9th Weber Symposium (2014)

Abstracts by corresponding author

1. Paolo Annibale (Speaker)
   3D orbital tracking of a DNA locus during transcription.
   Abstract

   Paolo Annibale and Enrico Gratton
   Laboratory for Fluorescence Dynamics, University of California, Irvine

   We report on the application of a 3D particle tracking method, namely orbital tracking, to a model cellular system allowing to simultaneously label an actively transcribing genetic locus and the synthesized mRNA using the EGFP-labeled MS2 coat protein. Orbital tracking in a 2-photon microscope allows collecting unprecedented sub-second temporal resolution, hour-long fluorescence trajectories of the newly transcribed mRNA.
   We observe distinct regions of active transcription, termed petals, displaying a well defined spatial organization, corralling the denser part of the genetic locus and rotating along its surface.
   We apply fluorescence fluctuation analysis to investigate the transcription kinetics of each of these petals; the comparison of experiments and simulations allows highlighting a wide range of polymerase elongation rates, ranging from as low as a few tens basepairs/s to hundreds of basepairs/s. Response of the elongation rate to a common transcription inhibitor is also demonstrated.
   Exploiting the trajectory information that we collect for each of the transcribing petals we provide, to our knowledge for the first time, an experimental measurement of the relationship existing between chromatin displacement and transcriptional output. A cross correlation signal is observed between petal rotations of a definite handedness and their transcriptional bursting.
2. Andrea Anzalone (Speaker)
   Gold nano-imaging of the cell nucleus.
   Abstract

   Andrea Anzalone1, Chiara Stringari2, and Enrico Gratton1
   1 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University California, Irvine
   2 Laboratory for Optics & Biosciences École Polytechnique Paris, France

   Metallic nanoparticles such as Gold Nanoparticles (GNPs) have recently become a common fluorescent microscopy tool because they do not bleach or blink upon continuous illumination. Gold nanoparticles are also characterized by an intrinsic feature, the plasmonic enhancement surface, which enhances fluorescence of molecules when in close proximity (few nanometers) to the particle. At the LFD it has been developed the 3D orbital tracking method that utilizes the characteristics of a 2-Photon microscope to study cellular processes. The 3D orbital tracking combined with the plasmonic enhancement of the GNPs provide a reliable method to observe sub-cellular structures, such as chromatin, with a nano-scale resolution. During the movement inside the cell nucleus the gold nanoparticles interact with the surroundings and give fluorescent enhancement of specific fluorophores with different emission spectra. The enhancement due to the proximity of gold NP and fluorophores inside the nucleus appears as emission spikes of different colors presumably according to the color of the fluorescent molecules in close proximity at the nanoparticles. The 3D reconstruction of the spikes inside the orbits gives us information about the position of the enhancement in the orbit. In this study we showed we are able to discriminate different color molecules interacting with the nanoparticle along the motion, and at the same time identify the position in the orbit of these emission spikes.
3. Beniamino Barbieri (Sponsor Talk)
   Single molecule detection for quantitative cell biology.
   Abstract

   Beniamino Barbieri
   ISS Inc, 1602 Newton Drive, Champaign, IL 61822

   The revolution in quantitative cell biology is here. In the past few years the application of functional genomics, proteomics and metabolomics to the single cell system has allowed the analysis of collectives of molecules and the structures they form within the single cell.
   In this talk we describe a laser scanning confocal microscope that incorporates several of the tools required by quantitative cell biology to identify and clarify the molecular dynamics processes and molecular interactions within the cell at the single molecule level sensitivity. The tools encompass the family of Fluorescence Fluctuations Spectroscopy measurements at single locations (FCS, PCH, FCCS, FLCS) and their extension to imaging (scanning FCS, Number and Brightness, Raster Imaging Correlation Spectroscopy) and the Fluorescence Lifetime Imaging (FLIM). The integration of these tools into a single platform allows for the user to rapidly switch between measurement modalities.
4. Francisco Barrantes (Speaker)
   Nanocluster organization and dynamics of the nicotinic acetylcholine receptor at the cell surface.
   Abstract

   Francisco J. Barrantes
   Lab. Molec. Neurobiology, Inst. Biomedical Research, UCA- CONICET, 1107 Buenos Aires, Argentina

   The efficiency of chemical synaptic transmission relies to a great extent on adequate neurotransmitter release in the pre-synapse and a suitable number of active receptors at the postsynaptic target cell. The latter depends, in turn, on the equilibrium between synthesis, delivery to and removal of receptor molecules from the cell membrane. Organization and maintenance of an adequate number of neurotransmitter receptors at the cell surface also depends on extrinsic factors, such as the lipid milieu in which the receptor proteins are embedded. We are interested in the supramolecular organization, dynamics and trafficking of neuronal and muscle-type nicotinic acetylcholine receptors (AChRs) and study the influence of lipids such as cholesterol on these proteins using a combination of ensemble averaging methods and single-molecule experimental techniques. Using STED optical nanoscopy we could resolve AChR nanoclusters of ~70 nm which increase in size upon ligation and are sensitive to cholesterol content (Kellner et al., 2007). The neutral lipid cholesterol can also modulate the default clathrin- and dynamin-independent AChR internalization mechanism (Kumari et al., 2008) and re-route it to a different, ligand-independent, Arf-6-dependent endocytic pathway (Borroni & Barrantes, 2011). Cholesterol also affects the surface dynamics of AChR: TIRF microscopy combined with single-particle tracking analysis disclosed the dependence of receptor lateral mobility on cholesterol content (Almarza et al., 2014). Single-molecule nanoscopy of live cells is currently enabling us to follow the trajectory of AChR nanoclusters beyond the diffraction barrier. The emerging picture is that the neutral lipid cholesterol and cortical actin dynamics act synergically to modulate the supramolecular organization of the AChR at the cell surface.
   [1] Almarza, G., Sánchez, F. & Barrantes, F.J. (2014). Transient cholesterol effects on nicotinic acetylcholine receptor surface mobility. PLOSOne (in press).
   [2] Borroni, V. and Barrantes, F.J. (2011) Cholesterol modulates the rate and mechanism of acetylcholine receptor internalization. J. Biol. Chem. 286:17122-17132.
   [3] Kellner, R. Baier, J., Willig, K.I., Hell, S.W. and Barrantes, F.J. (2007). Nanoscale organization of nicotinic acetylcholine receptors revealed by STED microscopy. Neuroscience 144:135-143.
   [4] Kumari, S., Borroni, V., Chaudhry, A., Chanda, B. Massol, R., Mayor, S. and Barrantes, F.J. (2008). Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway. J. Cell Biol. 181:1171-1193.
5. Frederick P Bellinger (Poster)
   Selenoprotein P is required for hippocampal synaptic Zinc release and brain Zinc homeostasis.
   Abstract

   AC Parubrub, RH Rueli, CW Shuttleworth, MJ Berry, and FP Bellinger

   Selenoprotein P (Sepp1) is a selenium-rich protein involved in extracellular transport of selenium (Se). Selenium can bind Zinc (Zn2+) with high affinity, and Sepp1 has metal binding properties. We investigated if genetic deletion of Sepp1 could alter Zn2+ levels and release from synaptic terminals. Timm-Danscher and N-(6-methoxy-8-quinolyl)-p-toluenesulphonamide (TSQ) staining revealed increased levels of intracellular Zn2+ in the Sepp1-/- hippocampus compared to the wild-type (WT) mice. Unexpectedly, live Zn2+ imaging of hippocampal slices with a selective extracellular fluorescent Zn2+ indicator (Fluozin-3) showed that Sepp1-/- mice have impaired Zn2+ release in response to KCl-induced neuron depolarization. Levels of key Zn2+-regulating proteins in the brain were affected by the absence of Sepp1, possibly in response to the elevated Zn2+ content. Taken together, our findings reveal that Sepp1 plays a crucial role in the maintenance of Zn2+ homeostasis in the hippocampus and for proper brain function. The identification of a naturally occurring Zn2+-chelator regulated by dietary selenium may significantly contribute to the treatment and prevention of Alzheimer’s disease (AD).
6. Quill Bowden (Poster)
   Utilising ensemble and single-molecule fluorescence techniques to investigate the dynamics of disease protein, α-synuclein and its interaction with molecular chaperones.
   Abstract

   Quill Bowden1, Alex MacMillan2, Katharina Gaus1, and Till Böcking1
   1 Centre for Vascular Research, UNSW, Sydney 2052
   2 Biomedical Imaging Facility, UNSW, Sydney 2052

   Many neurodegenerative disorders are caused by a pathological accumulation of misfolded or aggregated proteins. In Parkinson’s Disease (PD), the protein α-Synuclein and its early stage oligomers have been implicated as the primary cause of neuronal toxicity. Molecular chaperones play an important role in neurological diseases as they maintain protein homeostasis and are pivotal in all stages of a proteins life cycle.
   Both chaperones and the α-synuclein substrate exist with transient intermediates and highly dynamic conformational states. This has presented problems in understanding the molecular mechanisms using traditional biochemical techniques. α-Synuclein is therefore an ideal substrate to investigate the mechanism of chaperone interactions through the use of both ensemble and single-molecule fluorescence techniques.
   We have utilised FLIM-FRET with phasor analysis and the Number and Brightness method (N&B). These techniques have allowed us to visualise dynamic subunit interactions and concentration dependant oligomeric fluctuations. To further understand the finer details of the α-synuclein oligomeric form we have used intrinsic tyrosine fluorescence to reveal the folded state at varied concentrations.
   We are currently working on studies of chaperone and co-chaperone interactions with the use of co-localisation and FRET in smTIRF. We will be able to gain further insight into the order of chaperone interactions, the stoichiometry of interacting species and the kinetics of the associations. A greater understanding of the mechanism of chaperone mediated disease modulation and the form of the toxic conformers in PD can provide solutions for a range of diseases and conditions which have a similar pathological origin.
7. Francesco Cardarelli (Speaker)
   Measuring short-range protein Brownian motion in the cytoplasm of living cells.
   Abstract

   Francesco Cardarellia
   Center for Nanotechnology Innovation, NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12 - 56127 Pisa, Italy

   An ubiquitous observation in cell biology is that translational motion of molecules is anomalous, i.e. diffusion is strongly suppressed compared to what observed in dilute solutions. By contrast, molecular rotation is less affected by this environment thus indicating that the close proximity of the molecule is aqueous. Theoretical models provide explanations for this apparent discrepancy pointing to the presence of macromolecular intracellular crowding. Yet, if dynamics is examined throughout the widely-different length and time scales that characterize local rotation and long-range translation, predictions drastically depend on the nanoscale organization assumed for crowding agents. High concentration of freely-diffusing solutes would suppress translational motion already at the molecular scale, while impenetrable, immobile structures would admit unobstructed (Brownian) motion up to the characteristic scale of crowding organization. Experimental discrimination between these two scenarios in the actual intracellular environment has remained elusive due to the lack of techniques that can probe crowding at the nanoscale. Here we show that fluorescence-fluctuation analysis of raster scans at variable time-scales can provide this information without a priori assumptions about the diffusive properties of molecules. By using monomeric GFP as a fluorescent tracer, we unveil the occurrence of i) unobstructed (Brownian) translational motion, as in dilute solutions, at the hitherto unexplored spatial scales from 25 to 100 nanometers, and ii) suppressed diffusion above 100 nm. The crossover between the two regimes identifies the characteristic scale at which GFP motion in the cytoplasm is affected by crowding. Experiments on model in cuvette systems confirm that the observed phenomenology can be explained as the result of excluded-volume due to immobile structures rather than to diffusing crowding agents. We argue that the experimental measurement of protein unobstructed Brownian motion in the cell cytoplasm can revolutionize the way biochemical reactions are modeled, with impact on our understanding of virtually all intracellular processes.
8. Yan Chen (Speaker)
   Dynamin 2-dependent biogenesis of Endophilin B1 containing vesicles.
   Abstract

   Jinhui Li1, Barbara Barylko2, Serkan Berk1,3, Joachim D. Mueller1,3, Joseph P. Albanesi2, and Yan Chen1
   1 School of Physics and Astronomy, University of Minnesota, Minneapolis, MN, 55455
   2 Pharmacology Department, UT Southwestern Medical Center, Dallas, TX, 75239
   3 Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, 55455

   Endophilins are SH3- and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2, which function in the early stages of endocytosis from the plasma membrane, exist almost exclusively as soluble dimers in the cell cytoplasm. Here we use fluorescence correlation spectroscopy and brightness analysis to show that a population of endophilin B1, which mediates intracellular membrane trafficking, is instead present in multiple copies on small and highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. In contrast, vesicle association of the endophilin B1 BAR domain was dynamin-independent, supporting previous in vitro experiments showing that full-length endophilins adopt an auto-inhibited conformation relieved by interaction of their SH3 domains with dynamins. Although dynamin 2 activity was important for the formation of endophilin B1-containing vesicles, it did not co-migrate with these vesicles. However, both dynamin 2 and synaptojanin 1, a phosphoinositide phosphatase required for uncoating of clathrin-coated vesicles, were found to associate with soluble, cytosolic endophilin A2 in living cells. These results suggest important mechanistic differences between the two closely related membrane-deforming proteins, endophilins A2 and B, in the cytoplasm of living cells.
9. Chi-Li Chiu (Weber Prize Guest)
   3D cell-ECM dynamics revealed by innovative fluorescence microscopy methods.
   Abstract

   Chi-Li Chiu

   The projects presented in this thesis targeted questions related to different aspects of the 3D cell-ECM interactions that are associated with cell migration mechanism, including focal adhesion dynamics, actin dynamics, and ECM remodeling. Focal adhesions play an important role in connecting ECM to actin cytoskeleton. In 2D, the coordination of focal adhesions binding/unbinding to the ECM and actin dynamics facilitates cell migration. Moving from 2D to the more physiologically relevant 3D environment, however, focal adhesions and actin cytoskeleton distribution change greatly. It is not clear whether the cell migration mechanism that involves focal adhesion and actin based on the observation of 2D cell monolayers applies to the cells migrating in the 3D ECM.
   To expand our understanding of cell-ECM dynamics in 3D, we developed novel fluorescence microscopy methods that are capable of capturing the protein dynamics in a live cell in 3D. These methods are based on nSPIRO and various FCS approaches. With the new methods that provide higher spatial-temporal resolution compared to other state-of-the-art techniques, we showed, for the first time, molecular level details of focal adhesion and actin dynamics in cells cultured in the 3D environment. Compared to the 2D cell monolayer, although the distribution of focal adhesion and actin may be different, their dynamics at molecular level share several similarities.
10. Christian Combs (Speaker)
    Intravital imaging of pH using ratiometric infrared two-photon excitation of the red fluorescent protein mKeima.
    Abstract

    Christian A. Combs1, Jeanho Yun2, and Toren Finkel2
    1 NHLBI Light Microscopy Facility, combsc@nih.gov
    2 NHLBI Laboratory of Molecular Biology, National Institutes of Health, Bethesda, Maryland 20892-1061

    Intracellular pH (pHi) is a critical modulator of cell function. It plays a role in many processes including mitochondrial metabolism, protein function, regulation of the cell cycle, lysosomal function, and apoptosis. Targeted pHi imaging in specific cellular compartments can be done with genetically encoded pH sensors. Most of these sensors are GFP based or rely on FRET between two proteins. In 2011, Tantama et al. (1) showed the utility of using RFP based ratio-metric imaging of pHi. Use of these RFP variants extended the color range of pHi imaging allowing for labelling or measuring other physiological parameters with other fluorescent proteins or dyes. In their work a sensor named pHRed showed an apparent pKa of 6.6 with a ratiometric dynamic range >10 when excited at 440 and 610nm . They also showed that single excitation two-photon FLIM could be used with these variants to measure pHi. Here we show that the RFP variant mKeima (targeted to mitochondria) can be ratiometrically imaged using (two) two-photon excitation lines (820nm and 1120nm) from a standard Ti:Sapph laser and an Optical Parametric Oscillator. This combination allows for intravital imaging with all of the benefits of standard two-photon excitation microscopy (TPEM) including depth of penetration in tissues, speed of imaging, and inherent optical sectioning. This technique is demonstrated on cells and freshly harvested tissues. In our work mKeima shows at least a > 5 fold dynamic range between pH 7.5 and 4.0 in permeabilized cells. We show a mitochondrial (high pH) and lysosomal (low pH) distribution of mKeima in mouse kidney, brain, muscle (heart and skeletal), and liver. Utility of this technique is discussed with an eye towards examining levels of autophagy, deep two-photon imaging, and avoidance of autoflurescence while imaging pHi using red shifted TPEM.
    [1] Tantama, M., Hung, Y.P, and Gary Yellen. 2011. JACS. 133: 10034-10037.
11. Christian Combs (Poster)
    Compact non-contact total emission detection for in-vivo multi-photon excitation microscopy.
    Abstract

    Christian A. Combs1, Aleksandr Smirnov2, Brian Glancy3, Glen Redford4, Karl Kilborn4, Jay R. Knutson2 and Robert S. Balaban3
    1 NHLBI Light Microscopy Facility
    2 NHLBI Laboratory of Molecular Biophysics
    3 NHLBI Laboratory of Cardiac Energetics, National Institutes of Health, Bethesda, Maryland 20892-1061
    4 Intelligent Imaging Innovations, Inc., Denver, CO 80216

    The inherent optical sectioning capability of multiphoton excitation microscopy allows for the collection of all of the excited light generated at the focal volume within a sample. Over the last two Weber conferences we have described variations along a theme of collecting emission light missed by the objective using a parabolic mirror [1,2]. We call this approach Total Emission Detection (TED). This work has culminated in a design that conforms to a standard upright two-photon microscope and focuses on capturing back scattered light using a relatively high N.A. water dipping lens traditionally used in high resolution two-photon imaging [3]. This is in contrast to other techniques which use low N.A. lens and capture light in the forward direction with index matching at the detector [4,5]. Our system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. We have shown that this design increases light collection from living tissues (mouse skeletal muscle, zebrafish, rat kidney and mouse mixed tissue including fat) imaged in vivo by a factor 2-5x using a standard water immersion lens with a N.A. of 1.0. Direct measurement of bleaching rates confirmed that the lower laser powers (enabled by greater light collection efficiency) yielded reduced photobleaching in vivo. Although we were able to capture more substantially more emission light we have not observed an ability to sharply image deeper into the types of samples we have examined to date. We suggest that this is likely due to other limiting factors which deteriorate the coherence and/or focus of the excitation wavefront at depth in the tissue. The potential benefits of increased light collection in terms of speed of imaging and reduced photo-damage, as well as the applicability of this device to other multi-photon imaging methods will be discussed.
    [1] Combs et al. 2007. J Microsc, 228: 330-337.
    [2] Combs et al. 2011. J Microsc, 241: 153-161.
    [3] Combs et al. 2014. J. Microsc, 252: 83-92.
    [4] Crosignani et al. 2012. J Biomed Opt, 17: 116023.
    [5] Crosignani et al. 2011. J Biophotonics, 4: 592-599.
12. Francesco Cutrale (Poster)
    A spectral phasor perspective in Zebrafish muscle development.
    Abstract

    Francesco Cutrale, Vikas Trivedi, Thai Truong, Le A. Trinh, and Scott E. Fraser

    Hyper- (Multi) spectral imaging provides the potential for assessing biochemical interactions in the zebrafish embryo in a label-free manner that extends beyond conventional morphological and molecular phenotyping. It takes advantage of the intrinsic wavelengths emitted or reflected from a sample without the need for extrinsic staining methods. The specific spectral signature from a sample can arise from chemical interactions, molecular bonds and macro-structural arrangements. A challenge in hyperspectral imaging is the large spectral data sets that result from acquiring a spectrum for every pixel within an image. Spectral Phasor offers an efficient representation of the spectral data as vectors in Fourier space, thereby condensing each spectrum into a single point in a 2-D plot. The Spectral Phasor has been successfully applied to hyperspectral data on protein samples, demonstrating changes in fluorescence signatures. This study proposes an application of Spectral Phasor to the zebrafish muscle development. The skeletal muscle system provides an attractive model for the proof-of- principle experiments in the implementation of Spectral Phasor. Skeletal muscle is a highly organized tissue with myofibrils as the functional unit that contributes to the repetitive segment of the myotome. The modularity of these units provides unique landmarks for anchoring the SP data. Our analysis of muscle suggest that SP can be used for staging the skeletal muscle development.
13. Xavier Darzacq (Speaker)
    Dissecting transcription regulation using single molecule imaging.
    Abstract

    Xavier Darzacq

    While membranes do not compartmentalise the nucleus, it shows a complex organisation at many scales. Spatial organisation of chromatin and transcription factors can modulate nuclear functions and in order to study this relation, we have developed methods to localise proteins and mRNAs at the single molecule level and with spatial resolutions in the range of a few nanometers (modifications and improvements of PALM, sptPALM and STORM using adaptive optics). Moreover, proteins move throughout the nucleus by diffusion, transiently and repetitively contacting their target sites. While DNA has been reported as a guide facilitating target search in the cell by restricting 3 dimensional explorations to a 1 dimensional search, such exploration modes were not envisioned mediated by protein-protein interactions. I will discuss chromatin and RNA polymerase II organisation in the nucleus as well as mechanisms guiding proteins to their targets in the nucleoplasm.
14. Lloyd M Davis (Poster)
    Microfluidic cell-sorter measures fluorescence lifetime, photobleaching, and brightness for use in developing improved red fluorescent proteins.
    Abstract

    Lloyd M. Davis1, Kevin M. Dean2, Premashis Manna3, Jennifer L. Lubbeck3, Amy E. Palmer2, and Ralph Jimenez3
    1 Center for Laser Applications, University of Tennessee Space Institute, Tullahoma, TN 37388
    2 BioFrontiers Institute and Dept. of Chemistry & Biochemistry, University of Colorado, Boulder, CO
    3 JILA and Dept. of Chemistry & Biochemistry, NIST and University of Colorado, Boulder, CO 80309

    Vigorous efforts are underway to develop fluorescent protein (FP) fusions with improved brightness for use in single-molecule-based super-resolution imaging within living cells. Commonly used development procedures include rational design of amino-acid residues at key positions in the protein and finding combinations of residues at supporting positions by directed evolution, which entails successive rounds of sorting live cells on the basis of their fluorescence properties. Although development of FPs with improved photostability is a high priority, it remains challenging, in part due to a lack of automated cell selection strategies that probe FP photophysics. Existing fluorescence activated cell sorters typically measure emission within selected bands, which depends more on the FP expression level than intrinsic brightness, and they use sub-microsecond cell transits that are insufficient for unraveling millisecond photobleaching dynamics. We have shown that permanent photobleaching may be distinguished from reversible dark-state conversion by exciting FPs with a sequence of laser pulses interspersed with millisecond dark intervals. We have incorporated this scheme in a microfluidic cell sorter, in which a hydrodynamically focused stream of cells flows through a series of several, 532-nm laser beams such that each cell undergoes a millisecond modulated excitation sequence. Real-time analysis of the fluorescence signals triggers the movement of a focused 1064-nm laser beam for selection of cells with enhanced photostability by use of optical gradient force switching (Lab Chip 13, 2320-2327 (2013)). Here, we describe a second-generation instrument that not only accomplishes faster assays by resolving cells that overlap in their transits through the beams, but moreover also screens on the basis of fluorescence lifetime, which is a measure of the quantum yield of the fluorophore, and which is independent of the level of expression. The capability to appraise quantum yield together with photostability is important because enhanced photostability can be achieved by lowering the quantum yield, whereas both factor into the overall brightness. To determine fluorescence lifetime, the first laser beam is intensity modulated at 29.5 MHz and the resulting millisecond burst of fluorescence as each cell passes through this beam is spatially isolated onto a photomultiplier (PMT). The in-phase and in-quadrature components of the modulation of this signal are resolved with a custom demodulator circuit to enable a precise evaluation of the fluorescence lifetime (coefficient of variation ~1.2%). The time-averaged signal is also digitized to determine the arrival time of each cell and its initial brightness. After the stream of cells pass through the beams for photobleaching, the signal from the final beam is spatially isolated on a second PMT. A computer running LabVIEW Real-time software matches the bursts from each PMT, calculates the ratio of their amplitudes to determine the photostability and tests if the lifetime, photostability, initial brightness and other measures are within user-adjustable limits. Analysis rates are ~10–30 cells/sec with cell selection rates of up to ~5 Hz, providing rapid measure of photophysical diversity and capability to screen libraries with 105 members in a matter of hours.
15. Richard Day (Speaker)
    Imaging protein network interactions inside living cells.
    Abstract

    Richard N. Day
    Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202 USA

    The development of non-invasive imaging techniques to visualize dynamic protein behavior inside living cells has become increasingly important for biomedical research. When coupled with the recent improvements in the photophysical qualities of the genetically encoded fluorescent proteins (FPs), these imaging approaches enable the quantitative measurement of protein behaviors and interactions in their natural environment. We are using fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) measurements obtained by fluorescence lifetime imaging microscopy (FLIM) to investigate the functional interactions between transcription factors and chromatin modifying proteins inside the living cell nucleus. This talk will provide an overview of how we are using FCS and FRET-FLIM to interrogate the functional interactions between the heterochromatin protein 1 α (HP1α), and the CCAAT/enhancer binding protein α (C/EBPα), a transcription factor that functions in the control of cellular differentiation. We propose that the HP1α-C/EBPα network of protein activities play critical roles in the epigenetic regulation of programs of cellular differentiation.
16. Alberto Diaspro (Speaker)
    Variations on the theme of fluorescence optical super resolution.
    Abstract

    Alberto Diaspro

    The advent of super resolution microscopy approaches, offering unlimited resolution in space, opens new window in the observation and study of cellular processes. We will discuss targeted and stochastic readout methods expanding to the concepts to multi-photon excitation(MPE), both in terms of resolution and localization precision accuracy. The interest lies in the access to 4D (x,y,z,t) information including the possibility of getting super resolution within thick biological objects like cell aggregates. To this end, individual molecule localization (IML) implemented within selective plane illumination microscopy (SPIM) will be addressed towards 3D super resolution imaging in thick biological samples including non linear photo-activation. Conventional and MPE-STED microscopy will be discussed reporting about the utilization of a single wavelength (SW) both for MPE and fluorescence depletion. A variety of architectures will be outlined and further variations on the super resolution theme addressed, including gated STED, correlative approach with atomic force microscopy (AFM) and potential of nanoscale lithography.
17. Michelle Digman (Speaker)
    Fluorescence lifetime mapping of p53 alterations in metabolism upon DNA damage.
    Abstract

    Swathi Bagilthaya1 and Michelle A. Digman1,2
    1 Department of Biomedical Engineering, University of California, Irvine
    2 University of New England, Armidale, Australia

    p53 is a tumor suppressor protein that regulates target genes involved in DNA damage migation and repair. If cells become stressed due to DNA damage, p53 will induce genes that trigger cell cycle arrest and/or apoptosis. p53 has a dual role in promoting oxidative phosphorylation (oxsphos) and glycolysis upon cellular stress. However, the metabolic function of p53 has not been fully explored. p53 metabolic activities may play an important role in normal growth, development, and tumor suppression, it might also be misused to help promote, rather than hinder, tumor development. To further assess p53 activity, we investigated the effect of metabolic changes under the same conditions and tested if the concentration of p53 influences the balance between apoptosis and DNA repair. We used the fluorescent lifetime imaging microscopy (FLIM) phasor approach to detect changes in oxsphos and glycolysis. Overall our data show the formation of p53 protein near the nucleolus and in areas near the inner regions of the nucleus. We also show that upon DNA damage the concentration of p53 increases by a factor of two independent of initial expression levels. The FLIM/Phasor data show that low concentrations of p53 is enough to trigger glycolytic response in the nucleus of the cells upon DNA damage with Cisplatin and under high expression levels, a new lifetime phasor is detected. This new lifetime correlates with dead phenotype cells and may be a new "apoptotic" lifetime signal. This work is supported in part by NIH grants P50 GM076516 and P41 GM103540.
18. Elliot Elson (Speaker)
    Relaxation of cellular metabolic steady states.
    Abstract

    Artem Melnykov, Yanfei Jiang, and Elliot Elson
    Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO 63110

    Differentiated cells maintain stable phenotypes over time and, after responding to stimuli, return to their baseline states. For example, a pancreatic islet β-cell responds to an increase in glucose concentration by secreting insulin and a neural signal causes an increase in the cytoplasmic calcium ion concentration to initiate an actin-myosin contraction in a skeletal muscle cell. After responding to these stimuli, these cells then relax to their pre-stimulus baseline states. We hypothesize that underlying these stable phenotypic states are biochemical reaction networks for metabolism and signaling that are in correspondingly stable non-equilibrium steady states (NESSs) and that will similarly relax to their baseline states after a concentration perturbation. We are attempting to discover whether specific cellular NESS systems are stable to perturbations, to measure the “energy landscapes” that govern transitions among different states of multi-stable NESS systems, and to determine the fluxes and kinetic rate constants that describe NESS systems in cells. One experimental approach to these objectives is to measure reaction fluxes using two-color fluctuation cross-correlation functions (Qian & Elson 2004; Qian & Elson 2010). This approach is technically difficult, however, and so we have developed a “light-jump” perturbation kinetic method. A pulse of light at a specified wavelength and of defined intensity and duration initiates transcription of a fluorescent protein. The transcription can be terminated by light at a different wavelength. We are using this approach to investigate the stability of a linear NESS system in which the rate of synthesis of the protein is controlled by light and the rate of ubiquitin-mediated degradation by the N-terminal amino acid (Bachmair, et al. 1986). We are also attempting to measure the kinetics of state switching in the bistable galactose signaling network (Acar, et al. 2005).
    [1] Acar, M., A. Becskei and A. van Oudenaarden (2005). "Enhancement of cellular memory by reducing stochastic transitions." Nature 435(7039): 228-32.
    [2] Bachmair, A., D. Finley and A. Varshavsky (1986). "In vivo half-life of a protein is a function of its amino-terminal residue." Science 234(4773): 179-86.
    [3] Qian, H. and E. L. Elson (2004). "Fluorescence correlation spectroscopy with high-order and dual-color correlation to probe nonequilibrium steady states." Proc Natl Acad Sci U S A 101(9): 2828-33.
    [4] Qian, H. and E. L. Elson (2010). Chemical fluxes in cellular steady states measured by fluorescence correlation spectroscopy. Single Molecule Spectroscopy in Chemical Physics and Biology. A. Graslund, R. Rigler and J. Widengren. New York, Springer. 96: 119-137.
19. Joerg Enderlein (Speaker)
    SOFI, MIET, and cryo-fluorescence: Alternative approaches to superresolution fluorescence microscopy.
    Abstract

    Joerg Enderlein

    The presentation introduces and discusses three unconventional and recently developed methods of superresolution fluorescence microscopy: Stochastic Optical Fluctuation Imaging, which uses the temporal fluctuations of emitters for improving spatial resolution; Metal-Induced Energy Transfer, which uses the electrodynamic coupling between fluorescent emitters and a metal surface for axial localization with nanometer resolution; and cryo-fluorescence microscopy, which allows localizing single molecules with sub-nm accuracy.
20. Patton Garay (Sponsor Talk)
    Application of advanced fluorescence techniques to study botulinum neurotoxin trafficking and activity in cultured mammalian neuronal cell lines.
    Abstract

    Patton E. Garay1, Nicholas G. James2, Justin A. Ross2, Chi-li Chiu2, David M. Jameson2 K. Roger Aoki1, and Ester Fernández-Salas1
    1 Department of Biological Sciences, Allergan Inc., Irvine, CA
    2 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawai`i at Manoa, Honolulu, HI

    Allergan, Inc., is a multi-specialty health care company focusing on pharmaceuticals in ophthalmology, dermatology, neurology, and urology . One of Allergan’s products is BOTOX®(onabotulinumtoxinA), which has been approved for a number of therapeutic and cosmetic procedures. Botulinum neurotoxin type A (BoNT/A) is the active ingredient in the complex utilized for BOTOX®. BoNTs are 150 kDa proteins consisting of three 50 kDa domains: a receptor binding domain (HC), a translocation domain (HN), and a metallo-protease domain (light chain, LC), which specifically cleaves one of three SNARE proteins to block neuromuscular transmission.
    In order to gain entry into a neuron, the HC binds a cellular receptor complex composed of proteins and gangliosides. Fibroblast growth factor 3 (FGFR3) and members of the synaptic vesicle glycoprotein family (synaptic vesicle protein or SV2) have been identified as potential cellular receptors for BoNT/A. A variety of advanced fluorescent techniques were employed to characterize the trafficking of the different BoNT/A domains within cultured neuronal cell lines. These neuronal cell lines stably express either a fluorescent FGFR3-Halo tag chimeric protein or a GFP fusion protein attached to various organelle markers. Using 3D particle tracking and fluorescence-lifetime imaging microscopy (FLIM), fluorescently labeled recombinant HC/A (rHC/A, 50 kDa) was observed to bind and travel with labeled FGFR3 on the plasma membrane. The intracellular trafficking of the rHC/A with FGFR3 through early endosomes and in synaptic vesicles was then analyzed using cross correlation raster image correlation spectroscopy (ccRICS), which examines the dynamic interaction of two fluorescent species. The cross correlation between the two particles peaked after 3 hours of treatment in SiMa cells. In addition, rHC/A was observed to traffic through the early endosome when the cells were placed in basal media. The movement of the rHC/A was also found to correlate with the movement of the synaptic vesicle when the cells were placed in depolarzing media. These observations suggest that the receptor and pathway that BoNT/A uses to gain entry into the cell is determined by the membrane potential of the cell. Overall, these data support a model where FGFR3 is part of a high-affinity receptor complex for BoNT/A on the cell surface.
21. Patton Garay (Poster)
    BoNT/A trafficking with FGFR3 in neuronal cell lines studied with cross correlation RICS.
    Abstract

    Patton E. Garay1, Nicholas G. James2, Justin A. Ross2, David M. Jameson2 K. Roger Aoki1, and Ester Fernández-Salas1
    1 Department of Biological Sciences, Allergan Inc., Irvine, CA
    2 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawai`i at Manoa, Honolulu, HI

    Botulinum neurotoxin serotype A (BoNT/A) is a versatile protein that is therapeutically used to treat a variety of neuromuscular and pain disorders. BoNT/A is a 150 kDa protein consisting of three domains, including a 50 kDa receptor binding domain (HC/A). To gain entry into neurons, the HC/A binds to a receptor complex composed of proteins and gangliosides. Members of the synaptic vesicle (SV2) glycoprotein family have been reported to mediate BoNT/A uptake by facilitating toxin binding and internalization during vesicle recycling in depolarized neurons. Previously, we identified the Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity component of the BoNT/A receptor complex. The interaction between FGFR3 and recombinant HC/A (rHC/A) was analyzed using Cross Correlation RICS (ccRICS). Differentiated SiMa, PC-12, and SH-SY5Y cells expressing a fluorescent FGFR3-HaloTag chimera were treated with Alexa-Fluor633-rHC/A. These proteins were observed to travel together on the membrane of SiMa and PC-12 cells with a diffusion rate of 3.69 µm2/s and, after vesicular endocytosis, the rHC/A-FGFR3 complex diffuses intracellularly at a rate of 0.2 µm2/s. The GccRICS (0, 0) of rHC/A and FGFR3 peaks in SiMa, PC-12, and SH-SY5Y cells 3, 6, and 9 hours post-treatment, respectively. We investigated the trafficking of rHC/A in PC-12 cells with GFP labeled markers for early endosomes and synaptic vesicles. The fluorescent signal from rHC/A cross correlated with early endosomes when the cells were treated in basal or depolarizing media. However, cross-correlation with synaptic vesicles was only observed when the cells were depolarized. These data suggest that BoNT/A interact with the endosomal and/or synaptic vesicle pathways depending on the cell’s polarization state. In summary, FGFR3 may be part of a high-affinity receptor complex for BoNT/A and the FGFR3-BoNT/A interaction may play a role in trafficking of the toxin in neuronal cell membranes.
22. Katharina Gaus (Speaker)
    Molecular insights into T cell signalling with single molecule localisation microscopy.
    Abstract

    Katharina Gaus
    Centre for Vascular Research, University of New South Wales, Sydney, Australia

    T cell activation begins with the formation of signalling complexes at the cell surface involving the T cell antigen receptor (TCR), the Src family kinase Lck and the adaptor protein, linker for activation of T cells (LAT). How early TCR signalling events are regulated to prevent inopportune signalling in resting T cells but efficient activation upon receptor ligation is poorly understood. We have established single molecule localization microscopy to determine how TCR engagement reorganizes signalling proteins on the molecular scale. Imaging single molecules in intact cells has provided new insights into the mechanisms of Lck clustering (Rossy et al. Nat Immunol 2013) and LAT recruitment (Williamson et al. Nat Immunol 2011) upon TCR activation. We are now extending this work to elucidate how the membrane environment (Owen et al. Nat Commun 2012) and topography (Owen et al. Biophys J 2013) affects protein interactions to better understand the molecular principles of TCR signalling regulation.
23. Enrico Gratton (Speaker)
    Chromatin structure and dynamics.
    Abstract

    Enrico Gratton
    Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine

    Chromatin structure, compaction and remodeling at the micro and nanometer scale have fundamental roles in many biological events. Chromatin compaction produces heterogeneity of the cell nucleus which results in structural and transport properties which have been only partially studied. Although the nucleosome structure has been in part deciphered, the topology of chromatin structure at the micron scale remains unresolved. In this work, based on the Gold Enhanced Nano Imaging (GENI) method we studied chromatin organization using the orbital 3D tracking technique applied to 20 nm gold nanoparticles (NPs). This method provides insight of local structure and transport properties at the nano scale by following the trajectories of gold nanoparticle that are trapped in the chromatin. Using two-photon excitation, the fluorescence of fluorophores that are in the nanometer proximity to the gold particle is strongly enhanced. We have recently shown that metallic NPs do not bleach or blink upon continuous illumination, are extremely stable, very bright and their luminescence spans over the visible spectrum. These characteristics allow us to track them for minutes thus providing 3D trajectories appreciably longer than those based on fluorescent proteins or quantum dots. For this study we have analyzed the motion of a large number of nanoparticles incorporated inside the nucleus of NIH3T3 live cells. By tracking with high precision in 3D these nanoparticles we can have ~5 – 30 minutes long trajectory. In ~30% of the cases, we have observed that the NPs remain in regions of apparent confined motion (clusters) and eventually they undergo a long (in the micrometer range) excursion. We have found that the NPs within the clusters move faster than when travelling between clusters. These results suggest a topology for the chromatin made of cavities where the NP can move fast. These cavities are connected by channels where the particles move slowly but in a constant direction. Additionally, we express histones in NIH3T3 cells with different colors of fluorescent proteins. We show that as the NP moves along the trajectory, the emission changes alternatively between the two colors indicating that the NP is sensing the local chromatin environment.
24. Enrico Gratton (Sponsor Talk)
    The SimFCS environment for analysis of fluctuations at the micro and nanoscale.
    Abstract

    Enrico Gratton
    Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine

    The software package SimFCS is a complete virtual machine for the acquisition, analysis and display of images acquired in different microscopy modalities. The major modules which are part of the SimFCS software comprise 1) the FCS module for analysis of single point FCS capable of analyzing almost all known data formats for diffusion, binding and PCH analysis. 2) A module for analysis of raster scan images for RICS and N&B determination of diffusion, binding, number and brightness from confocal microscope images. 3) A module for analysis of diffusion and MSD (mean square displacement) for images from fast cameras obtained in the TIRF and SPIM mode of operation. 4) A module for 3D particle tracking based on the orbital tracking approach. 5) A comprehensive module for the analysis of FLIM images acquired by most microscopes operating in the time and frequency domain. This part of the software is also used for the analysis of camera-based FLIM instruments from various manufacturers. The FLIM analysis is performed using the phasor approach pioneered by the LFD. This module has been recently improved with the addition of several new “phasor” based analyses for spectral phasor, z-phasor and diffusion phasors. Every year Globals organizes a workshop in Irvine during the last week of October with lectures and demonstrations about the various analysis methods offered by the SimFCS software. In this talk I will discuss some recent advances in the algorithms used by SimFCS for the analysis of camera based fluctuations for obtaining the MSD form images with applications to biological systems.
25. Taekjip Ha (Speaker)
    Advances in FRET at the single molecule level.
    Abstract

    Taekjip Ha
    University of Illinois at Urbana-Champaign

    FRET is a powerful biophysical technique that can measure distances and distance changes in the few nanometer length scale with precision as high as 3-5 angstroms. Late Robert Clegg pioneered the application of FRET into nucleic acids, for example determining the structure of DNA four-way junction. My laboratory has developed the single molecule FRET method and its extension into three and four colors and its combination with optical trap. Some of the recent applications of single molecule FRET in nucleic acids and nucleic acids-protein interactions will be presented including those of the DNA and RNA four-way junction, and nucleosome.
26. Per Niklas Hedde (Weber Prize Guest)
    Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells.
    Abstract

    Per Niklas Hedde1,2,6, Rene M. Doerlich2, Rosmarie Blomley2, Dietmar Gradl3, Emmanuel Oppong4, Andrew C.B. Cato4 & G. Ulrich Nienhaus2,4,5
    1 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA
    2 Institute of Applied Physics, Karlsruhe Institute of Technology & Center for Functional Nanostructures, 76131 Karlsruhe, Germany
    3 Zoological Institute, Department of Cell and Developmental Biology, Karlsruhe Institute of Technology, 76128 Karlsruhe, Germany
    4 Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76021 Karlsruhe, Germany.
    5 Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
    6 Correspondence should be addressed to phedde@uci.edu

    Raster image correlation spectroscopy (RICS) is an excellent way to study molecular movement such as protein diffusion and transport, or, receptor–ligand interactions within living cells and tissues [1]. From rasterscanned microscopy images, fluctuations in fluorescence intensity can be analyzed with spatio-temporal correlations revealing molecular motions in a spatially resolved manner. However, due to the diffraction-limit, conventional raster image correlation spectroscopy can only separate larger regions of interest; also RICS requires low fluorophore concentrations in the nanomolar range. Here, we applied stimulated emission depletion microscopy (STED) [2] to RICS in order to overcome these limitations. With imaging experiments on model membranes and live cells, we demonstrate that due to the enhanced spatial resolution, STED-RICS offers insight into micron-sized regions of interest as well as access to 10–100 times higher fluorophore concentrations [3].
    [1] Digman, M. A. et al., Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys. J. 89, 1317–1327 (2005).
    [2] Klar, T. A. et al., Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000).
    [3] Hedde, P. N. et al., Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells. Nat. Commun. 4, 2093 (2013).
27. Elizabeth Hinde (Speaker)
    Imaging the diffusive route of molecules in live cells by fluctuation analysis.
    Abstract

    Elizabeth Hinde, Enrico Gratton, and Katharina Gaus

    Dynamic changes in higher order chromatin structure modulate the accessibility of DNA toward transcription, DNA repair and replication. The spatiotemporal scale upon which these dynamics occur however, render them ‘invisible’ to live cell imaging. Here we present a novel method to measure the real time accessibility of chromatin that is based on pair correlation analysis of EGFP (an inert tracer) molecular flow throughout the chromatin network. From detected changes in the arrival time of EGFP molecules into and out of the chromatin, this method detects millisecond changes in protein access to condensed chromatin with sub-micron resolution. Using this method we have probed the naturally occurring changes in chromatin compaction during the cell cycle and in combination with laser micro-irradiation, monitored for the first time the in vivo structural rearrangements of chromatin during repair. The pair correlation method thus offers a unique opportunity to probe the directionality of intra-nuclear traffic, by measuring the accessibility of the nuclear landscape and the role it plays in determining the diffusive routes adopted by molecules. Given that pair correlation analysis can be performed anywhere in the cell, combined with other optical information or performed as a two channel acquisition to detect the molecular flow of interacting molecules, more recently we have employed this method to look at intracellular traffic in other sub-cellular compartments as well as cell uptake of external molecules in the context of drug delivery.
28. Ken Jacobson (Speaker)
    How Dengue virus enters cells via the DC-SIGN receptor.
    Abstract

    Ping Liu1, Marc R. Ridilla12, Aravinda M. de Silva2,3, Nancy L. Thompson4, and Ken Jacobson1,2
    1 Department of Cell Biology and Physiology
    2 Lineberger Comprehensive Cancer Center
    3 Department of Microbiology and Immunology
    4 Department of Chemistry, University of North Carolina at Chapel Hill

    DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) is a pattern recognition receptor which binds to the mannose or fucose structures present on a variety of pathogens and stimulates diverse immune responses. DC-SIGN forms domains on cell surfaces which function as portals for virus entry into dendritic cells, including HIV, Ebola, dengue and hepatitis C. In particular, dengue is a mosquito-borne viral infection, which has become a rapidly growing global health threat and half of the world’s population is now at risk. Many reports have shown that ectopically expressed DC-SIGN enhances productive dengue infection in different human cell types; however, detailed molecular-level studies on interactions between DC-SIGN membrane assemblies on cells and dengue virus (DENV) at the initial binding and internalization stages are lacking. By employing immunostaining, confocal imaging, single domain tracking, super-resolution direct stochastical optical reconstruction microscopy (dSTORM) imaging and flow cytometry assays, we show that cell surface DC-SIGN micro/nano-domains are sufficient to capture the small sized DENV (50 nm diameter), leading to efficient virus internalization and productive infection of the host cells. DC-SIGN nanodomains exhibit several different classes of lateral mobility, including unusual highly directed and rapid transport. At the initial binding stage, DENV is highly colocalized with cell surface DC-SIGN domains, indicating DC-SIGN is the major receptor for DENV; and, binding of DENV markedly increased the lateral diffusion of not only those nanoclusters to which it is bound but also other DC-SIGN nanoclusters in distal locations in a apparent global effect. Increased lateral diffusion may enhance the probability of bound DENV to encounter sites of internalization which occurred within a few minutes after incubating virus with NIH3T3 cells expressing DC-SIGN. This internalization led to massive new viral particle synthesis 24h after infection. By contrast, no internalization and virus replication were observed on wt NIH3T3 cells even after 72h of incubating with DENV. The results indicate that DC-SIGN capturing of DENV leads to rapid internalization of the viruses and productive infection thereafter.
    Supported by NIH R01 GM 41402 (KJ & NLT) and NIAID R01 AI 107731 (AMS).
29. Ankur Jain (Weber Prize Guest)
    Probing cellular protein complexes using single-molecule pull-down.
    Abstract

    Ankur Jain

    Cellular processes result from dynamic interactions between biomolecules. The gold standard method for investigating interaction between biomolecules is the pull-down assay. We have extended the conventional pull-down assay to its ultimate limit: the analysis of single biomolecular complexes. We achieve this goal by performing the pull-downs directly on to the surface of microscope slides and visualizing the captured biomolecules at single-molecule resolution using total internal reflection fluorescence (TIRF) microscopy. We name this technology single-molecule pull-down or SiMPull. Using SiMPull we are able to discriminate between multiple association states of a protein as well as determine the stoichiometry of interaction. This technology is widely applicable to an array of biological contexts, and is suitable to analysis of endogenous protein complexes from animal tissue. In particular, we have used SiMPull to investigate the architecture and assembly of mechanistic target of rapamycin complexes. The complexes captured from cell extracts on to our imaging chambers retain their functional activities: thus SiMPull can be used as a preparatory tool for single-molecule biochemical analysis on proteins that cannot be readily purified or reconstituted. Lastly, we extend this approach to the analysis RNA-protein complexes.
30. Nicholas James (Speaker)
    Analysis of functional interactions of LRRK2 in live cells.
    Abstract

    Nicholas G. James, Matthew S. Goldberg, Joseph P. Albanesi, and David M. Jameson
    1 Department of Cell and Molecular Biology, University of Hawaii at Manoa, JABSOM, 651 Ilalo St, Honolulu, HI
    2 Department of Pharmacology, UTSouthwestern Medical School, 5323 Harry Hines BLVD, Dallas, TX, 75390

    Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the most common genetic causes of Parkinson's Disease (PD) and have been shown to induce a phenotype that is identical to idiopathic PD. LRRK2 is a 280 kDa protein that catalyzes both GTPase and kinase reactions. The most common disease-linked mutations occur within the activation loop of the kinase catalytic domain and result in enhanced activity. Despite intensive searches for relevant substrates and for mutation-linked phenotypes, the precise cellular function(s) of LRRK2 remain to be discovered. Nevertheless, efforts at developing therapeutic strategies against PD have focused on methods to diminish LRRK2 kinase activity. Prior studies have shown that LRRK2 activity is stimulated upon self-association, which occurs preferentially on membranes. Based on our data and those of others, we hypothesize that LRRK2 cycles between a cytosolic, low-activity state and a membrane-bound, high-activity state in response to stimulus –dependent changes in post-translational modifications. To begin to test this hypothesis in live cells, we used number and brightness (N&B) analysis of cells transfected with LRRK2-mEGFP. This analysis revealed that LRRK2 was predominantly monomeric in the cytosol but preferentially associated into dimers and higher order oligomers on the plasma membrane (PM). Because LRRK2 lacks an obvious trans-membrane domain, we tested whether it could undergo palmitoylation, the covalent attachment of 16-carbon fatty acids to specific cysteines. LRRK2 was indeed palmitoylated in cells and, moreover, chemical inhibition of palmitoylation reduced its redistribution to the PM and partially suppressed its dimerization. Future studies will be aimed at applying N&B analysis to confirm and quantify the biochemical results obtained with palmitoylation inhibitors. To examine potentially relevant hetero-interactions of LRRK2, we co-expressed LRRK2-eGFP with mCherry-tagged synuclein, another protein implicated in PD pathogenesis. Cross-correlation RICS (ccRICS) analysis demonstrated that these two proteins co-migrate, suggesting that they are in the same macromolecular complexes. Prior studies from several groups had demonstrated a functional interaction between LRRK2 and synuclein, and our data suggest that they may interact physically as well. Further work will be required to determine if the interaction between the two proteins is direct or indirect, and to define the effects of post-translational interactions on its affinity. Taken together, our results support a model in which multiple mechanisms are used to control LRRK2 subcellular targeting, stability, activity and, potentially, neurotoxicity.
31. David M. Jameson (Speaker)
    The seminal contributions of Gregorio Weber to modern fluorescence spectroscopy and to protein chemistry.
    Abstract

    David M. Jameson
    Dept. Cell and Molecular Biology, John A. Burns School of Medicine, 651 Ilalo Road, BSB222, University of Hawaii, Honolulu, Hawaii 96813 USA. e-mail: djameson@hawaii.edu

    Gregorio Weber is acknowledged to be the person responsible for many of the more important theoretical and experimental developments in modern fluorescence spectroscopy. In particular, Weber pioneered the application of fluorescence spectroscopy to the biological sciences. His list of achievements includes: the synthesis and use of dansyl chloride as a probe of protein hydrodynamics; the extension of Perrin’s theory of fluorescence polarization; the first spectral resolution of the fluorescence of the aromatic amino acids and of intrinsic fluorescence of proteins; the first demonstration that both FAD and NADH make internal complexes; the first report on anilino-naphthalene sulfonates (ANS); the first description of the use of the fluorescence of small molecules as probes for the viscosity of micelles, with implications for membrane systems; a general formulation of depolarization by energy transfer; the discovery of the “red-edge” effect in homo-energy transfer; the development of modern cross-correlation phase fluorometry; the synthesis of several novel fluorophores, including pyrenebutyric acid, IAEDANS, bis-ANS, PRODAN and LAURDAN, designed to probe dynamic aspects of biomolecules.
    Gregorio Weber’s original and life-long motivation was to use fluorescence methods to probe the nature of proteins and in addition to his contributions to the fluorescence field, he was one of the true pioneers of protein dynamics. A study of his papers from the 1960's demonstrates that even then he regarded proteins as highly dynamic molecules. He rejected the view, common at that time after the appearance of the first x-ray structures, that proteins had a unique and rigid conformation. His contributions to the field of protein chemistry were recognized by the American Chemical Society in 1986, which named Weber as the first recipient of Repligen Award for the Chemistry of Biological Processes.
    In addition to these seminal contributions, Gregorio Weber also trained and inspired generations of spectroscopists and biophysicists who went on to make important contributions to their fields, including both basic research as well as the commercialization of fluorescence methodologies and their extension into the clinical and biomedical disciplines.
32. David M. Jameson (Speaker)
    Applications of phasors to in vitro systems including proteins, nucleic acids and membranes.
    Abstract

    David M. Jameson
    Dept. Cell and Molecular Biology, John A. Burns School of Medicine, 651 Ilalo Road, BSB222, University of Hawaii
    Honolulu, Hawaii 96813 USA, e-mail: djameson@hawaii.edu

    The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions, such as solvent relaxation and Förster Resonance Energy Transfer. Phasors can be calculated using either frequency domain or time-domain data. A principal advantage of the phasor method is that it provides a model-less approach to time-resolved data amenable to visual inspection. The phasor approach has become a valuable tool for both in vitro studies and for fluorescence lifetime imaging microscopy (FLIM) studies with live cells. In addition to intensity decay, e.g., lifetime data, time-resolved fluorescence can be used to provide information on the rotational mobilites of molecules. This information is acquired in the time-domain using time-decay anisotropy or its frequency domain equivalent known as dynamic polarization, and the phasor method can also be applied to these data. In this talk I shall discuss the basic phasor approach and illustrate its application to diverse systems, including proteins, nucleic acids and membranes.
    KEYWORDS: lifetimes, phasors, FRET, time-decay anisotropy, FLIM
    [1] Jameson, D.M., Gratton, E., and Hall, R.D. (1984) App. Spectros. Rev. 20:55-106. The Measurement and Analysis of Heterogeneous Emissions by Multifrequency Phase and Modulation Fluorometry.
    [2] Stefl, M., James, N.G., Ross, J.A. and Jameson, D.M. (2011) Anal. Biochem. 410:62-69. Application of Phasor Plots to In Vitro Time-Resolved Fluorescence Measurements.
    [3] James, N.G., Ross, J.A., Stefl, M. and Jameson, D.M. (2011) Anal. Biochem. 410:70-76. Application of Phasor Plots to In Vitro Protein Studies.
33. Jay Robert Knutson (Poster)
    Super-resolution via transiently activated quenching (STAQ) - A route to multicolor, low-power nanoscopy.
    Abstract

    Tilman Rosales, Jianhua Xu ,Michael Brenner, and Jay R. Knutson
    Optical Spectroscopy Section, LMB, BBC, NHLBI, NIH, Bethesda 20892

    Nanoscopy is becoming a common tool in cell biology, especially given the wide utility and simple instrumentation needs for stochastic localization methods like PALM and STORM. The pointillist painting approach is not limited to still life, but methods like STED (and related RESOLFT) are much faster. The latter require somewhat more complex optics and expense, but can provide true video rate imaging. They have also suffered from the low and seldom overlapped cross sections of the dyes, making STED illumination approach damage limits and making multicolor STED imaging a rarity.
    We have developed a general scheme for pairing luminescent dyes with quenching partners that are innocuous unless excited themselves; they provide a low-power “off switch” for dyes throughout the visible. We demonstrate resolution near 50nm using only 12mW of many-picosecond wide pulses at focus for 775nm NIR light. This “donut” beam is simply constructed with a spiral phase plate.
    Two-color images of microtubules are shown, and we will also discuss the development of an inexpensive laser diode-driven nanoscope.
34. Markéta Kubánková (Poster)
    Molecular rotors as novel probes of protein aggregation.
    Abstract

    Markéta Kubánková and Marina K. Kuimova
    Department of Chemistry, Imperial College London, United Kingdom

    Molecular rotors are a group of small synthetic fluorophores which act as sensors of microenvironmental restriction. The fluorescence intensities and lifetimes of these molecules strongly depend upon the viscosity of their immediate surroundings. We took advantage of the environment-sensitive photophysics of molecular rotors to investigate protein assembly into amyloid fibrils. Amyloid materials are a hallmark of several serious human diseases, nevertheless they are also known to have important functional roles in organisms. The physicochemical conditions that lead to protein aggregation are still poorly understood and the characterisation of the pathways of assembly is an important goal amongst researchers. We monitored in vitro protein aggregation by measuring fluorescence decays of molecular rotors using time-resolved fluorescence spectroscopy and microscopy. Phasor analysis of the data revealed mechanistic details of aggregation that were not observed previously with alternative techniques. The approach also permits quantitative measurements of the mechanical properties (i.e. the viscosity) of the aggregates. As such, our results demonstrate that molecular rotors are a powerful tool for investigating protein aggregation.
35. Joanna Kwiatek (Poster)
    Fluorescence microscopy approaches and phasor analysis for detecting nanoscale lipid phase separation.
    Abstract

    Joanna Kwiatek*1, Aleš Benda1,2, Dylan M. Owen3, and Katharina Gaus1
    1 ARC Centre of Advanced Molecular Imaging and Australian Centre for NanoMedicine, School of Medical Sciences
    University of New South Wales, Sydney, NSW, Australia
    2 Biomedical Imaging Facility, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, NSW, Australia
    3 Department of Physics and Randall Division of Cell and Molecular Biophysics, King’s College London, London, UK
    *E-mail: j.kwiatek@student.unsw.edu.au

    The lipid raft hypothesis postulated the existence of highly dynamic, nano-scaled lipid domains in cellular membranes that are regulated by the dynamic lipid phase behaviour. Phase segregation into liquid ordered phase (Lo) and liquid disordered phases (Ld) yields biophysically and biochemically discrete platforms. However, the existence and the role of the nanoscale lipid phases in cell membranes remain controversial, mainly because of the technical difficulties in directly detecting small and transient lipid domains. Here, we will present evidence of different lipid phases in model membranes and live cell membranes acquired with different microscopy methods. We combined phasor analysis of spectrally resolved fluorescence lifetime imaging (FLIM) and fluorescence spectrally resolved correlation spectroscopy (FSCS) with the membrane order-sensitive dye. We acquired information about the local polarity and dipole relaxation rate as well as evidence for the coexistence of two lipid phases in cell membranes.
    KEY WORDS: membrane order, membrane order sensitive dyes, FLIM and phasor analysis, fluorescence correlation spectroscopy.
36. Melike Lakadamyali (Speaker)
    Single molecule protein counting with photoactivatable and photoconveritble fluorescent proteins.
    Abstract

    Nela Durisic, Lara Laparra Cuervo, Angel Sandoval Alvarez, Joseph Steven Borbely, and Melike Lakadamyali:
    ICFO-Institut de Ciencies Fotoniques

    Single molecule localization based super-resolution microscopy methods (Stochastic Optical Reconstruction Microscopy, STORM, Photoactivated Localization Microscopy, PALM and others) have generated great excitement in the recent years. These methods allow far-field fluorescence imaging at the nanoscale beyond the diffraction limit. To break the diffraction limit, these methods take advantage of distinct molecular states (dark and bright) of fluorescent probes. They have provided valuable information about sub-cellular structures and macromolecular assemblies. One dream is to be able to not only image the nanoscale organization of proteins with these methods, but to also determine protein stoichiometries and count protein numbers at the nanoscale. However, this goal has been confounded by the complex photophysics of fluorescent probes that lead to over and under-counting artefacts. We have used a nanotemplate with well-defined stoichiometry to characterize these complex photophysics and found that most fluorescent proteins have moderate photoactivation efficiency (50-60%). Our analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy and we propose best-performing fluorescent proteins for quantitative measurements.
37. Hongjiang Li (Poster)
    Polarity maintenance by clustering and extracellular matrix in Arabidopsis.
    Abstract

    Hongjiang Li1.2, Satoshi Naramoto2,3, Krzysztof Wabnik2,1, Riet De Rycke2, Nasser Darwish-Miranda1, Jan Dettmer2,4, and Jiří Friml1,2,*
    1 Institute of Science and Technology Austria (IST Austria), Am Campus 1, 3400 Klosterneuburg, Austria
    2 Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, B-9052 Gent, Belgium
    3 Molecular Membrane Biology laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan
    4 Present Address: Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
    5 Present Address: Cell Biology Division, Department of Biology, University of Erlangen-Nuremberg, Staudtstrasse 5, 91058 Erlangen, Germany
    *Correspondences: jiri.friml@ist.ac.at

    Cell polarization is fundamental for plant growth and morphogenesis. The PIN auxin transporters are trans-membrane proteins required for polarity maintenance in Arabidopsis. The clustering of PIN polar cargos within the plasma membrane (PM) has been proposed to be crucial for polarity maintenance in root cells. However, the characteristics of PIN clusters, the mechanisms controlling PIN clustering and polarity, and the structural requirements of clustering remain unclear due to the technical challenges in the past.
    In this work, immunogold electron microscopy, laser scanning confocal microscopy and spinning disc confocal microscopy had been used to observe and characterize PIN clusters. In addition, based on the plant-specific cell wall structure regulates polarity and constrains PM protein diffusion, chemical interference with cell wall and cytoskeleton structures, mutants with defects in the cell wall and cytoskeleton had been analyzed for altered PIN protein clustering and polarity maintenance.
    Moreover, we developed software to analyze and track the clusters with sizes ranging the resolution limits of optical microscopy on the face of the membranes that points towards the direction of growth, which is perpendicular to the focal plane. In this case, spinning disc microscopy was chosen because of its relatively good time resolution, compatible with the plant growth dynamics.
    Taken together, this work uncovers novel cell polarity maintenance mechanisms in plants, and provides a conceptual framework for the interpretation of polarity maintenance in other systems.
38. Lei Li (Poster)
    Application of soluble Pyridinium salts as visible fluorescence reagents in protein structure and dynamics.
    Abstract

    Lei Li, Mengfang Chang, Xiaodan Cao, Menghui Jia, Sanjun Zhang, Haifeng Pan*, and Jianhua Xu
    State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai, China
    Corresponding author: hfpan@phy.ecnu.edu.cn

    Many fluorescent labels have been widely used to investigate conformational dynamics and molecular interactions, or track the movement of proteins in order to gain insight into their biological functions. In our study, some water soluble pyridinium salts were used as fluorescent probes (excitation at 450nm, emission from 500nm to 650nm) for detection of proteins (such as BSA) by interacting with Trp (or other amino groups) through π-π stacking and hydrophobic effect. Increasing the concentration of BSA solution, the steady state fluorescence intensities of pyridinium salts were shown a large range of enhancement accompanied by the blue-shift of emission peaks. From the time resolved fluorescence measurements by TCSPC technique and DAS analysis, it is found that the single short lifetime becomes multiexponential decay with two longer different lifetime components, which indicates two disparate interacting sites of BSA protein to the pyridinium salt probes. Moreover, the proportion of these two long lifetimes appears bigger and blue-shifted compared to the intrinsic short lifetime of the probe. Candidate mechanisms and the analysis of time resolved emission spectra will be discussed.
39. Leslie M Loew (Speaker)
    Two-photon fluorescence study of electrical compartmentation in dendritic spines.
    Abstract

    Erika Hoyos-Ramirez, Corey D. Acker, Ping Yan, and Leslie M. Loew
    University of Connecticut Health Center, Farmington, CT 06030

    Dendritic spines are the sites of excitatory post-synaptic input in many mammalian brain regions. They are pleomorphic membrane protrusions connected to the dendrite through a spine neck that is variable in diameter and length. Spines have been widely studied as biochemical compartments that can individually process patterns of synaptic inputs; spines are also likely to play a key role in the transfer of electrical signals to the dendritic shaft and the soma, but the ability of spines to serve as electrical compartments has been controversial. This represents a fundamental question in neuroscience since the postsynaptic responses to synaptic input, as well as the likelihood that a synapse will undergo long-term plasticity, will largely depend on the interplay between biochemical and electrical signals within individual dendritic spines. Our laboratory has previously developed single-voxel 2-photon voltage sensitive dye imaging to measure voltage changes directly from single spines in acute brain slices. In the present study, we combine this method with 2-photon MNI-glutamate uncaging and fluorescence recovery after photobleaching (FRAP), in order to measure the voltage responses in individual spines to unitary synaptic stimuli, and to study the effect of spine neck resistance (Rneck) on the excitatory post-synaptic potential (EPSP) amplitudes. Our results show a linear relation between the amplitude of the EPSP measured at the spine and Rneck. These data provide experimental evidence for previous theoretical predictions that large Rneck can enhance the EPSPs locally. However, our directly measured spine voltage responses are generally of lower amplitude than previous estimates inferred from restricted diffusion through the neck or spine calcium influx. Our experimental results were recapitulated in a compartmental model of a L5 pyramidal neuron, which could also reproduce the spine to soma EPSP amplitude ratio observed experimentally. These results suggest that Rneck is the primary determinant of the amplitude of the EPSP measured in the spine and also determines the attenuation of the EPSP measured at the soma.
40. Jose Luiz de Souza Lopes (Speaker)
    Steady-state and time-resolved fluorescence investigations on DGAT1 lipid binding sites.
    Abstract

    Jose L.S. Lopes, Ana P.U. Araujo, and David M. Jameson
    1 Institute of Physics of Sao Carlos, University of Sao Paulo, Sao Carlos, SP, Brazil
    2 Dept. Cell and Molecular Biology, University of Hawaii at Manoa, Honolulu, HI, USA
    e-mail: zeluiz@ifsc.usp.br

    Diacylglycerol acyltransferase 1 (DGAT1) is the enzyme, in many living organisms, recruited in the final step of the reaction that converts acyl-CoA and diacylglycerol into triglyceride. In mammals, DGAT1 is a large transmembrane protein, which is seen as a potential target for treating obesity, but with a three-dimensional structure not yet determined. In this study, peptides corresponding to the putative binding sites of DGAT1 to the substrates were synthesized to investigate their interactions with membrane models and lipid substrates, using fluorescence methodologies. Three small linear peptides with 13, 15 and 22 amino acid residues, containing one, two and three tryptophan residues, respectively, were studied in aqueous solution and in the presence of lipids. The binding of these peptides to the lipids was assayed using polarization, quenching, and time-resolved analyses, showing dependence with the net charge on surface of the membrane model. After binding, the local mobilities of the Trp residues were decreased; their maximum emission shifted to lower wavelengths, and the lifetimes increased. Phasor plots were employed to analyze peptide-lipid binding and resulted in a trajectory that lies along a line connecting the points of the unbound and bound peptide. The application of phasor plots to investigate this system will be discussed.
41. Yves Mély (Speaker)
    Site-selective characterization of protein-nucleic acid interactions by using environment-sensitive nucleotide and amino-acid analogues.
    Abstract

    Yves Mély1, Marianna Sholokh1, Viktoriia Postupalenko
    1 Andrey Klymchenko1, Vasyl Pivovarenko 2 Alain Burger3,and Yitzhak Tor4
    1 Biophotonics and Pharmacology laboratory, UMR 7213 CNRS, Université de Strasbourg, 74 route du Rhin, ILLKIRCH, France.
    2 Institut de Chimie de Nice, UMR 7272, Université de Nice, Parc Valrose, Nice, France
    3 Department of Chemistry, National Taras Shevchenko University of Kyiv, Kyiv, Ukraine
    4 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA

    Interactions between proteins and nucleic acids are critical in most cell functions. Therefore, to better understand the mechanisms governing these cell functions, there is a strong need to characterize both qualitatively and quantitatively these interactions. Due to their exquisite sensitivity, fluorescence-based techniques are ideally suited to obtain general information on the binding and dynamics of interacting partners. However, they are more limited in providing site-selective information on a given amino-acid or nucleotide. To fill this gap, we have developed original nucleotide and amino acid analogues based on 3-hydroxychromone (3HC) probes that show a two band emission highly sensitive to minute changes in their environment. These analogues were used to characterize the interaction of the nucleocapsid protein NCp7, a key HIV-1 protein, with its nucleic acid targets. Two amino-acid analogues were developed and found to perfectly mimic the highly conserved Trp37 residue that plays a key role in NCp7 structure and activity. These conservative Trp substitutes were observed to sensitively and site-selectively report on the interaction of the labelled protein with oligonucleotides, through changes in the ratio of their two emission bands, as a result of local hydration changes that could be quantified. We also designed a 3HC-based fluorescent nucleotide analogue that possesses the characteristics of a universal base and that is up to 50-fold brighter than the “gold standard” 2-aminopurine, when inserted in oligonucleotides. This nucleotide analogue was successfully applied to monitor local conformational changes upon interaction with NCp7 and obtain new information on the mechanism of the nucleic acid chaperone activity of NCp7. Finally, we used and characterized thieno-deoxyguanosine (thG), a nearly perfect fluorescent substituent of the guanine base. Being included in the loop of a nucleic acid target of NCp7, it provided key information on the interaction of the substituted G with the Trp37 residue, as well as on its subsequent local changes.
    This work was supported by ANR (07-BLAN-0287), FRM (DCM20111223038), ANRS and the European Project THINPAD (FP7 – Grant Agreement 601969).
42. Alex Macmillan (Poster)
    Spectral phasor approaches for imaging drug delivery by nanoparticles.
    Abstract

    Alex Macmillan1, Elizabeth Hinde3, Hien T.T. Duong2, Cyrille Boyer2 & Renee Whan1
    1 Biomedical Imaging Facility, University of New South Wales, Australia 2052
    2 Australian Centre for Nanomedicine and Centre for Advanced Macromolecular Design School of Chemical Engineering, University of New South Wales, Sydney, Australia 2052
    3 Centre for Vascular Research, University of New South Wales, Sydney, Australia 2052

    Recent publications have illustrated the advantages of fluorescence lifetime imaging (FLIM) and phasor analysis to monitoring drug delivery by nanoparticle [1-3]. FLIM has provided a means to directly monitor and demonstrate time-release of the anti-cancer drug Doxorubicin (DOX) from polymeric and iron nanoparticles. Importantly, it allows us to distinguish between the free and conjugated doxorubicin localisation within live cells. Phasor analysis provides an alternative to the more complicated traditional exponential fitting of data which can be difficult to interpret when multiple components are present [4]. Although, FLIM phasor analysis simplifies the previously “expert only” technique, it still requires pulsed laser sources and expensive timing electronics in order to record the time delays at each pixel of the FLIM image. In this presentation we will discuss the application of spectral phasor analysis [5] in monitoring drug release which can be implemented on confocal microscopes with spectral detectors.
    References
    1. Basuki JS, Duong HTT, Macmillan A, Erlich RB, Esser L, Akerfeldt MC, Whan RM, Kavallaris M, Boyer C, Davis TP: Using Fluorescence Lifetime Imaging Microscopy to Monitor Theranostic Nanoparticle Uptake and Intracellular Doxorubicin Release. ACS Nano 2013.
    2. Sagnella SM, Duong HTT, Macmillan A, Boyer C, Whan RM, McCarroll JA, Davis TP, Kavallaris M: Dextran based Doxorubicin Nanocarriers with Improved Tumour Penetration. Biomacromolecules 2013.
    3. Basuki JS, Duong HTT, Macmillan A, Whan R, Boyer C, Davis TP: Polymer-Grafted, Nonfouling, Magnetic Nanoparticles Designed to Selectively Store and Release Molecules via Ionic Interactions. Macromolecules 2013, 46(17):7043-7054.
    4. Digman MA, Caiolfa VR, Zamai M, Gratton E: The Phasor Approach to Fluorescence Lifetime Imaging Analysis. Biophysical Journal 2008, 94(2):L14-L16.
    5. Fereidouni, F., A.N. Bader, and H.C. Gerritsen, Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images. Optics Express, 2012. 20(12): p. 12729-12741.
43. Leonel Malacrida (Speaker)
    Phasor plots for lifetime and spectrum analysis of LAURDAN and PRODAN emissions in membrane: A new perspective for membrane biophysics studies.
    Abstract

    Leonel S. Malacrida1,2, Rosina Toledo-Gallo1, Arturo Briva1, Ana Denicola3, Enrico Gratton4, and David M. Jameson5
    1 Área de Investigación Respiratoria (AIR, )Departamento de Fisiopatología, Hospital de Clínicas, Universidad de la Republica, Montevideo, Uruguay
    2 Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, Uruguay.
    3 Laboratorio de FisicoquímicaBiológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
    4 Laboratory for Fluorescence Dynamics, University of California, Irvine, CA, USA
    5 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI, USA

    Since its introduction by Weber [1], fluorophores in the PRODAN series have contributed to our understanding of hydration and packing in biological membranes. Based in their solvatochromism properties PRODAN and LAURDAN have long red shifts associated with the dipolar relaxation of the environmental water molecules around the probe. Exploiting this property, here we apply methods based on lifetime determinations and phasor plots as well as steady-state measurements using the spectral phasor approach, for analysis of the behavior of LAURDAN and PRODAN in multilamellar vesicles. The lifetime Phasor approach (Jameson et al., [2]) uses a plot of M.sin(Φ) versus M.cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. With Spectral phasors, introduced by Fereidouni et al [3], the steady-state fluorescence spectrum is Fourier transformed, resulting in two coordinates in x and y (cosine and sine, respectively) used for a scatter plot (Spectral phasor).
    The temporal Phasor approach shows significant improvement compared with older methods as regards discrimination of the effects of temperature, cholesterol content and drug addition, in our membrane model systems. This approach is very convenient for characterization of complex systems wherein lifetime heterogeneity and relaxation processes are present.
    The Spectral phasor approach is a very useful method for characterization of subtle changes in membrane hydration and packing, induced by temperature or cholesterol concentration.
    The major advantage of both methods is that they provide a model-less approach, which is relevant to complex studies on native systems, where endogenous fluorescence can introduce undesired mistakes. On the other hand, all the basic properties of the vector and Fourier series can be used to determined proportion of states (i.e., linearity, higher harmonics). Examples of the application of both methods to membrane systems will be given.
    [1] Weber et al, Biochemistry, 1979.
    [2] Jameson et al, Appl. Spectosc. Rev., 1984.
    [3] Fereidouni et al, Opt. Express, 2012.
44. Gerard Marriott (Speaker)
    New optical probes to quantify and manipulate target proteins and their macromolecular complexes.
    Abstract

    Gerard Marriott, Alexander Hoepker, and Yuling Yan

    Fluorescence techniques provide extraordinary high levels of sensitivity, specificity and selectivity and they are well-suited for high-throughput screening and imaging of target proteins and drugs in biological samples. One of most effective methods to quantify protein complexes is to use fluorescence anisotropy (FA) techniques to measure a difference in the hydrodynamic properties of the free and target-bound states of a suitable FA probe. We will introduce new genetically encoded fluorescent protein that are specifically designed for FA-based detection and imaging of target proteins. The new encoded FA sensors are unique in having only 40%~67% of the mass of GFP and far longer fluorescence lifetimes that result in low FA values (<0.2) for the free state of the sensor and much higher values, to the limiting anisotropy value, in a target bound complex. We will present suitable examples to demonstrate the effectiveness of these new genetically encoded probes as FA and FRET based sensors, including new sensors for small GTPases, for in vitro and in vivo analysis of target proteins.
    In the latter part of my talk I will introduce new optical switch probes and associated approaches to reversibly control with high spatial resolution the physico-chemical micro-environment of stem cells.
45. David Millar (Speaker)
    Conformational dynamics of single G protein coupled receptors.
    Abstract

    Jeffrey J. Liu, Rajan Lamichhane, Raymond C. Stevens, and David P. Millar
    Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

    G protein coupled receptors are the integral membrane proteins that detect extracellular ligands and mediate signal transduction. Binding of ligands triggers a cascade of conformational changes, which are recognized by intracellular effectors. Chemically distinct ligands often trigger different signaling responses or activate different signaling pathways. Despite the availability of high-resolution crystal structures of receptor-ligand complexes, the molecular basis for ligand-dependent signaling responses is still unknown. We developed a single-molecule fluorescence system to observe the β2-adrenergic receptor (β2AR) switching between inactive and active conformations in real-time within a native-like membrane environment. Receptor constructs, labeled in specific trans-membrane helices with a bright and photostable dye, were reconstituted in phospholipid nanodiscs, tethered to a quartz surface and monitored over time by TIRF microscopy. The ligand-free receptor was observed to spontaneously fluctuate between two discrete intensity states, assigned to inactive and active conformations, and the lifetimes in each state were determined from dwell time analysis. The receptor was also examined in the presence of a series of β2AR ligands spanning a range of pharmacological efficacy. Two-state fluctuations were observed in all cases, but the lifetimes of each conformational state and the equilibrium conformer distribution were different for each ligand. Agonist ligands were observed to stabilize the active receptor conformation, while an inverse agonist stabilized the inactive conformation. These observations highlight the intrinsically dynamic character of β2AR and reveal a linkage between the pharmacological efficacy of a ligand and it’s ability to stabilize specific receptor conformations.
    Supported by NIH Road Map grant P50 GM073197.
46. Pierre Moens (Speaker)
    Modes of diffusion of Cholera toxin bound to GM1 on live cell membrane by image mean square displacement (iMSD) analysis.
    Abstract

    Pierre DJ Moens, Michelle A Digman, and Enrico Gratton

    The image-Mean Square Displacement technique (iMSD) applies the calculation of the mean square displacement (MSD) used in single molecule tracking to images without resolving single particles. The iMSD plot obtained is similar to the MSD plot obtained using the single-particle tracking technique and is used to reconstruct the protein diffusion law. From these plots, we can determine whether the labeled molecules are undergoing pure isotropic, restricted, corralled, transiently confined or directed diffusion. In our study total internal reflection fluorescence (TIRF) microscopy images were taken of Cholera toxin subunit B (CtxB) membrane labeled cells. We mapped the modes of CTxB diffusion when bound to GM1 in live cell membranes and found different types of diffusion across the cell including isotropic, confined and transiently confined. However, no directed diffusion could be observed. We show that repeated illumination of the same area of the cell leads to a transition of diffusion type from confined to isotropic diffusion mode, highlighting a potential artefact that can be produced by performing FRAP experiments to investigate diffusion processes at the cell membrane. We also combined the iMSD analysis with number and brightness analysis to evaluate the influence of cluster formation on the diffusion of CTxB/GM1. We find that CTxB/GM1 are binding to immobile structures or are incorporated in areas of extremely slow diffusion in the cell membrane and that CTxB/GM1 are dynamically partitioning in membrane nanodomains as small as 60 nm in diameters. We show that the formation of clusters favors isotropic diffusion and reduces the rate of diffusion. We also report that in confined diffusion regime we observe a large range of diffusion rates demonstrating that the size of the confinement does not determine the diffusion rates and suggesting that the mechanism of confinement is different from the mechanism controlling CtxB/GM1’s diffusion rates.
47. Joachim Mueller (Poster)
    Optical dissection of peripheral membrane proteins by fluorescence fluctuation spectroscopy.
    Abstract

    Elizabeth Smith, Jared Hennen, Yan Chen, and Joachim D. Mueller
    School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455

    Peripheral or extrinsic membrane proteins associate temporarily with the membrane to perform a variety of cellular processes including signal transduction, cytoskeletal membrane interactions, membrane trafficking and enzymatic activities like phospholipid metabolism and catabolism. Membrane association and dissociation of peripheral membrane proteins provide a mechanism for triggered conformational changes that serve to regulate protein-protein interactions and biological activity. Since peripheral membrane proteins associate reversibly with the membrane, they often exist in two pools: a membrane-bound form and a free soluble form in the cytoplasm. The challenge for quantitative fluorescence microscopy is to uniquely distinguish the signal contributions from each layer to identify the concentration and assembly state of each pool of proteins. Z-scan fluorescence fluctuation spectroscopy allows us to untangle the fluorescence contributions of the different pools of proteins and establish their concentrations and assembly state. We establish the theoretical foundation of the technique and verify the method experimentally before applying it to the study of retroviral assembly. The Gag polyprotein plays a pivotal role in the assembly and release of retroviruses. Recent studies indicated fundamental differences between the early assembly events of two retroviruses, human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1). While HIV-1 Gag requires the onset of cytoplasmic Gag-Gag interactions to promote translocation to the inner leaflet of the plasma membrane, HTLV-1 Gag appears to engage the plasma membrane as a monomer. Since the MA domain of Gag is the primary driver of Gag association with the inner leaflet of the plasma membrane, this study investigates the peripheral membrane protein MA by z-scan FFS to shed light on the ability of HTLV-1 Gag to bind to the membrane as a monomer and to identify its oligomeric state at the membrane. Supported by NIH (R01GM064589) and NSF (PHY-0346782).
48. Sua Myong (Speaker)
    Single Molecule Study on telomeres.
    Abstract

    Sua Myong
    University of Illinois at Urbana-Champaign

    Human telomeres terminate in a single-stranded 3’ overhang that plays a pivotal role in chromosome end protection. The G-rich overhang serves as the substrate for telomerase which extends telomeres by adding tandem TTAGGG repeats. The G-rich single stranded DNA at the 3’ end of human telomeres can self-fold into G-quaduplex (GQ). However, telomere lengthening by telomerase or the recombination-based alternative lengthening of telomere (ALT) mechanism requires protein loading on the overhang. We used single molecule fluorescence detection to study the conformational dynamics in telomeric overhang and how this modulates its accessibility to proteins associated with telomere maintenance and lengthening. We have also established a platform that enables detection of real-time telomerase extension.
49. Kazuhiro Oiwa (Poster)
    In vitro dynamic alignment and vortex formation of microtubules driven by axonemal dyneins.
    Abstract

    Naoki Kanatani, Hiroaki Kojima, and Kazuhiro Oiwa

    In vitro motility assays, in which individual fluorescently-labeled protein filaments are observed moving on protein motor-coated surface, provide a powerful tool for studies on ensemble behavior of self-propelled particles. With using the in vitro motility assay, we have found self-organization phenomena of a large number of microtubules driven by axonemal dyneins. The materials we used are inner arm dynein subspecies c and g of Chlamydomonas flagella, which are single-headed dynein composed of one heavy chain and two types of light chains, actin and p28 (dyein c) or centrin (dynein g). These dyneins adsorbed on a glass surface at densities higher than 1000 molecules µm-2 are capable of moving microtubules on glass surface at velocities of 6-12 µm/sec in the presence of 1 mM Mg-ATP at 23 ˚C. Microtubules with length of 24 ±12 µm (n = 95) did not move in the absence of ATP but bound to dynein-coated glass surface being slightly aligned by flow. Upon the addition of 1 mM Mg-ATP, microtubules started moving smoothly. Collisions between moving microtubules were often observed. Upon these collisions, microtubules often aligned each other. These interactions between microtubules lead to nematic alignment. Within a few minutes, streams of moving microtubules spontaneously appear, which show meandering motions with a very large distance (longer than 400 µm) compared to the microtubule's length and size of a dynein molecule. The number of microtubules in a stream increased over time (>1 µm in width and >300 µm in length). Finally, these microtubule streams grew into vortices within tens of minutes. These results combined with numerical simulations suggest that non-white noise and nematic interactions of self-propelled particles can create the large-scale ordered structures.
50. Uwe Ortmann (Poster)
    With photon patterns towards species selective microscopy.
    Abstract

    Felix Koberling, Volker Buschmann, Benedikt Krämer, Steffen Ruettinger, Marcelle König, Sebastian Tannert, Matthias Patting, Uwe Ortmann, and Rainer Erdmann

    Fluorescence Correlation Spectroscopy is nowadays a standard tool in biophysics and more and more used also in complex environments, like in cell biology and mutilabel applications. Common problems which complicate these experiments, like detector afterpulsing and spectral crosstalk, have recently be overcome by looking at the nanosecond arrival time of the detected photons after pulsed excitation. By comparing the nanosecond arrival time of every photon with reference decay patterns it is possible to identify artifact signals and to distinguish and separate photons coming from species with different emission lifetime properties.
    We will show current results for absolute concentrations measurements of diffusing proteins in live cells as well as dual colour FCCS binding studies. Especially in dual colour applications when two pulsed lasers are not available the decay pattern analysis allows quantitatively to separate the pulsed laser excited fluorescence from the cw excited one to overcome spectral bleedthrough problems.
    In our latest work we generalize the concept of single photon classification beyond one-dimensional patterns and correlation spectroscopy in diffusion. A simplified approach for photon ensembles is a decay pattern matching analysis describing the photon ensemble decay with a linear combination of reference decays which can be highly multi-exponential and do not have to be described itself mathematically. We will show this approach to be a powerful alternative to multi-exponential decay fitting based FLIM and consider it even applicable to burst classification in single molecule experiments.
51. Dylan Owen (Speaker)
    A new Bayesian analysis method for cluster identification in single-molecule localisation microscopy data.
    Abstract

    Patrick Rubin-Delanchy, Garth Burn, Nick Heard, Andrew Cope, and Dylan M Owen

    Single-molecule identification-based super-resolution microscopy techniques such as photo-activated localisation microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) produce pointillist data sets of molecular coordinates. While many algorithms exist for the identification and localisation of molecules from the raw image data, including Bayesian approaches, methods for interrogating the resulting point-patterns for statistical parameters such as clustering has remained relatively under-studied. Here we present the first model-based Bayesian approach to evaluate molecular cluster assignment proposals which in this case were generated from Ripley’s K-function-derived analysis. The method is also the first to take full account of the individual localisation precisions calculated for each emitter. The technique is validated using simulated data and then demonstrated using experimental data acquired by direct (d)STORM imaging from which we characterise the clustering behaviour of the kinases Csk and ZAP70 in resting T cells and at the T cell immunological synapse.
52. Suman Ranjit (Speaker)
    Mapping diffusion in a living cell using the phasor approach.
    Abstract

    Suman Ranjit1, Luca Lanzanò2, and Enrico Gratton1*
    1 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California Irvine, Irvine, California 92697
    2 Department of Nanophysics, Italian Institute of Technology, Genoa, Italy And Department of Physics, University of Genoa, Genoa, Italy

    Grant - NIH-P41 GM103540 and NIH P50-GM076516
    The diffusion of a fluorescent protein within the cell has been measured by using either the fluctuation based techniques (FCS, RICS) or through particle tracking or through FRAP. However none of these methods enable us to measure the diffusion of the fluorescent particle at each pixel of the entire image. Measurement using the conventional single point FCS at every individual pixel results in the long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation we have developed a new method of scanning while constructing fluorescent image of the cell. In this new method of modified raster scanning, the laser scans each individual line multiple times before moving to the next line. This continues until the complete area is scanned. The difference from the RICS approach is in RICS the data is acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition required for this method is much shorter than the time required for the traditional FCS analysis at each pixel. However, at single pixel the acquired intensity time sequence is short; requiring a non-conventional analysis of the correlation function to extract information about the diffusion. The phasor approach, a fit-free method that was originally developed for the analysis of FLIM images was applied to analyze the obtained correlation functions. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus a detail diffusion map of the cell can be created.
53. Suman Ranjit (Poster)
    Mapping diffusion in a living cell using the phasor approach.
    Abstract

    Suman Ranjit1, Luca Lanzanò2, and Enrico Gratton1*
    1 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California Irvine, Irvine, California 92697.
    2 Department of Nanophysics, Italian Institute of Technology, Genoa, Italy And Department of Physics, University of Genoa, Genoa, Italy

    Grant - NIH-P41 GM103540 and NIH P50-GM076516
    The diffusion of a fluorescent protein within the cell has been measured by using either the fluctuation based techniques (FCS, RICS) or through particle tracking or through FRAP. However none of these methods enable us to measure the diffusion of the fluorescent particle at each pixel of the entire image. Measurement using the conventional single point FCS at every individual pixel results in the long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation we have developed a new method of scanning while constructing fluorescent image of the cell. In this new method of modified raster scanning, the laser scans each individual line multiple times before moving to the next line. This continues until the complete area is scanned. The difference from the RICS approach is in RICS the data is acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition required for this method is much shorter than the time required for the traditional FCS analysis at each pixel. However, at single pixel the acquired intensity time sequence is short; requiring a non-conventional analysis of the correlation function to extract information about the diffusion. The phasor approach, a fit-free method that was originally developed for the analysis of FLIM images was applied to analyze the obtained correlation functions. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus a detail diffusion map of the cell can be created.
54. Suman Ranjit (Poster)
    Three photon excitation FCS of Phosphofructokinase (PFK).
    Abstract

    Suman Ranjit1, Alexander Dvornikov1, David M. Jameson2, Greg D. Reinhart3, and Enrico Gratton1
    1 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA, USA
    2 Department of Cell and Molecular Biology, University of Hawaii, Honolulu, HI, 96822 USA
    3 Biochemistry & Biophysics, Texas A&M University, College Station, TX 77843

    In this work, three photon excitation fluorescence correlation spectroscopy (FCS) was used to measure oligomerization equilibrium of human phosphofructokinase (PFK). Three photon FCS can provide a new method to study oligomerization of unlabeled proteins by using the endogenously fluorescent tryptophan residues, excitable using a commercially available Ti-Sapphire laser. The fluorescence intensity produced by the three photon excitation was measured using the DIVER microscope set up, originally designed for deep tissue imaging. In this modified home built upright microscope, a large area photomultiplier tube is used as a detector and is placed directly below the sample. The lack of optical elements in the detection path of the microscope results in much improved detection efficiency in the UV spectral region up to about 300 nm, which nicely matches the emission from tryptophan and gives DIVER its extraordinary sensitivity. The three photon excitation autocorrelation decays obtained for Phosphofructokinase (PFK) in presence of fructose-6-phosphate showed the presence of large oligomers. In absence of fructose-6-phosphate and in the presence of ATP, the large oligomers dissociate, as evidenced by the decrease in the amplitude of the autocorrelation functions. This technique was further extended to measure autocorrelation decays of F1 phage virus. The three photon excitation process was verified from the slope of the plot of log(intensity) against the log(excitation power). Thus three photon excitation FCS represents a new method of obtaining diffusion and oligomerization information for the biological molecules without the requirement of extrinsic labeling of the system.
55. Gregory D Reinhart (Speaker)
    A new perspective on an old friend: rat liver Phosphofructokinase.
    Abstract

    Gregory D. Reinhart, David Holland, Junjie Zhang, and Jeng-Yih Chang
    Department of Biochemistry and Biophysics, Texas A&M University and Texas A&M AgriLife Research, College Station, TX 77843-2128

    The enzyme phosphofructokinase (PFK) is ubiquitously present in prokaryotic and eukaryotic cells that can utilize glucose as an energy source. Consequently it is subject to allosteric regulation that provides feedback regulation relative to the energy state of the cell. The regulation of PFK from liver is more complex since the liver must synthesize glucose during times of fasting so as to maintain blood sugar homeostasis. Hence, regardless of the liver's own energy needs, PFK must be inhibited during times of low glucose supply. It has long been recognized that this additional mechanism of inhibition is likely triggered by the hormone glucagon which is secreted by the pancreas during times of fasting (i.e. glucose deprivation). The binding of glucagon initiates a cascade that leads to the loss of fructose 2,6-bisphosphate – a potent activator of liver PFK. However, what is often overlooked is that even physiologically high concentrations of fructose 2,6-bisphosphate do not provide for PFK activity at physiological concentrations of its substrate, fructose 6-phosphate (F6P) according to in vitro studies. Clearly an activation mechanism is still required to relate the in vitro activity of the enzyme to its in vivo behavior. Activation of PFK invariably involves increasing the affinity the enzyme displays for F6P. One notable difference between the in vitro conditions in which the kinetics of PFK are studied and the physiological milieu is the PFK concentration, which for technical reasons is much more dilute in vitro than in vivo. Over 30 years ago we demonstrated that both F6P and fructose 2,6 bisphosphate stabilized or promoted the self-association of rat liver PFK at near-physiological PFK concentrations to species much larger than the smallest active oligomer (which is a tetramer with molecular weight equal to 320,000). The tetramer form of the enzyme is presumed to predominate under the dilute concentrations of PFK used for enzyme kinetics studies in vitro. These results were largely deduced by the fluorescence polarization behavior of pyrene-labeled rat liver PFK. Using a Weber linkage argument, we proposed that the enzyme likely consists of highly self-associated species in the cell and that these species likely have a higher affinity for the F6P than does the tetramer. In essence self-association is the overlooked activation mechanism. However, those experiments suffered by limited enzyme quantities that were available and by the ensemble nature inherent in the fluorescence measurements. We have recently begun to revisit this hypothesis using the techniques of fluorescence correlation spectroscopy (FCS) and electron microscopy with rat liver PFK cloned and expressed in E. coli. 2-photon FCS has been performed on rat liver PFK labeled with alexa-488. The results suggest a complex self-association behavior in the presence of F6P. Electron microscopy reveals that, in the presence of F6P, rat liver PFK can form long flexible fibrils that are comprised of up to 36 PFK tetramers. Because of the large size this enzyme can attain, and the fact that each subunit contains 10 tryptophans, 3-photon FCS of rat liver PFK was attempted in collaboration with Dave Jameson and members of the LFD. The preliminary results qualitatively agree with the 2-photon FCS experiments. Funding by NIGMS grant GM 033216.
56. Jeremie Rossy (Poster)
    Using RICS to look at what happens outside T cell microclusters and understand how Lck finds engaged T cell receptors.
    Abstract

    Yui Yamamoto, Elizabeth Hinde, Katharina Gaus, and Jeremie Rossy

    Because the spatial organisation of signalling proteins is central to T cell activation, single molecule localisation microscopy (SMLM) techniques such as PALM and STORM are opening a whole new path in the study of T cell signalling. Using this approach in a previous study, we established a link between the conformational state of the kinase Lck and its surface patterning, with the active/open form inducing and the inactive/closed form preventing clustering. We also demonstrated that clusters of Lck interact with clusters of the activated T cell receptor (TCR). However, how molecules of Lck discriminate between engaged and resting TCR remains yet to be elucidated. As SMLM is essentially providing information about non-diffusing particles, we used Raster Image Correlation Spectroscopy (RICS) to investigate the impact of Lck conformational state on its diffusion and propensity to interact with TCR. In this study, we first show that RICS can be used to study the diffusion of essential T cell signalling proteins. We focused on the diffusion of Lck molecules to get a better grasp at their behaviour while they are not retained in clusters. We show that although they display the same diffusion rate, open/active Lck molecules have a higher probability to diffuse together than their closed counterparts. Further, in opposition to what we observed previously within clusters using SMLM, only a minor fraction of diffusing Lck interacts with TCRζ, demonstrating that the association of Lck and TCR happens only when they are engaged in a signalling process. Finally, focusing on how the co-receptor CD4 contributes to Lck distribution, we show that more than 60% of Lck molecules diffuse together with CD4.
    These data confirm that the conformational state of Lck governs its propensity to self-associate. They also demonstrate that a large proportion of Lck molecules diffuse in association with CD4 and, more importantly, that Lck and TCRζ have a low level of interaction outside of signalling clusters, supporting a model in which CD4 and Lck travel together until they encounter TCR clusters.
57. Hays Rye (Speaker)
    Epsin induced membrane fission resolved by single particle fluorescence burst analysis.
    Abstract

    Arielle Brooks1, Lauren Kustigian1, Daniel Shoup1, Jason Puchalla2, Chavela M. Carr1, and Hays S. Rye
    1 Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
    2 Department of Physics, Princeton University, Princeton, New Jersey 08544

    Elucidating the molecular mechanisms that underlie intracellular membrane dynamics is fundamental for an understanding of such central cell-biological processes as cell growth, vesicle trafficking, immunology and synaptic transmission. All of these vital processes require the formation of membrane-enclosed compartments, known as vesicles, which carry molecules to specific stations in the cell. The ultimate step that completes the formation of each vesicle requires the activity of highly specialized proteins, which must cleave and reseal membranes in a process known as membrane fission. The mechanism of membrane fission, as well as the assignment of fission activity to specific proteins, remain subjects of ongoing research. Here we use single-particle burst analysis spectroscopy (BAS) to study the process of membrane fission induced by the potent, fission-active ENTH domain of epsin. In addition to obtaining time-resolved measurements of vesicle intermediates and products generated by the ENTH domain, we also discovered that full-length epsin has measurable fission activity, arguing that the membrane-fission activity observed previously with the ENTH domain is indeed a native function of the full-length epsin protein.
58. Claus Seidel (Speaker)
    Watching complexes and transient states of proteins by high-precision FRET in vitro and in vivo.
    Abstract

    Claus A. M. Seidel
    Chair for molecular physical chemistry, Heinrich-Heine-Universität Düsseldorf, Germany, E-mail: cseidel@hhu.de

    So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many detailed static snapshots of protein structures. Recent experiments by single-molecule and other techniques have shown the heterogeneity and flexibility of biomolecular structures and questioned the idea that proteins and other biomolecules are static structures. The visualization of transiently populated conformational states and the identification of exchange pathways are key steps to understand enzyme function.
    I will present novel tools for quantitative FRET analysis to be applied in imaging and biophysics: (i) species and component analysis for FRET-FLIM. (ii) comprehensive toolkit for Förster resonance energy transfer (FRET)-restrained modeling of proteins and their complexes for quantitative applications in structural biology [1-3] and cell biology [4, 5] referred to as FRET positioning and screening (FPS).
    Useful experimental input can be obtained by multi-parameter fluorescence detection on the single-molecule level and for complexes imaged in live cells. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. We introduce FRET-guided “screening” of a large structural ensemble created by computer simulations to proteins and their complexes. The precision and confidence levels of the models are calculated by rigorous error estimation. In planta FRET-FLIM measurements and hybrid studies of the large GTPase hGPB1 using FRET, SAXS and EPR will be presented.
    [1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010)
    [2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011)
    [3] Kalinin et al. Nat. Methods 9, 1218-1225 (2012).
    [4] Stahl, Y., et al. Current Biology 23, 362–371 (2013).
    [5] Kravets, E., et. al. J. Biol. Chem. 287, 27452–27466 (2012).
59. Chiara Stringari (Speaker)
    Fluorescence lifetime microscopy (FLIM) to monitor tumor microenvironment and metabolism in vivo.
    Abstract

    Chiara Stringari
    Laboratory for Optics and Biosciences, École Polytechnique, Palaiseau, France

    Cellular metabolism plays a crucial role in tumor progression and formation. Observing cancer metabolism and microenvironment with sub-cellular resolution in vivo is fundamental for the understanding of cancer development and progression.
    Here we develop a label-free and non-invasive method to measure the metabolic phenotype of single cancer cells within the native microenvironment of tumors in vivo. By using NADH as optical biomarker and Fluorescence Lifetime microscopy (FLIM) we identify cancer metabolism related to different rates of glycolysis, cell growth and proliferation. We perform FLIM on living and actively-perfused tumors. We are able to distinguish collagen fibers, the tumor stroma, adipocytes, blood vessels and single cancer cells within the living tumor. By measuring NADH lifetime, we quantify the relative concentration of free and bound NADH in single cancer cells, which reflects the cellular redox NADH/NAD+ ratio and balance of oxidative phosphorylation and glycolysis. We investigate the tumor microenvironment by characterizing the distribution of the metabolic fingerprint of single colon cancer cells and by mapping the three-dimensional metabolic heterogeneity of the tumor at different distances from blood vessels.
    Our method permits to non-invasively measure single cancer cell metabolism in a living intact tumor microenvironment and to monitor the spatial and temporal dynamics of tumor metabolism upon different physiological conditions such as blood flow, tissue oxygenation levels, nutrient availability and drug delivery.
60. Kerry Swift (Poster)
    Structure-function properties of anti-fluorescein mAb 43.1 directed to the Carboxyphenyl moiety.
    Abstract

    Kerry M. Swift, Susan Gayda, and Sergey Y. Tetin
    Diagnostics Research, Abbott Diagnostics Division, Abbott Park, IL 60064

    We report the three-dimensional structure and structure-function analysis of mAb 43.1 in comparison with widely known mAbs 4-4-20 and 9-40. The mouse monoclonal antibody 43.1 was developed in the early 90s by D.S. Linthicum at Texas A&M University using fluorescein-BSA immunogen in which fluorescein was conjugated to the protein carrier through its xanthene moiety. Thus, the antibody is primarily directed to the carboxy-phenyl ring and binds fluorescein with the KD~60 pM. The binding site comprises a wide groove-like entrance area that reaches deep into a cavity. The two sections complementarily fit the ligand’s xanthene and carboxyphenyl moieties, with the phenyl ring resting in the cavity. In contrast to previously reported anti-fluorescein antibodies, in mAb43.1 the carboxyphenyl is tightly fixed by multiple hydrogen bonds, originating from His35H, Arg99H, and a water molecule that bridges to Arg51L. Even though this water molecule is the only one close to the carboxyphenyl ring, the cavity is remarkably polar. In contrast, the groove surrounding the xanthene ring is hydrophobic except for the two positive charges from arginine side chains close to both edges. Its aromatic interface is partially stacked against the side chain of Tyr37L and further faces Leu55L and the carbon side chain of Arg99H, providing an nonpolar milieu. Such stacking interaction provides an opportunity for the electron transfer Tyr-FL-Arg that results in shifting fluorescein’s absorption maximum to 513nm and 99.6% fluorescence quenching if excited at 470nm. Interestingly, mAb43.1 - fluorescein complex is much more resistant to high hydrostatic pressure than other anti-fluorescein antibodies, demonstrating a smaller volume change upon ligand dissociation. The affinity of mAb 43.1 to fluoresceinamine is 0.6 nM and to HPF < 1uM.
61. Sergey Y Tetin (Speaker)
    Structural basis for spectroscopic effects observed in anti-fluorescein monoclonal antibody 43.1.
    Abstract

    Sergey Y. Tetin
    Diagnostics Research, Abbott Diagnostics Division, Abbott Park, IL 60064

    Unlike previously investigated anti-fluorescein monoclonal antibodies 4-4-20 and 9-40, mAb 43.1 is directed to the carboxyphenyl moiety of fluorescein. It tightly binds fluorescein with the KD~60 pM but does not bind its derivatives that lack either the whole carboxyphenyl or just the carboxyl group. The X-ray structure of mAb 43.1 has been solved at 2.5Å which allows for structural interpretations of spectral effects observed in mAb 43.1 – fluorescein complex. In the binding site of mAb 43.1, the xanthene ring of fluorescein stacks between Tyr37L and Arg99H providing an opportunity for electron transfer directed from Tyr to FL and to Arg that results in formation of a specific tri-molecular complex with characteristic absorption and emission spectra and fluorescence lifetime. We use this speculation to explain spectroscopic effects observed in other known anti-fluorescein antibodies.
62. Sergey Y Tetin (Speaker)
    A water channel in the binding site of a high affinity anti-Methotrexate antibody.
    Abstract

    Sergey Y. Tetin
    Diagnostics Research, Abbott Diagnostics Division, Abbott, Abbott Park, IL 6006

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as the specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1x107 M-1s-1) and slow (4x10-5 s-1) dissociation rates determined for mAb ADD056, resulting in a very strong binding with a KD ~ 3.6 pM at 20°C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region, which suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering.
    Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.
63. Valeriya Trusova (Poster)
    Total internal reflection fluorescence microscopy imaging of lysozyme-induced structural deformations of lipid membrane.
    Abstract

    V.M. Trusova, G.P. Gorbenko, I. Akopova, J.G. Molotkovsky, I. Gryczynski, and Z. Gryczynski

    Total internal reflection fluorescence microscopy (TIRFM) technique has been utilized to explore the topological transformations of supported lipid bilayer (SLB) made from zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL), evoked by basic protein lysozyme (Lz). To visualize the bilayer defects, SLB was doped by trace amounts of fluorescent lipid analog Bodipy-PC (ByPC). Addition of Lz to PC bilayers did not affect SLB geometry as can be judged from invariance of ByPC fluorescence. Contrary to this, Lz binding to CL-containing SLB led to the appearance of large areas with increased surface fluorescence. These areas are not vesicular structures and in general remain in the plane of the SLB, being in focus at the level of the SLB, and producing a stepwise change in fluorescence intensity. The step size between the areas of different intensity is discrete and approximately equals the intensity level of the parent bilayer pointing to stacking of several monolayers upon each other. The formation of lipid stacks was found to depend on time and CL content. The observation of this effect only in CL-containing SLBs suggests that formation of bilayer stacks is electrostatically-driven process. The obtained results may have implication not only with respect to Lz action against Gram-positive and Gram-negative bacteria containing CL, but also from the viewpoint of cell membrane structural deformations induced by proteins.
64. Renee Whan (Poster)
    FLIM a useful tool for determining drug release from nanoparticles.
    Abstract

    Alex MacMillan1, Sharon Sagnella2,3, Hien Duong2, Nicole Bryce4, May Lim5, Pall Thordarson2,6 Cyrille Boyer2,5, Renee Whan1,2 Maria Kavallaris2,3,and Thomas Davis7,8
    1 Biomedical Imaging Facility, Mark Wainwright Analytical Centre, UNSW Australia, Sydney, Australia 2052
    2 Australian Centre for Nanomedicine, School of Chemical Sciences and Engineering, UNSW Australia, Sydney, Australia 2052
    3 Children’s Cancer Institute Australia, Lowy Cancer Research Centre, UNSW Australia, PO Box 81, Randwick, Australia 2031
    4 Sydney Medical School, Blackburn Building, University of Sydney, Sydney, Australia 2006
    5 School of Chemical Engineering, UNSW Australia, Sydney, Australia 2052
    6 School of Chemistry, UNSW Australia, Sydney, Australia 2052
    7 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Melbourne, Australia
    8 Department of Chemistry, University of Warwick, United Kingdom

    Understanding how a nanoparticle accumulates in the cell and whether it actually reaches its biological target to deliver its cargo has been a challenge to determine for a number of years. Light and optical microscopy plays an important role in understanding a fluorescent compounds movement in 2 and 3 dimensional cell cultures and in vivo. Fluorescence Lifetime Microscopy (FLIM) is one tool that allows spectrally similar fluorescent components to be identified and separated in different molecular environments and as such, provides a means to determine whether a carrier molecules cargo has been released under various stimuli.
    We have previously reported the use of FLIM for monitoring the accumulation and release of both free and nanoparticle conjugated Doxorubicin.1-4 This body of work demonstrates the shift from traditional lifetime analysis where each species is identified on a pixel by pixel basis and resolved by fitting the data to an exponential decay to analysis via the Phasor method.5 The phasor analysis provides a straightforward means to visualise nanoparticle distribution in cells and where they release there active moieties. We have studied a number of nanoparticle systems using this method and drug or fluorophore release can be monitored for a number of release mechanisms whether it involves stimulation by pH or light or involves release from physical encapsulation, direct ionic or covalent bonding, or has quenching contributions to the fluorescence profile.1,2,4,6-8
    [1] H. Duong, F.Hughes, S. Sagnella, M. Kavallaris, A. Macmillan, R. Whan, J. Hook, T. Davis, C. Boyer. (2012) Mol. Pharmaceutics, 9 (11), 3046–61
    [2] J. Basuki, H. Duong, A.Macmillan, R. Erlich, L. Esser, M. Akerfeldt, R. Whan, M. Kavallaris, C. Boyer, T.Davis. (2013) ACS Nano, 7(11), 101710189
    [3] N.S Bryce, B. Pham, N. Fong,N. Jain, E.Pan, R. Whan,T. Hambley, B. Hawkett. (2013) Biomater. Sci., (1), 1260-1272
    [4] S.Sagnella, H.Duong, A. MacMillan, C.Boyer, R.Whan, J.McCarroll, T.Davis, M.Kavallaris. (2014) Biomacromolecules 15 (1), pp 262–275
    [5] Digman, M. A., Caiolfa, V. R., Zamai, M. & Gratton, E. (2008) Biophys. J. 94, L14-L16.
    [6] A. Dunn, D. Dunn, A. Macmillan, R. Whan, T. Stait-Gardner, W. Price, M. Lim, C. Boyer. (2014) Polym. Chem.,5, 3311-3315
    [7] Basuki, J.; Duong, H.; Macmillan, A.; Whan, R.; Boyer, C; Davis, T.(2013) Macromolecules 46(17) 7043-54
    [8] W. Truong, S. M. Sagnella, A. Macmillan, R. M. Whan, J. A. McCarroll, M. Kavallaris, P.Thordarson. submitted 2014
65. Paul Wiseman (Speaker)
    Podosome protein dynamics in immune dendritic cells revealed by STICS, radial and pair correlation analysis.
    Abstract

    Paul W. Wiseman
    Departments of Chemistry & Physics, McGill University

    Image correlation methods are an extension of fluorescence fluctuation spectroscopy that can measure protein-protein interactions and macromolecular transport properties from input fluorescence microscopy images of living cells. These approaches are based on space and time correlation analysis of fluctuations in fluorescence intensity within images recorded as a time series on a laser scanning or total internal reflection fluorescence (TIRF) microscope. We previously introduced spatio-temporal image correlation spectroscopy (STICS) which measures vectors of protein flux in cells based on the calculation of a spatial correlation function as a function of time from an image time series. Here we will describe the application of time window STICS and its two color extension, spatio-temporal image cross-correlation spectroscopy (STICCS), for measuring cellular waves of adhesion related macromolecules talin and vinculin as well as cytoskeletal actin between assembling and disassembling podosomes in dendritic immune cells. Podosomes are cylindrical membrane complexes with an integrin adhesive ring and an actin rich core that are associated with cellular migration and invasion in specific cell types. EM and super-resolution microscopy of cells shows radial actin filaments that connect neighboring podosomes so we applied radial and podosome pair correlation analysis to further characterize the transport waves within connected podosome clusters. These analyses combined with pharmalogical perturbation experiments show that pododosome turnover is coordinated within local clusters in cells.
66. Thorsten Wohland (Speaker)
    Light sheet based imaging fluorescence correlation spectroscopy measures structure and dynamics in 2D and 3D.
    Abstract

    Thorsten Wohland

    Fluorescence Correlation Spectroscopy (FCS) is a ubiquitously used quantitative tool for the determination of molecular parameters, including concentrations, diffusion and transport coefficients, and dissociation constants. FCS possesses single molecule sensitivity and in its confocal implementation can be used in 3D living system. However, due to the confocal mode it bleaches the sample and can collect one or at best a few spots simultaneously. Light-sheet based excitation, either in a total internal reflection (TIR) or a single plane illumination mode (SPIM), illuminates a whole cross section of a sample. This allows collecting data from hundreds or thousands of points in parallel. It thus illuminates only parts of a sample, which are observed, reducing photobleaching per measurement, and allowing FCS multiplexing. In particular, because of the reduced photobleaching we can acquire at least 10-12 imaging FCS measurements on a single living cell using fluorescent proteins without visible morphological changes or other indicators of cell damage, yielding several thousand data points. This allows producing either 3D diffusion maps or time lapse FCS movies. In this seminar we will discuss technical and sample requirements for imaging FCS and demonstrate imaging FCS measurements in 2D and 3D on lipid bilayers, giant unilamellar vesicles, and live cells.
67. Belinda Wright (Poster)
    Analysis of endogenous nuclear NADH in differentiating myoblasts using the spectral phasor approach.
    Abstract

    B. K. Wright1, M. R. Jones1, M. A. Digman2 and E. Gratton2
    1 University of Western Sydney, School of Science and Health, Richmond, New South Wales, Australia
    2 Laboratory of Fluorescence Dynamics (LFD), University of California, Biomedical Engineering Department, Irvine, CA, 92697

    Analysis of endogenous autofluorescent energy metabolites, for example NADH in the nuclear regions of live cells, aids in determining a cells metabolic status. Detecting the spectral differences of free and bound NADH is currently limited since the NADH spectrum could change depending of the protein that is binding NADH; however applying Spectral Phasor to identify discrete wavelength shifts and discriminated at a pixel level overcomes the need for the a prior knowledge of the spectra. For this investigation, live progenitor rat myoblast cells (L6 cells), were stimulated to differentiate through the reduction of foetal bovine serum (2%) in the cell culture media. Spectral data was acquired using the lambda mode of a Zeiss LSM 710 confocal microscope, exciting the cells at 740nm and analysed with SimFCS. Two emission wavelength maxima; 473nm and 480nm, were selected in both undifferentiated myoblasts and those induced to differentiate. The 473nm wavelengths observed in undifferentiated myoblasts were detected centrally in the nucleus, while the 480nm wavelength localised around the nuclear periphery. After 1 hour of stimulated differentiation, the distribution of NADH had changed compared to the undifferentiated L6 cells. The myoblasts in this investigation demonstrated regional clustering of the 473nm wavelengths predominately in one area of the nuclear periphery, while the 480nm were localised throughout the remainder of the nucleus. We have demonstrated the application of Spectral Phasor to identify discrete wavelengths and their distribution, can be identified in the nucleus of live cells undergoing differentiation.
68. Bin Wu (Speaker)
    Imaging the stimulation dependent localization of single endogenous mRNAs to dendritic spines.
    Abstract

    Bin Wu, Young J Yoon, Carolina Eliscovich, and Robert H. Singer

    RNA localization and local protein synthesis play an essential role in synaptic plasticity as well as learning and memory. However it has not been possible to image the dynamics of mRNAs under the influence of synaptic signaling in a live neuron. Here, we develop a fluorescence microscopy method to visualize endogenous mRNAs with single molecule sensitivity and at the same time to deliver synaptic stimulation at a single synapse. To visualize endogenous mRNA, we used a transgenic mouse in which 24xMS2 binding sites had been knocked into the 3’ untranslated region (3’ UTR) of the essential β-actin gene. We introduced fluorescent protein tagged MS2 coat proteins in the cultured primary hippocampal neuron to label the endogenous β-actin mRNA. We built a microscope that is able to stimulate a single dendritic spine with photo-uncaging of glutamate. We verified that repeated synaptic stimulation by photo-uncaging resulted in long lasted structure plasticity of a single spine. We investigated the dynamics of β-actin mRNA under the influence of synaptic stimulation. We found that single β-actin mRNAs localized to the base of the stimulated spines. Blocking NMDA receptors resulted in a reduction of localized mRNA, suggesting a link between synaptic activity and mRNA transport. Addition of a translation inhibitor had no effect on localization implying that dendritic control of mRNA localization and local translation are decoupled. The localized mRNA persisted at the stimulated dendrites for more than 120 minutes. Our results show that synaptic activity is responsible for dendritic β-actin mRNA localization and docking at spines undergoing plasticity.
69. Yuling Yan (Poster)
    Dynamin 2 dependent biogenesis of Endophilin B1 containing vesicles.
    Abstract

    Jinhui Li1, Barbara Barylko2, Serkan Berk1,3, Joachim D. Mueller1,3, Joseph P. Albanesi2, and Yan Chen1
    1 School of Physics and Astronomy, University of Minnesota, Minneapolis, MN, 55455
    2 Pharmacology Department, UT Southwestern Medical Center, Dallas, TX, 75239
    3 Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, 55455

    Endophilins are SH3- and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2, which function in the early stages of endocytosis from the plasma membrane, exist almost exclusively as soluble dimers in the cell cytoplasm. Here we use fluorescence correlation spectroscopy and brightness analysis to show that a population of endophilin B1, which mediates intracellular membrane trafficking, is instead present in multiple copies on small and highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. In contrast, vesicle association of the endophilin B1 BAR domain was dynamin-independent, supporting previous in vitro experiments showing that full-length endophilins adopt an auto-inhibited conformation relieved by interaction of their SH3 domains with dynamins. Although dynamin 2 activity was important for the formation of endophilin B1-containing vesicles, it did not co-migrate with these vesicles. However, both dynamin 2 and synaptojanin 1, a phosphoinositide phosphatase required for uncoating of clathrin-coated vesicles, were found to associate with soluble, cytosolic endophilin A2 in living cells. These results suggest important mechanistic differences between the two closely related membrane-deforming proteins, endophilins A2 and B, in the cytoplasm of living cells.
70. Robert T Youker (Speaker)
    Proteasome inhibition alters dynamics of CFTR aggregation measured by N&B analysis.
    Abstract

    Robert T. Youker1, Chris Rabold1, Jeffrey L. Brodsky2, and Ora A. Weisz1
    1 Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
    2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260

    The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a polytopic protein that traffics to the apical surface of epithelial cells where it transports chloride. The folding and synthesis of CFTR in the endoplasmic reticulum (ER) is inefficient, and up to 80% of wild type protein may be degraded prior to reaching the cell surface. A common mutation in CFTR is the deletion of a single amino acid that enhances misfolding, leads to the complete degradation of the protein, and is a major cause of Cystic Fibrosis (CF). Prolonged inhibition of the proteasome, a multi-subunit cytosolic protease, results in the accumulation of CFTR into perinuclear insoluble aggregates termed aggresomes. Fixed image microscopy and in vitro biochemical studies have provided insight into the composition of CFTR aggresomes but there is little information on the dynamics of aggresome formation. It is important to understand the dynamics of CFTR misfolding and aggregation in order to develop strategies to improve folding of CFTR in the hopes of curing CF. We have employed number and brightness (N&B) analysis to investigate the dynamics of early CFTR misfolding intermediates. We expressed low levels of EGFP-CFTR in MDCK cells, a renal epithelial cell line, and monitored the CFTR oligomeric status in the absence and presence of the proteasome inhibitor, MG132. We found that the EGFP-CFTR can form clusters and these clusters contained on average 6 fluorescent molecules. The size of these clusters increased to ~15 fluorescent molecules after several hours of proteasome inhibition. Cluster formation in proteasome inhibited cells occurred at expression levels that were 5 fold lower compared to control cells. Interestingly, CFTR clusters colocalized with the ER marker Sec61-RFP but not with a marker for ER exit sites and were not perinuclear. Washout of MG132 led to a reduction of the CFTR clusters after approximately 3 hours, suggesting these CFTR puncta were not insoluble aggresomes but could represent “pre-aggresome” intermediates. These preliminary N&B data suggest that inhibition of the proteasome alters the equilibrium between clustered and non-clustered EGFP-CFTR by shifting the expression threshold at which cluster formation can occur.
71. Robert T Youker (Poster)
    Biophysical studies to dissect the role of oligomerization in apical protein trafficking.
    Abstract

    Robert T. Youker, Jennifer R. Bruns, and Ora A. Weisz
    Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261

    The sialomucin endolyn is a protein of unknown function that cycles between the apical surface of mammalian kidney epithelial cells and lysosomal compartments. Proper apical targeting of endolyn is required for normal pronephric kidney development and function in zebrafish. Efficient apical sorting of endolyn is dependent on N-glycosylation in its cysteine rich domain but how this post-translational modification regulates endolyn sorting is not completely understood. Recent work from our group has demonstrated that siRNA knockdown of galectin-9 reduces the apical sorting efficieny of endolyn [Mo et al. MBoC (2012) 23, 3636-3646]. Galectins are lectins that can crosslink proteins at the plasma membrane of cells to create protein lattices. Oligomerization of membrane proteins can modulate their apical sorting and we hypothesize that galectin-9 mediated clustering of endolyn is required for its efficient apical sorting. Using number and brightness (N&B) analysis to study endolyn oligomerization status in vivo, we found that wild type endolyn forms higher-order oligomers in the trans-Golgi network (TGN). Endolyn is known to traffic through a rab11a positive endosomal compartment en route to the apical surface. Similarly, large oligomers of endolyn were observed in rab11a positive compartments. A soluble version of endolyn (ensol) that is delivered to the apical surface via a glycan independent mechanism did not form higher-order oligomers. Interestingly, siRNA knockdown of galectin-9, or treatment of cells with the mannosidase I inhibitor kifunensine caused a reduction in endolyn oligomer size in the rab11a compartment but not in the TGN. Taken together, these results suggest that endolyn clustering into higher-order oligomers is important for apical sorting and that the mechanism of clustering may be different in the TGN versus in rab11a-positive endosomes. Further studies will be performed using cross-correlation N&B in living cells to identify the subcellular location where galectin-9 binds to endolyn.
72. Sanjun Zhang (Poster)
    Studying protein dynamics by time-resolved fluorescence spectroscopy and microscopy.
    Abstract

    Sanjun Zhang, Haifeng Pan, and Jianhua Xu

    Time resolved fluorescence spectroscopy and imaging nowadays play an important role in the study of both structure and conformational dynamics of proteins. Depending on the interested time scale, the people usually use two typical techniques. One is the picosecond resolved fluorescence spectroscopy based on time correlated single photon counting (TCSPC), and another one is the femtosecond resolved fluorescence based on optical frequency upconversion. In this presentation, the experimental setups and particulars of these two time resolved fluorescence techniques are discussed as well as the applications in protein dynamics. An instrument response standard (quenched fluorescein sodium) was developed to improve the time resolution of TCSPC based spectroscopy and imaging measurements. Picosecond and femtosecond resolved fluorescence of a dipeptide Trp-Trp and its derivatives were measured through the TCSPC and upconversion systems. Decay associated spectra analysis reveals the intramolecular interaction among the neighboring Trp residues. Moreover, time resolved spectroscopy and imaging of the fluorescent proteins were performed in vitro and vivo to study the dynamics and interactions in various environments.
73. Yi Zuo (Poster)
    Imaging phospholipid phase separations on a droplet.
    Abstract

    Yi Y. Zuo1, Hongtao Chen2, and David M. Jameson3
    1 Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, HI 96822
    2 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA 92697
    3 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813

    Self-assembled phospholipid monolayer at the air-water interface is a commonly used surface thermodynamic model for studying two-dimensional phase transitions and separations. To date, nearly all of these studies rely on the classical Langmuir-type film balance. However, due to its design limitations, the classical Langmuir balance has little or no capacities in controlling experimental conditions, especially the temperature at which the surface phase transitions occur. We have developed a miniaturized Langmuir film balance, called the constrained drop surfactometer (CDS), by shrinking the liquid subphase into a droplet of only 10 microliter. This system miniaturization permits a precise temperature control of the entire air-water interface with a customized environmental control chamber. Using the CDS, we have measured compression isotherms of dipalmitoyl phosphatidylcholine (DPPC) monolayers under rigorously controlled temperatures (± 0.1 oC), which allows us to study the detailed surface thermodynamics of DPPC phase transitions. More importantly, we have integrated a confocal laser scanning microscope with the CDS, which permits direct in situ imaging of phospholipid phase separations on a droplet. We will show that the CDS can be used as a miniaturized film balance to replace the traditional Langmuir-type film balance in studying insoluble monolayers.