7th International Weber Symposium (2008)

Abstracts by corresponding author

1. Matthew Antonik (Poster)
   Determining the fluorescence intensity distribution from a signal containing a poisson background using multiparameter fluorescence techniques.
   Abstract

   M Antonik. Physics and Astronomy Dept, U of Kansas, Lawrence.

   Single molecule measurements can provide excellent contrast between the signals of different components of dynamic or heterogeneous systems, which has made single molecule techniques increasingly popular. Protein folding and conformational dynamics, enzymology, ribozyme function, bacterial light harvesting, and protein-nucleic acid interactions are just a few examples of complex systems that have benefited from single molecule techniques. However the experimental methods have advanced faster than the analytical techniques, and the full potential of single molecule measurements has not been achieved due the lack of a rigorous understanding of the fluorescence signal. A particular example is the difficulty in extracting a fluorescence intensity distribution from a signal distribution which contains a Poisson background signal. Background signals represent an ever-present signal component which has its own spectral, polarization, and lifetime characteristics. In order to avoid background effects, intensity threshold limits are typically imposed which exclude signals which are dominated by background. This approach is not optimal, first because it reduces the available data for analysis, and second because it does not account for the background which still exists in the remaining signal, which introduces an intesity dependent systematic error into the analysis. Furthermore, if different components of the same signal have different fluorescence brightnesses due to quenching or environemtal changes, thresholding preferentially selects data from the brighter species. Optimally, a proper analysis of these signals would include a method by which the fluorescence intensity profile can be determined for each component, even in the presence of background.
   This poster presents an analytical method by which the fluorescence intensity profile of a homogeneous system with a Poisson background can be estimated, and subsequently used as a calibration profile for the whole experiment. The new technique analyzes the traditional signal intensity distribution, but also can exploit the additional information contained in multi-parameter fluorescence techniques, which simultaneously measure the spectral, polarization, and lifetime characteristics of the signal. The error in such estimates of the fluorescence intensity distribution are discussed, as well as techniques for applying this method to heterogeneous systems without a prior calibration experiment.
2. Matthew Antonik (Poster)
   Defining the precision of structural and dynamics measurements via fluorescence resonance energy transfer.
   Abstract

   M Antonik. Physics and Astronomy Dept, U of Kansas, Lawrence.

   Recently, a probability distribution analysis (PDA) framework was introduced which successfully describes the characteristic shapes of photon counting histograms in single molecule fluorescence resonance energy transfer (FRET) experiments. That work suggested an ultimate precision of ~2% in determining the transfer efficiency E, and a similar precision in determining heterogeneous variations. Here, the question of achievable precision is approached using statistical methods to determine the maximum temporal and spatial resolution in such measurements. The ability to detect dynamics with FRET measurements are also evaluated. The ultimate precision under typical experimental conditions is found to be less than length of typical flexible dye linkers, which would make dye motions a limiting factor in the precision of FRET measurements. Calculations are supported by simulations.
3. Nicolas Auduge (Poster)
   Imaging the fraction of hyperacetylated Histone H4 in living cells by multifocal multiphoton FLIM-FRET quantification.
   Abstract

   N Audugé, S Padilla-Parra, M Tramier, and M Coppey-Moisan. Institut Jacques Monod, CNRS, France.

   Quantitative analysis in Förster Resonance Energy Transfer (FRET) experiments in live cells for protein interaction studies is still a challenging issue. In a two component system (FRET and no FRET donor species), fitting of Fluorescence Lifetime Imaging Microscopy (FLIM) data gives the fraction of donor molecules involved in FRET (fD) and the intrinsic transfer efficiency. But when fast FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolution in living cells, photon statistic and time resolution is limited. In this case, fitting procedures are not reliable, even for single lifetime donors. We introduce the new concept of a minimal fraction of donor molecules involved in FRET (mfD), coming from the mathematical minimization of fD. We find particular advantage in the use of mfD because it can be obtained without fitting procedures and is derived directly from FLIM data. mfD constitutes an interesting quantitative parameter for live cell studies because it is related to the minimal relative concentration of interacting proteins. We applied this methodology for imaging the interaction between the bromodomains of TAFII250 and acetylated histones H4 in living cells at high resolution. We used the wide field time correlated single photon counting method (TCSPC), and multifocal multiphoton microscopy (TRIM) coupled to a fast-gated CCD to obtain fluorescence lifetime images of the donor, EGFP-H4, and to determine the fraction of donor that interacts with the acceptor, mCherry-BD. Confocal maps of the fraction of acetylated EGFP-H4 (bound to mCherry-BD) reveal a punctuate nuclear pattern at a spatial resolution of 500 nm in live HEK293 cells, the highest acetylated chromatin regions being located at the nuclear periphery. We show that the variation of the average fraction of acetylated EGFP-H4 observed from cell to cell corresponds to both a change in the proportion of positive FRET pixels per nucleus and the level of acetylated EGFP-H4 per pixel.
4. Luis A Bagatolli (Speaker)
   Direct visualization of lipid domains in human skin stratum corneum’s lipid membranes: effect of pH and temperature.
   Abstract

   L A Bagatolli. MEMPHYS, U of So Denmark, Odense.

   The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol and free fatty acids. Three unsolved key questions are (i) whether the stratum corneum extracellular lipid matrix is constituted by a single gel-phase or by coexisting crystalline (solid) domains, whether (ii) a separate liquid crystalline phase is present, and iii) whether pH has a direct effect on the lipid matrix’ phase-behavior? In this talk I will present experiments that focus in the lateral structure of membranes composed of lipids extracted from human skin stratum corneum[1]. These studies were done in a broad temperature range (10 to 90C) using different techniques such as differential scanning calorimetry (DSC), fluorescence spectroscopy and two-photon excitation and laser scanning confocal fluorescence microscopy. Additionally, a new model system consisting in giant membrane structures composed of hydrated bilayers of human skin stratum corneum lipids will be presented. These giant lipid structures can be directly visualized using the aforementioned fluorescence microscopy techniques. Interestingly, this aforementioned model system expresses morphology with dimensions corresponding to the global 3D morphology of the stratum corneum extracellular space. At skin physiological temperatures (28 - 32C) the phase state of these hydrated bilayers correspond microscopically (radial resolution limit 300 nm) to a single gel-phase at pH 7, coexistence of different gel-phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral structure and stability.
   [1]Plasencia-Gil, L. Norlen,and L.A: Bagatolli. 2007. Biophys. J 93(9): 3142-3155.
5. Francisco J Barrantes (Speaker)
   Nanodomain organization and dynamics of nicotinic acetylcholine receptor at the cell membrane.
   Abstract

   F J Barrantes. UNESCO Chair of Biophysics and Molecular Neurobiology/ Instituto Investigaciones Bioq. de Bahia Blanca, U Nac. Sur, Argentina.

   The nicotinic acetylcholine receptor (AChR) function and distribution is quite sensitive to cholesterol (Chol) levels in the plasma membrane (reviewed in Barrantes, J. Neurochem. 2007). A combination of experimental techniques (patch-clamp single-channel recording, high-resolution fluorescence microscopy, confocal fluorescence recovery after photobleaching (FRAP), particle statistical analysis) has been used to analyze the supramolecular organization of the AChR, the dynamics of the receptor at the cell surface, and the kinetics of receptor internalization. In particular, the effect of changes in cholesterol (Chol) content on AChR organization and dynamics were studied in CHO-K1/A5 cells, a mammalian cell line stably expressing adult murine AChR. Exposure to methyl-beta-cyclodextrin (CDx) dramatically accelerated AChR internalization from the cell surface. This was accompanied by gain-of-function changes observed in single-channel patch-clamp recordings. Wide-field and confocal microscopy revealed AChR submicron-sized (240-280 nm) domains that remain stable over a period of hours at the cell membrane. Domains could be resolved into AChR “nano-clusters” with a peak size distribution of ~55 nm by STED microscopy. CDx-mediated Chol depletion resulted in a smaller number of nanoclusters, increase in their size, and changes in the supramolecular “social” organization of the nanoclusters on larger scales (0.5-3.5 microns). FRAP experiments in the confocal mode provided information on the dynamics of AChR nanoclusters, the mobility of the AChR and its sensitivity to Chol at the cell surface. Plasma membrane AChR exhibited limited mobility and depletion of membrane Chol reduced mobility even more; the fraction of mobile AChR fell from 50% to 20% in Chol-depleted cells. Latrunculin treatment disclosed the dependence of receptor mobility on cortical cytoskeleton. Chol content at the plasmalemma may thus modulate cell-surface organization and dynamics of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute AChR losses from the cell surface.
6. Christopher L Berger (Speaker)
   Interaction of bimane and tryptophan: a spectroscopic ruler below 25 Å.
   Abstract

   M L Moffett1, A M Branagan1, M I Fajer2, P G Fajer3, and C L Berger4. 1 Depts of Biochemistry and 4 Mol Physiology and Biophysics, U of Vermont, Burlington, 2 Dept of Chem and Biochem, U of California at San Diego, 3 Dept of Biological Sci, Inst of Molecular Biophysics, and Natl. High Magnetic Field Lab, Florida State U, Tallahassee.

   Förster resonance energy transfer (FRET) is a powerful technique for monitoring conformational changes in proteins as they occur in solution. The ability to make FRET measurements below 20 Å is limited, however, by the 1/r6 dependence of the interaction on distance and by the relatively large size of the fluorescent probes. In an effort to expand the range over which meaningful distance measurements can be made, we investigated and quantified the interaction between bimane, a small extrinsic fluorophore, and tryptophan. Bimane fluorescence is sensitive to solvent polarity and is also quenched by proximal tryptophan residues with an exponential dependence on distance over a range of 5 to 25 Å via photoinduced electron transfer (PET). Use of a bimane:tryptophan probe pair provides a unique advantage over other PET probe pairs in that it requires only one extrinsic labeling site for bimane, as tryptophan is incorporated into the protein naturally or by genetic engineering. We have empirically derived an equation that describes the interaction between the two probes as a function of both solvent polarity and interprobe distance. This novel equation makes it possible to quantitatively determine the distance between bimane and tryptophan as a protein undergoes conformational changes in solution.
7. Keith M Berland (Invited talk)
   Investigating the self-assembly of amyloid peptides using two-photon microscopy.
   Abstract

   Y Liang, D G Lynn, and K M Berland. Emory U., Atlanta.

   While the growth and structure of amyloid fibers with beta-sheet secondary structure has been widely investigated in recent years, the mechanism of self-assembly remains poorly understood. Multiple intermediate species have been proposed to play important roles in the self assembly process, yet many of these remain poorly defined or have not been clearly observed. To better understand the fundamental mechanisms involved in peptide self-assembly we have used a series of 6 to 15 amino acid fluorescent peptides that model the core of the amyloid-beta peptide together with two-photon imaging, FCS, and FRET measurements to investigate amyloid fiber growth. We report on our observations of early intermediate structures, conformational changes in peptides upon self assembly, and investigations of the dynamics of peptide exchange within mature fibers.
8. Jan Willem Borst (Invited talk)
   ATP effects on FRET biosensors.
   Abstract

   J W Borst1,2, R Slijkhuis2, M Willemse4, J Franssen4, A van Hoek1,3, and A Visser1,2. 1 Microspectroscopy Centre, Labs of 2 Biochemistry and 3 Biophysics, Wageningen U, Dreijenlaan, The Netherlands; 4 Dept. of Cell Biology, NCMLS, Radboud U, Nijmegen Med Centre,The Netherlands.

   The development of Förster Resonance Energy Transfer (FRET) biosensors has allowed researchers to achieve real-time visualization of metabolite behavior or signal transduction events in living cells. The most commonly used biosensors are genetically encoded sensors with metabolite, second messenger or protein-modification specific binding domains sandwiched between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). FRET biosensors are designed to monitor changes in conformation of the fused sensory domain leading to changes in FRET signal. During the development of an ATP sensor, the group of Fransen encountered an unusual effect of ATP on their control sensors. These control sensors showed a change in YFP/CFP signal by addition of physiological concentrations of ATP. The aim of this research was to examine the molecular mechanism of the ATP effects on FRET sensors using time resolved fluorescence spectroscopy techniques. This approach was used to eliminate artifacts which can be encountered using intensity based measurements. Addition of ATP to the control FRET sensor showed an increase of the donor fluorescence lifetime of about 10 % which confirmed the intensity based data. An increase in the fluorescence lifetime was also measured on purified ECFP only, but no changes were observed on free YFP molecules. As a possible model for the ATP effects on FRET sensors we propose that ATP might bind to ECFP via an electrostatic interaction with the Histidine 148 residue.
9. Jean-Claude Brochon (Speaker)
   Steady-state and time-resolved luminescence of quantum dots upon one and two photon excitation.
   Abstract

   N LI1, V D Chinh2, P T Nga2, E Henri1, H N Tran2, and J C Brochon1. 1 Lab de Biotech et Pharmacol génétique Appliquée, CNRS, Ecole Normale Sup de Cachan, France; 2 Lab of Laser Spectroscopy, Inst of Physics and Electronics, VAST, Hanoi, Vietnam.

   Quantum dots (or semi-conductor nanocrystal) are inorganic nanoparticles where charges are confined in tiny spaces. They show some rather unique photophysical properties such as size dependent emission wavelength, large absorption spectra allowing common excitation wavelength, sharp and symmetrical emission spectra, good photo-stability. QDs have been used as biological probes and bio-sensors mainly through steady-state luminescence measurement and particularly upon two photon excitation process because the corresponding absorption cross section are higher than of organic fluorophores. Another interest in QDs is to be FRET donors because of possible reduced cross-talked between donor-acceptor signals and the possibility to shift excitation wavelength apart of acceptor absorption. In biological system change in luminescence decay upon FRET is a way to escape from artefact in FRET measurement such as scattered light or static quenching(1).
   Time-resolved luminescence properties of CdSe-ZnS in toluene and CdSe-ZnS hydrosoluble QDs have been studied upon one-photon and two-photon excitation. The emission spectra remain unchanged under one-photon or two-photon excitation, the excitation spectra upon TPE is flat in comparison with one-photon excitation process. Time-resolved luminescence of QDs is complex, they have been analysed in terms of sum of five exponentials ranging from 1.0 ns to 100.0 ns. The average lifetime was 21ns for the hydrosoluble QD and 4.0 ns for the toluene soluble emitting at 550 nm. This heterogeneity can be partly attributed to the size heterogeneity as detected by FCS measurements. Further experiments using lifetime discrimination in autocorrelation function calculation may help to clarify the size parameter in luminescence kinetics heterogeneity.
   (1) M. T. Fernández-Argüelles et al. (2007) Nano Lett. 7, 2613-2617.
10. Long Cai (Winner of Weber Prize)
    Life at low copy number: a single molecule adventure in live cell gene expression.
    Abstract

    L Cai, N Friedman, and X S Xie. Caltech, Dept. of Biology, Pasadena.

    In a living cell, gene expression, the transcription of DNA to mRNA followed by translation to protein, occurs stochastically, as a consequence of the small copy numbers of DNA and mRNA molecules involved. These stochastic events of protein production are difficult to observe directly with measurements on large ensemble of cells due to lack of synchronization among cells. Measurements to date on single cells, however, lack the sensitivity to resolve individual events of protein production.
    We demonstrate a microfluidics based assay that allows real-time observation of the expression of ß-galactosidase in living Escherichia coli cells with single molecule sensitivity. We observe that protein production occurs in bursts, with exponentially distributed burst sizes. Application of this assay to probe gene expression in individual budding yeast and mouse embryonic stem cells demonstrate its generality.
    We note that many proteins involved in important cellular decisions, such as cell fate and differentiation, are expressed at low levels. We utilize single molecule detection to study the role of low copy numbers lac permease in committing E.coli to bistable metabolic states. We show conclusively that one molecule of permease is insufficient to trigger induction and elucidate the molecular mechanism for decision making in the lac operon.
    From the experimentally observed burst statistics, we develop an analytic model showing that the copy number distribution of a protein in a population of cells is Gamma distributed. This model allows us to establish that the two kinetic parameters of protein expression, the burst size and frequency, can be extracted directly from the “noise” in a steady state population measurement and is equivalent to those measured in the real time experiments.
11. Yan Chen (Speaker)
    Probing nucleocytoplasmic transport with fluorescence fluctuation spectroscopy and two-photon activation of photoactivable GFP.
    Abstract

    Y Chen, B Wu, and J Müller. Physics Dept, U of Minnesota, Minneapolis.

    Large proteins and macromolecular complexes have to enter and leave the nucleus in an efficient and selective manner. Macromolecules that are greater than 40 kD are transported actively across the nuclear envelope through nuclear pore complexes using soluble transport factors or carrier molecules that cycle between the cytoplasm and nucleus. Interestingly, the carrier proteins themselves interact with each other in order to transport cargo proteins across the nuclear pore complexes. In this work, we apply dual-color time-integrated fluorescence cumulant analysis (TIFCA), a fluorescence fluctuation spectroscopy technique, to investigate the protein interactions of the carrier proteins directly in cells. In addition, we apply two-photon activation to directly examine the nucleocytoplasmic transport of photoactivable GFP tagged carrier proteins. With these two approaches, we are able to probe the nucleocytoplasmic transport process of NTF2 directly inside cells and under equilibrium conditions. We investigate the oligomerization of NTF2 in cells and its transport properties when crossing the nuclear pore complexes.
12. Andrew Clayton (Invited talk)
    Reinventing the fluorescence wheel: the polarized AB-plot for the frequency-domain analysis and representation of fluorophore rotation and resonance energy homotransfer.
    Abstract

    A H A Clayton. Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Australia.

    The graphical representation of single-frequency phase-modulation fluorescence lifetime imaging data, referred to as the AB-plot (phasor plot or polar plot), is extended to take into account measurements of the polarized components of the fluorescence. For a hindered rotator model (characterised with a single excited-state lifetime, a single rotational correlation time and limiting initial and final anisotropies) the rotational correlation time and the excited lifetime can be determined from the AB-plot of any two of the following emission components; parallel, perpendicular, total emission or combinations thereof. A strategy for resolving the component hindered rotations and lifetimes for mixtures of two hindered rotators from measurements of the total, parallel and perpendicular components of the emission is developed. The analysis does not require prior knowledge of the initial limiting anisotropy or of the steady-state anisotropy or of the excited state lifetime. Plots in polarized AB space derived for heterogeneous systems are constructed to aid interpretation of frequency-domain dynamic depolarization imaging microscopy (rFLIM) experiments. These plots can be used to distinguish spatially-dependent rotational correlation time heterogeneity from heterogeneity in limiting anisotropies. The effects of aperture depolarization are discussed. It is anticipated that the polarized AB plot will provide a useful adjunct to existing methods for visualising and analysing dynamic polarization phenomena arising from molecular dynamics and homo-energy transfer in single-frequency microscopy applications.
13. Christian A Combs (Invited talk)
    Multi-photon imaging with total emission detection (TED).
    Abstract

    C A Combs1, A V Smirnov2, J R Knutson2, R S Balaban3. 1 NHLBI Light Microscopy Facility; 2 NHLBI Lab of Molecular Biophysics; 3 NHLBI Lab of Cardiac Energetics, Natl Institutes of Health, Bethesda.

    Intrinsic to multi-photon excitation microscopy is the optical sectioning that occurs during excitation at the diffraction-limited spot. Fluorescence generated at the focal spot propagates in all directions. Recently we have constructed and tested a device that maximizes the probability of collecting all of the scattered and ballistic light generated at the focal spot of multi-photon excited emissions (MPE) to optimize the signal-to-noise ratio (SNR) for micro-imaging. The main part of the device is a parabolic reflector (that surrounds the sample and top of the objective) that is optically coupled to a pair of collimating lenses (above the sample) that redirect emitted light to a separate detector. This combination of additional optics, in conjunction with normal light collection with the objective, has allowed the total emission detection (TED) condition to be approached. This device has now been tested on several tissue types (heart, kidney and brain) expressing non-muscle myosin labeled with GFP. The results are consistent with simulations suggesting that the gain in light collection is a function of the NA of the objective and also a function of the tissue properties (primarily thickness). Light collection gains as high as 8.9 fold more were observed when using a 20x (0.75 NA) objective and up to 5x when using a 20x (0.95 NA) water objective in the tissues tested. Although this increase in SNR can be used to improve time resolution and to reduce laser power requirements/photodynamic damage we have not observed 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.
14. Maïté Coppey-Moisan (Speaker)
    Time regulated relationship between cell strengthening of extracellular matrix - cytoskeleton linkage and external rigidity: a quantitative study by multiple optical tweezers.
    Abstract

    M Allioux-Guerin and M Coppey-Moisan. Institut Jacques Monod, Paris, France.

    We investigated cellular response modulation to low external loads by applying small asymmetrical tensions on the cortex of 3T3 fibroblasts. Using latex beads functionalized with fibronectin and trapped in multiple optical tweezers, we studied single cell reaction to a rigidity differential, such as force generation, adhesion reinforcement and CSK reorganization. Our system allows a quantitative study of the spatio-temporal regulation of the cell response. Here, we show that a cell can feel heterogeneities of even weak substrate rigidities, and adapt to it in a local and time dependent response. Small mechanical stresses induce a local development of adhesion sites and an acto-myosin complex reorganization. Close to the adhesion, the actin recruitment occurs proportionally to the force developed by the cell on the bead, which itself increases at a rate proportional to the external rigidity. This demonstrates that the cell sensing of a rigidity differential is translated into a time regulated localized CSK strengthening. Moreover, at any time the cell forces are modulated following the substrate rigidity to conserve a constant bead movement. It gives us clues on how cells control their mechanotransduction process. These results highlight the importance of a dynamic regulation of the cell response to differential rigidities.
15. Lloyd Davis (Poster)
    Actively-controlled electrokinetic delivery of single fluorescent biomolecules in fluidic nanochannels.
    Abstract

    L M Davis1, B Canfield1, X Li1, W Hofmeister1, I Lescano1, B Bomar1, Z Sikorski1, W Robinson1, J King1, J Germann1, G Shen1,2, J Wikswo3, D Markov3, P Samson3, and C Daniel4. 1 Ctr for Laser Apps, U of Tennessee Space Inst, Tullahoma; 2 Presently at Luna Innovations, Inc., Blacksburg, VA; 3 Vanderbilt Inst. for Integrative Biosystems Res and Education, Vanderbilt U, Nashville; 4 Oak Ridge National Lab, Oak Ridge.

    Time-resolved fluorescence studies of conformations and interactions of individual proteins would benefit from decreased probe volume, increased residence time, high-speed fluorescence photon counting, low background, and rapid collection of statistical data on many individuals. To address these issues, we have fabricated sub-100-nanometer-sized channels between two closely-spaced microchannels in a fused silica lab-on-a-chip device and have conducted experiments that demonstrate detection of single fluorescently-labeled proteins in buffer solution within a nanochannel with high signal and low background. The nanochannel constrains the molecule to move along one dimension and the net rate of diffusion is observed to be slowed compared to that in bulk solution. Electrodes have been placed into the microchannels for electrokinetic transport of molecules along the nanochannels. The fluorescent biomolecules are driven along the nanochannel until one passes into a two-focus laser irradiation zone. Pulse-interleaved excitation and time-resolved single-photon detection within a custom-built confocal fluorescence microscope enables the position of the biomolecule to be estimated. Our goal is to actively control the fluidic transport in response to the molecule position to reposition the molecule to the center of the two laser beams, where the irradiance is approximately constant, and to achieve rapid delivery of each new biomolecule to the sensing zone, following the completion of a measurement, or the photobleaching of the prior molecule. We have used Monte Carlo computer simulations that include Brownian diffusion and photophysical effects such as triplet crossing and photobleaching of the label to test control algorithms, which are being implemented in a custom field-programmable-gate-array (FPGA) circuit for the active control of the electrokinetic voltages. The measured photon signal is also analyzed by FCS, wherein the autocorrelation function (ACF) of the stream of photons yields a peak whose width gives the mean residence time of molecules within the dual-laser-beam focal volume. Initial experiments with a fresh nanochannel device indicate that active control of the electrokinetic voltage results in an increase in the ACF width, as might be expected when molecules are trapped, and a decrease in the amplitude, indicative of decreased time between new molecules entering the focal volume. Unfortunately, the problem of fluorescent molecules sticking to the walls of the nanochannels presently limits the useful life of a nanochannel device to a few hours. Ongoing work involves the use of polarization and emission-wavelength resolved single-photon detection to study binding interactions of individual protein molecules that have been labeled with photoactivatable switching molecules.
    This project is supported in part by DARPA grant W911NF-07-1-0046, by the Center for Laser Applications, and by the Vanderbilt Institute for Integrative Biosystems Research and Education. A portion of this research was conducted at the Center for Nanophase Materials Sciences and the SHaRE User Facility, which are sponsored by the Division of Scientific User Facilities, Office of Basic Energy Sciences, U.S. Department of Energy.
16. Michelle A Digman (Speaker)
    Huntingtin (Htt) oligomer formation in live cells.
    Abstract

    M A Digman1, G Ossato1, E Frasnelli1, G Buonaventura2, C Aiken3, J L Marsh3, and E Gratton1. 1 U of California, Irvine, Dept of Biomed Engineering; 2 U of Catania, Italy, Dept. of Pharmacology; 3 U of California, Irvine, Dev and Cell Biology, Sch of Biological Sciences, Irvine.

    The process of protein multimerization can elicit crucial signaling cellular events to ensure the survival of the cell or lead to devastating effects such as progressive degenerative diseases. Current methods to measure aggregation of proteins are few and limited to few pairs of proteins for examples using FRET, Homo-FRET or limited to measuring one point at a time in live cells such as in photon counting histogram analysis. We have developed a novel method, i.e., the Number and Molecular Brightness (N&B) method, based on moment analysis to map the number and size of aggregates in live cells. The N&B analysis can distinguish with pixel resolution, the aggregation state of proteins by calculating the average intensity and the variance of the molecular fluctuations. We use this approach to study the aggregation of the huntingtin protein (Httex1 97QP-eGFP, Httex1 46Qp-eGFP and Httex1 25QP-eGFP) as a function of time in living cells. Our results indicate that the huntingtin aggregates are highly dynamic associating and dissociating in many regions of the cell. We also show that they form aggregates of discrete sizes which seem to be in three stages: small aggregates of about 10 units, larger aggregates of 50-100 units and the nucleation of plaques. The plaque, located close to the membrane, recruits most of the oligomeric huntingtin protein in the cell. Using the N&B method, we have developed a model for huntingtin aggregation in live cells.
17. Elliot Elson (Speaker)
    Detection and mechanical effects of nanodomains in lipid bilayer model membranes.
    Abstract

    K M Pryse1, W B McConnaughey1, Y Li3, J Whisler2, E Fried2, J Dolbow4, G Genin2, and E L Elson1. 1 Dept of Biochem and Mol Biophysics, 2 Dept of Mechanical and Aerospace and Structural Eng, 3 Dept of Physics, Washington U, St. Louis; 4 Dept of Mechanical Eng, Duke U.

    Lipid rafts are small (~100 nm) specialized nanodomains in biological membranes, enriched in specific membrane lipids such as sphingomyelin and cholesterol, that serve as platforms for collecting signaling molecules and as sites for the assembly of specialized structures such as enveloped viruses (Pike 2004). To understand the factors that control the stability and dynamic properties of rafts we are studying nanodomain properties in membrane model systems, “giant unilammelar vesicles” (GUVs), with simplified compositions that can be precisely controlled. Much earlier work on model membranes including GUVs composed of higher and lower melting lipids as well as cholesterol has demonstrated the existence of separate phases enriched in one or the other of the lipid species (Veatch 2005). In these GUVs the distinct domains can be specifically labeled with fluorescent probes that preferentially sequester into one or the other of the two types of domains. The domains are resolvable by fluorescence microscopy and therefore must have dimensions of the order of a μm or more. To detect and measure nanodomains below the optical resolution limit we are using methods based on FCS. There is already a considerable volume of data on bilayer phase behavior using this and related approaches (Korlach 1999; Ruan 2004; Kahya 2005; 2006; Celli 2008). We will use both the diffusion rate as well as the extent of clustering of the fluorescent lipid probes to determine the size of the nanodomains.
    According to a simple model the sizes of membrane domains should be determined by the balance between surface energy (line tension) and the cohesive energy that binds together the molecules of a specific phase (Frolov 2006). The mechanical properties of a membrane, however, can influence its phase separations (Baumgart 2005). Phase separation is influenced by curvature changes in phase domains (Reigada 2005). Our aim is to examine the relationship between phase separation and membrane mechanical properties using a chemomechanical model. Beginning with a 2-component system, the energy of the membrane is estimated as the sum of a chemical contribution of a Cahn-Hilliard type (Cahn 1958) and a mechanical energy that depends on the difference between the curvature of the membrane and its spontaneous curvature. This leads to an expression for the chemical potential for one species relative to the other and ultimately to an evolution equation for the local densities of the two components over time. Our long range goal is to discover the factors that control the distributions of sizes, stabilities and evolution rates of nanodomains in GUVs of defined compositions. (See E. Elson for list of literature cited.)
18. Zeno Földes-Papp (SPONSOR)
    What is the meaningful time for studying a single molecule in solution and a live cell without immobilization or hydrodynamic/electrokinetic focusing?.
    Abstract

    Z Földes-Papp1, J Liao2, Terpetschnig2, E Pilger3, G Kostner4, and B. Barbieri2. 1 Visiting professor at ISS2; 2 ISS, Inc., Champaign; 3 Dept of Internal Medicine and 4 Inst of Molecular Biology and Biochemistry, Med U of Graz, Austria.

    We present ideas for increasing the observation time of a single molecule free in solution without the need for immobilization or hydrodynamic/electrokinetic focusing [1, 2]. By using hidden, continuous-time numerical Markov models for evaluation of our new concepts, we compare our way with normal fluctuation experiments. We first give answers to the questions:
    (1) What is the time and average entering frequency C, respectively, for a molecule to find the probe volume DV at an initial molar concentration in the bulk?
    (2) What is the probability (d/dt)p>0(Dt) that the entering molecule is the original molecule as function of the time Dt from last entry and the concentration of other molecules of the same kind in the bulk?
    (3) What is the number of meaningful re-entries of the original molecule?
    Approaches (1) – (3) give the meaningful time Tm in the probe region DV allowing to study just a single molecule in solution or a live cell without immobilization or hydrodynamic/electrokinetic focusing. Tm is physically defined by the stochastic variable waiting time Dt for the next entry of another molecule than the original one.
    We discuss and describe how our work differs from the results of other groups that have so far addressed the reentry problem of a single molecule.
    [1] Földes-Papp, Z. (2007). Fluorescence fluctuation spectroscopic approaches to the study of a single molecule diffusing in solution and a live cell without systemic drift or convection: a theoretical study. Curr. Pharm. Biotechnol. 8, 261-273.
    [2] Földes-Papp, Z. (2007). ‘True’ single-molecule observations by fluorescence correlation spectroscopy and two-color fluorescence cross-correlation spectroscopy. Exp. Mol. Pathol. 82, 147-155.
    (*Financial support: FWF Austrian Science Fund Research Project P20454-N13, Medical University of Graz, Austria.)
19. Marcella Gilmore (Allergan Sponsor Talk)
    Daret assays of botulinum neurotoxin proteolytic activity with a fully recombinant substrate.
    Abstract

    M A Gilmore, D Williams, Y Okawa, P Kim, B Holguin, J A Webber, M F Verhagen, K R Aoki, and L E Steward. Allergan, Inc., Irvine, CA.

    The DARET (Depolarization After Resonance Energy Transfer) assay is a homogenous, coupled FRET-fluorescence polarization (FRET-FP) assay for Botulinum neurotoxin Type A or Type E proteolytic activity that relies on a fully recombinant substrate. The substrate consists of BFP and GFP proteins flanking SNAP-25 residues 134-206. In the assay, the substrate is excited with polarized light at 379 nm, which primarily excites the BFP, while emission from the GFP at 509 nm is monitored. Energy transfer from BFP to GFP in the intact substrate results in a substantial depolarization of the GFP emission. Upon cleavage by toxin, the BFP and GFP domains are spatially separated, the energy transfer is eliminated, and the polarization of the GFP emission increases. This increase in polarization can be monitored to assay the proteolytic activity of low picomolar concentrations of Type A and Type E toxin in real time. The assay is amenable to high-throughput applications, and the substrate can also be used in a traditional FRET assay.
20. David Michael Grant (Poster)
    Multiplexed FRET for imaging cell signaling and high speed optically sectioned FLIM for high throughput screening applications.
    Abstract

    D M Grant1, W Zhang2, E J McGhee1, C B Talbot1, J McGinty1, T D Bunney2, I Munro1, C Dunsby1, P Courtney3, M Katan2, M A A Neil1, and P M W French1. 1 Dept of Physics, Imperial College London, UK; 2 Div Cell and Mol Biology, Inst of Cancer Res, UK; 3Perkin Elmer Inc, UK.

    Förster Resonance Energy Transfer (FRET) is a powerful technique for studying molecular interactions which when implemented in the microscope can provide information on both spatial and temporal aspects of these interactions in single cells. Fluorescence lifetime imaging (FLIM) can provide a robust read-out of FRET and using this we have developed an automated FLIM multi-well plate reader for high throughput screening applications. This system combines wide field time gated FLIM with a Nipkow disc confocal scan head for fast optically sectioned FLIM imaging of cell populations. We are also combining our wide field time gated FLIM technology with spectral ratiometric imaging for read-outs of multiple FRET sensors expressed in single cells. Recent developments have led to an increased number of fluorescent proteins towards the red end of the visible spectrum with potential to image additional FRET pairs alongside the commonly used CFP/YFP pair. By combining spectral ratiometric imaging of a CFP/Venus calcium sensor with FLIM of a longer wavelength FRET pair we have been able to image the signal from two distinct FRET sensors within the same single cell. We are currently applying this instrument to dual studies of calcium signalling and small GTP-ase Ras activation within single living cells.
21. Enrico Gratton (Speaker)
    Exploiting fluctuations to determine protein aggregation and stoichiometry in cells.
    Abstract

    E Gratton. Laboratory for Fluorescence Dynamics, U of California, Irvine.

    The study of protein interactions in live cells is a major challenge in cell biology. We have developed a method for detecting and quantifying when and where two different proteins interact during complex cellular processes. Our method detects these complexes and also provides the fraction of molecules that reside in complexes as well as their rates of diffusion or binding kinetics. In this analysis, we compute the cross-correlation of temporal fluctuations in the fluorescence intensity from molecules labeled with two different fluorescence tags. The novelty of the method is that the cross-correlation is obtained simultaneously across an entire image and thereby produces a detailed, cellular map of protein interactions. This method is robust and applicable to images obtained on commercial laser scanning confocal microscopes and therefore is of general interest cell biologists. Unlike FRET, the technique is not constrained by the distance of the fluorophores; this makes it more generally applicable than FRET. It also complements single molecule methods, like PALM, which reveal distances, but not structural interactions. Thus, while PALM might be considered a “super resolution” technique, our technique reveals unexpected, “super information” that is inherent in scanning confocal images. (Supported in part by the NCRR of the NIH (P41-RR003155), and by the NIGMS through the Cell Migration Consortium (U54-GM064346) and the Center for Complex Biological Systems (P50-GM076516).)
22. Enrico Gratton (SPONSOR)
    The SimFCS environment: from simulation to data acquisition and analysis.
    Abstract

    E Gratton. U of California, Irvine.

    SimFCS is a complex program for simulation, data acquisition, data analysis and modeling of fluorescence data. In simulation mode, SimFCS can provide FCS, scanning FCS, RICS N&B and particle tracking data to test models and functions. In data acquisition, SimFCS support several of the popular data acquisition cards based on National Instrument drivers and also from other manufactures. In the data acquisition mode, SimFCS can also move the scanner in x, y and z coordinates along predetermined patterns. The scan pattern can be changes on the fly for real time particle tracking in 3D. In the analysis mode, several modules are available for the analysis of FCS data, a comprehensive toolbox for scanning FCS with a large number of libraries. For image analysis, most of the image correlation methods have been implemented including RICS, tICS, STICS and N&B. In the particle tracking mode, several modes of tracking are also implemented and the library for the analysis of trajectories is comprehensive of many common models. Many more analysis modules are available. Please, come and ask about how SimFCS can help with your research.
23. Zygmunt (Karol) Gryczynski (Speaker)
    Plasmons assisted fluorescence - emerging fluorescence applications.
    Abstract

    Z Gryczynski, J Borejdo, N Calander, E Matveeva, R Luchowski, M Szabelski, P Sakar, and I Gryczynski. Ctr for Commercialization of Fluorescence Technologies, Fort Worth.

    This presentation will give an overview of new emerging plasmonic nanotechnology utilizing metallic nanostructures and optical phenomena for applications in biomedical imaging and sensing. Combination of nanotechnology, plasmonics, and photonics opens new possibilities for developing novel ultrasensitive assays for medical and chemical uses. Plasmons enhanced fluorescence (PEF) presents incredible potential for use in microscopy, biological assays, immunoassays, genomics, and for studying biophysical properties of macromolecules on a nanoscale level. In this presentation we will discuss novel nanoplasmonic approaches that open new capabilities for single molecule detection and makes feasible development of nanophotonic assays based on single molecule system.
24. German Gunther (Poster)
    Interaction of human erythrocyte with MMS detergent: changes in membrane fluidity reported by Laurdan.
    Abstract

    G Gunther1, C Toro1, A Zanocco1, M Rifo2, E Gratton3, and S A Sánchez3. 1 Lab. de Cinética y Fotoquímica, Facultad de Ciencias Químicas y Farmacéuticas, U. de Chile, Santiago, Chile; 2 Universidad Tecnológica de Chile. Instituto Profesional Centro de Formación Técnica, INACAP, Santiago, Chile; 3 Lab for Fluorescence Dynamics, UC-Irvine, Irvine.

    LAURDAN is a membrane probe that gives information about the water content in the bilayer that is related with the membrane fluidity. The spatial resolution necessary for cellular work is given by the use of 2-photon microscopy and dual channel detection.
    In this report we present the changes observed in fluidity of the lipid bilayer when it interacts with detergents using 2-photon Laurdan GP imaging.
    The solubilization of biological membranes by detergents has been used as the main method for the isolation and purification of membrane proteins and other constituents and no much information in the literature related with the fluidity changes during the process is found. We present here results of the fluidity changes in the solubilization process of artificial and natural systems produced by the sucrose monoester of myristic acid, b-D-Fructofuranosyl-6-O-myristyl-a-D-Glucopyranoside (MMS). In POPC liposomes, the solubilization process is accompanied by changes in the size of the liposomes until it completely disappears without changes in the water content of the bilayer. For erythrocytes, two processes can be observed with time of incubation: first the echinocytes (erythrocytes with regularly spaced spicules on their surface) change to a round shape with a decrease in the water penetration in the membrane, later the cells will suffer an increase in volume and an abrupt increase in water penetration. Financial support for E.G. and S.S. NIH RR03155 and Fondecyt 1040573 for C.T and G. G.
25. Taekjip Ha (Speaker)
    Playing extreme sports with biological molecules in singulo.
    Abstract

    T Ha. U of Illinois at U-C and Investigator, HHMI, Urbana.

    Single molecule measurement techniques have revolutionized biological inquiries by providing previously unobtainable data on elementary molecular processes. However, most studies thus far have been limited to single isolated molecules even though these molecules do not function in isolation in the cell. To emulate the cellular conditions better, more complex systems with multiple components need to be looked at. This requires major improvements in measurement and analysis techniques which is a subject of this talk. I will present our attempts to develop ‘extreme in vitro single molecule techniques’ in order to maximize information content in single molecule analysis.
26. George Hanson (Poster)
    Creating new age fluorescent reagents for cell biology applications.
    Abstract

    G T Hanson, D Thompson, R Batchelor, P Melquist, and M O’Grady. Invitrogen, Madison.

    Fluorescent molecules are indispensable tools for cell biologists. Whether monitoring calcium mobilization in living cells or highlighting subcellular compartments using antibodies in fixed cells, organic fluorophores have their utility. However with the advances in genetically encoded auto-fluorescent proteins (FPs) over the past decade, there has been an explosion of applications describing the spatial and temporal resolution of cellular processes using FPs. The challenge no longer lies in creating probes for cellular applications, but in the more simple aspects such as delivering these probes to living cells in a non-invasive fashion. We describe the development of modern fluorescent reagents for cell biology applications. These reagents combine the utility that is afforded by FPs with the versatility of the BacMam delivery technology. BacMam is the use of an insect cell virus, baculovirus to deliver genes to mammalian cells. We have created both active indicators of calcium and cytoskeletal structure as well as myriad of passive fluorescent organelle markers. We demonstrate the use of these fluorescent reagents by screening for H1 histamine G protein-coupled receptor agonists and antagonists and imaging of live human primary cells by fluorescent microscopy. Overall these reagents wonderfully combine the content of FPs with the delivery of BacMam.
27. Christian Hellriegel (Invited talk)
    Latest data from 3D single particle tracking.
    Abstract

    C Hellriegel and E Gratton. U of California, Dept of Biomedical Engineering, Irvine.

    3D Particle tracking is used to characterize the motion of a particle (labelled protein, bead, organelle, etc...). At no extra cost, these trajectories also map substrates (membranes, surfaces, fibers) with high-resolution (2-20 nm) when the particles move in close proximity to those structures. The possibility to record intensity, spectrum and lifetime of the particle's emission - simultaneously while tracking - opens the possibility to characterize the interactions between particle and the substrate even further. In this contribution will be show the latest examples that illustrate these capabilities of the tracking method.
28. Jelle Hendrix (Poster)
    Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy.
    Abstract

    J Hendrix1, C Flors2, P Dedecker2, J Hofkens2, Y Engelborghs1. 1 Lab of Biomol Dynamics, Dept of Chemistry, Katholieke U Leuven, Belgium; 2 Lab of Photochem and Spectros and Inst for Nanoscale Physics and Chemistry (INPAC), Dept of Chemistry, Katholieke U Leuven, Belgium.

    Monomeric red fluorescent proteins (mRFPs) have become indispensable tools for studying protein dynamics, interactions and functions in the cellular environment. Their emission spectrum can be well separated from other fluorescent proteins and their monomeric structure preserves the natural function of fusion proteins. However, previous photophysical studies of some RFPs have shown the presence of light-induced dark states that can complicate the interpretation of cellular experiments. In this article, we extend these studies to mRFP1, mCherry and mStrawberry by means of fluorescence correlation spectroscopy (FCS) and prove that this light-driven intensity flickering also occurs in these proteins. Furthermore, we show that the flickering in these proteins is pH-dependent. Single molecule spectroscopy revealed reversible transitions from a bright to a dark state in several timescales, even up to seconds. Time-resolved fluorescence spectroscopy showed multiexponential decays, consistent with a “loose” conformation. We offer a structural basis for the fluorescence flickering using known crystal structures and point out that the environment of Glu-215 is critical for the pH dependence of the flickering in RFPs. We apply dual-colour FCS inside live cells to prove that this flickering can seriously hamper cellular measurements if the timescales of the flickering and diffusion are not well-separated.
29. Jelle Hendrix (Poster)
    How a human protein becomes a hostage of the HIV - fluorescence techniques for studying live cell protein dynamics reveal strong chromatin tethering of HIV-1 Integrase by human LEDGF/p75.
    Abstract

    J Hendrix1, Z Debyser2, and Y Engelborghs1. 1 Lab of Biomolecular Dynamics, Dept of Chemistry, Katholieke U Leuven, Belgium; 2 Lab of Molecular Virology and Gene Therapy, Katholieke U Leuven, Flanders, Belgium.

    The retroviral replication cycle includes a step where the viral genome is incorporated into that of the host cell, by the action of the enzyme integrase (IN). Nowadays it is accepted that the integrase of HIV-1, the virus that causes AIDS, has a human co-factor, LEDGF/p75. In contrast to the cellular function of LEDGF/p75, a transcriptional co-activator, the exact viral function is poorly understood.
    We studied eGFP-tagged LEDGF/p75 with cellular fluorescence correlation spectroscopy (FCS), to gain insight in the cellular dynamics of the protein. In the nucleus the diffusion of the protein was a lot slower than in the cytoplasm, and two-thirds of the protein population was present in a second slow component. We concluded that LEDGF/p75 is slowed down by dynamic binding to the chromatin. This result is in line with known information on transcription factors. We then investigated the dynamics of the non-chromatin-bound protein using domain-deletions and site-directed mutagenesis. We found that free LEDGF/p75 shows anomalous diffusion both in the cytoplasm and nucleus, most likely due to its large size and predicted partially unfolded structure.
    In an attempt to study the intracellular interaction of LEDGF/p75 and HIV-1 integrase (IN), we used fluorescence cross-correlation spectroscopy (FCCS). When co-overexpressing mRFP1-tagged IN, the diffusion time of the intranuclear complex of the two proteins is situated on the hundreds-of-milliseconds timescale, resulting in strong photophysical bleaching of the fluorescence. By specifically decreasing the affinity of the complex for the chromatin using site-directed mutagenesis of the main chromatin-binding domain of LEDGF/p75, the apparent diffusion time of the protein complex decreased, allowing for the quantification of the interaction with FCCS. These results show the possibility to dissect diffusion and exchange kinetics when performing FCS or FCCS.
30. Martin Hof (Speaker)
    The compaction mechanism of intermediate-sized DNA molecules elucidated by fluorescence lifetime correlation spectroscopy.
    Abstract

    J Humpolíčková, A Benda, L Beranová, and M Hof. Dept of Biophysical Chemistry, J. Heyrovský Inst of Physical Chemistry, Acad of Sciences of the Czech Republic.

    The compaction of DNA plays a role in the nuclei of several types of cells and becomes important in the non-viral gene therapy. Using single molecule fluorescence microscopy it was shown, that spermine-induced compaction of large DNA molecules (30 kbp-170 kbp) occurs in a discrete “all-or-non” regime, where the coexistence of free and folded DNA molecules was observed. In the case of intermediate-sized DNA molecules (~10 kbp), so far, it was stated that the mechanism of folding is continuous. We show that neither a standard benchmark techniques nor a single molecule technique such as fluorescence correlation spectroscopy, can decide what kind of mechanism is undertaken in the compaction process. Besides, we introduce an application of a new approach - fluorescence lifetime correlation spectroscopy. The method takes an advantage of a subtle lifetime change of an intercalating dye PicoGreen® during the titration with the condensing agent and based on that, it reveals the mechanism of the process and gives information on the equilibrium state transition dynamics of the compaction process. The method is used to compare the condensation process induced by different condensers. (Supported by Ministry of Education of the Cz Rep, grant LC06063.) See Dr. Hof for list of references cited.
31. Alan Rick Horwitz (Speaker)
    The assembly, maturation, and disassembly of adhesions in migrating cells.
    Abstract

    C K Choi1,2, M Vicente-Manzanares2, J Zareno2, C Brown2, A Mogilner3, M A Digman4, D Kolin5, P W Wiseman5, E Gratton4, and A R Horwitz2. 1 Dept of Biomed Engineering and 2 Cell Biology, U of Virginia, Charlottesville; 3 Dept of Neurobiol, Physiol and Behavior and Dept of Mathematics, U of California, Davis; 4 LFD and Dept of Biomed Engineering, U of California, Irvine; 5 Dept of Chemistry and Physics, McGill U, Montreal, Canada.

    Cell migration is driven by actin polymerization and the formation and turnover of adhesions at the cell front and the disassembly of adhesions and retraction at the cell rear. Using dual label imaging and high resolution TIRF microscopy, we have shown that nascent adhesions assemble and become stable within the lamellipodium of migrating cells. The assembly is myosin II-independent, requires actin polymerization, and most components enter simultaneously. As the back of the lamellipodium moves past the nascent adhesions, they either disassemble as the protrusion continues to advance or begin to grow and elongate as the protrusion pauses. Adhesion maturation is guided by an α-actinin-actin template and the crosslinking properties of myosin IIA. We have developed a toolbox of fluctuation methods for studying adhesion formation and disassembly at high spatial and temporal resolution during migration. Measurements of the dynamics and brightness of adhesion components, using correlation microscopy and number & brightness (N&B) analyses show that adhesions tend to assemble by the rapid addition of monomers, or small aggregates, while mature adhesions disassemble as large, more slowly exchanging aggregates. Two color cross-correlation shows that these complexes are comprised of several different molecules, and that they disperse quickly upon leaving the adhesion. Preliminary analyses suggest a uniform brightness for components in the complexes. All of this suggests that adhesions are comprised of subcomplexes and that they disassemble through the mass action once released from the adhesion. From our studies, a model emerges for adhesion assembly and maturation that clarifies the relative contributions of actin polymerization and organization and reveals a subunit based mechanism for adhesion disassembly.
32. Ken Jacobson (Speaker)
    “Post-raft” era nano- and microdomains.
    Abstract

    K Jacobson1,2, Y Chen2,3, A Neumann1,2. 1 Dept of Cell and Developmental Biology, 2 Lineberger Comphrensive Cancer Center and 3 Dept of Biomedical Engineering U of North Carolina-Chapel Hill.

    Lipid rafts as an organizing principle for biomembrane functionality has gained prominence but the form of the putative microdomains in cell membranes is an area of healthy controversy. However, when glycosyl-phosphatidylinositol-anchored proteins (GPIAPs) are induced to aggregate, distinctive domains form with interesting properties. Using single particle tracking (SPT), we found that by cross-linking several GPIAPs under antibody conjugated 40-nm gold particles, transient anchorage of the gold-labeled clusters occurred for periods ranging from 300 ms to 10 seconds. Transient anchorage was markedly reduced by (1) cholesterol depletion, (2) addition of Src family kinase (SFK) inhibitors, (3) knocking down the transmembrane protein, Csk binding protein (cbp), and (4) by expressing the dominant negative cytoskeletal adaptor, EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol dependent, SFK-regulatable linkage between a transmembrane cluster sensor, cbp, and the cytoskeleton that is mediated by the adaptor EBP50. Second, the dynamics and processing of stable DC-SIGN domains on dendritic cells (DC) will be described. These microdomains are critical for the uptake of pathogens by DC. Supported by NIH GM 41402.
33. Nicholas James (Poster)
    Incorporation of 5-hydroxytryptophan into transferrin and its receptor allows assignment of pH induced change in intrinsic fluorescence.
    Abstract

    N G James, S L Byrne, and A B Mason. U of Vermont, Burlington.

    Human serum transferrin (hTF) is a bilobal glycoprotein that binds and delivers iron to cells. A single ferric iron is coordinated within each lobe (N- and C-lobe) by 4 amino acids, including an aspartic acid, two tyrosines, and a histidine residue. The coordination sphere is completed by two oxygen atoms donated by the synergistic anion, carbonate. At pH 7.4 diferric hTF preferentially binds to its specific receptor (TFR) on the surface of cells and undergoes receptor mediated endocytosis inside a clathrin coated pit. Iron is released from hTF when the pH is lowered (~5.6) after fusion of the vesicle with an endosome (and in the presence of a chelator). Iron free (apo) hTF remains bound to the TFR and is recycled back to the surface to sequester more iron.
    Coordination of the ferric iron in each lobe severely quenches the intrinsic tryptophan fluorescence of hTF due to energy transfer to an absorption band that is produced by tyrosine-metal interaction. Under “endosomal conditions” (low pH and with a chelator and salt present) the recovery in the intrinsic fluorescence actually provides a sensitive assay to determine iron release rate constants. Fluorescence spectroscopy in combination with recombinant hTF and soluble TFR allow us to evaluate the role of TFR in release from the two lobes as a function of pH and salt “in vivo”. A concern in monitoring iron release from an hTF/TFR complex via tryptophan fluorescence is whether or not the TFR might be contributing to the signal. The soluble portion of the TFR contains 11 Trp residues per monomer; it is therefore possible that one or more of these Trp residues might undergo a pH induced change in quantum yield. To convincingly show that the TFR is or is not contributing to the change in signal attributed to iron release from the hTF/TFR complex, we have incorporated 5-hydroxytryptophan into both hTF and sTFR. This allows selective excitation of each individual protein during the iron release process.
34. David M Jameson (Speaker)
    Fluorescence investigations of Dynamin interactions – both in vivo and in vitro.
    Abstract

    D M Jameson1, Y Chen2, J Müller2, J A Ross1, B Barylko3, and J Albanesi3. 1 Dept Cell and Mol Biology, U of Hawaii, Honolulu, HI; 2 Physics Dept, U of Minnesota, Minneapolis; 3 Pharmacology Dept., UT SW Med Ctr, Dallas.

    Dynamin is a large (98kDa) GTPase involved in receptor mediated endocytosis, vesicle trafficking and assembly of the actin cytoskeleton. In vitro studies have indicated that dynamin can undergo a tetramer/monomer equilibrium and can also self-assemble to form large molecular weight aggregates. The oligomerization state of dynamin in vivo, though, has not heretofore been addressed. In the present work GFP was linked to the N-terminal domains of dynamin isoforms 1 and 2. The fluorescent proteins, expressed in COS cells, were studied using two-photon fluorescence correlation spectroscopy (FCS). The FCS data were analyzed using the Photon Counting Histogram (PCH) method which allowed for determination of the intrinsic “brightness” of the labeled protein complexes. In this way the aggregation state of both dynamin isoforms were determined in both nuclear and cytoplasmic regions of the cell. The results indicated that both dynamin 1 and dynamin 2 exist as a higher order oligomer in the cytoplasm. In the cell nucleus, the oligomerization of dynamin 1 and 2 is greatly reduced, and they exist predominantly as monomers. We also examined the dynamin-binding protein endophilin, also GFP-tagged on the N-terminus. Straight endophilin monomers self-associate into bent homodimers, which are believed to induce curvature in vesiculating membranes. It has been suggested that endophilin dimerization occurs only after endophilin associates with membranes. However, our studies using FCS and analytical ultracentrifugation support the view that dimerization precedes membrane binding.
    Supported in part by NIH grant RO1GM076665 (DMJ and JPA) and NIH grant R01GM064589 (JM).
35. David M Jameson (Opening Lecture)
    The seminal contributions of Gregorio Weber to modern fluorescence spectroscopy and to protein chemistry.
    Abstract

    D M Jameson. Dept of Cell and Molecular Biology, U of Hawaii, Honolulu.

    Gregorio Weber's career had two distinct periods. In the first, from 1947 to 1975, he contributed to the development of fluorescence instrumentation and to the theory and procedures of determination of quantum efficiency, fluorescence polarization, fluorescence spectra, and also to the analytical determination of the number and character of the components of composite fluorescence. He also introduced several novel and important fluorescence probes, including dansyl chloride, ANS, pyrene butyric acid, IAEDANS and PRODAN. The fluorescence polarization techniques developed by Weber have been applied to numerous problems of clinical investigation and to the diagnostic determination of drugs and metabolites in the blood. Weber devoted a great deal of time and effort to improving the determination of fluorescence lifetimes and created the "cross correlation" technique of phase fluorometry that forms the current basis of the phase measurements of lifetimes. In recent times it has lead to applications in microscopy and even to the macroscopic imaging of tissues. From 1975 onwards, initially in collaboration with H.G. Drickamer, he applied high pressure fluorescence spectroscopy to the study of molecular complexes and proteins. These observations confirmed the power of the fluorescence techniques to resolve questions of structure, and particularly dynamics at the molecular level. Weber and collaborators, in papers published from 1980 to 1997, demonstrated that most proteins made up of subunits can be dissociated by application of hydrostatic pressure, and opened in this way a new method of study of protein aggregates, which is influencing the approach to problems of both physiology and pathology. In these studies quite unexpected properties of protein aggregates have been revealed. For example, Weber and his collaborators in Urbana, Rio de Janeiro and Göttingen demonstrated the possibility of destroying the infectivity of viruses without affecting their immunogenic capacity and have thus opened the possibility of developing viral vaccines that contain without covalent modification all the antigens present in the original virus. In 1972 Weber introduced a general approach to the thermodynamics of multiple ligand binding to proteins which has greatly facilitated such studies and which has been extended to enzyme kinetics. His views on protein thermodynamics were summarized in his 1992 book entitled Protein Interactions, which he dedicated to: “Those who put doubt above belief.”
36. Chirlmin Joo (Weber Prize Honorable Mention)
    Real-Time observation of RecA filament nucleation and dynamics.
    Abstract

    C Joo1, S A McKinney2, I Cisse3, B Okumus4, M Nakamura3, I Rasnik5, S Myong3, and T Ha3. Present addresses: 1Seoul Natl U, South Korea; 2 Janelia Farm, HHMI; 3 U of Illinois at UC, Dept of Physics and IGB, Urbana; 4 Harvard Med School, Boston; 5Emory U, Atlanta.

    The RecA protein helps maintain genomic integrity through recombination. Using new single-molecule fluorescence assays and hidden Markov modeling, we first show the most direct evidence that a RecA filament grows and shrinks primarily one monomer at a time and only at the extremities. Both ends grow and shrink, contrary to expectation, but a higher binding rate at one end is responsible for directional filament growth. Next, we estimate that about five monomers are sufficient for filament nucleation. We further find that the nucleation is likely to occur through the binding of a pre-assembled oligomer rather than the simultaneous binding of several monomers, using a vesicle encapsulation assay.
37. Sunil Kumar (Poster)
    Multifocal multiphoton microscopy with time correlated single photon counting fluorescence lifetime imaging microscopy.
    Abstract

    S Kumar1, C Dunsby2, P A A De Beule2, D M Owen1, U Anand3, P M P Lanigan2, R K P Benninger2, D M Davis4, M A A Neil2, P Anand5, C Benham6, A Naylor6, and P M W French2. 1 Chem. Biology Cent, Imperial College London; 2 Photonics, Dept of Physics, Imperial College London; 3 Cancer Res. UK, Lincoln's Inn Fields; 4 Div of Cell and Mol Biology, Imperial College London; 5 Div of Neurosci and Mental Health, Imperial College London; 6 GlaxoSmithKline, New Frontiers Science Park, UK.

    A multifocal multiphoton fluorescence lifetime imaging (FLIM) microscope that enables the collection of time correlated single photon counting (TCSPC) data at 16 points on the sample simultaneously is presented. This allows the possibility of greatly increased acquisition speeds compared to single beam multiphoton TCSPC FLIM. A Ti:Sapphire laser (MaiTai, Spectra Physics) provides excitation that is split by a combined beam multiplexer and scanner (TriMScope, LaVision Biotec), and the resulting fluorescence is imaged onto a linear multi-anode PMT (PML16, Becker & Hickl). Fluorescence is collected in a nondescanned configuration combined with 1D stage stepping, and photon counting data is obtained via a TCSPC module (SPC-830, Becker & Hickl). If multiple TCSPC detection modules were to be employed, then this system has the potential to achieve photon pile-up limited count rates as high as 64 MHz, with the count rate increasing with the number of foci used.
    We believe that this technique offers the optimal means of obtaining multiphoton excited 3D FLIM data. Multiphoton excitation minimises out-of-plane photobleaching, which is extremely important when acquiring high resolution 3D FLIM data sets consisting of a large number of optically sectioned images. In addition, TCSPC provides direct electronic detection of the fluorescence lifetime signal with a high photon detection efficiency. We have applied this system to FLIM of live cell autofluorescence with the goal of detecting changes in cellular metabolism through the fluorescence lifetime of NADH. In addition to this, high resolution 3D FLIM datasets of KIR-GFP at immunological synapses formed between NK cells and their surveillance targets have been taken at increased speeds, allowing for higher imaging throughput, and we are working on increasing imaging speeds towards those that would be needed for live cell 3D FLIM.
38. Don C Lamb (Speaker)
    On the trail of single particles: 3D single particle tracking in live cells.
    Abstract

    Y Katayama, O Burkacky, S Lange, C Bräuchle, R P Jansen, E Gratton, and D C Lamb. Ludwig-Maximilians-Universität München, Germany.

    Imagine tracking a tourist in Rome using GPS. From the resulting trajectory, you can gather information over what mode of transportation this person prefers, what their daily routine is and what interests she or he has. In the last decade, a revolution has occurred in fluorescence microscopy, making it possible to follow individual molecules and particles with nanometer accuracy. We can gather similar information by following individual biomolecules within living cells using single particle tracking. Intracellular mRNA localization is a common mechanism for creating asymmetric distributions of proteins in live cells. The mRNAs are transported to the translocation site and the proteins are produced “on site” rather than having to be transported through the cell. Using yeast as a model system, we have investigated how different mRNA species are transported to their final destination. We have developed a novel labeling strategy that allows the labeling two different mRNAs in vivo. Hence, we could show that different mRNA types are transported together within the same particles.
    In the second portion of my talk, I will discuss a novel 3D tracking microscope that has been developed in my laboratory. The laser beam is orbited about the particle and from the phase and modulation of the fluorescence signal, the position determined in two dimensions. For the third dimension, two confocal planes, immediately below and above the particle, are monitored simultaneously. The position of the tracked particle is determined in 3D in real time and the orbit of the laser beam is recentered on the particle using a feedback loop. In addition to orbital tracking, a wide-field detection system is incorporated into the microscope to allow visualization of the environment about the tracked particle. We have utilized this system to investigate the entry pathway of artificial viruses.
39. Robert Learmonth (Poster)
    Membrane fluidity regulation in yeasts.
    Abstract

    R P Learmonth1 and B A Butcher2. 1 Centre for Systems Biology, U of Southern Queensland, Toowoomba, Australia; 2 Contract Manufacturing, Progen Industries, Darra, Australia.

    Membrane fluidity modulation appears to be an important mechanism facilitating yeast environmental adaptation. To elucidate underlying processes that may contribute to fluidity modulation we compared responses in yeasts mutants lacking the lipid desaturase ole1 or membrane-associated hsp30, to response of the parent strain. We assessed membrane fluidity, composition and cell physiological parameters during aerobic and anaerobic batch culture, as well as in response to glucose, a metabolite that stimulates marked change in cell physiology. Fluidity modulation was assessed by determination of Generalized Polarization of the membrane probe laurdan, via steady state fluorescence spectroscopy.
    During batch growth the parent strain showed characteristic patterns of membrane fluidity, membrane phospholipid and fatty acyl composition, growth rate, glucose or ethanol consumption and viability. However the ole and hsp30 deletants appeared to be out of balance with less efficient metabolism, lower cell division rates and lower viability. The ole defect could be ameliorated by addition of unsaturated fatty acids. The hsp30 defect could not be ameliorated externally and had particular impact over the transition at glucose exhaustion. The hsp30 and ole deletants both showed abnormal changes in fluidity on exposure to glucose. In summary the fluidity regulation responses in the mutants were different to the parent strain, being unbalanced by ablation of the activities of either membrane-associated protein.
40. Marcia Levitus (Invited talk)
    Applications of fluorescence correlation spectroscopy to the study of nucleic acid conformational dynamics.
    Abstract

    M Levitus, Biodesign Inst, Tempe.

    Fluorescence correlation spectroscopy (FCS) is a technique based on the measurement of the spontaneous fluctuations of the fluorescence signal of a small number of molecules. Fluorescence fluctuations are typically measured in an optically restricted sub-micron observation volume, and then analyzed statistically to reveal kinetic information about the processes that lead to these fluctuations. Such processes include concentration fluctuations via molecular diffusion, chemical reactions, photophysical processes, etc. I will discuss recent applications of FCS to the study of conformational dynamics in nucleic acids. Much of the effort in this regard has been focused on dealing with the problem of separating the contributions due to conformational dynamics from those due to molecular diffusion. We have recently developed a new methodology that combines FCS and FRET (fluorescence energy transfer) and allows conformational dynamics to be analyzed independently of the fluctuations caused by diffusion. I will discuss this new approach and present recent experimental applications to the study of conformational dynamics in nucleosomes.
41. Joachim Müller (Poster)
    Resolution of protein mixtures in living cells with brightness analysis.
    Abstract

    B Wu, Y Chen, and J Müller. School of Physics and Astronomy, U of Minnesota, Minneapolis.

    Quantitative information about protein-protein interaction in vivo is crucial for building physical models of cellular processes. Fluorescence fluctuation spectroscopy (FFS) is a promising experimental technique for addressing protein-protein interaction in living cells. However, the signal / noise ratio of traditional FFS analysis is insufficient to resolve the heterogeneous mixtures found in the cell environment. A powerful approach for increasing the signal /noise ratio of FFS experiments is the extension of the analysis to long sampling times by time-integrated fluorescence cumulant analysis (TIFCA). EGFP and mCherry currently represents the most promising pair of fluorescent proteins for dual-color FFS studies. However, our results show that mCherry exists in more than one brightness state. Taking the complexity of mCherry into account we demonstrate that dual-color TIFCA provides enough sensitivity to directly resolve a mixture of interacting proteins in living cells. With dual-color TIFCA we are able to study the hetero-oligomerization between full-length nuclear receptors and their coactivator. Our results suggest that the coactivator acts as scaffold that assembles nuclear receptors prior to their interaction with the DNA.
42. Joachim Müller (Invited talk)
    The study of viral-like particles with fluorescence fluctuation spectroscopy.
    Abstract

    J Müller. School of Physics and Astronomy, U of Minnesota, Minneapolis.

    Enveloped viruses contain an encapsulating membrane that the virus acquires from the host cell during the budding process. The presence of the enveloping lipid membrane complicates the physical characterization of the proteins assembled within the virus considerably. Here we present a method based on fluorescence fluctuations that quantifies the copy number of proteins within an enveloped viral particles. We choose the viral protein Gag of the human immunodeficiency virus (HIV) type 1 as a model system, because Gag expressed in cells is sufficient to produce viral-like particles (VLPs) of the same size as authentic virions. VLPs harvested from cells that express fluorescently labeled Gag were investigated by two-photon fluorescence fluctuation spectroscopy. Our data show that while the hydrodynamic size of the fluorescent VLPs agrees with the literature, the Gag copy number per VLP is much lower than expected. Our data imply that the assembly of HIV particles is not adequately described by the prevailing model.
43. Georgianna L Martin (Poster)
    Fluorescence characterization of polymer-enzyme interactions.
    Abstract

    G L Martin1, J A Ross2, D M Jameson2, and M J Cooney1. 1 Hawaii Natural Energy Inst, U of Hawaii, Honolulu , 2 John Burns Med Schl, U of Hawaii, Honolulu.

    Within the context of biosensor and bio-fuel cell development, enzyme immobilization has several important goals: (1) to extend of the enzyme’s active lifetime[1], (2) to increase enzyme activity[2], (3) to avoid catalyst loss in flow systems, and (4) to increase enzyme loading[3]. Choosing an immobilization material that best suits the needs of the enzyme as well as the industry requires careful characterization of the materials and their interaction with the enzyme of choice. Here we have chosen to study chitosan, a polycationic, biodegradable, non-toxic biopolymer[4]. These characteristics along with the biological nature of the polymer, its abundance and low cost, make it an attractive material for applications that require long term stability, such as enzymatic biofuel cells. In recent work, Klotzbach et al. have further enhanced the immobilization properties of the chitosan polymer by replacing a portion of the amine groups on the chitosan backbone with hydrophobic side chains[5]. This modification has been shown to create amphiphilic micellular structures that increase in the activity of entrapped glucose oxidase2. The purpose of this study is to characterize the micelleular nature the modified polymer and it interaction with different enzymes. Using various techniques, fluorescence microscopy can provide a detailed picture of the polymer-enzyme interaction. Here we present some initial studies and their results. The authors gratefully acknowledge support from the AFOSR Multidisciplinary University Research Initiative program (award number, FA9550-06-1-0264) under the Fundamentals and Bioengineering of Enzymatic Fuel Cells award.
    [1] Savett, S. C., J. R. Atkins, et al. J. Electrochem. Soc. 149 (2002).A1527-A1532.
    [2] Moore, C. M., N. L. Akers, et al. Biomacromolecules 5 (2005) 1241 - 1247.
    [3] Cooney, M. J. and B. Y. Liaw (2006). P. S. Wang and J. B. Kim, eds.,American Chemical Society.
    [4] Krajewska, B. Enzyme and Microbial Technology. 35 (2004) 126–139.
    [5] Klotzbach, et al., Journal of Membrane Science, 282 (2006) 276-283.
44. Tomoko Masaike (Poster)
    Domain motions of F1-ATPase that drive rotation of the central shaft revealed by TIRF microscopy with polarization modulation.
    Abstract

    T Masaike1, F Koyama1, K Oiwa2, M Yoshida3, and T Nishizaka1. 1 Dept of Physics, Gakushuin U, Tokyo, Japan. 2 Kobe Advanced ICT Research Ctr, Natl Inst of Info and Comm Tech, Japan. 3 Chem Resources Lab, Tokyo Inst of Tech, Japan.

    Observation of conformational changes of proteins is a key to understanding mechanisms of enzymatic functions. The TIRF microscopy with polarization modulation is a powerful tool for this strategy. By using this optical system, we demonstrate here that the catalytic subunit in the enzyme FoF1-ATPase synthase changes its conformations upon chemical reaction steps to drive rotation of the central shaft. FoF1-ATPase synthase catalyzes synthesis of ATP from ADP and inorganic phosphate (Pi) in living cells. Its F1 portion can catalyze the reverse reaction, ATP hydrolysis, and hence is called F1-ATPase. The α3β3γ catalytic core of F1-ATPase (hereafter denoted F1) is the smallest rotary molecular motor ever known in which the central γ subunit rotates against the surrounding α3β3 cylinder during ATPase reactions. Conformational changes of the catalytic subunit β are assumed to be closely related to rotation of the γ subunit, but timing and extent of motions in these different subunits have not been correlated clearly in the functioning molecules. In the present study, orientation of a fluorescent probe linked to the β subunit in F1 immobilized on a glass surface was measured by TIRF microscopy in which the excitation polarization of green laser was modulated and fluorescence of the probe was monitored in time (Ha, T., 1996, Nishizazka, T., 2004). Meanwhile, stepping rotation of beads attached to γ in the same single molecule was observed under the dark-field microscopy. As a result, polarization dipole of the fluorescent probe linked to beta showed three distinct angles, 0˚, ~4