Internal Fluorescence Research Articles

Differential Expression of Cannabinoid Receptors, CB1R and CB2R, in Prostate Tumor Samples with Perineural Invasion

Perineural invasion (PNI) affects over 20% of patients with prostate cancer who often recur with metastasis primarily to the bones. Despite its association with the patient's poor prognosis and dissemination of various cancers, knowledge of the regulatory mechanisms that drive invasion of prostate cancer cells through and around nerves is limited. Cannabinoid receptors (CBRs) are highly enriched in prostate cancer cells and exogenous application of CBR ligands attenuates the cell's survival, migration and invasion ability. Our objective is to define whether activation of the endocannabinoid system (ECS) associates with PNI and to identify the endogenous regulatory mechanisms through which the ECS promotes prostate cancer cell invasion through nerves. We used immunohistochemistry technique to determine the expression levels of the CB1R and the CB2R in formalin‐fixed paraffin embedded (FFPE) prostate tumor samples with PNI. Preliminary data from our laboratory show that CB1R is highly enriched in FFPE prostate tumor samples with PNI when compared to CB2R. Given the increase of the CB1R in PNI FFPE samples, we used total internal fluorescence reflection microscopy (TIRFm) to elucidate in vivo the trafficking kinetics of CB1Rs in highly invasive versus less invasive prostate cancer cells, PC3 and 22RV1, respectively. We found that PC3 cells display constitutive CB1R trafficking. However, this was not the case for the less invasive prostate cancer cells, 22RV1. Our results suggest that the elevated CB1R levels detected in the FFPE PNI positive samples is not coincidental, but rather an indication of an association of the ECS and PNI. Ongoing studies aim to determine how nerve‐PC tumor interactions encourage PNI by using an in vitro model in which we can modulate the ECS.Support or Funding InformationGrants: RCMI‐NIMHD 8G12MD007600 to JFO.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Coupling electrochemistry and TIRF-microscopy with the fluorescent false neurotransmitter FFN102 supports the fluorescence signals during single vesicle exocytosis detection

Applications of the Fluorescent False Neurotransmitter FFN102, an analog of biogenic neurotransmitters and a suitable probe for coupled amperometry and TIRFM (total internal reflexion fluorescence microscopy) investigations of exocytotic secretion, were considered here. The electroactivity of FFN102 was shown to very likely arise from the oxidation of its phenolic group through a CE (Chemical-Electrochemical) mechanism. Evidences that the aminoethyl group of FFN102 is the key recognition element by BON N13 cells were also provided. Amperometric measurements were then performed at the single cell level with carbon fiber electrode (CFE) or Indium Tin Oxide (ITO) surfaces. It proved the disparity of kinetic and quantitative parameters of FFN102-stained cells acquired either at cell top and bottom. Moreover, coupled analyses of FFN102 loaded vesicles allowed us to classify three types of optical signals that probably arise from secretion releases thanks to their concomitant detection with an electrochemical spike. Finally, preliminary benefits from the coupling involving FFN102 were reported in terms of origins of overlapped amperometric spikes or assignment of fluorescence extinctions to real exocytotic events.

CellSpecks: A Software for Automated Detection and Analysis for Calcium Channels in Live Cells

With the intent to create a new approach to single channel recording that could couple the fidelity of patch-clamp recording with a more high-throughput screening capability, we have pioneered the development of a novel approach for single channel recording, we named Optical patch clamp recording. By combining the use of highly-sensitive fluorescent Ca2+ indicator dyes in conjunction with total internal fluorescence microscopy (TIRFM) techniques, we monitor the flux of Ca2+ ions passing through the pores of a single open ion channels. This new approach provide channel gating information with time resolution of < 2 ms , with the great advantage of being massively parallel providing simultaneous and independent recording from thousands of ion channels. However, manual analyses of these data by visual inspection, presents severe challenges as each video recording can include 15,000 or more images. To overcome this major drawback, we develop a computational image processing and analyses framework, we named CellSpecks, capable of detecting and fully analyzing kinetics of ion channel contained in a video sequence. By using a randomly generated synthetic data, we tested the ability of CellSpecks to rapidly and efficiently detect and analyze the activity of thousands of ion channels. Here, we report the use of CellSpecks for the analyses of experimental data acquired by imaging muscle nicotinic acetylcholine receptors (nAChRs), and Alzheimer¯s disease associated/multi-conductance levels Amyloid beta pore (Aβ42) inserted in the plasma membrane of Xenopus laevis oocytes. CellSpecks rapidly and efficiently, was able to generated maps of their locations, create raw and processed time-dependent fluorescence traces, histograms distributions of open and close dwell-times, and open probability. While, we process and analyze fluorescence data from Ca2+ channels, CellSpecks is equally applicable to fluorescence imaging of other channels and molecules.

Quantification of Single-Molecule FRET between Quantum Dots and Organic Dyes

Single-molecule Förster resonance energy transfer (FRET) is a useful technique for studying inter- and intra-molecular dynamics in biophysics. FRET efficiency is highly sensitive to distance, with half-maximal energy transfer occurring at an inter-dye distance on the order of 5 nm, which makes it possible to quantify distances changes on the molecular scale. FRET efficiency also depends on the spectral overlap and quantum yield of the donor and acceptor molecules, and therefore the choice of dye pair is critical to the success of any experiment. Most organic dyes that are used for single-molecule imaging have bleaching lifetimes in the tens of seconds at most, which limits their use to relatively fast processes. Quantum dot nanocrystals can emit for thousands of seconds without entering a long-lived dark state, which should make them good candidates for use as a FRET donor for longer single-molecule experiments, but there has been little quantitative study of the energy transfer efficiency at the single-molecule level. In this work, we use dsDNA to couple individual quantum dot donors to fluorescent dye acceptors. We then immobilize these FRET pairs on a functionalized glass coverslip and image them using total internal fluorescence (TIRF) microscopy with additional optics that allow us to image the donor and acceptor simultaneously. The length of the DNA between the pair can be varied from 11 to 32 base pairs, allowing us to observe and quantify the energy transfer efficiency over a range of distances on the nanometer scale. Our goal is to develop the use of quantum dots as donors for quantitative single-molecule FRET experiments.

Lentinan Exerts its Anti-Inflammatory Activity by Suppressing TNFR1 Transfer to the Surface of Intestinal Epithelial Cells through Dectin-1 in an in vitro and mice model

It has been reported that lentinan, β-1,3; 1,6-glucan derived from Lentinula edodes, suppresses intestinal inflammation and ameliorates symptoms of colitis. However, the mechanism of intestinal anti-inflammatory activity of lentinan and how it is recognized by intestinal epithelial cells remains largely unclear. The receptor for lentinan on intestinal epithelial cells was identified using an in vitro gut inflammation model consisting of Caco-2 and RAW264.7 cells. Colitis was induced in 7 to 8 week-old wild-type (WT) or knockout (KO) mice by the free intake of water containing 2% dextran sulfate sodium (DSS) for 7 days. Lentinan was administered daily via intragastric administration. Tumor necrosis factor receptor 1 (TNFR1)-GFP complex was constructed to monitor its movement in Caco-2 cells using confocal and total internal fluorescence microscopy. The results indicated that lentinan suppressed DSS-induced body weight loss, shortening of colon length, histological score, and inflammatory cytokine mRNA expression in the inflamed tissues of WT mice, whereas these suppressive effects of lentinan were not observed in Dectin-1 KO mice. Furthermore, lentinan reduced TNFR1 expression in intestinal epithelial cells of WT mice but not those from Dectin-1 KO mice. Using TNFR1-GFP constructs, it was confirmed that lentinan reduced TNFR1 expression on Caco-2 cell membranes through Dectin-1 ligation. Our study revealed that lentinan suppressed intestinal inflammation by Dectin-1-mediated regulation of TNFR1 transfer to the surface of intestinal epithelial cells.

Sensors and Biosensors for C-Reactive Protein, Temperature and pH, and Their Applications for Monitoring Wound Healing: A Review.

Wound assessment is usually performed in hospitals or specialized labs. However, since patients spend most of their time at home, a remote real time wound monitoring would help providing a better care and improving the healing rate. This review describes the advances in sensors and biosensors for monitoring the concentration of C-reactive protein (CRP), temperature and pH in wounds. These three parameters can be used as qualitative biomarkers to assess the wound status and the effectiveness of therapy. CRP biosensors can be classified in: (a) field effect transistors, (b) optical immunosensors based on surface plasmon resonance, total internal reflection, fluorescence and chemiluminescence, (c) electrochemical sensors based on potentiometry, amperometry, and electrochemical impedance, and (d) piezoresistive sensors, such as quartz crystal microbalances and microcantilevers. The last section reports the most recent developments for wearable non-invasive temperature and pH sensors suitable for wound monitoring.

Temporal Effects on Internal Fluorescence Emissions Associated with Aflatoxin Contamination from Corn Kernel Cross-Sections Inoculated with Toxigenic and Atoxigenic Aspergillus flavus.

Non-invasive, easy to use and cost-effective technology offers a valuable alternative for rapid detection of carcinogenic fungal metabolites, namely aflatoxins, in commodities. One relatively recent development in this area is the use of spectral technology. Fluorescence hyperspectral imaging, in particular, offers a potential rapid and non-invasive method for detecting the presence of aflatoxins in maize infected with the toxigenic fungus Aspergillus flavus. Earlier studies have shown that whole maize kernels contaminated with aflatoxins exhibit different spectral signatures from uncontaminated kernels based on the external fluorescence emission of the whole kernels. Here, the effect of time on the internal fluorescence spectral emissions from cross-sections of kernels infected with toxigenic and atoxigenic A. flavus, were examined in order to elucidate the interaction between the fluorescence signals emitted by some aflatoxin contaminated maize kernels and the fungal invasion resulting in the production of aflatoxins. First, the difference in internal fluorescence emissions between cross-sections of kernels incubated in toxigenic and atoxigenic inoculum was assessed. Kernels were inoculated with each strain for 5, 7, and 9 days before cross-sectioning and imaging. There were 270 kernels (540 halves) imaged, including controls. Second, in a different set of kernels (15 kernels/group; 135 total), the germ of each kernel was separated from the endosperm to determine the major areas of aflatoxin accumulation and progression over nine growth days. Kernels were inoculated with toxigenic and atoxigenic fungal strains for 5, 7, and 9 days before the endosperm and germ were separated, followed by fluorescence hyperspectral imaging and chemical aflatoxin determination. A marked difference in fluorescence intensity was shown between the toxigenic and atoxigenic strains on day nine post-inoculation, which may be a useful indicator of the location of aflatoxin contamination. This finding suggests that both, the fluorescence peak shift and intensity as well as timing, may be essential in distinguishing toxigenic and atoxigenic fungi based on spectral features. Results also reveal a possible preferential difference in the internal colonization of maize kernels between the toxigenic and atoxigenic strains of A. flavus suggesting a potential window for differentiating the strains based on fluorescence spectra at specific time points.

Visualization of ligand-induced dopamine D2S and D2L receptor internalization by TIRF microscopy

G protein-coupled receptors (GPCRs), including the dopamine receptors, represent a group of important pharmacological targets. Upon agonist binding, GPCRs frequently undergo internalization, a process that is known to attenuate functional responses upon prolonged exposure to agonists. In this study, internalization was visualized by means of total internal reflection fluorescence (TIRF) microscopy at a level of discrete single events near the plasma membrane with high spatial resolution. A novel method has been developed to determine the relative extent of internalized fluorescent receptor-ligand complexes by comparative fluorescence quantification in living CHO cells. The procedure entails treatment with the reducing agent sodium borohydride, which converts cyanine-based fluorescent ligands on the membrane surface to a long-lived reduced form. Because the highly polar reducing agent is not able to pass the cell membrane, the fluorescent receptor-ligand complexes located in internalized compartments remain fluorescent under TIRF illumination. We applied the method to investigate differences of the short (D2S) and the long (D2L) isoforms of dopamine D2 receptors in their ability to undergo agonist-induced internalization.

Phyllanthus taxodiifolius Beille Extract Disrupted Microtubule Dynamics in Glioma Cells

Glioma is the most common, life threatening, malignant primary brain tumor. This tumor has the ability to spread and invade through surrounding areas in the brain. Cell migration and adhesion are the most important processes involved in cancer cell spreading and invasion. Phyllanthus taxodiifolius Beille extract has been studied for its anti‐cancer action in breast and lung cancer cells. However, its mechanisms of action are still unclear. In addition, the effect of Phyllanthus taxodiifolius Beille extract on brain cancer cell viability and invasiveness, especially in glioma cells, has never been determined. Here we investigated the effect of Phyllanthus taxodiifolius Beille extract on glioma cell migration and adhesion using wound‐healing cell migration and cell spreading assays, respectively. We also determined its effects on microtubule structure and dynamics using both in vitro microtubule polymerization assay, and live cell imaging techniques with Total Internal Fluorescence Microscope (TIRFM). The results showed that Phyllanthus taxodiifolius Beille extract reduced glioma cell viability, disrupted cell adhesion and delayed cell migration. In addition, the extract rapidly disrupted microtubule structure and dynamics in glioma cells. Real time imaging of microtubule end‐binding protein (EB3) in glioma cells demonstrated that the extract disrupted the binding and dynamics of EB3 at the tips of microtubules. This study provided supporting evidences for the anti‐cancer properties of Phyllanthus taxodiifolius Beille extract. It is also the first study to demonstrate that the Phyllanthus taxodiifolius Beille extract interfered microtubule structure and dynamics, and suggested the possibility to further study and develop Phyllanthus taxodiifolius Beille derived compounds into a novel anti‐cancer drug targeting microtubules.Support or Funding InformationThis work was supported by a grant from the Center of Excellence on Environmental Health and Toxicology (EHT) to WS. We also gratefully acknowledge support from the Faculty of Science and Faculty of Graduate Studies, Mahidol University (to WS and JK) and support from Sritrangtong scholarship program (to JK).

Detailed Study of BSA Adsorption on Micro- and Nanocrystalline Diamond/β-SiC Composite Gradient Films by Time-Resolved Fluorescence Microscopy

The adsorption of bovine serum albumin (BSA) on micro- and nanocrystalline diamond/β-SiC composite films synthesized using the hot filament chemical vapor deposition (HFCVD) technique has been investigated by confocal fluorescence lifetime imaging microscopy. BSA labeled with fluorescein isothiocyanate (FITC) was employed as a probe. The BSAFITC conjugate was found to preferentially adsorb on both O-/OH-terminated microcrystalline and nanocrystalline diamond compared to the OH-terminated β-SiC, resulting in an increasing amount of BSA adsorbed to the gradient surfaces with an increasing diamond/β-SiC ratio. The different strength of adsorption (>30 times for diamond with a grain size of 570 nm) coincides with different surface energy parameters and differing conformational changes upon adsorption. Fluorescence data of the adsorbed BSAFITC on the gradient film with different diamond coverage show a four-exponential decay with decay times of 3.71, 2.54, 0.66, and 0.13 ns for a grain size of 570 nm. The different decay times are attributed to the fluorescence of thiourea fluorescein residuals of linked FITC distributed in BSA with different dye-dye and dye-surface distances. The longest decay time was found to correlate linearly with the diamond grain size. The fluorescence of BSAFITC undergoes external dynamic fluorescence quenching on the diamond surface by H- and/or sp2-defects and/or by amorphous carbon or graphite phases. An acceleration of the internal fluorescence concentration quenching in BSAFITC because of structural changes of albumin due to adsorption, is concluded to be a secondary contributor. These results suggest that the micro- and nanocrystalline diamond/β-SiC composite gradient films can be utilized to spatially control protein adsorption and diamond crystallite size, which facilitates systematic studies at these interesting (bio)interfaces.

A Dual-Color Reporter Assay of Cohesin-Mediated Gene Regulation in Budding Yeast Meiosis.

In this chapter, we describe a quantitative fluorescence-based assay of gene expression using the ratio of the reporter green fluorescence protein (GFP) to the internal red fluorescence protein (RFP) control. With this dual-color heterologous reporter assay, we have revealed cohesin-regulated genes and discovered a cis-acting DNA element, the Ty1-LTR, which interacts with cohesin and regulates gene expression during yeast meiosis. The method described here provides an effective cytological approach for quantitative analysis of global gene expression in budding yeast meiosis.

Structural and functional dynamics of tyrosine amino acid in phycocyanin of hot-spring cyanobacteria: A possible pathway for internal energy transfer

Phycocyanin (PC) is the most abundant accessory light-harvesting pigmented protein found in cyanobacteria. In the present investigation, prominent roles of tyrosine residue (Tyr) in excitation energy transfer between chromophores in PC of hot-spring cyanobacteria were studied. The expression pattern of codons suggests that Tyr was most abundant amino acids in α subunits as compared to β subunits in total aromatic amino acids. The α subunits of PC model (PDB 3O18) have one chromophore at cysteine (Cys) α-84 position (~10 Tyr) and β subunits contains two chromophores located at β-84 (~5 Tyr) and β-155 (~3 Tyr) in the cyanobacterium Thermosynechococcus vulcanus. The hydrophobic and hydrogen bond interaction was most favored between Tyr and buried amino acids near α-84 chromophore. The aromatic-aromatic interaction, aromatic-sulphur interaction, cation-pi interaction were mostly linked with Cys (α-84) as compared to β-84 and β-155. Phenylalanine (Phe) was demonstrating fewer interactions with chromophores due to nature of weak internal fluorescence. The coordinates of PC chromophore showed that internal fluorescence was very high close to Cys (α-84) residue which transmits excited photonic energy between intra-monomeric subunits Cys α84adg-Cys β84bch and inter-monomer Cys β84b-Cys β84c-Cys β84h. The lateral transmission of energy between adjacent hexamer-hexamer was feasible by Cys β155 chromophore. In contrast to total aromatic amino acids, tyrosine appeared exclusive for internal energy transfer by property of high internal fluorescence. These findings provide the illustrated role of tyrosine amino acids in rapid energy transfer mechanism inside PC which may be incorporated for making energy devices and various biotechnological sciences.

A novel upconversion@polydopamine core@shell nanoparticle based aptameric biosensor for biosensing and imaging of cytochrome c inside living cells

Herein, a novel upconversion@polydopamine core@shell nanoparticle (termed as UCNP@PDA NP) -based aptameric biosensor has been fabricated for the quantitative analysis of cytochrome c (Cyt c) inside living cells, which comprises an UCNP@PDA NP, acting as an internal reference and fluorescence quenching agent, and Cy3 modified aptamer enabling ratiometric quantitative Cyt c measurement. After the hybridization of Cy3 labeled aptamer with amino-terminated single DNA on the UCNP@PDA NP surface (termed as UCNP@PDA@AP), the fluorescence of Cy3 can be efficiently quenched by the PDA shell. With the spontaneous cellular uptake of UCNP@PDA@AP, the Cyt c aptamer dissociates from UCNP@PDA NP surface through formation of aptamer-Cyt c complex, resulting in concomitant activation of the Cy3 fluorescence. High amount of Cyt c leads to high fluorescence emission, enabling direct visualization/measurement of the Cyt c by fluorescence microscopy/spectroscopy. The steady upconversion luminescent (UCL) signals can be employed not only for intracellular imaging, but also as an internal reference for evaluating intracellular Cyt c amount using the ratio of fluorescence intensity of Cy3 with the UCL intensity of UCNP. The UCNP@PDA@AP shows a reasonable detection limit (20nM) and large dynamic range (50nM to 10μM, which covers the literature reported values (1–10μM) for cytosolic Cyt c in apoptotic cells) for detecting Cyt c in buffer with excellent selectivity. In addition, the UCNP@PDA@AP has been successfully used to monitor etoposide induced intracellular releasing of Cyt c, providing the possibility for cell-based screening of apoptosis-inducing drugs.

A ratiometric theranostic probe for tumor targeting therapy and self-therapeutic monitoring.

Feedback imaging-guided precise photodynamic therapy (PDT) can facilitate the development of personalized medicine. In this work, a Förster resonance energy transfer (FRET) based theranostic probe was fabricated for simultaneous tumor targeting PDT and ratiometric imaging of the therapeutic effect. The theranostic probe (designated as P-PpIX) was comprised of a targeting moiety, a caspase-3 responsive linker, a FRET fluorophore pair and a photosensitizer. It was found that P-PpIX exhibited low intrinsic background fluorescence due to the high FRET quenching efficiency. The Arg-Gly-Asp (RGD) targeting moiety allowed P-PpIX to selectively accumulate in αvβ3 integrin overexpressed tumor cells. Upon photo irradiation, the PDT effect of P-PpIX could induce cell death with apoptosis related mechanism, and the activated caspase-3 would subsequently cleave the Asp-Glu-Val-Asp (DEVD) peptide sequence to terminate the intramolecular FRET process. The activated caspase-3 expression and the real time therapeutic efficacy could be precisely assessed in situ by the fluorescence intensity ratio of the released 5(6)-carboxylfluorescein (FAM, reporter fluorescence) and protoporphyrin IX (PpIX, internal reference fluorescence). This novel ratiometric theranostic probe could provide the real-time feedback for precise PDT.

A biomimetic system to reconstitute and test the actomyosin-dependent mechanosensitive protein complexes

Many biological processes are governed by mechanical cues. For example, during migration, cells sense and respond to the mechanical stress of their environment by remodeling their shape, dynamics and adhesion to the extracellular matrix (ECM). Focal adhesions (FAs) sense the force generated by the actomyosin cytoskeleton and respond by recruiting new actin binding proteins ( ABPs). Although up to date several mechanosensitive path­ways have been identified, the biochemical characterization of the individual mechanosensitive proteins is currently limited by the available techniques. We developed a microscopy assay, with the ABP talin immobilized in 5-μm-diameter discs, regularly spaced by 35 μm and micropatterned on a glass coverslip. The self-organized actomyosin network exerts force on ABP and in response talin reveals cryptic vinculin-binding sites (VBSs). Finally, we observed that the activation of vinculin by talin induces a positive feedback that strengthens the actin-talin-vinculin association. This biomimetic system and the activity of the fluorescent proteins are visualized by total internal refection fluorescence (TIRF) microscopy. This technique can be used to study the ABPs that sense and respond to the mechanical force generated by the actomyosin cytoskeleton in a variety of physiological and pathological processes or for screening pharmacological inhibitors of mechanosensitive protein interactions.

Mitochondrial oxidative phosphorylation complexes exist in the sarcolemma of skeletal muscle.

Although proteomic analyses have revealed the presence of mitochondrial oxidative phosphorylation (OXPHOS) proteins in the plasma membrane, there have been no in-depth evaluations of the presence or function of OXPHOS I-V in the plasma membrane. Here, we demonstrate the in situ localization of OXPHOS I-V complexes to the sarcolemma of skeletal muscle by immunofluorescence and immunohistochemistry. A portion of the OXPHOS I-V complex proteins was not co-stained with MitoTracker but co-localized with caveolin-3 in the sarcolemma of mouse gastrocnemius. Mitochondrial matrix-facing OXPHOS complex subunits were ectopically expressed in the sarcolemma of the non-permeabilized muscle fibers and C2C12 myotubes. The sarcolemmal localization of cytochrome c was also observed from mouse gastrocnemius muscles and C2C12 myotubes, as determined by confocal and total internal resonance fluorescence (TIRF) microscopy. Based on these data, we conclude that a portion of OXPHOS complexes is localized in the sarcolemma of skeletal muscle and may have non-canonical functions. [BMB Reports 2016; 49(2): 116-121]

Sensing of tryptophan by a non-toxic cobalt(ii) complex

The first report of a cobalt(ii) based non-toxic, hemocompatible, fluorescent probe that sense Trp and BSA by reducing internal fluorescence quenching of Trp in aqueous solution.

Abstract A165: Engineering approaches to uncover the mechanism of apoptotic cell clearance by a conserved signaling system

Abstract Introduction: Efficient apoptotic cell clearance after injury, chemotherapy, and other triggers of programmed cell death is required to avoid inflammation and further tissue damage. However the behaviors of engulfment signaling molecules and their contributions towards non-inflammatory resolution are poorly understood. In Drosophila glial cells the receptor Draper is proposed to drive engulfment of apoptotic neurons. An orthologous receptor, MEGF10, has been proposed to work in mammalian glia. Both Draper and the T cell receptor complex (TCR) are proposed to recruit downstream molecules to the plasma membrane but functional similarities and differences between T cell and engulfment signaling have not been described. We have reconstituted engulfment in order to uncover the mechanism of this important, conserved signaling system. Experimental systems: By expressing Draper in otherwise inept phagocytes we have dramatically increased the engulfment of apoptotic cells or physiological mimics that expose natural ligands. We have also generated an inducible ligand-receptor system that provides a platform to monitor rapid responses of Draper signaling that are otherwise hard to capture. We utilize a supported lipid bilayer system and total internal fluorescence (TIRF) imaging to monitor the localization of Draper and downstream signaling molecules at the plasma membrane. Results: Remarkable recent work in T cells has led to the development of chimeric antigen receptors (CARs) that link specific ligands to heterologous intracellular signaling pathways in order to kill tumor cells. We have taken a similar approach to engulfment signaling by engineering chimeric engulfment receptors. An inducible heterologous receptor-ligand pair is used in our chimeric engulfment receptor system. Like Draper, the engineered chimeric receptor recruits signaling machinery to the plasma membrane and drives engulfment. Our results indicate ligand flexibility in the system and that the mechanism of activation required for endogenous Draper can be mimicked by engineered chimeric receptors. Using our TIRF assay we have discovered that Draper forms dynamic clusters similar to TCR and that clustered receptors recruit cytosolic signaling proteins to the plasma membrane. Importantly, simultaneous imaging of engulfment receptors and filamentous-actin (F-actin) has revealed that engulfment receptors drive F-actin remodeling that is distinct from behavior seen during TCR signaling. Our TIRF work has revealed both similarities and significant differences between the cellular changes set into motion by Draper signaling as compared to T cell triggering. Conclusions: We have built reconstitution platforms to study engulfment signaling and are in a unique position to understand the essential factors and rate-limiting steps of clearance. We have discovered that engineered chimeric receptors drive efficient engulfment and that engulfment signaling is sparked by formation of dynamic clusters of signaling molecules. We are hopeful that the lessons we learn from our reconstitution systems can be applied to activate non-inflammatory clearance of apoptotic cells after injury or extensive cell death. Citation Format: Adam Williamson, Ron Vale. Engineering approaches to uncover the mechanism of apoptotic cell clearance by a conserved signaling system. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A165.

Adiponectin is released via a unique regulated exocytosis pathway from a pre-formed vesicle pool on insulin stimulation.

Adiponectin, a hormone secreted from adipocytes and released at a high rate into the circulation, plays a pivotal role in maintaining insulin sensitivity at the whole-body level. Despite the importance of this adipokine in metabolic homoeostasis, the mechanism of its secretion from adipocytes remains largely unclear. In the present study, we investigated the subcellular localization of adiponectin, and its secretion regulation in 3T3-L1-differentiated adipocytes, using biochemical methods and fluorescence microscopic imaging. We show that adiponectin is localized in vesicular compartments with no apparent overlap with the Golgi apparatus or endosomes. Moreover, adiponectin-containing vesicles are enriched in two distinct pools: one at the plasma membrane (PM) and the other co-fractionating with endoplasmic reticulum membranes. When viewed under a total internal refection fluorescence microscope, a subset of adiponectin-Venus vesicles is readily observed in proximity to PMs, and could be released in response to insulin. Insulin-stimulated adiponectin release appears to be from a pre-existing pool of vesicles, and is not dependent on new protein synthesis, because adiponectin mRNA levels remain unchanged over a 6-h period of insulin treatment, and inhibition of protein synthesis has no effect on adiponectin release. Disruption of insulin signalling by inhibitors of phosphoinositide 3-kinase and protein kinase B (Akt)-1/2 abrogates the stimulated release of adiponectin. Taken together, our results show that adiponectin is stored in a unique vesicular compartment, and released through a regulated exocytosis pathway that is dependent on insulin signalling.

γ-Secretase Modulators and Inhibitors Induce Different Conformational Changes of Presenilin 1 Revealed by FLIM and FRET.

Elucidation of γ-secretase structure and dynamic conformational changes is of importance to drug discovery targeting this enzyme. Electron microscopy analyses provided important structural information, but the dynamic changes of γ-secretase in cells need to be explored further. We found that PS1 internal fluorescence resonance energy transfer (FRET) probes can incorporate into γ-secretase complex and possess secretase activity. Our results from fluorescence lifetime image microscopy (FLIM) and acceptor photobleaching FRET show different PS1 internal FRET when PS1 fluorescent probes expressed alone or with other secretase subunits Aph1aL, Nicastrin, and Pen2, indicating that PS1 internal FRET could be applied for probing conformational change of γ-secretase complex. Further, we accessed whether γ-secretase activity interfering compounds induced different conformational changes of PS1. Our results show that both γ-secretase modulators and inhibitors affect PS1 internal FRET but in different manners. These results demonstrate that FLIM and acceptor photobleaching FRET could be applied to monitor different PS1 conformational changes in γ-secretase.