that causes TRPM4 overactivation in the stressed heart.

Generation and Evaluation of Transgenic Mice Expressing FRET-based Tension Sensor

Generation and Evaluation of Transgenic Mice Expressing FRET-based Tension Sensor

Abstract 14925: Mechanical Tension in Syndecan-1 is Regulated by Extracellular Mechanical Cues and Fluidic Shear Stress

Syndecan-1 (SDC1) is a transmembrane proteoglycan that mediates the shear stress-induced signaling and inflammatory phenotypes of endothelial cells. While SDC1 is known to be involved mechanically in regulating the behavior of cells, it remains unknown how SDC1 responds to extracellular mechanical cues on the molecular level. We designed a set of FRET-based SDC1 tension sensors including full-length SDC1 (SDC1TS), SDC1 with deleted ectodomain (ΔEcto), SDC1 with deleted glycosylation sites (ΔGAG) and SDC1 with a deleted cytoplasmic tail (ΔCyto). We transduced these constructs into ECs and validated the constructs by WB and confocal imaging. When cultured on glass-bottom coverslips, we found that the baseline tension in SDC1TS was significantly higher than that in Ecto and GAG mutants. When cultured on engineered substrates, we demonstrated that tension in SDC1 is modulated by micropatterned surfaces and nanotopographical cues. To study if SDC1 directly responds to substrate stiffness, transduced ECs were cultured on 0.2 kPa and 25 kPa substrates. We found that there was significantly decreased tension in the Ecto construct compared to the full-length SDC1TS construct on 0.2 kPa substrates but no significant changes were observed on the 25 kPa substrates. To examine the effect of substrate stiffness on the association of SDC1 with adhesion-related proteins, we performed IP and WB on ECs expressing HA-tagged SDC1 cultured on 0.2 kPa or 25 kPa substrates. We found there were increases in SDC1 binding to action and myosin IIb, while there were decreased in SDC1 binding to Src, PKA, and FAK in cells grown on stiff substrates versus on soft substrate. To study the effect of shear stress on the tension of SDC1, ECs expressing the constructs were cultured on static or under 12dyn/cm 2 fluidic shear stress. We found that a gradient developed in the tension of SDC1 in all four constructs. There was higher tension of SDC1 in the upstream region in comparison to the downstream region. IP and WB results showed that shear stress-induced SDC1 association with actin, Src, myosin IIb, cortactin, calmodulin, and integrin beta3. In summary, our results demonstrate that SDC1 is mechanically responsive to substrate mediated biophysical cues and shear stress.