Total Internal Fluorescence Microscopy Research Articles

Single-Molecule Approaches to Study DNA Condensation.

Proteins drive genome compartmentalization across different length scales. While the identities of these proteins have been well-studied, the physical mechanisms that drive genome organization have remained largely elusive. Studying these mechanisms is challenging owing to a lack of methodologies to parametrize physical models in cellular contexts. Furthermore, because of the complex, entangled, and dense nature of chromatin, conventional live imaging approaches often lack the spatial resolution to dissect these principles. In this chapter, we will describe how to image the interactions of λ-DNA with proteins under purified and cytoplasmic conditions. First, we will outline how to prepare biotinylated DNA, functionalize coverslips with biotin-conjugatedpoly-ethylene glycol (PEG), and assemble DNA microchannels compatible for the imaging of protein-DNA interactions using total internal fluorescence microscopy. Then we will describe experimental methods to image protein-DNA interactions in vitro and DNAloop extrusion using Xenopus laevis egg extracts.

The microtubule-severing protein UNC-45A preferentially binds to curved microtubules and counteracts the microtubule-straightening effects of Taxol

Uncoordinated protein 45A (UNC-45A) is the only known ATP-independent microtubule (MT)-severing protein. Thus, it severs MTs via a novel mechanism. Invitro and in cells, UNC-45A-mediated MT severing is preceded by the appearance of MT bends. While MTs are stiff biological polymers, in cells, they often curve, and the result of this curving can be breaking off. The contribution of MT-severing proteins on MT lattice curvature is largely undefined. Here, we show that UNC-45A curves MTs. Using invitro biophysical reconstitution and total internal fluorescence microscopy analysis, we show that UNC-45A is enriched in the areas where MTs are curved versus the areas where MTs are straight. In cells, we show that UNC-45A overexpression increases MT curvature and its depletion has the opposite effect. We also show that this effect occurs is independent of actomyosin contractility. Lastly, we show for the first time that in cells, Paclitaxel straightens MTs, and that UNC-45A can counteracts the MT-straightening effects of the drug.

Calcium signaling consequences of RyR2-S4938F mutation expressed in hiPSC-CMs.

Ryanodine receptor 2 (RyR2) is the major Ca2+ release channel of cardiac sarcoplasmic reticulum (SR) that regulates the rhythm and strength of the heartbeat, but its malfunction generates arrhythmia leading to heart failure. Here we report on a catecholaminergic polymorphic ventricular tachycardia (CPVT1)-associated RyR2 mutation (S4938F) located in RyR2 carboxyl-terminal that was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying S4938F mutation were studied using total internal fluorescence microscopy (TIRF) and patch clamp technique. ICa densities measured either by depolarizations to zero mV or repolarizations from +100 mV to −50 mV in mutant hiPSC-CMs were comparable to WT cells, but their accompanying Ca2+-transients detected by Fura-2 (cytosolic Ca2+) or ER-GCaMP6 (SR Ca2+ release) were significantly suppressed, suggesting ICa induced Ca2+ release (CICR) was compromised. Even though 5mM caffeine triggered Fura-2 or ER-GCaMP6 signals and the frequency of spontaneously generated calcium transients of mutant cells were comparable to those of WT cells, the 4-CmC triggered cytosolic or SR Ca2+ signals and magnitude of spontaneously occurring Ca2+-transients were significantly suppressed. Spontaneously occurring Ca2+ sparks in S4938F hiPSC-CMs had longer durations but their frequency was comparable to those of WT cells. Isoprenaline increased the beating frequency that led to arrhythmic beating and cessation of excitability. Our data suggests that S4938F-RyR2 mutation does not significantly affect Ca2+content of SR, but decreases its Ca2+ sensitivity resulting in suppressed ICa-gated and spontaneous Ca2+ release from SR. Supported by NIH grant R01HL153504.

Light trapping by porous TiO2 hollow hemispheres for high efficiency photoelectrochemical water splitting.

Photocatalytic water splitting has recently received increasing attention as a green fuel source. The controlled nano-geometry of the photocatalytic material can improve light harvesting. In this study, as a proof of concept, hollow hemisphere (HHS)-based films of TiO2 material were created by a conventional electrospray method and subsequently applied for photoelectrochemical (PEC) water splitting. To preserve the morphology of the HHS structure, a hydrolysis precipitation phase separation method (HPPS) was developed. As a result, the TiO2 HHS-based thin films presented a maximum PEC water splitting efficiency of ca. 0.31%, almost two times that of the photoanode formed by TiO2 nanoparticle-based films (P25). The unique morphology and porous structure of the TiO2 HHSs with reduced charge recombination and improved light absorption are responsible for the enhanced PEC performance. Light scattering by the HHS was demonstrated with total reflection internal fluorescence microscopy (TRIFM), revealing the unique light trapping phenomenon within the HHS cavity. This work paves the way for the rational design of nanostructures for photocatalysis in fields including energy, environment, and organosynthesis.

Confocal and TIRF microscopy based approaches to visualize arrestin trafficking in living cells.

Arrestins are key proteins that serve as versatile scaffolds to control and mediate G protein coupled receptors (GPCR) activity. Arrestin control of GPCR functions involves their recruitment from the cytosol to plasma membrane-localized GPCRs and to endosomal compartments, where they mediate internalization, sorting and signaling of GPCRs. Several methods can be used to monitor trafficking of arrestins; however, live fluorescence imaging remains the method of choice to both assess arrestin recruitment to ligand-activated receptors and to monitor its dynamic subcellular localization. Here, we present two approaches based on Total Internal Fluorescence (TIRF) microscopy and confocal microscopy to visualize arrestin trafficking in live cells in real time and to assess their co-localization with the GPCR of interest and their localization at specific subcellular locations.

Optical tweezer and TIRF microscopy for single molecule manipulation of RNA/DNA nanostructures including their rubbery property and single molecule counting.

Life science is often focused on the microscopic level. Single-molecule technology has been used to observe components at the micro- or nanoscale. Single-molecule imaging provides unprecedented information about the behavior of individual molecules in contrast to the information from ensemble methods that average the information of many molecules in various states. A typical feature of living systems is motion. The lack of synchronicity of motion biomachines in living systems makes it challenging to image the motion process with high resolution. Thus, single-molecule technology is especially useful for real-time study on motion mechanism of biomachines, such as viral DNA packaging motor, or other ATPases. The most common optical instrumentations in single-molecule studies are optical tweezers and single molecule total internal refection fluorescence microscopy (smTIRF). Optical tweezers are the force-based technique. The analysis of RNA using optical tweezer has led to the discovery of the rubbery or amoeba property of RNA nanoparticles for compelling vessel extravasation to enhance tumor targeting and fast renal excretion. The rubbery property of RNA lends mechanistic evidence for RNAs use as an ideal reagent in cancer treatment with undetectable toxicity. Single molecule photobleaching allows for the direct counting of biomolecules. This technique was invented for single molecule counting of RNA in the phi29 DNA packaging motor to resolve the debate between five and six copies of RNA in the motor. The technology has subsequently extended to counting components in biological machines composed of protein, DNA, and other macromolecules. In combination with statistical analysis, it reveals biomolecular mechanisms in detail and leads to the development of ultra-sensitive sensors in diagnosis and forensics. This review focuses on the applications of optical tweezers and fluorescence-based techniques as single-molecule technologies to resolve mechanistic questions related to RNA and DNA nanostructures.

Single-Cell Probe Force Studies to Identify Sox2 Overexpression-Promoted Cell Adhesion in MCF7 Breast Cancer Cells.

The replacement of the cantilever tip by a living cell in Atomic Force Microscopy (AFM) experiments permits the direct quantification of cell–substrate and cell–cell adhesion forces. This single-cell probe force measurement technique, when complemented by microscopy, allows controlled manipulation of the cell with defined location at the area of interest. In this work, a setup based on two glass half-slides, a non-fouling one with bacterial S-layer protein SbpA from L. sphaericus CMM 2177 and the second with a fibronectin layer, has been employed to measure the adhesion of MCF7 breast cancer cells to fibronectin films (using SbpA as control) and to other cells (symmetric vs. asymmetric systems). The measurements aimed to characterize and compare the adhesion capacities of parental cells and cells overexpressing the embryonic transcription factor Sox2, which have a higher capacity for invasion and are more resistant to endocrine therapy in vivo. Together with the use of fluorescence techniques (epifluorescence, Total Internal Fluorescence Microscopy (TIRF)), the visualization of vinculin and actin distribution in cells in contact with fibronectin surfaces is enabled, facilitating the monitoring and quantification of the formation of adhesion complexes. These findings demonstrate the strength of this combined approach to assess and compare the adhesion properties of cell lines and to illustrate the heterogeneity of adhesive strength found in breast cancer cells.

Dissociation of β-Arrestin-Dependent Desensitization and Signaling Using 'Biased' Parathyroid Hormone Receptor Ligands

While structurally distinct ligands can “bias” the efficiency with which G protein-coupled receptors (GPCRs) activate different downstream effectors, it is thought that the events downstream of a single effector are inseparable. We tested a panel of type 1 parathyroid hormone receptor (PTH1R) ligands for their effects on two β-arrestin-mediated processes, receptor desensitization and β-arrestin-dependent signaling. Unlike conventional agonists, which caused desensitization while activating β-arrestin-dependent signaling, we found that β-arrestin biased agonists that did not activate G protein signaling or desensitize the receptor paradoxically retained the ability to signal via β-arrestins. Total internal fluorescence microscopy, bimolecular fluorescent complementation between PTH1R and β-arrestin2, and intramolecular β-arrestin2 FlAsH-BRET conformational profiling demonstrated that these ligands transiently recruit β-arrestin2 to the receptor and induced distinctive changes in β-arrestin2 conformation without a requirement for stable binding. Our results demonstrate that β-arrestin-dependent signaling can be dissociated from receptor desensitization and establish a novel profile of β-arrestin-dependent efficacy.

Surface plasmon resonance imaging of excitable cells

Surface plasmons (SPs) are surface charge density oscillations occuring at a metal/dieletric interface and are highly sensitive to refractive index variations adjacent to the surface. This sensitivity has been exploited successfully for chemical and biological assays. In these systems, a surface plasmon resonance (SPR)-based sensor detects temporal variations in the refractive index at a point. SPR has also been used in imaging systems where the spatial variations of refractive index in the sample provide the contrast mechanism. SPR imaging systems using high numerical aperture (NA) objective lenses have been designed to image adherent live cells with high magnification and near-diffraction limited spatial resolution. Addressing research questions in cell physiology and pharmacology often requires the development of a multimodal microscope where complementary information can be obtained.In this paper, we present the development of a multimodal microscope that combines SPR imaging with a number of additional imaging modalities including bright-field, epifluorescence, total internal reflection microscopy and SPR fluorescence microscopy. We used a high NA objective lens for SPR and TIR microscopy and the platform has been used to image live cell cultures demonstrating both fluorescent and label-free techniques. Both the SPR and TIR imaging systems feature a wide field of view (~300 µm) that allows measurements from multiple cells whilst maintaining a resolution sufficient to image fine cellular processes. The capability of the platform to perform label-free functional imaging of living cells was demonstrated by imaging the spatial variations in contractions from stem cell-derived cardiomyocytes. This technique shows promise for non-invasive imaging of cultured cells over very long periods of time during development.

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

To couple the fidelity of patch-clamp recording with a more high-throughput screening capability, we pioneered a, to our knowledge, novel approach to single-channel recording that we named “optical patch clamp.” By using highly sensitive fluorescent Ca2+ indicator dyes in conjunction with total internal fluorescence microscopy techniques, we monitor Ca2+ flux through individual Ca2+-permeable channels. This approach provides information about channel gating analogous to patch-clamp recording at a time resolution of ∼2 ms with the additional advantage of being massively parallel, providing simultaneous and independent recording from thousands of channels in the native environment. However, manual analysis of the data generated by this technique presents severe challenges because a video recording can include many thousands of frames. To overcome this bottleneck, we developed an image processing and analysis framework called CellSpecks capable of detecting and fully analyzing the kinetics of ion channels within a video sequence. By using randomly generated synthetic data, we tested the ability of CellSpecks to rapidly and efficiently detect and analyze the activity of thousands of ion channels, including openings for a few milliseconds. Here, we report the use of CellSpecks for the analysis of experimental data acquired by imaging muscle nicotinic acetylcholine receptors and the Alzheimer’s disease-associated amyloid β pores with multiconductance levels in the plasma membrane of Xenopus laevis oocytes. We show that CellSpecks can accurately and efficiently generate location maps and create raw and processed fluorescence time traces; histograms of mean open times, mean close times, open probabilities, durations, and maximal amplitudes; and a “channel chip” showing the activity of all channels as a function of time. Although we specifically illustrate the application of CellSpecks for analyzing data from Ca2+ channels, it can be easily customized to analyze other spatially and temporally localized signals.

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.

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).

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]

Abstract 4994: RET isoforms differentially contribute to focal adhesion formation

Abstract RET is a receptor tyrosine kinase crucial for the development of the kidney, and some neuroendocrine tissues. RET activation by its ligand, glial cell-line derived neurotrophic factor (GDNF), results in autophosphorylation of several key tyrosine kinase residues in the intracellular region, which allow binding of adaptor and signalling proteins, and consequently activation of various cellular processes, including survival, proliferation and migration. RET has two major protein isoforms, called RET9 and RET51 after the number of unique amino acids at the C-terminus, generated by alternative splicing. Although the RET isoforms are highly conserved across species, and both isoforms are normally co-expressed, they have major molecular and functional differences, including different transforming abilities, and trafficking properties. RET is implicated in several human diseases. Gain-of-function RET mutations result in the cancer syndrome multiple endocrine neoplasia type 2, that affects neuroendocrine tissues, somatic RET rearrangements are found in papillary thyroid carcinoma, and lung adenocarcinoma, and aberrant wild-type RET activation occurs in many cancers, including breast and pancreatic cancer. Focal adhesion protein complexes are involved in interactions of cells with the extracellular matrix, and are crucial for controlling cancer-related processes, including cell migration and invasion. The contributions of RET isoforms to the processes of focal adhesion formation and dynamics have not yet been investigated. To explore the roles of RET isoforms in focal adhesion formation, we used total internal resonance fluorescence (TIRF) microscopy to assess localization of various focal adhesion proteins, including paxillin, vinculin and zyxin in response to GDNF in a cell-based model system. Our results suggest that RET51 promotes more early (ie. paxillin) and late (ie. zyxin) stage focal adhesion formation than does RET9. Additionally, we demonstrate that the kinase dead form of RET51 results in a decrease of the number of focal adhesions compared to RET51 wild-type, suggesting that the increase in focal adhesion formation in the presence of RET51 is phosphorylation-dependent. Inhibition of SRC and FAK led to a reduction in focal adhesion formation, indicating that these pathways play roles in this process. Ongoing studies are investigating the focal adhesion dynamics of the RET isoforms and further characterizing the molecular differences underlying the ability of RET51 to induce greater focal adhesion formation than RET9. Citation Format: Piriya Yoganathan, Eric Lian, Lois M. Mulligan. RET isoforms differentially contribute to focal adhesion formation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4994. doi:10.1158/1538-7445.AM2015-4994

Effect of receptor dimerization on membrane lipid raft structure continuously quantified on single cells by camera based fluorescence correlation spectroscopy.

Membrane bound cell signaling is modulated by the membrane ultra-structure, which itself may be affected by signaling. However, measuring the interaction of membrane proteins with membrane structures in intact cells in real-time poses considerable challenges. In this paper we present a non-destructive fluorescence method that quantifies these interactions in single cells, and is able to monitor the same cell continuously to observe small changes. This approach combines total internal fluorescence microscopy with fluorescence correlation spectroscopy to measure the protein’s diffusion and molecular concentration in different sized areas simultaneously. It correctly differentiates proteins interacting with membrane fences from proteins interacting with cholesterol-stabilized domains, or lipid rafts. This method detects small perturbations of the membrane ultra-structure or of a protein’s tendency to dimerize. Through continuous monitoring of single cells, we demonstrate how dimerization of GPI-anchored proteins increases their association with the structural domains. Using a dual-color approach we study the effect of dimerization of one GPI-anchored protein on another type of GPI-anchored protein expressed in the same cell. Scans over the cell surface reveal a correlation between cholesterol stabilized domains and membrane cytoskeleton.

Lamellar sheet exfoliation of single lipid vesicles by a membrane-active peptide.

Using total internal fluorescence microscopy, highly parallel measurements of single lipid vesicles unexpectedly reveal that a small fraction of vesicles rupture in multiple discrete steps when destabilized by a membrane-active peptide which is in contrast to classical solubilization models. To account for this surprizing kinetic behaviour, we identified that this vesicle subpopulation consists of multilamellar vesicles, and that the outermost lamella is more susceptible to rupture than unilamellar vesicles of even smaller size. This finding sheds light on the multiple ways in which membrane configuration can influence strain in the bilayer leaflet and contribute to nm-scale membrane curvature sensing.