10th Weber Symposium (2017)

Abstracts by corresponding author

1. Luis A. Bagatolli (Speaker)
   Dynamical coupling between intracellular water relaxation and oscillating glycolysis in yeast cells.
   Abstract

   Luis A. Bagatolli

   We detected very strong coupling between the oscillating concentration of ATP and the dynamics of intracellular water during glycolysis in Saccharomyces cerevisiae [1]. Our results indicate that: i) dipolar relaxation of intracellular water is heterogeneous within the cell and different from dilute conditions, ii) water dipolar relaxation oscillates with glycolysis and in phase with ATP concentration, iii) this phenomenon is scale-invariant from the subcellular to the ensemble of synchronized cells and, iv) the periodicity of both glycolytic oscillations and dipolar relaxation are equally affected by D2O in a dose-dependent manner. These results offer a new insight into the coupling of an emergent intensive physicochemical property of the cell, i.e. cell-wide water dipolar relaxation, and a central metabolite (ATP) produced by a robustly oscillating metabolic process [2].
   [1] Seir Thoke H., Tobiasen A., Brewer J., Lyngs Hansen P., Stock R. P., Folke Olsen L. and Bagatolli L.A. 2015. "Tight coupling of metabolic oscillations and intracellular water dynamics in Saccharomyces cerevisiae" PLoS ONE 10(2): e0117308.
   [2] Bagatolli, L.A. and Stock, R. P. 2016. "The cell as a gel: material for a conceptual discussion" Physiological Mini Reviews 9(5): 38-49.
2. Francisco J. Barrantes (Speaker)
   Nanoscopy of nicotinic acetylcholine receptors: cholesterol effects on supramolecular topography and dynamics.
   Abstract

   Alejo Mosqueira, Pablo Camino and Francisco J. Barrantes
   Lab. Molec. Neurobiology, Inst. Biomedical Research, UCA- CONICET, 1107 Buenos Aires, Argentina

   The organization and maintenance of an adequate number of neurotransmitter receptors at the cell surface depends on a multiplicity of factors, fundamental among these being the membrane environment in which the receptor proteins are embedded. We employed stochastic optical reconstruction microscopy (STORM), a form of single-molecule localization microscopy, to study the effects of cholesterol on the supramolecular arrangement and dynamics of nicotinic acetylcholine receptor (nAChR) molecules. Application of Bayesian analyses (Rubin-Delanchy et al., 2015) enabled us to characterize a variety of descriptors (cluster/non-cluster distribution, number of single-molecules in the two populations, size, interparticle distances, density) of the nanocluster assemblies in fixed cells. Single-particle tracking (SPT) applied within minutes of changes in cholesterol levels in live cells enabled the extraction of time series of individual molecule trajectories and ensemble averages. A probabilistic Bayesian inference approach which automatically tests competing, multiple hypotheses (Monnier et al., 2012) identified various motional modalities associated with the experimentally determined translational diffusional parameters. The emerging picture is that the nAChR operates a complex variety of motional regimes, including anomalous diffusion, and that these are subject to cholesterol modulation at the cell surface.
3. Mário Berberan-Santos (Speaker)
   Phasors, spectral phasors and complementary approaches.
   Abstract

   Fábio Fernandes, Sandra Pinto, Liliana Martelo, and Mário Berberan-Santos
   CQFM, Instituto Superior Tecnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal

   The phasor approach [1-6] is discussed for several decay laws [7,8] and in its application to three-state systems [9,10]. The spectral phasor [11] and generalized polarization [12] are also discussed and compared with alternative treatments.
   [1] D. M. Jameson, E. Gratton, R. D. Hall, Appl. Spectrosc. Rev. 20 (1984) 55-106.
   [2] M. Berberan-Santos, J. Lumin. 50 (1991) 83-87.
   [3] M. A. Digman, E. Gratton, in Fluorescence Lifetime Spectroscopy and Imaging: Principles and Applications in Biomedical Diagnostics, L. Marcu, P. M. W. French, D. S. Elson eds., CRC Press, Boca Raton, 2012.
   [4] B. Valeur and M. N. Berberan-Santos, Molecular Fluorescence. Principles and Applications, Wiley-VCH, Weinheim, 2nd ed., 2012.
   [5] D. M. Jameson, Introduction to Fluorescence, CRC Press, Boca Raton, 2014.
   [6] Y. Engelborghs, A. J. W. G. Visser eds., Fluorescence Spectroscopy and Microscopy, Humana Press, New York, 2014.
   [7] M. N. Berberan-Santos. Chem. Phys. 449 (2015) 23-33.
   [8] L. Martelo, A. Fedorov, M. N. Berberan-Santos, J. Phys. Chem. B 119 (2015) 10267-10274.
   [9] L. Martelo, A. Fedorov, M. N. Berberan-Santos, J. Phys. Chem. B 119 (2015) 15023-15029.
   [10] L. Martelo, A. Fedorov, M. N. Berberan-Santos, J. Lumin. in press
   [11] F. Fereidouni, A. N. Bader, H. C. Gerritsen, Opt. Express 20 (2012) 12729-12741.
   [12] T. Parasassi, G.D. Stasio, A. d'Ubaldo, E. Gratton, Biophys J. 57 (1990) 1179-1186.
4. Carlos Lenz César (Speaker)
   Fluorescence correlation spectroscopy in crude oils and Quantum Dots.
   Abstract

   C.L. César, A.A. de Thomaz, V.B. Pelegati, H.F. Carvalho, C.S., Carneiro, F. Nepomuceno, and J. Mendes Filho

   FCS allows the measurement of the hydrodynamical radius of molecules, which is important to discriminate crude oil components, especially asphaltenes, for oil classification purposes. Moreover, it can provide the hydrodynamical radius of capped quantum dots which are larger than the electron confinement radius. The presence of blinking in the Quantum Dots, however, requires some corrections to obtain the right sizes.
5. Samir Pereira da Costa Campos (Poster)
   Yellow fever virus infects and modulates cell viability and metabolism of megakaryo-cyte progenitors.
   Abstract

   S.P.C. Campos, M.G.Castro, C.V. Gomes, M.F. Caldeira, M.F. Rodrigues, J.L. Silva, A.M.O. Gomes, and A.C. Oliveira
   Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brasil

   Introduction: Yellow Fever Virus (YFV) has great importance in economy and public health in Africa and South America. In Brazil, this year, we had an important outbreak, with deaths and economic expenditure by the vaccination on areas of great risk of infection. YFV causes an acute hemorrhagic fever related to hemo-stasis dysfunction, with thrombocytopenia, both related to the disease severity. Platelets are the final stage of megakaryoblast differentiation and alterations on megakaryoblasts could be related to the pathogenesis and explain in part the thrombocytopenia. Viral infections can lead to cell death and metabolic dysfunctions such as in the mitochondrial metabolism. The cell metabolic profile can also be modulated, and it can be followed by fluorescence lifetime imaging microscopy, using for example NADH fluorescence.
   Aim: Study the interaction between YFV and megakaryoblasts to better understand the processes by which YFV infection could leads to thrombocytopenia.
   Materials and Methods: We infected MEG-01 cells (Human Megakaryoblastic cell line) with YFV 17DD in a multiplicity of infection 1 and followed infection by plaque assays and fluorescence confocal microscopy. Mitochondrial physiology was analyzed in intact cells through high-resolution respirometry and the free/bound NADH ratio by fluorescence lifetime microscopy, using the phasor analysis.
   Results: We confirmed that YFV infects MEG-01 cells, with increasing production of infectious particles until 96 hours post-infection (p.i.), followed by a decrease. We observed increased cell death in YFV-infected cells from 120 hours p.i. as compared to control. We also observed increased apoptosis from 120 h p.i. on infected cells by TUNEL assay. Through high-resolution respirometry, we observed decreased rou-tine, ATP synthesis coupled oxygen consumption and maximum capacity of infected cells electron transport system 144 hours p.i. By analyzing the free/bound NADH ratio, we observed that since 72 hours p.i., infection induces a lower free NADH fraction than in control cells, indicating a more oxidative profile compared to control cells.
   Conclusion: Our data suggest that YFV infects and replicates in MEG-01 cells, induces cell death and apoptosis from 120 hours p.i.. We also observed that YFV infection modulates MEG-01 metabolism by modulating mitochondrial physiology with decreased respiration and a shift to oxidative profile.
   Keywords: Yellow Fever, Megakaryocyte, Platelet, Apoptosis
   Financial Support: CAPES, CNPq, FAPERJ, INBEB/CNPq
6. Francesco Cardarelli (Speaker)
   Probing the dynamic fingerprint of insulin secretory granules in living cells by spatiotemporal fluctuation spectroscopy.
   Abstract

   Gianmarco Ferri 1, Luca Digiacomo 2, Lorenzo Scipioni 3, Marco Bugliani 4, Luca Lanzano 3, Piero Marchetti 4, Giulio Caracciolo 2, and Francesco Cardarelli 1
   1 NEST - Scuola Normale Superiore, Istituto Nanoscienze - CNR (CNR-NANO), Pisa, Italy
   2 Department of Molecular Medicine, "La Sapienza" University of Rome, Italy
   3 Nanoscopy, Nanophysics Dept., Istituto Italiano di Tecnologia, Genoa, Italy
   4 Department of Clinical and Experimental Medicine, University of Pisa, Italy

   Insulin secretory granules (ISGs) are cytoplasmic organelles (200–400 nm in linear size) containing a dense core consisting of insulin molecules arranged in crystalline-like arrays. They are naturally present in the β-cells of Langerhans islets within pancreas and in several tumor-derived cell lines commonly used as models of β-cell biology. They exert the complex function of insulin secretion through tight regulation of key structural and functional properties, such as size, speed, mode of motion, number, etc. Quantitative description of these parameters in living matter is crucial for our understanding of insulin granule function in physiology and its alterations in pathology. Yet, despite the efforts, a systematic characterization of the dynamic behaviour of ISGs remains largely elusive. We address this hot topic by fluctuation microscopy. In particular, we start from a fluorescence-based spatiotemporal fluctuation analysis method that makes possible to probe the actual molecular “diffusion law” directly from imaging, in the form of a mean square displacement (MSD) versus time-delay plot (image-derived MSD, hereafter referred to as iMSD) [1,2] with no need to preliminarily assume any interpretative model. By fitting the iMSD curve relevant parameters describing the average dynamic behavior of ISGs can be extracted, such as short- and long-range diffusion coefficients, confinement strength (in case of confined diffusion), anomalous diffusion coefficient (α), and an estimate of the ISG average size. From each analyzed cell, the aforementioned set of parameters is extracted and represented in a parametric space, in such a way that that each cell corresponds to one point in the plot. Thus, the clustering of single-cell points readily depicts what we define as the “dynamic fingerprint” of ISGs at the whole-cell level. To prove the potency of this experimental strategy, we show some relevant examples, including the effect of ISG labelling (by selected genetically encoded proteins), cholesterol overloading, and cytoskeleton perturbation on the ISG dynamic fingerprint. Also, in order to study the dynamic fingerprint of ISGs in a mostly unperturbed intracellular environment, we dissociated human pancreatic islets and transfected the obtained isolated cells with Insulin-EGFP. In our knowledge, this is the first time that the cytoplasmic dynamic behavior of ISGs in Human primary cells is described. Of note, our analysis reveals sensibly lower values of both size and diffusivity in Human primary β-cell ISGs as compared to INS-1 ones. Our fluctuation microscopy approach adds a dynamic quantitative dimension to standard fluorescence imaging of ISGs in living matter and paves the way to the cell-based, rapid and robust screening of fundamental kinetic and thermodynamic parameters over wide spatial and temporal scales, in physiology and pathology.
   [1] C. Di Rienzo et al., PNAS (2013). 12307-12312.
   [2] L. Digiacomo et al., Acta biomaterialia (2016). 189-198.
7. Maria Soledad Celej (Speaker)
   On the structure and membrane-binding properties of oligomeric species of the amyloidogenic protein α-synuclein.
   Abstract

   Maria Soledad Celej
   Dto. de Química Biológica, CIQUIBIC-CONICET, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina

   Amyloid deposits of the presynaptic protein α-synuclein (AS) are pathognomonic of Parkinson's disease, a neurodegenerative movement disorder associated with axon degeneration of dopaminergic nigral neurons. Prefibrillar AS oligomers (oAS) are highly neurotoxic since they might damage synapses and dendrites by both altering the physiological function of AS and acting as active pathogenic species. However, little is known about their structure or molecular basis of biological activity. We used a combination of ATR-FTIR spectroscopy and site-specific fluorescence to interrogate about the β-sheet organization within oAS. We showed that oAS adopt a distinct antiparallel β-sheet structure, as opposed to the parallel cross- fibrillar fold [1] and that they are ordered assemblies possessing a well-defined pattern of intermolecular contacts [2]. Exploring the β-strand remodeling during amyloidogenesis of AS and its physiological N-terminally acetylated form, we found that antiparallel β-sheet intermediates are built-up at early stages of aggregation, which then evolve to parallel β-sheet fibrils through helix-rich/disordered species [3]. Currently, we are exploring the loss-of-function that might be associated to the conversion of AS from its monomeric functional state to its pathological oligomeric form by evaluating the impact of AS oligomerization on its membrane-curvature sensitivity. We are using Fluorescence Correlation Spectroscopy to obtain quantitative information on the interaction between monomeric and oligomeric AS and vesicles varying in sizes [4]. Preliminary results indicate that oAS still retains a somehow higher affinity for small than for large unilamellar vesicles. We anticipate that our work is of interest for the understanding of the molecular bases underlying AS physiopathology.
   Acknowledgements: Agencia-FONCyT, CONICET, SECyT-UNC, Fundación Florencio Fiorini, ISN-CAEN, Fulbright-CONICET, CONICET-FNRS and MINCyT-FNRS for financial support. We thank Dr. Enrico Gratton for your valuable help in FCS measurements and data processing.
   References
   [1] Celej MS et al, Biochem. J. 2012, 719.
   [2] Gallea JI et al, J. Biol. Chem. 2014, 26733.
   [3] Gallea JI et al, Biochim. Biophys. Acta. 2016, 501.
   [4] Gallea JI, Mas C, Ambroggio EE, James N, Jameson D & Celej MS, unpublished.
8. Amitabha Chattopadhyay (Speaker)
   Applications in membrane and cell biology.
   Abstract

   Amitabha Chattopadhyay

   The origin of dipole potential in an organized molecular assembly is the electrostatic potential difference within the assembly due to the nonrandom arrangement of amphiphile dipoles and solvent (water) molecules at the assembly interface. I will focus my talk on the measurement of dipole potential using electrochromic fluorescent probes, and highlight applications in a number of molecular assemblies and cellular systems. In addition, I will extend the concept of membrane dipole potential to other organized molecular assemblies such as micelles. Our results show that subtle changes in membrane dipole potential could be crucial in understanding the modulation of membrane lipids, and the interplay between membrane lipids and proteins in the cellular milieu.
9. Sijie Chen (Poster)
   Real-time imaging of cell behaviors in living organisms by a mitochondria-targeting AIE fluorogen.
   Abstract

   Sijie Chen, Bo Situ, and Ben Zhong Tang
   Karolinska Institutet, Hong Kong

   Fluorescence-based techniques offer high sensitivity, low background noises and broad dynamic ranges, which have been widely used in microscopy, biotechnology, and clinical diagnostics. Most of the conventional organic conjugated fluorescent molecules are highly emissive in dilute solutions but become weakly or even non-emissive when aggregated or in the solid state. Such “aggregation-caused quenching” (ACQ) effect has greatly limited their practical applications. Different from the “self-quenching” ACQ dyes, the fluorogens with aggregation induced emission (AIE) properties are in the dark state when molecular dissolved, but are turned on when aggregates are formed. Mechanistic study suggested that in the aggregate state or solid state, the molecular motions of the AIE fluorogens are restricted, promoting the radiative decay. The non-planar conformations of the AIE fluorogens also prevent the π-π interaction formation. These two correspond to the strong emission of AIE dyes in the highly concentrated aggregate state. The unique properties of AIE fluorogens make them excellent candidates for sensitive biosensors and emissive bioprobes.
   In this work, we report a biocompatible AIE fluorogen, TPE-PyN3, for noninvasive bio-imaging and sensing within living systems. TPE-PyN3 exhibits unique an AIE attributes and high affinity to mitochondria, enabling it to achieve specific mitochondrial imaging and long-term cellular observing with excellent photostability both in vitro and in vivo. The high membrane penetrability of TPE-PyN3 allows the cells within the living zebrafish embryos to be morphologically visualized and reconstructed in 3D. The findings presented here provide a simple and noninvasive tool for studying behaviors of cell populations in vivo by a small-molecule AIE probe.
10. Rachel Perlas Cinco-Hedde (Speaker)
    Spatial characterization of bioenergetics and metabolism of primordial to preovulatory follicles in whole ex vivo murine ovary.
    Abstract

    Rachel Cinco, Michelle A. Digman, Enrico Gratton, and Ulrike Luderer
    University of California, Irvine, USA

    Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic by- products in culture media. However, culture conditions perturb the native state of the follicle, and these methods do not distinguish between metabolism occurring within oocytes or granulosa cells. We applied the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) at 740-nm two-photon excitation to examine the spatial distri- bution of free and protein-bound nicotinamide adenine dinucleotide hydride (NADH) during primordial through preovulatory stages of follicular development in fresh ex vivo murine neonatal and gonadotropin stimulated prepubertal ovaries. We obtained subcellular resolution phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD+) sepa- rately for oocyte nucleus and oocyte cytoplasm. We found that dynamic changes in oocyte nucleus free/bound NADH paralleled the developmental maturation of primordial to primary follicles. Immunohistochemistry of NAD+-dependent deacetylase SIRTUIN 1 (SIRT1) in neonatal ovary revealed that increasing SIRT1 expression in oocyte nuclei was inversely related to decreasing free/bound NADH during the primordial to primary follicle transition. We characterized oocyte metabolism at these early stages to be NADH producing (glycolysis/Krebs). We extended the results of prior studies to show that cumulus and mural granulosa cell metabolism in secondary through preovulatory follicles is mainly NADH producing (glycolysis/Krebs cycle), while oocyte metabolism is mainly NADH consuming (oxidative phosphorylation). Taken together, our data characterize dynamic changes in free/bound NADH and SIRT1 expression during early follicular development and confirm results from previous studies defining antral and preovulatory follicle metabolism in culture. Cinco, R., Digman, M. A., Gratton, E. & Luderer, U. Spatial Characterization of Bioenergetics and Metabolism of Primordial to Preovulatory Follicles in Whole Ex Vivo Murine Ovary. Biology of Reproduction 95, 129 (2016).
    Supported by National Institutes of Health (NIH) R01ES020454 to U.L., NIH F31AG048733 to R.C., NIH P41GM103540 and NIH P50GM076516 to E.G. and M.A.D., and NIH P50-GM076516 to R.C. from UCI Center for Complex Biological Systems.
11. Rachel Perlas Cinco-Hedde (Poster)
    Characterization of NADH/NAD+ ratio during early stages of ovarian follicular activation in live murine neonatal ovaries.
    Abstract

    Rachel Cinco, Michelle A. Digman, Enrico Gratton, Ulrike Luderer
    University of California, Irvine, USA

    Follicular bioenergetics is a critical determinant of oocyte quality and ovarian aging. Ovarian bioenergetics has largely focused on defining follicular metabolism by measuring metabolic byproducts in cultured media. However, culture conditions required for these data compromise the native biological cell state. Furthermore, there are few data characterizing early follicular metabolism before the primary stage, likely due to limitations in small follicle isolation.
    Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH play a central role in bioenergetics as one of the main energy currencies of the cell. The NADH/NAD+ ratio has significant impact on energy production, proliferation, cell survival, and aging. NADH is naturally fluorescent and its free and bound states can be measured by fluorescence lifetime imaging microscopy using 740nm 2-photon excitation. The development of the phasor approach to fluorescence lifetime imaging microscopy (Phasor FLIM) offers a straightforward approach to interpretation of lifetime differences at the pixel level. Phasor FLIM analysis of relative free/bound NADH provides a quantitative measure that approximates the intracellular NADH/NAD+ ratio. This method has been extensively used to characterize the spatial distribution of free/bound NADH in living tissues.
    Here, we apply Phasor FLIM at 740nm 2-photon excitation to examine the spatial distribution of free/bound NADH during early stages of follicular development in live mouse neonatal ovary. We hypothesized dynamic changes in NADH/NAD+ ratio to parallel the developmental maturation of primordial thru activated primary follicles. We obtained subcellular resolution phasor FLIM 2D and 3D data and assessed the NADH/NAD+ ratio in oocyte nucleus and oocyte cytoplasm within primordial to primary follicles. Furthermore, we assessed the NADH/NAD+ ratio in granulosa cell nuclei of transitional follicles containing mixtures of morphologically flat and cuboidal granulosa cells. Primordial to primary follicles showed no statistically significant differences in NADH/NAD+ in the oocyte cytoplasm, suggesting little change in metabolism during these stages of development. However, we observed primordial follicle nuclei had significantly higher NADH/NAD+ ratios compared to activated transitional and primary staged follicles. We furthermore characterized the developmental expression of NAD+ dependent deacetylase SIRTUIN1 (SIRT1) in neonatal ovary by immunohistochemistry and found SIRT1 expression in oocyte nuclei to parallel observed decreases in free/bound NADH. Cell phasor analysis of free/bound NADH in transitional follicle granulosa cells revealed three populations of granulosa cells characterized by statistically significant differences in NADH/NAD+ ratios. Taken together, our data characterize a dynamic change in NADH/NAD+ and SIRT 1 expression during early follicular development that has not been previously characterized. Supported by NIH ES020454 to UL and NIH/NIGMS 8P41 GM103540-28 to EG and MD.
12. Ibrahim Cissé (Speaker)
    Capturing genome regulation with single molecule resolution in live mammalian cells.
    Abstract

    Ibrahim Cissé
    Massachusetts Institute of Technology, USA

    Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.
13. Xavier Darzacq (Speaker)
    Single molecule imaging of morphogen gradients during early fly development.
    Abstract

    Xavier Darzacq
    University of California, Berkeley, USA

    Morphogen gradients direct the spatial patterning of developing embryos, however, the mechanisms by which these gradients are interpreted remain elusive. Here we perform in vivo single molecule imaging of the transcription factor Bicoid that forms a gradient along the anteroposterior axis of the early Drosophila melanogaster embryo. We observe that Bicoid binds to DNA with a rapid off-rate, such that its average occupancy at target loci becomes on-rate dependent, a property required for concentration-sensitive regulation. Surprisingly, we also observe abundant specific DNA binding in posterior nuclei, where Bicoid levels are vanishingly low. Live embryo imaging reveals "hubs" of local high Bicoid concentration that are dependent on the ubiquitous maternal factor Zelda. We propose that localized modulation of transcription factor on-rates via clustering provides a general mechanism to facilitate binding to low-affinity targets under limiting factor conditions, and that this may be a prevalent feature directing other developmental transcription networks.
14. Rupsa Datta (Weber Prize Guest)
    Label-free fluorescence lifetime imaging microscopy (FLIM) to study metabolism and oxidative stress in biological systems.
    Abstract

    Rupsa Datta and Enrico Gratton

    Study of cellular metabolism and its influence on physiological functions and pathology along with investigation of oxidative stress in pathogenesis are essential for fundamental biology as well as biomedical research. Optical imaging offers the opportunity to assess these indices non-invasively. In this work we apply two photon fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores for label-free metabolic and oxidative stress imaging in a wide range of biological samples. Analysis of FLIM data was performed by applying the ‘fitfree’ phasor approach where each pixel of the image is transformed to its corresponding phasor on the phasor plot. Biological systems are a rich resource of autofluorescent biomolecules. Their fluorescence lifetimes are sensitive to alteration of normal physiology, making them attractive endogenous probes. We discovered one such endogenous fluorophore with characteristic long fluorescent lifetime. We hypothesized these long lifetime species (LLS) to be fluorescent products of lipid oxidation by reactive oxygen species (ROS), rendering them biomarkers of oxidative stress. To correlate the long lifetime species (LLS) with lipid droplets, we performed simultaneous FLIM and two coherent nonlinear microscopy techniques: third harmonic generation (THG) imaging microscopy and coherent anti-Stokes Raman scattering (CARS) microscopy that are sensitive to lipids. We went one step further to characterize the chemical nature of this discovered species by classical Raman spectral analysis. We show application of this technique in cancer, induced pluripotent stem cell derived cardiomyocytes, as well as in freshly excised mice adipose tissue. The identified endogenous biomarker unfolds opportunities of performing non-invasive measurements of oxidative stress in vivo.
    We also exploited the autofluorescent coenzyme reduced nicotinamide adenine dinucleotide (NADH), an endogenous probe extensively used for metabolic imaging. We performed NADH-FLIM to study the metabolic status of a vascularized three-dimensional tumor microenvironment in a microfluidic based platform. We could identify metabolically dissimilar regions, as well as identify metabolic response to anticancer drug.
    Finally, we explored NADH-FLIM of a different class of organisms – bacteria. We show for the first time, FLIM-phasor fingerprint of clinically important bacteria. We discovered interesting bacterial phasor trajectories at different growth phases as well as response to antibiotics, all at single cell resolution.
15. Rupsa Datta (Poster)
    Intrinsic biomarker for oxidative stress by fluorescence lifetime imaging microscopy (FLIM).
    Abstract

    Rupsa Datta and Enrico Gratton
    Laboratory for Fluorescence Dynamics, Biomedical Engineering, University of California, Irvine

    Reactive oxygen species (ROS), when present above normal physiological levels, causes oxidative stress (OS), leading to damage of proteins and DNA and lipid peroxidation. OS has been associated with myriad of diseases like inflammation, diabetes mellitus, cardiovascular diseases, and cancer. Hence an OS biomarker would have immense clinical importance. There is a dearth of nondestructive techniques to measure OS in vivo. This work shows non-invasive detection of OS by fluorescence lifetime imaging microscopy (FLIM) of an intrinsic probe. Recognition of this endogenous biomarker enables label-free OS imaging. We identified this as fluorescent product of lipid oxidation by ROS. Using FLIM-phasor analysis, we can identify the presence and spatial location these species with a characteristic long lifetime (LLS). We could correlate the LLS to lipid droplets in the oleic acid treated HeLa cells using third harmonic generation (THG) imaging and coherent anti-Stokes Raman scattering (CARS) microscopy. For chemical analysis, we performed single point Raman spectroscopy which revealed typical lipid droplet Raman-bands. LLS FLIM signature was also observed in mouse perigonadal white adipose tissue (WAT) lipid droplets, which co-localized with THG signal. This further confirms the association of LLS to lipid droplets. We show application of LLS as OS biomarker in cardiology and cancer. An increase of LLS was observed in induced-pluripotent-stem-cell-derived cardiomyocytes under OS due to hypoxia. This correlates to the increase in ROS production due to hypoxia. LLS FLIM signature was also observed in melanoma, a ROS driven cancer. LLS were mostly located in the dendritic projection of the melanoma cells. Comparatively, only a small amount of LLS was displayed by melanocytes. This label-free, non-invasive FLIM-phasor method is a promising imaging technique for OS detection in biological systems, especially for in vivo measurements.
    Work supported in part by NIH grants P50GM076516,P41GM103540 and UH3-TR000481.
16. Michelle A. Digman (Speaker)
    Imaging techniques to measure spatio-temporal dynamics and metabolic alterations of p53 upon DNA damage.
    Abstract

    Michelle Digman
    Biomedical Engineering, University of California, Irvine, USA

    For biological reaction-diffusion systems, live single cell spatiotemporal analysis of protein dynamics provides a mean to observe stochastic biochemical signaling which may lead to better understanding of cancer cell invasion, stem cell differentiation and other fundamental biological processes. This talk will describe methodologies used to investigate p53 activity and alteration of the metabolic pathway upon DNA damage. p53 is a tumor suppressor protein that regulates target genes involved in DNA damage migration and repair. If cells become stressed due to DNA damage, p53 will form tetramers in specific chromatin sites and active genes that trigger cell cycle arrest and/or apoptosis. We used the Number and Molecular Brightness (N&B) method to map aggregation processes in the entire cell upon DNA damage with cisplatin (a chemotherapy agent); thus revealing the spatial distribution of events, the site of tetramer formation and the time sequence of the aggregation events with quantitative information about the distribution and size of any intermediate aggregates that are formed. To gain information regarding fast dynamic processes we use laser beam line scanning on a convention confocal microscope to reveal the transient binding dynamics across the nucleus. Given that p53 has a dual role in promoting oxidative phosphorylation (oxsphos) and glycolysis upon cellular stress and may play an important role in normal growth, development, and tumor suppression, we investigated the effect of metabolic changes upon DNA damage under the same treatment 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. 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. Overall, our findings demonstrate that by multiplexing these techniques we have the ability to spatially and temporally quantify p53 activation and map p53’s influence in the metabolic pathway. This work is supported in part by NIH grants P50 GM076516 and P41 GM103540.
17. Michelle A. Digman (Poster)
    Fluorescence diffusion tensor maps p53 activation acquired with SPIM.
    Abstract

    Michelle A. Digman 1,2, Lukas Rottschäfer 1, Enrico Gratton 1, and Nik Hedde 1
    1 Laboratory for Fluorescence Dynamics, Biomedical Engineering, University of California, Irvine, USA
    2 Department of Developmental and Cell Biology, University of California Irvine, USA

    The transcription factor, p53, can activate the DNA repair or apoptotic pathway in response to cellular stress. Specific binding sites must be accessible to active either pathway. Thus, specific chromatin location as well as accessibility are required for stable tetramers of p53 to active either response pathway. To gain information regarding p53 fast dynamic binding interaction and diffusion to localized nuclear regions, we used single plane illumination microscopy (SPIM) to acquire high speed frame rate images with a time resolution below 1 ms. Cells were treated with either anisomyocin or cisplatin to induce cellular stress. To obtain maps of protein diffusion and directional local motion the fluorescence Diffusion Tensor Imaging (fDTI) method, inspired by the Diffusion Tensor imaging in the MRI field, was used. This powerful technique provides maps of molecular diffusivity and protein interactions in the context of their endogenous environments. The fDTI is based on spatial pair correlations between adjacent pixels in a given number of radial angles that are explored in a given point to determine the anisotropic diffusion. The time points for the pCF correlation function are calculated in a log scale using by dividing the tau axis in 32 segments equally spaced in the log scale. Thus diffusion and connectivity maps are obtained by calculating pair correlation functions of each voxel with all the neighbor voxels. For this work we show changes in molecular diffusion and alterations of protein connectivity post activation of the cell stress response mechanism and are able to construct molecular connectivity maps of p53. This type of analysis can provide a well-informed picture of spatial temporal p53 regulation throughout the living cell. This work is supported in part by NIH grants P50 GM076516 and P41 GM103540.
18. Michelle A. Digman (2nd Poster)
    Focal adhesion protein reorganization and tension on nanotopography.
    Abstract

    Emma Jane Mah 1, Albert F. Yee 1,2, and Michelle A. Digman 1,2
    1 Chemical Engineering and Materials Science, University of California, Irvine, USA
    2 Biomedical Engineering, University of California, Irvine, USA

    Surfaces with nanostructures have been shown to modulate cell contact points (e.g. focal adhesions, FAs) and have the potential to create selective adhering surfaces for implants. Previouse studies have shown that cells on nanolines form FAs and migrate parallel to the structures. In addition, FAs on nanostructures are seen to form on the edges of the features due to increased tension in these regions. However, the architecture of the FA complex on nanostructures and how this a ects cell tension have yet to be studied. Here, we utilized the z-phasor analysis to measure the distance between FA proteins, integrin and paxillin, within breast cancer cell line MDA-MB-231. Tension at the FAs were measured done with a vinculin and talin FRET tension sensor (VinTS and TalTS, respectively). Insight on the mechanosensing signals which occur in these cells will help further the understanding of environmental signals that trigger metastasis.
19. Elliot Elson (Speaker)
    Measurement of the kinetics of protein synthesis and degradation in non-equilibrium steady states using “Light Jumps”.
    Abstract

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

    The concentrations of proteins are regulated in a cell to suit their functions as enzymes, signaling molecules, and structural components. These concentrations vary with the changing circumstances of the cell as it differentiates and responds to signals. A differentiated cell in stable conditions, however, maintains a stable phenotype in which we expect the concentrations of proteins to remain more or less constant. Concentrations higher or lower than suited to the state of a cell can distort its physiological function, e.g., the products of oncogenes can cause unregulated cell multiplication. Conversely, the presence of a protein at a concentration higher or lower than normal indicates that the cell is an abnormal state. Hence, the mechanisms that maintain stable protein concentrations are worth study.
    In a living cell a protein is maintained at constant concentration by balancing its rates of synthesis and degradation. That is, the protein is in a non-equilibrium steady state (NESS) in which energy is consumed and there is a net flux of molecules along a sequence of reactions from synthesis to degradation. To understand the mechanisms by which the NESS is maintained it is useful to measure the rates of synthesis and degradation of specific proteins. One experimental approach is to measure reaction fluxes using two-color fluctuation cross-correlation functions (Qian and Elson 2004, Qian and 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 testing this approach in yeast cells by measuring the rates of synthesis and degradation of fluorescent proteins. The protein concentration and its fluorescence increase and then relax back to the initial steady state after transcription is first initiated and then terminated by light jumps at different frequencies. The time course of the fluorescence changes supplies estimates of effective rate constants by fitting simple kinetic models. We are also using the light jump method to measure the kinetics of state switching in the bistable galactose signaling network (Acar, Becskei et al. 2005).
20. Debora Foguel (Speaker)
    The role of microglia and neutrophils in amyloidoses.
    Abstract

    Estefania Azevedo, Thyago C. Pires, Heitor Paula-Neto, Elvira Saraiva, Fernando L. Palhano, and Debora Foguel
    Instituto de Bioquímica Médica - Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil

    Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with elastase and histones to entangle pathogens. We showed that amyloid fibrils from three different sources (α-synuclein, Sup35 and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. We observed in situ, NETs in amyloidotic deposits from amyloidotic patients, which co-localized with amyloid deposits. These data reveal that NETs can be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species. In an animal model, NETs were also observed when amyloid fibrils were injected in the peritoneum. In a second approach, we have investigated the pathogenic mechanism behind oculoleptomeningeal amyloidosis (OA) caused by fibrillation of A25T-TTR. We have showed that fibrils of A25T activate microglia leading to secretion of TNF-α, IL-6 and nitric oxide. A25T amyloid fibrils induce NFκB translocation to the nucleus of microglia. Exposure of neuronal cultures to conditioned media of fibril-activated microglia causes synapse loss culminating in apoptosis of neurons. Finally, intracerebroventricular injection of A25T fibrils caused microgliosis and cognitive deficits in mice, which could be prevented by minocycline. These results indicate that A25T fibrils act as inflammatory agents, activating microglia and neuronal death.
21. Maria Garcia-Parajo (Speaker)
    Spatiotemporal organization of biological membranes using nanophotonic tools.
    Abstract

    Maria Garcia-Parajo
    ICFO-Institute of Photonic Sciences, Barcelona, Spain

    The quest for optical imaging of biological processes at the nanoscale has driven in recent years a swift development of a large number of nanoscopy techniques based on far-field optics. These, so called, super-resolution methods are providing new capabilities for probing biology at the nanoscale by fluorescence. While these techniques conveniently use lens-based microscopy, the attainable resolution and/or localization precision severely depend on the sample fluorescence properties. True nanoscale optical resolution free from these constrains can alternatively be obtained by interacting with fluorophores in the near-field. Indeed, near-field scanning optical microscopy (NSOM) using subwavelength aperture probes is one of the earliest approaches sought to achieve nanometric optical resolution. More recently, photonic antennas have emerged as excellent alternative candidates to further improve the resolution in the near-field ! by enhancing electromagnetic fields into regions of space much smaller than the wavelength of light. In this contribution, I will describe our efforts towards the fabrication of different nanoantenna probe configurations as well as 2D antenna arrays for applications in nano-imaging and spectroscopy of living cells with unprecedented resolution and sensitivity. In particular, I will show that in-plane dimer antennas provide giant fluorescence enhancement factors up to 104−105 times, together with nanoscale detection volumes in the 20 zL range. Moreover, we have taken advantage of the superior optical performance of these in-plane antennas arrays together with their extreme planarity to enquire on the nanoscale dynamics of multicomponent lipid bilayers. Our results reveal for the first time the coexistence of fluctuating nanoscopic domains on both Liquid order and Liquid disorder phases of mimetic membranes, in the microsecond scale and with characteristic sizes be! low 10nm. These nanoscale assemblies might be reminiscent to those nat urally occurring in living cells that in the absence of proteins and/or other stabilizing factors, are poised to be highly transient.
22. Andre Marco de Oliveira Gomes (Speaker)
    Cell metabolism, membrane organization and virus entry during Arboviruses infections.
    Abstract

    Andre Marco de Oliveira Gomes

    “Arbovirus” (Arthropod-Born viruses) is the term used to refer to an ensemble of hundreds of RNA viruses that are transmitted by arthropods, mainly mosquitoes and ticks. Arboviruses present complex replication cycles involving vertebrates such as mammals or birds and blood-feeding vectors. Most of the arboviruses remain in their sylvatic cycles and humans are usually accidental hosts. Only a few arboviruses such as the alphavirus chikungunya and flaviviruses such as dengue and West Nile had caused clinically significant human diseases. Recent outbreaks of the Zika (ZIKV) and Chikungunya (CHIKV) viruses in Central and South America have caught the attention of the world and led to the release of an alert by the WHO abut the risks of Zika virus spread. While ZIKV and CHIKV have only recently shown themselves to the world, Dengue (DENV) and Yellow Fever (YFV) have been making thousands of victims over the years around the world. At the same time, other arboviruses, such as Mayaro virus (MAYV) remain in their sylvatic cycles in the endemic regions in amazon, awaiting the chance to adapt to an urban cycle that could lead them to the center of this discussion. Understanding how arboviruses interact with their host cells has great implications for understanding how the infection process leads to disease and death. Our objective is to investigate the interaction of viruses with living cells of vertebrate and invertebrate hosts in order to understand the infection process regarding the distribution of their components in the cell, entry strategies, changes caused to cell metabolism and in the formation of membranes for viral replication and formation of new enveloped viral particles. We use Laurdan spectroscopy, FLIM and biochemical techniques for analyzing the metabolism and lipid composition of the cells and the envelopes of the viral particles. Our results with the alphavirus MAYV and the flavivirus YFV and ZIKV show that viral infection affects the energy metabolism of cells and their lipid composition in both mammalian and insect cells, providing a suitable environment for viral replication and formation of new infectious particles. Metabolic changes caused by YFV may be related to the fate of platelet-forming cells, megakaryoblasts, leading to thrombocytopenia. In Mayaro virus infection, our results show that the virus is able to modify the lipid composition of the cells, thus modulating the lipid composition of the viral envelopes and compensating for the lack of cholesterol in the insect cells. Time lapse confocal microscopy images of cells infected with labeled-MAYV show that the virus may use either clathirin-mediated endocytosis or caveolae-derived vesicles to gain entry into their host cells. Using these techniques, we compared the infection of insect and mammal cells by Zika virus through the metabolic changes measured by FLIM with the use of the Phasor approach. Arbovirus can use different strategies to invade the host cells and change their metabolism, changing the original characteristics of these cells and modulating the cell environment to favor virus replication in different organisms. Our results have the potential to help finding new methods to interfere with the cell infection process by the design of rational antiviral strategies.
23. Enrico Gratton (Speaker)
    Correlation of molecular diffusion in live cells.
    Abstract

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

    The coordination of cell functions requires that molecules move in the cell interior to find their partners. In the cell, the mechanisms for molecular motion are poorly understood. While in an isotropic fluid, diffusion is the default mechanism of motion, in the cell interior diffusion is hindered by barriers and by transient binding. Also molecules can move by active transport. One universal transport processes which is still debated is the shuttling of molecules between the cell membrane and other locations where the molecules will deliver a signal. Although we have made important progresses in understanding directed motion, less is understood in regard to paths for diffusion and in general the connectivity of the cell interior. In this presentation I will discuss the development of tools that could help us in measuring the path that molecules follow in the cell to reach their target. Among the tools needed for measuring diffusion on the entire cell, or at least in a plane of the cell is a microscope capable of acquiring images fast and in 3D over large field of view. I will discuss different techniques available at the LFD capable of providing fast acquisition over a relatively large field of view from which the correlation of molecular motions can be evaluated.
24. Per Niklas Hedde (Speaker)
    Selective plane illumination microscopy in the conventional inverted microscope geometry.
    Abstract

    Per Niklas Hedde
    Laboratory for Fluorescence Dynamics, Biomedical Engineering, University of California, Irvine, USA

    During the last decade, selective plane illumination microscopy (SPIM) has proven to be one of the most suitable techniques for three-dimensional time lapse imaging. By confining the excitation light to a sheet, SPIM combines axial sectioning capability with minimal light exposure and fast, camera-based image acquisition. However, the typical arrangement of two objective lenses perpendicular to each other provides a number of challenges in terms of instrument design and sample geometry, especially if the use of high numerical aperture (NA) lenses is desired. A popular approach is to dip into the sample container from the top, both lenses at a 45 degree angle with respect to the sample plane. Instead, our new design is based on a regular inverted microscope where the sample is illuminated from the side via an accessory. A custom designed chamber is used to allow side illumination. This way, all microscope ports remain available for other purposes and there is unrestricted access from the top. Without the need of dipping into the sample container, smaller sample volumes (<1 ml) can be realized and the use of high NA lenses is facilitated. Also, isolation of optics and sample allows imaging of sealed sample containers when demanded, e.g., for samples treated with potent toxins. Further, in this design, the orientation of the imaging plane is parallel to the surface of the sample container which is desirable for flat samples where it maximizes the field of view.
25. Per Niklas Hedde (Poster)
    Fluorescence anisotropy imaging in 3D with single plane illumination microscopy.
    Abstract

    Per Niklas Hedde, Suman Ranjit, and Enrico Gratton
    Laboratory for Fluorescence Dynamics, Biomedical Engineering, University of California, Irvine, USA

    Anisotropy imaging is a powerful tool to study the organization, composition and dynamics of molecules in biological systems. Changes in the organization and conformation of biomolecules within cells and tissues can be visualized at the single pixel level. Fluorescence anisotropy is typically probed with laser scanning and epifluorescence-based techniques. However, those techniques are limited in either axial resolution, image acquisition speed, or, by photobleaching. In the last decade, selective plane illumination microscopy (SPIM) has emerged as the best choice for three-dimensional time lapse imaging since it combines axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure [1,2]. We demonstrate how SPIM can be used for three-dimensional fluorescence anisotropy imaging of live cells [3]. To verify the accuracy of our SPIM system, we first measured and compared the anisotropy of Rhodamine110 solutions with different water/glycerol ratios with results obtained using a spectrofluorimeter. Then, we visualized the aggregation states of different fusion proteins in live cells. Further, we were able to show by 3D time lapse anisotropy imaging that formation of membrane protrusions and paxillin aggregates precedes focal adhesion assembly. Work supported in part by NIH grants P50 GM076516 and P41 GM103540.
    [1] Huisken, J. et al., Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy. Science 305, 1007¬–1009 (2004).
    [2] Hedde, P.N. et al., Rapid Measurement of Molecular Transport and Interaction inside Living Cells Using Single Plane Illumination. Sci. Rep. 4, 7048 (2014).
    [3] Hedde, P.N. et al., 3D fluorescence anisotropy imaging using selective plane illumination microscopy. Opt. Express 23, 22308–22317 (2015).
26. Christian E. Hellriegel (Sponsor Talk)
    Controls and calibrations - details that matter.
    Abstract

    Christian Hellriegel
    Carl Zeiss Microscopy GmbH, Jena, Germany

    One of the most important, yet tedious (and therefore perhaps often underappreciated) aspects in quantitative fluorescence microscopy has to do with equipment calibration, measurement corrections and control experiments. However, these seemingly laborious and long-winded parts need to be in place if not only to check reality or to make sure that the instrument is performing properly. In this short presentation we propose a series of small experiments and point to some details to look after when conducting quantitative fluorescence experiments in the context of microscopy.
27. Yuning Hong (Poster)
    Measuring macromolecular crowding in cells through fluorescence anisotropy imaging.
    Abstract

    Hamid Soleimaninejad, Trevor Smith, and Yuning Hong
    La Trobe University, Melbourne, Australia

    Macromolecular crowding effect is ubiquitously occurring in almost all types of cell and influences such processes as protein diffusion, folding, association, aggregation and so on. Direct visualisation of the crowding effect with spatiotemporal resolution would be desirable to determine the impact of excluded volume on the biological processes in cells. I will present a simple and general approach we design based on fluorescence anisotropy imaging with an aggregation-induced emission (AIE) fluorogen to measure the crowding effect in the heterogeneous and dynamic cellular milieu. The utilization of the AIE dye for investigating the intracellular environment will also be introduced.
28. David M. Jameson (Speaker)
    The seminal contributions of Gregorio Weber to modern fluorescence spectroscopy and to protein chemistry.
    Abstract

    David M. Jameson
    University of Hawaii, Hawaii, USA

    During the last few decades, fluorescence spectroscopy has evolved from a narrow, highly specialized technique into an important discipline widely utilized in the biological, chemical and physical sciences. As in all scientific disciplines, the development of modern fluorescence spectroscopy has benefited from the contributions of many individuals from many countries. However, one individual, Gregorio Weber, can be singled out for his outstanding and far-reaching contributions to this field. This lecture will briefly outline aspects of Gregorio Weber’s life and times and discuss some of his more important contributions to the fluorescence field. Some of his more important contributions to the field of protein chemistry will also be discussed. In addition to the facts of Weber’s life and work, I shall also interject several anecdotes from my personal experience with him, which will serve to illustrate his outstanding personality and character.
29. Luca Lanzanò (Speaker)
    Phasor analysis of fluorescence dynamics in quantitative nanoscale optical microscopy.
    Abstract

    Luca Lanzanò
    Istituto Italiano di Tecnologia, Genoa, Italy

    Fluorescence dynamics can be used to enhance the content of optical microscopy. I will discuss how the phasor analysis of fluorescence spectra can be used to (i) improve spatial resolution and (ii) provide quantitative information inside cells at the nanoscale.
    Most of the super-resolution fluorescence methods provide sub-diffraction resolution by transiently switching the fluorophores between optically distinguishable states, in a stochastic or targeted mode. However, super-resolution can also be obtained with a very different strategy: by encoding spatial information into a temporal channel and decoding it after the transmission through the microscope setup. Accordingly, the challenge of increasing the spatial resolution of an optical microscope is translated into the task of resolving the signal into its dynamics components. This strategy can be easily implemented on a STED microscope and the components resolved by phasor analysis [1]. I will show that this approach provides background-free nanoscale imaging of subcellular structures.
    In order to extract quantitative values from these images, I will introduce an algorithm based on local Image Correlation Spectroscopy (ICS). ICS is a well-established analysis method capable of extracting the average size and number of particles in images. However, when ICS is applied to a highly heterogeneous system, it can fail to retrieve, from a single correlation function, the characteristic size and the relative amount associated to each subspecies. I will describe a fast, unbiased, and fit-free algorithm based on the phasor analysis of multiple local image correlation functions, capable of mapping the sizes of elements contained in a heterogeneous system [2]. I will use this method to extract the size of subcellular structures, from both confocal and super-resolved images.
    [1] L. Lanzanò et al. “Encoding and decoding spatio-temporal information for super-resolution microscopy” Nature Communications 6, 6701 (2015).
    [2] L. Scipioni et al, “Phasor Analysis of Local ICS Detects Heterogeneity in Size and Number of Intracellular Vesicles” Biophysical Journal 111, 619-629 (2016).
30. Zeno Lavagnino (Weber Prize Guest)
    Improving selective plane illumination microscopy towards enhanced spatial resolution and imaging depth performances.
    Abstract

    Zeno Lavagnino
    Washington University in St. Louis, Missouri, USA

    In optical fluorescence microscopy, the main components contributing to the imaging performance are represented by the resolution of the optical system, the wavelength of the light used, and the sample investigated.
    The properties of the specimen can heavily limit the achievable resolution due to light-matter interactions which cause scattering and aberrations.
    For this reason, the overall resolution and the achievable contrast are often worse than the theoretically expected resolution, because they both depend on the optical thickness and on the heterogeneities of the sample.
    Moreover, when looking at live specimens, the influence of the light on the sample is extremely relevant. Photo-bleaching is the most predominant phenomenon, resulting in a loss of signal, because the fluorescent molecule loses its capability to emit photons. Furthermore, live specimens are dynamic systems, and the image acquisition speed of a microscope has to keep up with the speed of the processes under investigation. The practical use of a microscope is therefore determined by both its physical parameters and its biocompatibility.
    Unfortunately, there is no perfect optical system, which allows for maximal spatial and temporal resolution. In fact, improving spatial resolution will always be accompanied by a resulting slower acquisition (i.e. scanning techniques, like confocal microscopy). This is eventually exacerbated by the recently developed super-resolution techniques, which also require very complex sample preparations and optical systems arrangements. At the same time, increasing the temporal resolution will often result in poorer spatial resolution, due to the minimal amount of photons acquired. A powerful tool with high spatial and temporal resolution capability, it’s represented by Total Internal Reflection Fluorescence Microscopy (TIRFM), but the technique is limited to few hundreds of nanometers close to the coverslip, making it a suitable technique for membrane dynamics studies. Thus, the “ideal” optical system will require high penetration depth, optical sectioning, multicolor capability, and low photobleaching rate to perform time-lapse experiments.
    Evaluating the needed characteristics, light-sheet microscopy results to be the optimal microscope to perform live, multicolor imaging of thick samples. The working principle of the light-sheet microscope is relatively simple. A Gaussian laser beam is scanned very fast (faster than the imaging rate of the camera) to create the illuminating sheet (or focused through a cylindrical lens). The sample is then scanned through the light-sheet in order to provide optical sectioning, with a widefield-like detection system. In a similar implementation, instead of scanning the sample through the light-sheet, an additional couple of scanning mirrors allows for scanning of the light sheet through the sample lying in a medium-filled water chamber.
    In this work, particular attention will be focused on three main aspects: the improvement of spatial resolution in thick samples, the extension of the imaging depth capability and the enhancement of the portfolio of samples to be investigated with a recently developed vertical architecture. Thanks to the new arrangements proposed, it has been proven the possibility to improve the resolution, the imaging depth capability and to increase the portfolio of accessible samples by a Selective Plane Microscope, now including for example tissues, brain slices or thick membranes like a retina.
31. Valeria Levi (Speaker)
    Mapping the dynamics of transcription factors in living cells and whole organisms.
    Abstract

    Valeria Levi
    Departamento de Química Biológica- IQUIBICEN. Facultad de Ciencias Exactas y Naturales-Universidad de Buenos Aires-Argentina

    Gene expression results from the dynamical recruitment of the transcription machinery and regulatory proteins to specific target genes. Many components of this machinery including transcription factors (TFs), coregulators, chromatin remodelers and RNA polymerases do not homogeneously distribute in the cell nucleus. Determining how the highly dynamical and heterogeneous distribution of transcription-related proteins within the nucleus modulates the expression profile remains as a central challenge in the cell biology field. Here, we show how fluorescence fluctuations based techniques could help us to understand this complex process.
    First, we use fluorescence correlation spectroscopy (FCS) to quantify the dynamics of TFs that controls pluripotency in single cells of developing mouse embryos and explored whether cell fates originate in a stochastic or predictable manner. Our study demonstrates that TFs undergo temporally distinct binding events with DNA in the living mouse embryo. Furthermore, developmentally regulated epigenetic changes create differences in the fraction of the TF Sox2 engaged in long-lived DNA–binding between 4-cell blastomeres. Finally, we demonstrate that these differences in Sox2–DNA binding predict the allocation of the first cells to the inner mass of the embryo, which subsequently contributes most cells of the body.
    In addition, we investigated the dynamical organization of TFs in living cells focusing on the glucocorticoid receptor (GR), a ligand activated transcription factor. We present evidences showing that GR and its coregulator NCoA-2 diffuse within the nucleus and interact with PML bodies, DNA-dependent foci or engage in interactions with chromatin targets. Cross-correlation analyses show that GR and NCoA-2 sequentially interact with foci and glucocorticoid response elements. Moreover, we show that the coregulator and the conformation of the receptor tune the distribution of the receptor among the nuclear compartment. Our results suggest that GR partition in different nuclear reservoirs ultimately regulates the concentration of receptor available for the interaction with specific targets.
32. Diane Lidke (Speaker)
    Imaging early events in membrane receptor signaling.
    Abstract

    Diane Lidke
    University of New Mexico, New Mexico, USA

    The recruitment of intracellular signaling molecules to membrane receptors is a critical step in signal transduction. While many of the proteins involved in signaling cascades are known, how the spatiotemporal response of these protein-protein interactions regulates signal propagation is still not well defined. We have applied single molecule and FRET imaging methods to study the protein dynamics that regulate FcεRI signaling. Using single molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. We found that Syk colocalizes transiently to FcεRI and that Syk-FcεRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (SykY130E) led to an increased Syk-FcεRI off-rate and impaired downstream signaling, highlighting the importance of finely-tuned protein-protein interactions in directing cellular outcomes. To gain further insight into FcεRI-Syk interactions, we have been developing the Fluorogen Activating Protein (FAPs) system as a donor for FRET with blue shifted fluorescent proteins. FAPs are genetically encoded tags made from single chain antibody fragments (scFv) designed to bind fluorogens with high specificity. The fluorogen Malachite Green (MG) has two excitation peaks, the maximum at 630 nm and a secondary peak at 450 nm. The emission spectra of blue-emitting fluorescence proteins, such as mCerulean, overlap with the MG secondary peak, generating a FRET pair with large Stokes shift emission. We have confirmed that FAP-MG is a good acceptor for FRET with mCerulean and YFP. This system provides a useful FRET pair for applications to the study of protein interactions in live cells and we are applying FAP-FRET to the study FcεRI-Syk interactions.
33. Keith Lidke (Speaker)
    Single objective light-sheet microscopy for improved whole-cell 3D super-resolution.
    Abstract

    Marjolein B.M. Meddens, Sheng Liu, Patrick S. Finnegan, Thayne L. Edwards, Conrad D. James, and Keith A. Lidke

    SM-SR in 3D throughout a whole cell is made more difficult by high background fluorescence that can degrade detection and localization precision. We have developed a technique, termed Single Objective Light-Sheet Microscopy (SO-LSM) that uses a common, single objective, inverted epi-fluorescence microscope along with a reflective surface to generate illumination only in the image focal plane. We use a reflective surface, placed at an angle of 45° on a cover slip to reflect the beam. This surface forms the side wall of a microfluidic channel incorporated into a microfluidic device. A light-sheet is generated through the objective and reflected such that it illuminates the in-focus plane of the cell. The light-sheet is scanned through the cell for whole-cell imaging and an astigmatic lens in the emission path enables the 3D localization of individual emitters, creating a whole-cell 3D super-resolution image. SO-LSM provide! s improved localization accuracy due to background reduction and reduced photobleaching of un-imaged, out-of-focus fluorophores. The microfluidics device provides a closed environment that allows for fast and automated buffer exchange, which is utilized to implement multi-structure imaging using a sequential approach.
34. Jose Luiz Lopes (Speaker)
    Exploiting the thermal stability and organic solvent tolerance of esterases.
    Abstract

    Jose L.S. Lopes 1, J.M.M. Martins 2, R.F. Gutierrez 2, P.S. Kumagai 2, D.M. Jameson 3, B.A. Wallace 4, and AP.U. Araujo 2
    1 Institute of Physics, University of Sao Paulo, Sao Paulo, SP, Brazil
    2 Institute of Physics of Sao Carlos, University of Sao Paulo, Sao Carlos, SP, Brazil
    3 Dept. Cell and Molecular Biology, University of Hawaii at Manoa, Honolulu, HI, USA
    4 Birkbeck College, University of London, London, United Kingdom

    Enzymes produced by thermophilic organisms are of remarkable interest in several industrial applications because of their high structural and thermal stabilities which allow them to remain stable and active at high temperatures, or in the presence of organic solvents and other extreme conditions. The investigation of the biochemical properties, thermal and structural behavior of esterases isolated from thermophiles have been studied by a combination of steady state and time resolved fluorescence spectroscopies and synchrotron radiation circular dichroism (SRCD) spectroscopy. The enzymes presented optimum hydrolytic activity at elevated temperatures (<55ºC) and kept their typical structural fold of esterases unchanged at these conditions. While temperature had minor effect on secondary and tertiary structures of these esterases, the use of high concentrations of chaotropic agents was able to promote the enzymes unfolding through apparently different mechanisms. Nonetheless, the changes caused on protein structure can be completely reversible, indicating the enzymes are able to reassume their active state. The preservation of the esterase hydrolytic activity is an important requirement when thinking on the potential use of theses enzymes in industrial processes.
35. Leonel S. Malacrida (Speaker)
    Water dipolar relaxation inside nucleus: taking advantage of ACDAN spectroscopic properties and spectral phasors.
    Abstract

    Leonel Malacrida, Suman Ranjit, and Enrico Gratton
    Laboratory for Fluorescence Dynamics, Biomedical Engineering, University of California, Irvine, USA

    Hetero and euchromatin are described at cell interphase as two different supramolecular organizations of the DNA and histones. Heterochromatin was described by electron transmission microscopy as a high electron density structure when compared to euchromatin. The information on the amount of global hydration and molecular crowding in hetero and euchromatin are still missing. These properties are relevant to understand biophysical and biochemical functions of the cell. In this study, we measure the water dynamic in hetero and euchromatin using the solvatochromic properties of ACDAN (6-acetyl-2-dimethylaminonaphthalene). The spectral phasor analysis of the ACDAN fluorescence at pixel level resolution revealed the water dynamic in the nucleus of NIH-3T3 cells. Furthermore, by using the 2D pair-Correlation-Function (so-called connectivity maps) analysis of the ACDAN fluorescence fluctuation, we were able to measure diffusion of A! CDAN in the nucleus at the interphase. To correlate the regions of different dipolar relaxation in the nucleus, we used a heterochromatin marker based on the H2B-dsRed fluorescent protein. The nucleus at interphase was the most polar structure of the cell as reported by ACDAN. In the nucleus, we were able to discriminate at least two populations based on dipolar relaxation rates. Using the correlation between the ACDAN and H2B-dsRed, we associated the heterochromatin phase with regions of restricted ACDAN dipolar relaxations. The connectivity map shows that ACDAN inside the nucleus has areas with preferential/restricted diffusion. At cell division, there is a dramatic change in the dipolar relaxation profile. Changes in the dipolar relaxation can be explained as physical changes in the nucleus (phase transitions/molecular crowding) driven by the water activity. (Supported by NIH P41-GM103540 and NIH P50-GM076516)
36. Leonel S. Malacrida (Poster)
    Water activity inside the nucleus: Some clues using ACDAN fluorescence and its implications in the chromatin supramolecular organization.
    Abstract

    Leonel Malacrida, Suman Ranjit, Enrico Gratton
    Laboratory for Fluorescence Dynamics, Biomedical Engineering, University of California, Irvine, USA

    Hetero and euchromatin are described at cell interphase as two different supramolecular organizations of the DNA and histones. Heterochromatin was described by electron transmission microscopy as a high electron density structure when compared to euchromatin. The information on the amount of global hydration and molecular crowding in hetero and euchromatin are still missing. These properties are relevant to understand biophysical and biochemical functions of the cell. In this study, we measure the water dynamic in hetero and euchromatin using the solvatochromic properties of ACDAN (6-acetyl-2-dimethylaminonaphthalene). The spectral phasor analysis of the ACDAN fluorescence at pixel level resolution revealed the water dynamic in the nucleus of NIH-3T3 cells. Furthermore, by using the 2D pair-Correlation-Function (so-called connectivity maps) analysis of the ACDAN fluorescence fluctuation, we were able to measure diffusion of ACDAN in the nucleus at the interphase. To correlate the regions of different dipolar relaxation in the nucleus, we used a heterochromatin marker based on the H2B-dsRed fluorescent protein. The nucleus at interphase was the most polar structure of the cell as reported by ACDAN. In the nucleus, we were able to discriminate at least two populations based on dipolar relaxation rates. Using the correlation between the ACDAN and H2B-dsRed, we associated the heterochromatin phase with regions of restricted ACDAN dipolar relaxations. The connectivity map shows that ACDAN inside the nucleus has areas with preferential/restricted diffusion. At cell division, there is a dramatic change in the dipolar relaxation profile. Changes in the dipolar relaxation can be explained as physical changes in the nucleus (phase transitions/molecular crowding) driven by the water activity. (Supported by NIH P41-GM103540 and NIH P50-GM076516).
37. Jochen Mueller (Speaker)
    Venturing into the nuclear envelope: Lessons learned.
    Abstract

    Jochen Muller
    University of Minnesota, USA

    The nuclear envelope (NE) is a double lipid bilayer that encloses the nucleus. These two bilayers, the inner and the outer nuclear membrane, are separated by a ~40 nm-wide space, which is called the perinuclear space or lumen. While the NE is well-known as a physical barrier that regulates transport between the cytoplasm and nucleus through nuclear pore complexes embedded in the NE, it has become increasingly clear that important chemical and mechanical signaling events are coordinated by proteins in the NE. However, the majority of NE proteins are poorly characterized, which in part reflects the lack of tools capable of quantifying protein-protein interactions within this subcellular compartment. We have started to address this issue by exploring the use of fluorescence fluctuation spectroscopy in the NE to characterize the formation of protein complexes. We found that applying fluorescence fluctuation spectroscopy in the NE leads to perplexing results, prompting us to take a closer look at the unique environment provided by the NE. We have found that fluctuations originating from the NE structure are influencing the observed fluorescence. In this talk I will give an overview of the progress made so far and present data on the oligomerization of NE proteins.
38. Maria Victoria Neguembor (Weber Prize Guest)
    Investigating the chromatin fiber at the nanoscale level.
    Abstract

    Maria Victoria Neguembor, Maria Aurelia Ricci, Maria Pia Cosma, and Melike Lakadamyali
    Centre for Genomic Regulation (CRG), Barcelona, Spain

    The epigenetic and three-dimensional organization of the chromatin fiber plays a fundamental role in the regulation of gene expression and thus is key for maintenance of cell identity and for cell differentiation.
    To gain insight into how nucleosomes are arranged in vivo, we combined STORM super-resolution microscopy with computer simulations to visualize and count nucleosomes along the chromatin fiber in single nuclei of many cell types.
    STORM imaging revealed that nucleosomes do not form a highly ordered and homogenous organization, as usually depicted, but rather arrange into discrete clusters of various sizes and densities and interspaced by nucleosome-depleted regions. We showed that changes in the assembly of nucleosomes resemble changes in cellular phenotype when passing from pluripotent to somatic state and vice-versa.
    In order to correlate chromatin states with transcriptional activity, we are currently developing CRISPR-based imaging tools to label specific genes in living cells and in super resolution. With this approach, we can investigate dynamic behavior of individual loci and extract quantitative information of their genetic features.
    Overall, we aim to visualize the chromatin organization of individual loci at the nanoscale level.
39. Gerd Ulrich Nienhaus (Speaker)
    Stimulated emission double depletion (STEDD) for background suppression in nanoscale imaging and fluorescence fluctuation spectroscopy.
    Abstract

    Gerd Ulrich Nienhaus
    Karlsruhe Institute of Technology, Karlsruhe, Germany

    Stimulated emission depletion (STED) fluorescence nanoscopy is a raster-scanning imaging technique that achieves subdiffraction resolution via confinement of fluorescence emission to the central subregion of an observation volume through de-excitation of fluorophores in the periphery via stimulated emission. In this talk, I will discuss stimulated emission double depletion (STEDD) as a powerful STED variant that selectively removes artificial background inherent to the STED method [1]. The conventional STED pulse is followed by a second, delayed Gaussian STED pulse that specifically depletes the central region, thus leaving only background. We use time-resolved detection, which enables us to remove background voxel by voxel by calculating the weighted difference of photons collected before and after the second STED pulse. Background-free STEDD imaging is particularly advantageous when imaging dense 3D structures. Moreover, STEDD allows us to significantly reduce the 3D observation volume in fluorescence fluctuation pair correlation spectroscopy techniques including FCS or RICS [2,3]. Of note, STEDD imaging allows the use of lower-power, less red-shifted depletion pulses, which provides enhanced flexibility in the choice of fluorophores and laser sources.
    [1] Gao, P., Prunsche, B., Zhou, L., Nienhaus, K., & Nienhaus, G. U., Nature Photon. 11 (2017) 163-169.
    [2] Dörlich, R. M., Chen, Q., Hedde, P. N., Schuster, V., Hippler, M., Wesslowski, J., Davidson, G., & Nienhaus, G. U. Sci. Rep. 5 (2015) 10149.
    [3] Hedde, P. N., Dörlich, R. M., Blomley, R., Gradl, D., Oppong, E., Cato, A. C. B., & Nienhaus, G. U., Nature Commun. 4 (2013) 2093.
40. Rory Nolan (Poster)
    An automatic detrending algorithm for image series with bleaching.
    Abstract

    Rory Nolan, Luis Alvarez, and Sergi Padilla-Parra
    Wellcome Trust Centre for Human Genetics, University of Oxford, United Kingdom

    All of the current algorithms which detrend to correct for photobleaching (exponential filtering, box-car, polynomial) require the user to select a parameter (the exponential filtering parameter tau, the size of the box car, the degree of the polynomial). Currently, this is done arbitrarily, however the choice of the parameter has a large impact on the quantitative analysis of the data. Here we present a method for selecting the optimal parameter automatically based on your data.
41. Murilo Martins Pedrote (Poster)
    The competent pre-amyloidogenic state of p53 is a wet molten-globule.
    Abstract

    Murilo M. Pedrote, Adriani L. Felix, Michelle F. Mota, Mayra de A. Marques, Iaci N. Soares, Elio A. Cino, Douglas R. Norberto, Jerson L. Silva, and Guilherme A. P. de Oliveira

    The p53 tumor suppressor is responsible for different types of cellular stresses such as DNA damage. The TP53 gene is the most frequently mutated gene in cancer. Single point mutations in the p53 core domain are related to phenotypes that contribute to tumor development, and some result in p53 loss- and, gain- of oncogenic function, contributing to cell survival and tumor progression. In addition, it has already been reported that amyloid-like aggregates of p53 are present in some types of cancer. Protein unfolding is a key determinant for the understanding of how p53 deviates from its native conformation to an aggregation pathway with implications to cancer progression. Spectroscopically, we can make use of physical and chemical tools such as high hydrostatic pressure and guanidine hydrochloride, with phasor plot analysis to investigate how p53 and its homologs p63 and p73 unfold and deviate to an aggregation pathway. Here, we show that low guanidine concentrations (0.8M) guide p53 to a competent pre-amyloidogenic and wet-molten globule state harboring a partially protected Trp residue. Our results demonstrate how the combination of physical and chemical agents, and the use of spectroscopic features, may help to uncover competent pre-amyloidogenic excited states of p53 participating in disease pathology. The ability to identify and intercept precursor states of p53 aggregation provides a unique approach to track aggregation of the p53 mutant and to test the efficacy of treatments that aim to stabilize the native form of p53.
42. Manuel José Estevez Prieto (Speaker)
    Advanced homo-FRET - Exact analytical solutions for tetramers and their application for retrieving structural information of the KcsA channel.
    Abstract

    M. L. Renart 1,3, A. Coutinho 1,2, J.A. Poveda 3, M. Giudici 3, A. Fedorov 1, M. Berberan-Santos 1, J. Gonzalez-Ros 3, and M. Prieto 1
    1 CQFM and IN, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
    2 DQB, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal
    3 IBMC, Universidad Miguel Hernández, Elche, Alicante, Spain

    Donor decay ensemble average in FRET studies of biological systems usually only allow qualitative information about topology and structure. However, in systems of high symmetry, exact analytical solutions containing information about specific distances can be obtained, and used to fit high quality time-resolved data (usually within a global analysis methodology).
    In this study, the tetrameric potassium channel KscA was engineered in order to have only a single tryptophan per monomer. An exact solution for homo-FRET within a square geometry was derived and fitted to anisotropy decays. The recovered distances will be compared with the ones from diffraction data, in both closed and open states, and for different ions. The advantage of this methodology over X-ray diffraction will be discussed.
    Acknowledgments: FCT (Portugal) for project FAPESP/20107/2014 and MEC (Spain) for BFU2011-25920
43. Rodrigo Alain Migueles Ramírez (Poster)
    Stoichiometry and spatio-temporal regulation of Polo-like kinase 4 in live cells.
    Abstract

    Rodrigo Alain Migueles Ramirez
    Laboratorio Nacional de Microscopía Avanzada, Instituto de Biotecnología, Universidad Nacional Autónoma de México

    The centrosome is a dynamic structure composed by two orthogonal centrioles surrounded by a peri-centriolar matrix. Unlike other organels, the centrosome is not membrane-bound and the exchange of material is therefore based on a diffusion and binding mechanism. Centrosomes duplicate once per cell cycle to form the mitotic spindle. Polo-like kinase 4 (Plk4) is the master regulator of centriole biogenesis. Spatio-temporal regulation of Plk4 is therefore crucial to assure correct chromosome segregation. Deregulation of Plk4's activity leads to aberrant centrosomes as well as multiple centrioles per cell, among other cellular pathologies. These phenotypes have been associated with illnesses such as cancer, microcephaly and other ciliopathies. Plk4 concentrates in the centrosome and two of its interacting partners in humans, Cep152 and Cep192, have been found to have a role in the spatial regulation of Plk4. However, the mechanism by which Plk4 is recruited to the centrosome remains poorly understood. Plk4 is activated by auto-phosphorylation in a trans mechanism, which suggests the formation of dimers. Although Plk4 dimerization has been reported using biochemical techniques and X-ray crystalography, this phenomenon has never been reported in live cells. This project aims to determine the stoichiometry of homo and hetero complexes formed by Plk4 and its binding partners Cep152 and Cep192 using the N&B method and to dilucidate whether these complexes form in the cytoplasm or exclusively in the centrosome using cross RICS. Furthermore, estimation of the diffusion constant of Plk4 in different parts of the cell using RICS will contribute to the understanding of the mechanisms by which Plk4 is recruited and retained at the centrosome.
44. Rajesh Ramachandran (Speaker)
    Cardiolipin-enriched microdomains sequester adaptors MiD49/51 and Mff to cooperatively regulate Drp1-mediated mitochondrial fission.
    Abstract

    Rajesh Ramachandran
    Case Western Reserve University, Ohio, USA

    Mitochondrial outer membrane (MOM)-anchored transmembrane proteins mitochondrial dynamics proteins of 49/51 kDa (MiD49/51) and mitochondrial fission factor (Mff) function as adaptors for the mitochondrial recruitment of the cytosolic mechanochemical GTPase, dynamin-related protein 1 (Drp1) toward mitochondrial fission. Whether these adaptors function independently or cooperatively in regulating Drp1 function remains controversial, as are their mechanisms in Drp1-catalyzed mitochondrial fission. MiD49/51and Mff can independently recruit Drp1 to the mitochondrial surface, yet mitochondrial fission is impaired in each adaptor's absence suggesting that their functions are cooperative and non-redundant. Using reconstitution approaches in model membranes and multiple fluorescence spectroscopic and imaging techniques, we demonstrate that both MiD49/51 and Mff preferentially partition into cardiolipin (CL)-enriched membrane microdomains characteristic of MOM-MIM contact sites that regulate cristae remodeling and cytochrome c release during apoptotic mitochondrial fission. Surprisingly, MiD49 and MiD51 utilize their soluble, cytosolic nucleotidyltransferase domains to specifically bind cone-shaped CL. The selective regulation of MiD51, but not MiD49, by pathophysiological ADP concentrations was also found to be CL-dependent. Moreover, the effect of MiD51 on solution and membrane-bound Drp1 was inversely correlated. MiD51 disassembled cytosolic Drp1 polymers into dimers in a ADP-regulated manner in solution, while promoting Drp1 dimer helical co-assembly with MiD51 on CL-containing membranes. Furthermore, the MiD51 nucleotidyltransferase domain co-assembled with Drp1 on CL-containing membranes to suppress Drp1 cooperative GTPase activity, an effect not observed with MiD49 at equivalent concentrations. MiD51 co-assembled with Mff and Drp1 to constitute a ternary copolymer that was morphologically distinct from Drp1 assembled in the presence of either adaptor. We propose that MiD51 functions to re-equilibrate solution Drp1 polymers into assembly-competent, membrane-bound Drp1 dimers. MiD51-Drp1 dimers then associate with Mff to constitute a ternary polymeric complex with reduced Drp1 cooperative GTPase activity that helps sustain the transition-state conformation of the Drp1 polymer on the membrane to effectively promote membrane constriction and fission. Thus, MiD49/51 and Mff cooperatively regulate Drp1 function in mitochondrial fission.
45. Gregory D. Reinhart (Speaker)
    Reconciling Weber’s protein-ligand ideas with contemporary data using fluorescence phasors.
    Abstract

    Xinxin Tian, Mauricio Lasagna and Gregory D. Reinhart
    Texas A&M University, Texas, USA

    Nearly 50 years ago, Gregorio Weber published a series of landmark papers in which he summarized the free energy constraints that must govern protein-ligand interactions. A focus of these discussions was the then newly proposed idea that proteins that bind different ligands with differing functional consequences do so by adopting one of two conformational forms that are respectively imbued with the different functional properties. Weber argued instead that systems must necessarily be more complex and that proteins are not “endowed with the mechanical properties that we reserve for macroscopic objects” but rather exhibit properties that are derived from “ordinary chemical equilibria” with the protein appearing as “one more reagent exhibiting the stochastic characteristics that we attribute to any molecular population”. A key element in Weber’s argument was that a protein that binds two mutually antagonistic ligands at distinct binding sites should be able to bind both ligands simultaneously with reasonable probability generating a potentially unique structural form with corresponding unique functional properties. Nonetheless, the idea that an allosterically regulated protein is one that can adopt one of only two available conformational states remains a popular presumption, and often data obtained from modern structural biology techniques such as NMR spectroscopy or X-ray crystallography are interpreted in ways that reinforce this notion. Prokaryotic phosphofructokinase from Thermus thermophilus is allosterically inhibited by phospho(enol)pyruvate (PEP) by reducing the affinity the enzyme displays for the substrate, fructose 6-phosphate (F6P) in a manner that has been traditionally interpreted with a 2-state model. We have used site-directed mutagenesis to introduce a single tryptophan side-chain into various positions of the structure by modifying pre-existing phenylalanine or tyrosine side-chains. These modifications did not appreciably alter the functional or regulatory properties of the enzyme. We then examined the time-resolved fluorescence response to ligand binding of the various single tryptophan variant enzymes using fluorescence phasors. The responses depend on the position of the tryptophan side-chain. Of eight tryptophan positions investigated, one proved to be insensitive to ligand binding. The fluorescence response of the tryptophan in 3 positions was influenced by ligand binding in a way that could reasonably be interpreted by a 2-state model. The fluorescence from the remaining 4 positions clearly shows that the fluorescence obtained when both F6P and PEP are bound simultaneously does not emanate from some combination of 2 limiting fluorescent (and hence conformational) states. We present a model that accounts for these observations in a straightforward manner. Supported by funding from NIH (GM033216) and Texas A&M AgriLife Research.
46. Susana Sánchez (Speaker)
    Using Laurdan and phasor analysis to follow solubilization of rabbit erythrocytes membranes by myristyl sucrose ester.
    Abstract

    Susana A. Sanchez 1, Joao Aguilar 1, Catalina Sandoval 2, and German Gunther 2
    1 Universidad de Concepción, Concepción, Chile
    2 Universidad de Chile, Santiago, Chile

    The use of micelle-forming surfactants is one of the preferred methods for isolation of specific constituents from membrane. Characterization studies on membrane properties using detergents are mainly performed on artificial membranes due to the complexity of natural membranes, where lipid composition and the presence of proteins and membrane domains make the scenario more complex. Among the natural membranes, the plasma membrane of red blood cells (erythrocytes) has been particularly useful as a model for studies of membrane structure due to their simplicity when compared with mammalian cells. Mammalian red blood cells do not contain nuclei or internal membranes, they are easy to manipulate, they have a size appropriate for imaging and their plasma membranes can be easily isolated for biochemical analysis. Most measurements on erythrocytes have been performed on erythrocyte ghosts to avoid the effect of hemoglobin that could interfere with the measurements. We studied the interaction of rabbit erythrocytes (whole and ghosts) with sucrose monoester, β-d-fructofuranosyl-6-O-myristyl-α-d-glucopyranoside (Mono myristyl sucrose, MMS). Sucrose esters correspond to a class of nonionic detergents that are non-toxic, skin compatible, non-polluting and biodegradable. Hemoglobin release into the incubation media (hemolysis) was measured by colorimetric and biochemical methods. Membrane properties were observed by two-photon excitation fluorescence microscopy (FLIM and spectral imaging) using Laurdan (6-lauroyl,1-2-dimethylamino naphthalene), a fluorescent dye designed and synthesized in 1979 by Gregorio Weber to study the phenomenon of dipolar relaxation, and data were analyzed using the phasor representation for FLIM and spectral analysis.
    Acknowledgements and Support: The authors would like to thanks to Fondecyt #1140454 and 1120196 for Financial Support, to CMA-Bio Bio, Universidad de Concepcion and Dr. Leonel Malacrida (LFD, UCI) for technical support.
47. Markus Sauer (Speaker)
    Super-resolution fluorescence imaging by dSTORM: Where next?.
    Abstract

    Markus Sauer
    Department of Biotechnology and Biophysics, University of Würzburg, Würzburg, Germany

    Super-resolution microscopy by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful high-resolution imaging techniques introduced in recent years, localization microscopy excels at it delivers single-molecule information about the distribution and, adequate controls presupposed, even absolute numbers of proteins present in subcellular compartments. This provides insights into biological systems at a level we are used to think about and model biological interactions. We briefly introduce basic requirements of localization microscopy, its potential use for quantitative molecular imaging, and discuss present obstacles and ways to bypass them. We demonstrate the advantageous use of dSTORM for quantitative imaging of synaptic proteins, the study of plasma membrane organization, and the molecular architecture of multiprotein complexes. Finally, we outline how dSTORM can be used advantageously to improve next generation medical therapies.
48. Jerson Silva (Speaker)
    Prion-like aggregation of proteins in cancer: the “Dr. Jekyll and Mr. Hyde” actions of p53.
    Abstract

    Jerson Silva
    Instituto de Bioquímica Médica Leopoldo de Meis, Instituto Nacional de Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    Protein misfolding results in devastating degenerative diseases and cancer. Among the culprits involved in these illnesses are prions and prion-like proteins, which can propagate by converting normal proteins to the wrong conformation. For spongiform encephalopathies, a real prion can be transmitted among individuals. In other disorders, the bona fide prion characteristics are still under investigation. Besides inducing misfolding of native proteins, prions bind nucleic acids and other polyanions. Key proteins involved in these diseases, such as Aβ, tau, α-synuclein, SOD1, and TDP43 can show a prion-like behavior. Studies by our group and others have demonstrated that prion-like behavior of proteins involved in cancer, which is the case of p53. p53 is a master regulatory protein that participates in cellular processes such as apoptosis, DNA repair and cell cycle control. The function of this tumor suppressor protein is lost in more than 50% of human cancers. Our studies have suggested that the formation of prion-like aggregates of mutant p53 is associated with loss-of-function, dominant-negative and gain-of-function (GoF) effects. Studies from our group have shown that p53 aggregation in a mixture of oligomers and fibrils that sequestrates the native protein into an inactive conformation, typical of a prionoid behavior. These aggregates are present in tissue biopsies of breast cancer especially in more aggressive ones. More recently, we have evaluated whether p53 aggregates may be also present in human glioblastoma cells and participate in the tumorigenic process. The prion-like properties of p53 aggregates are considered potential targets for drug development. We will discuss how nucleic-acid binding might influence protein misfolding for both disease-related and benign, functional prions and why the line between bad and good amyloids might be more subtle than previously thought. On the pathological side, there are no effective therapies against diseases involving the prion-like aggregation of proteins. An approach that focuses on protein-nucleic acid interactions, which are the key characteristic of these diseases, might reveal new therapeutic targets. Nucleic acids can have opposing effects on protein aggregation, depending on the specific cellular context, either in function or pathology. Thus, this relationship suggests a molecular personification of Dr. Jekyll and Mr. Hyde. (Supported by CNPq, FAPERJ, FINEP and CAPES).
49. Fernando D. Stefani (Speaker)
    Nanometer resolution single molecule localization with minimal photon fluxes.
    Abstract

    Francisco Balzarotti 1,2,3, Yvan Eilers 1,2,3, Klaus C. Gwosch 1,2,3, Arvid H. Gynnå 4, Volker Westphal 1,2,3, Fernando D. Stefani 5,6, Johan Elf 4, and Stefan W. Hell 1,2,3
    1 Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    2 Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany
    3 Optical Nanoscopy Division, German Cancer Research Center (DKFZ), Heidelberg, Germany
    4 Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Sweden

    MINFLUX is a new concept for localizing photon emitters in space using the minimum number of photons [1]. The emitter position is probed by subsequent exposures to local intensity minima of excitation light. In comparison to popular centroid-localization, MINFLUX attains identical precision using 22 times less photons.
    Fluorescence imaging with ~1-nm resolution precision is routinely performed with MINFLUX. An experimental demonstration where single molecules only 6 nm apart are fully resolved will be shown. Since 1 nm is the typical size of quantum transition dipoles in the visible, MINFLUX has reached the ultimate, physically meaningful resolution limit for an optical method.
    Also for single molecule tracking MINFLUX is greatly advantageous. The lower number of photons required for localization translates in a 100-fold increase in temporal resolution and the number of localizations per trace, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.
    [1] Balzarotti et al. Science 355, 606–612 (2017).
50. Chiara Stringari (Speaker)
    Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing.
    Abstract

    Chiara Stringari 1, Lamiae Abdeladim 1, Guy Malkinson 1, Pierre Mahou 1, Sébastien Brizion 2, Jean-Baptiste Galey 2, Willy Supatto 1, Renaud Legouis 3, Ana-Maria Pena 2, and Emmanuel Beaurepaire
    1 Laboratory for Optics and Biosciences, Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau cedex, France
    2 L'Oréal Research and Innovation, 93600 Aulnay sous Bois, France
    3 Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France

    Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate)(NAD(P)H),flavin adenine dinucleotide(FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750-1040nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041nm, an additional equivalent two-photon excitation wavelength at 879nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging(FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C.elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.
51. Marian L. Waterman (Speaker)
    Wnt signaling links to metabolism in cancer and stem cells.
    Abstract

    George T. Chen 1, Chiara Stringari 2, Mary Lee 3, Kira T. Pate 1, S. Sprowl-Tanio 1, Eric Puttock 3, Kehui Wang 4, Yung Lyou 1, John Lowengrub 4, Michelle Digman 2, Robert A. Edwards 4, Enrico Gratton 2, and Marian L. Waterman 1
    1 Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, USA
    2 Laboratory of Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, USA
    3 Department of Mathematics, University of California, Irvine, USA
    4 Department of Pathology, University of California, Irvine, USA

    LEF/TCF-ß-catenin complexes are transcriptional mediators of Wnt signaling and much attention has focused on their regulation of cell cycle progression and proliferation, an activity with high relevance for cell transformation. We have recently connected Warburg metabolism (aerobic glycolysis) to Wnt-ß-catenin signaling by the discovery of at least two direct Wnt target genes for glycolysis, PDK1 an inhibitory kinase for mitochondria and SLC16A1, a lactate transporter. Using Fluorescence Lifetime Imaging Microscopy (FLIM) to monitor the metabolic biomarker NADH we can show that glycolysis correlates with Wnt signaling in intestinal crypts and tumors. In normal intestine these signatures suggest new hypotheses for why the intestinal epithelium renews rapidly. In xenograft tumors, these signatures have revealed a new layer of complexity, which is that ß-catenin, LEF1, PDK1, and other factors linked to metabolism are heterogeneous. Using mathematical modeling and image analysis, we are studying the mechanisms that create Turing-like patterns of heterogeneity in tumors to understand how Wnt signaling might be modified by cross-talking signals in normal intestine and cancer.
52. Pegah Sadat Nabavi Zadeh (Poster)
    Immobilization of enzymes in mesoporous silica particles. Spectroscopic studies of protein mobility and pore microviscosity.
    Abstract

    Pegah S Nabavi Zadeh and Björn Åkerman
    Chalmers University of Technology, Göteborg, Sweden

    Mesoporous silica particles are used for immobilization of enzymes in order to increase enzyme stability, facilitate product purification and reuse of enzymes. Many efforts have been done to investigate how the environment inside the pores changes after enzyme immobilization and how it can affect activity and stability of immobilized enzymes. One of these environmental changes can be viscosity inside the pores and comparing the difference with the bulk solution.
    Here, we investigate viscosity inside mesoporous silica particles, SBA-15, 1µm diameter with 9nm pore diameter, based on two different cyanine fluorophores which are sensitive to viscosity, Cy3 and Cy5 using a ratiometric method. The probes can bond covalently to lipase, the enzyme that is used, in two different conditions: a. both probes attach on the same enzyme, or b. they can be attached to the enzyme separately. We work with a ratiometric method to measure only viscosity and no other solvent effects. In this study, Cy5 can be used to normalize for polarity other than viscosity. In addition, we take advantage of the energy transfer (FRET) between probes when they are attached on the same enzyme and measure the distance between them.