Elvin A. Alemán (Invited talk)
2-Aminopurine single-molecule fluorescence.
Elvin A. Alemán and David Rueda. Department of Chemistry, Wayne State University, Detroit MI 48202
Base flipping is a local conformational change that results in the rotation of a nucleotide out of its helical structure. This is frequently observed in numerous nucleic acid structural rearrangements, such as in DNA-protein interactions and in different RNA enzymes. 2-Aminopurine (2AP) is a fluorescent nucleotide analog extensively used to probe local conformational changes in nucleic acids in bulk experiments, but no single molecule approaches have been developed to study these mechanisms using 2AP. One challenge towards this goal is that 2AP fluorescence is very sensitive to the interaction with neighboring nucleotides, and its emission spectrum partially overlaps with that of tryptophan. We have developed new click-chemistry-based surface immobilization approach that enables us to monitor, in real time base flipping in DNA (Aleman EA, Pedini HS, & Rueda D (2009) Covalent-bond-based immobilization approaches for single-molecule fluorescence. ChemBioChem 10(18):2862-2866).
We have characterized the fluorescence properties of single 2AP labelled DNAs. Our results show that nucleotides are very dynamic in a single- or double-stranded DNA and that bases can flip out of the helix, suggesting that the base flipping occurs frequently and in a much slower time scale that previously believe. This new assay can also be used to study other local conformational changes in nucleic acids at the single-molecule level by using 2AP as the base-flipping probe.
Unlocking the intranuclear dynamics of RNA: an image derivative approach.
L. M. Andrews1, M. R. Jones1, E. Hinde2, M. A. Digman2 and E. Gratton2. 1School of Natural Sciences, University of Western Sydney, Australia. 2Laboratory for Fluorescence Dynamics (LFD), University of California, Biomedical Engineering Department, Irvine, California, 92697
Despite technological advances, the dynamics of RNA species within the nucleus and cytoplasm are not widely characterised. We have developed a model to analyse the spatial and temporal behaviour of RNA in live cells based on in situ staining of RNA, Raster Image Correlation Spectroscopy (RICS) to map diffusion and trajectory as well as an image derivative analysis to identify regions of transcriptional barriers. For this investigation, Chinese Hamster Ovary K1 cells (CHO-K1) were transfected with eGFP. Selected cell samples were then dual labelled with the fluorescent probe Pyronin Y to aid in the visualisation of RNA. Image data was captured with a Zeiss LSM 710. A RICS analysis assisted in the production of correlations which were algorithmically fit to determine the diffusive properties of each analysed region of interest within the cells. The eGFP alone was identified to diffuse between 3.85 - 8.71 µm2/s, coincident with the literature values, including elevated diffusion rates within the nucleus compared to the cytoplasm. The diffusive properties of the RNA in the CHO-K1 cells appeared to be within 0.410 – 5.65 µm2/s depending on the spatial positioning of the analysis. We further demonstrate that image derivative analysis enabled the identification of spatial barriers within the nucleus where size exclusion appears to be occurring. This analysis indicated discrete regions of transcriptional activity within the nucleus where eGFP appeared to undergo exclusion from regions of RNA where transcription was occurring.
Luis A. Bagatolli (Speaker)
LAURDAN generalized polarization studies in planar membrane systems.
Luis Bagatolli, Univ. Southern Denmark, Odense, Denmark
The feasibility of applying multiphoton excitation fluorescence microscopy related techniques in planar membrane systems, such as monolayers at the air water interface (Langmuir films)1 and lipid bilayers2, is presented and discussed. This approach, allows correlating spatially and temporally resolved information in both systems by exploiting the fluorescent properties of the LAURDAN probe. The fact that LAURDAN exhibits homogeneous distribution in membranes, particularly in systems displaying domain coexistence, overcomes a general problem observed when “classical” fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions1. Also, the well described photoselection effect caused by excitation light on LAURDAN allows inferring lipid tilting information in the membranes2. Particularly for Langmuir films this approach allows to visualize 3D membranous structures at the film’s collapse pressure. Upon compression, LAURDAN GP values in Langmuir films were compared with those obtained in compositionally similar planar bilayer systems. A strong correlation between the lateral packing of monolayers and bilayers was quantitatively performed when the lateral pressure of the monolayer is about 30 mN/m as predicted previously from theoretical approaches1.
1 J. Brewer, J. Bernardino de la Serna , K. Wagner and L.A. Bagatolli. “Multiphoton excitation fluorescence microscopy in planar membrane systems”, 2010, Biochim. Biophys. Acta. 1798(7):1301-1308;
2 U. Bernchou, J. R. Brewer, H. S. Midtiby, J. H. Ipsen, L. A. Bagatolli and A. C. Simonsen, “Texture of lipid bilayer domains” 2009, Journal of the American Chemical Society 131(40):14130-1431.
Direct correlation of protein structure and function by high-pressure X-ray crystallography.
Buz Barstow, Nozomi Ando, Chae Un Kim and Sol M. Gruner
A protein molecule is an intricate system whose function is highly sensitive to small external perturbations. However, few examples that directly correlate protein function with continuous, progressive sub-angstrom structural perturbations have thus far been presented. In order to elucidate this relationship, we have investigated the Aequorea yellow fluorescent protein Citrine as a model system under high-pressure perturbation. Citrine has been compressed by high pressure to produce deformations of its beta-barrel scaffold and light absorbing and emitting center, the chromophore, by applying a novel high pressure cryo-cooling technique.
A closely spaced series of high-pressure X-ray crystallographic structures of Citrine from 0.1 to 500 MPa reveal that the chromophore undergoes a progressive deformation of up to 0.8 Å at an applied pressure of 500 MPa. It is experimentally demonstrated that deformation of the chromophore is directly correlated with a progressive shift of the fluorescence peak of Citrine from yellow to green under these conditions.
The re-orientation of the Citrine chromophore is actuated by the differential motion of two clusters of atoms that compose the beta-barrel scaffold of the molecule, resulting in a bending or buckling of the beta-barrel. The high-pressure structures also reveal a perturbation of the hydrogen bonding network stabilizing the excited state of the Citrine chromophore that is implicated in the reduction of fluorescence intensity of the molecule under high pressure.
The blue-shift of the Citrine fluorescence spectrum resulting from the bending of the beta-barrel provides structural insight into the transient blue-shifting of isolated yellow fluorescent protein molecules under ambient conditions and suggests mechanisms to alter the time-dependent behavior of Citrine under ambient conditions.
The results presented here demonstrate that the fluorescence spectrum of Citrine is highly sensitive to sub-angstrom deformations and its fluorescent function must be understood at the sub-angstrom level. These results provide important general lessons for the structure-function relationship of enzymes, and may have significant implications for protein function prediction and biomolecule design and engineering as they suggest methods to tune protein function by modification of the protein scaffold.
Investigating amyloid nucleation and growth with fluorescence lifetime and fluctuation microscopy.
Keith Berland, Emory University, Atlanta, GA
We have used Fluorescence Fluctuation Spectroscopy (FFS) to investigate the fundamental mechanisms and structural pathways involved in the nucleation and growth of amyloid structures. Understanding the assembly pathway is of significant importance due to its relevance in human diseases, and also for the use of amyloid formation to develop functional nanomaterials. We will introduce how FFS methods have allowed us to determine that growth of Amyloid-ß (16-22) nanotubes nucleates within unstructured peptide aggregates, and elongates via monomer addition at the ends of growing tubes. New evidence from fluorescence lifetime microscopy will be presented demonstrating distinct fluorescence lifetime values within amyloid aggregates and nanotubes indicating structural heterogeneity. The possibility to observe structural differences in real time in solution provides new opportunities to follow molecular level details in the mechanisms of nucleation, growth, and structural maturation. We discuss our recent findings of structural heterogeneity along single amyloid nanotubes, evidence for nucleating cores within unstructured peptide aggregates that may represent nucleation events, and the relevance of these observations in biomedicine and nanotechnology.
Francesco Cardarelli (Speaker)
Tracking nuclear pore complexes in live cells: single molecule transport and beyond.
Francesco Cardarelli, Luca Lanzano, Enrico Gratton
Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the nucleus and the cytoplasm. No methods proposed thus far, however, have the capability to measure the transport of single molecules through single pores in intact cells. Hence fundamental questions remain as to how molecules physically perform facilitated transport and which is the nanomechanical basis of their selective gating through the NPC in live cells.
To address these issues we combine fluorescence correlation spectroscopy (FCS) to real-time tracking of the center of mass of single NPCs to study the nanomechanical basis of nucleocytoplasmic transport in live cells. The center of mass tracking allow us to create an “Einstein trap” in which the thermal motion of the entire pore is compensated so that we can observe the shuttling of single molecules in the reference frame of the pore. Using this experimental protocol we are able to demonstrate that the transport of Karyopherin-β1 (Kapβ1) receptor is regulated so as to produce a characteristic narrow correlation time at the NPC, which is the signature of directed motion. Furthermore we show that the back and forth components of Kapβ1 movement within the pore are coupled by an energy-consuming process.
Based on our results, we propose that molecular transport through the intact NPC is powered by a supramolecular spring-and-ratchet mechanism. As far as we know, this is the first time that a selective gating model is proposed on the basis of experimental evidences obtained in live, unperturbed cells.
Anna Celli (Invited talk)
Detection of Calcium gradients in live ex vivo human epidermis using phasor analysis of two-photon excitation fluorescence lifetime imaging (FLIM).
Anna Celli, Susana Sanchez, Enrico Gratton, Theodora Mauro
Ionic gradients regulate the function and the structure of many tissues and organs. However, traditional techniques used to measure ionic concentrations in tissue (such as Proton induced X-ray Emission or ion capture cytochemistry) are often highly invasive (requiring fixation and dehydration of the specimen) and offer poor spatial resolution. In skin, Ca2+ gradients have been shown to regulate epidermal barrier function, homeostasis and repair after perturbation, but a clear understanding of the mechanisms regulating such gradients is still lacking. We applied the phasor analysis approach to two-photon excitation FLIM measurements of Calcium Green 5N to quantify and localize Ca2+ pools in human epidermis. The high spatial resolution offered by this method and the data analysis performed allowed us to measure intracellular average calcium concentrations directly in tissue and detect a previously unknown heterogeneous distribution of Ca2+ concentrations in the proliferative epidermal basal layer. More importantly, this approach revealed that the extracellular space in epidermis is much narrower than previously thought. In fact, most epidermal Ca2+ localizes to intracellular stores, not extracellular space. Moreover, Ca2+ loss from these intracellular stores, not from the extracellular space, directs epidermal barrier repair after experimental abrogation. These new findings suggest that long-held hypotheses addressing Ca2+ control of differentiation and barrier formation require revision.
Ultra-high resolution optical trap with single fluorophore sensitivity.
Matthew J. Comstock1, Taekjip Ha1,2,3 and Yann R. Chemla1,2.
1Department of Physics and Center for the Physics of Living Cells, and 2Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 3Howard Hughes Medical Institute, Urbana, IL
We present a new single-molecule instrument that combines ultra-high resolution optical tweezers with single-fluorophore fluorescence microscopy. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (e.g., single base-pair stepping of DNA translocases) along with the detection of fluorescently labeled protein properties (e.g., internal configuration). The optical tweezers portion of the instrument is based on a timeshared dual optical trap design and is interlaced with a confocal fluorescence microscope. In a demonstration experiment, individual single-fluorophore labeled DNA oligonucleotides can be observed to bind and unbind to complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, coincident angstrom-scale changes in tether extension can be clearly observed. Further, we will report on measurements by the instrument of the activity of the UvrD helicase.
The oligomerization of endophilin B1 and its interaction with BAX.
Yan Chen1, Jinhui Li1, Joachim Mueller1, Barbara Barylko2, Joseph Albanesi2. Physics Department1, University of Minnesota, Minneapolis, MN 55455; Pharmacology Department2, University of Texas Southwestern Medical Center, Dallas, Texas, 75239
Endophilin belongs to a family of proteins that are involved in the membrane vesiculation processes. Endophilin consists two important domains, an amino-terminal N-BAR (Bin-Amphiphysin-Rvs) domain and a carboxy-terminal SH3 (Src-homology 3) domain. The N-BAR domain is required for binding to the lipid bilayer and the subsequent induction of membrane curvature, while the SH3 domain is known for binding to proteins that have a proline-rich motif. Based on sequence similarity, this family of proteins can be categorized into two groups, the Endophilin A and Endophilin B families. Endophilin A is a key player in clathrin-mediated endocytosis and recycling of synaptic vesicles at nerve terminals. Endophilin B, on the other hand, is known to bind to BAX (Bcl-2–associated X protein), and to regulate the membrane dynamics of various intracellular compartments, such as the control of mitochondrial morphology and autophagosome formation in fibroblasts. The oligomerization of endophilin A has been characterized in cells and shown to be a very tight dimer by fluorescence fluctuation spectroscopy. Here, we are interested in examining the oligomerization of endophilin B. To our surprise, endophilin B displays much higher oligomerization than endophilin A. We demonstrate that the oligomerization of endophilin B is not controlled by its self association, but rather by binding to lipid membrane. Furthermore, we examined protein-protein interactions of endophilin B and BAX with dual color heterospecies partition analysis. We found that endophilin B interacts with BAX in solution even in healthy cells, which suggests that endophilin B pre-forms a complex with BAX prior to apoptosis. This work is supported by the National Institutes of Health (R01 GM64589).
Mathias P. Clausen (Poster)
Parallel and fast tracking of quantum dots in plasma membranes.
Mathias P. Clausen, Eva A. Christensen, B. Christoffer Lagerholm. MEMPHYS – Center for Biomembrane Physics, University of Southern Denmark, Campusvej 55, DK-5230, Odense M
We here report a novel method based on tracking of single quantum dots (QDs) that allows for the parallel investigation of up to four different molecular species in the plasma membrane of live cells. By using QDs all excited at the same wavelength, but emitting at well separated colors, we have applied the method to simultaneous image the lateral dynamics at video rate (25 Hz) of three supposed lipid raft markers; a lipid (GM1), a lipid-anchored protein (CD59), and a transmembrane protein (EGFR) in the same region of the plasma membrane under various cell treatments. All membrane species show heterogeneous diffusive behavior with single trajectory diffusion coefficients ranging between 0.00 µm2/sec and 0.3 µm2/sec independent of the cell condition. The mean diffusion coefficient was changed upon cholesterol depletion for EGFR, and CD59, while it was unchanged for GM1.
Using the same wide-field microscopy set-up but a different EMCCD camera, we have also showed that it is possible to simultaneously image up to two different molecular species at an image rate of 1750 Hz with an image integration time of 0.5 msec. Imaging at this speed reveals a hopping-like diffusion behavior where the molecules jumps between areas of 100 nm in diameter. The behavior is observed for both lipids and proteins, and has previously been reported, but by using Ø40 nm gold particles. Further studies are required to fully understand the role that the QD label has in these results, although the QDs have half the diameter of the gold particles.
Robert M. Clegg (Speaker)
Bioassays, FLIM and FRET.
Robert M. Clegg, University of Illinois, Department of Physics, Urbana, IL 61801-3080
This talk will discuss some bioassays are based on FRET and lifetime measurements (FLIM) for measuring intracellular oxidation reduction potentials and the enzymatic activity of a matrix metalloproteinase. Fluorescent protein hybrids have played a central role in the development of these bioassay probes. An overview with examples will also be given of some analysis techniques that can be coupled with FLIM in order to provide rapid and detailed multi-parameter analysis of cellular based measurements.
Christian A. Combs (Speaker)
epiTED: a new tool for improving multi-photon light collection from living tissue.
Christian A. Combs, Aleksandr Smirnov, Jay R. Knutson, Robert S. Balaban
Multi-photon imaging of live animals presents many challenges that require modification of standard microscopes. In particular efficiency of light emission light collection can be improved. This opportunity arises from the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. Given that the field is scanned and that the fluorescence emission is incoherent but comes from one spot at a time it is possible to collect all of the photons possible from each excited spot without the need for ancillary apertures in the emission path. This allows for new detection schemes that efficiently funnel all attainable emission photons to detector pathways. We have previously shown [1] that parabolic mirrors and condensers can be combined to collect the totality of solid angle of emission around the excited spot for tissue blocks, leading to ~8-fold signal gain. To address live imaging modalities we have developed an in vivo Total Emission Detection (epiTED) instrument modified to make non-contact images from living tissue [2]. Simulations suggest that a ~4-6 fold enhancement is possible with this approach depending on objective characteristics, imaging depth and the optical properties of the sample being imaged. We show that two fold improvements in emission collection were from mouse brain and skeletal muscle as well as the rat kidney using a variety of fluorophores with no compromise of spatial resolution. These results show that epiTED emission detection can maintain image SNR at greater than two times the scan rate or at more than a 30% reduction in laser excitation power to minimize photo-damage. These results will be discussed in relation to other advances in in vivo multi-photon imaging.
[1] Combs, C. A., et al. (2007). J Microsc 228, 330-337.
[2] Combs et al. (2011). J. Microsc. 241, 153-161.
Probing the properties of supramolecular assemblies in live cells with single-molecule imaging and manipulation.
Patrice Dionne1, Marianne Renner2, Jean-Baptiste Masson3, Maxime Dahan1. 1Physics Departement and Institute of Biology, 2Ecole normale supérieure, Paris France; 3Institute of Biology, Ecole normale supérieure, Paris France; Institut Pasteur, Paris France
The organization and functions of live cells is largely controlled by the regulated properties of supramolecular assemblies. Single molecule techniques now make it possible to quantitatively study their composition, structure and dynamics in their cellular environment. Thereby, these techniques offer fresh insight on the mechanisms that underlie the dynamic architecture of live cells. Here I will first present recent results on the membrane organization of membrane receptors and their associated scaffolding clusters. In live neurons, we implemented a high-density tracking assay of transmembrane proteins, susceptible to interact or not with anchoring gephyrin proteins. With novel analytical methods, we were able to extract a large-scale energy and diffusivity landscape governing the lateral dynamics of the membrane proteins. By doing so, we get novel insight on the binding energies of gephyrin clusters.
When further combining these measurements with single molecule stochiometric analysis and 3D super-resolution images, we obtain structural information suggesting that gephyrin clusters are organized as two-dimensional submembrane assemblies.
Novel fluorescence properties of the anti-cancer drug Combretastatin A4 Disodium Phosphate.
Varsha Daswani, Berenice Venegas, and Parkson Chong. Temple University, Department of Biochemistry, Philadelphia, PA
With this research, we aim to gain a deeper understanding of the chemical and physical properties of the antivascular drug combretastatin A4 disodium phosphate (CA4P) in order to improve its clinical efficacy. Vascular-disrupting agents (VDAs) damage tumor microvasculature and cause a swift extensive shutdown of tumor blood flow producing the widespread death of cancerous cells. Due to its efficacy at one tenth of its maximum tolerated dosage, CA4P is considered the most prominent form of VDA. CA4P is the water soluble prodrug of combretastatin A4 (CA4). After administration, CA4P is rapidly cleaved to active CA4 by endogenous non-specific phosphatases. CA4P is a tubulin depolymerizing agent which functions by disrupting the formation of microtubules essential for cell motility, protein trafficking and mitosis. The antivascular effects of CA4P and CA4 have been demonstrated both in animal models and human cancer patients. Administration of CA4P alone induces extensive necrosis in a wide variety of preclinical cancer models, and when used in conjunction with conventional chemotherapy and radiotherapy has proven effective against a broad range of tumor tissue.
Here we report a novel fluorescent property of CA4P, which may lead to a higher therapeutic efficacy. We have found that once altered, the change in the fluorescence behavior of CA4P is irreversible with all concentrations. And small but significant differences in the excitation, emission, and absorption spectra ensue. We have also found distinction between the effects of the two forms of the drug on killing human breast cancerous cells (MCF-7). We hypothesize that we have discovered the induction of a conformational change in CA4P and this phenomenon may also be mimicked in CA4 and its derivates in clinical trial such as AVE8062 (avantis). The molecular behavior behind the production of this new form of CA4P remains unknown and all previous and current usage of the drug is confined to the unaltered form. The outcome of this study, may lead to an increase in the anti-microtubule activity of CA4P, thereby making it more effective against cancerous cells. With this work we aim to elucidate the mechanism behind the alteration in intrinsic fluorescence of CA4P, and describe a model for the phenomenon in other vascular disrupting agents.
Three-Dimensional flow manipulation in a microfluidic device for single-molecule trapping and characterization using four-beam fluorescence cross-correlation.
Lloyd M. Davis, Brian K. Canfield, Jason K. King, James A. Germann, William N. Robinson; Center for Laser Applications, University of Tennessee Space Institute, Tullahoma, TN 37388
A single fluorescent molecule in solution may be readily detected in a confocal microscope, but Brownian diffusion restricts the residence time of the molecule within the probe volume and hence limits the maximum observation time. However, an Anti-Brownian ELectrokinetic (ABEL) trap provides a means to counteract diffusion of a single molecule in solution for prolonged observations of its dynamics and photophysics. Cohen et al. have demonstrated trapping in two dimensions with a device in which a sub-micron layer of solution is held between two fused silica substrates and motion of the molecule in the plane of the device is electrokinetically controlled by adjusting the voltages at four electrodes. Also, we have demonstrated trapping in one dimension (i.e., a 1-D ABEL trap) using a fluidic nanochannel with two electrodes to regulate motion along the channel. In either case, the trapped molecule undergoes frequent collisions with the walls and is subject to sticking to the interfaces. To address these limitations, here we characterize a microfluidic device for electrokinetically controlling the motion of a single molecule in three dimensions, so that interfacial collisions may be avoided. The device consists of two vertically offset, crossed microchannels. Three-dimensional adjustment of the electrokinetically-driven flow velocity near the center of the crossed region of the channels can be made by tuning voltages applied to terminals at the four ends of the channels providing a possible method to counteract Brownian diffusion for trapping of a single fluorescent molecule in three dimensions. In related work, we are developing a method for simultaneously measuring the x, y, and z components of the solution flow velocity using fluorescence cross-correlation spectroscopy (FCCS), which may be used to characterize the flow in the crossed region for different voltage settings. To this end, we have set up a four-channel FCCS experiment that uses two-photon excitation, pulse-interleaved excitation with four foci arranged in a tetrahedral pattern, and time-gated photon counting. We have developed Monte Carlo simulations to study the effectiveness of globally fitting the set of 4 × 4 correlation functions to determine the three velocity components while accounting for temporal cross-talk between the four channels and other limitations in the experimental hardware.
Isabelle Deschênes (Invited talk)
A novel strategy to study disease causing cardiac sodium channels.
Isabelle Deschênes PhD, MetroHealth Campus, Case Western Reserve University, Cleveland, OH
The voltage-gated cardiac sodium channel is one of the fundamental molecules that underlie the electrical function of the heart, being responsible for the initiation of the cardiac action potential. Mutations in the cardiac sodium channel gene have been linked to multiple types of inherited arrhythmias such as Long QT and Brugada Syndromes. To further our understanding of cardiac sodium channels and the pathophysiological mechanisms of diseases of excitability, we have developed a strategy to study the dynamic 3D structure of these channels by creating a sodium channel construct containing a pair of fluorescent proteins to be used for FRET. This approach is used to study folding defects seen in Brugada Syndrome sodium channel mutations. In Brugada Syndrome, mutations in the sodium channel result in a reduction in sodium currents, mainly due to trafficking defects. A common cause of trafficking deficient channels is misfolding and retention by the quality control machinery in the ER. FRET was used to examine folding of wild-type and trafficking-deficient mutant sodium channels. This novel strategy allows us to “visualize” the folding state of these sodium channels.
Michelle Digman (Speaker)
Fluorescence correlation spectroscopy in 3D and in live tissue.
Michelle A. Digman1, Jose S. Aguilar3, Enrico Gratton1, Atsushi Suetsugu2,3 and Robert M. Hoffman2,3. 1Department of Biological Engineering, University of California Irvine, CA; 2AntiCancer Inc., San Diego, CA; 3Department of Surgery, University of California San Diego, CA
Recent developments in fluctuation correlation spectroscopy using laser based imaging have had a large impact in obtaining quantitative spatio-temporal information of protein dynamics at the level of single cells. The study of cells in tissues requires additional methods to penetrate in tissues and for compensation of rhythmic movements in live animals. The spatiotemporal fluctuation approach is in principle non-invasive. It can capture complex biological functions in real time, give insights into microscopic single cellular states and if done in whole organisms, maintain crucial biological network intact without altering cellular fate. We have developed new nano-imaging method which is based on steering a two-photon laser beam around cellular protrusions to look at protein dynamics in cells during extravasation. We are using a mouse model in which we inject cancer cells in the venous blood stream. The cancer cells (MB MDA231) have a cytoplasmic (Paxillin-EGFP) and/or a nuclear marker (H2B-mCherry). We use a skin flap model in which we can visualize blood vessels and recognize the position of the cells. A feedback mechanism controls the center of the laser orbit to be at the center of mass of the fluorescence distribution with nanometer precision, a crucial step to compensate for motion in the living animal. A program reconstructs in 3D the shape of the protrusion. During the orbit, the second channel of the microscope measures the mCherry signal. We plan to correlate the appearance of bright fluorescence spots on the protrusion surface with the points of contact of the protrusion with the surrounding matrix. In a 3D model system we have detected the diffusion of paxillin and binding and unbinding in real time and space as the cells attach to the blood vessel wall and migrate into the outside tissue. In the animal model the H2B reporter allows us to follow the position and shape of the cell nucleus. Our goal is to determine if at the end of the long protrusion both in 3D model systems and in the live animal we can observe the formation of focal adhesions and to see how the dynamics of focal adhesion turnover rate is orchestrated in the microenviroment of the mouse model. Preliminary results in the live animal show that when cells start to adhere to the blood vessel wall they form focal adhesions as determined by the characteristic elongated features observed in the paxillin-EGFP channel. Using this nano-imaging method will enable studying biological process related to diseases at the macro-, micro and nano scales.
Bioelectromechanics of engineered heart tissues.
Traci Abney1, Tetsuro Wakatsuki2, Guy Genin1, and Elliot Elson3.
1Department of Mechanical, Structural, and Aerospace Engineering, Washington University, St. Louis, MO; 2Department of Physiology and Biotechnology and Biotechnology Center, Medical College of Wisconsin, Milwaukee, WI; 3Department of Biochemistry and Molecular Biophysics, Washington University , St. Louis, MO
Heart muscle contains not only muscle cells (cardiomyocytes, CMs) but also a comparable number of much smaller fibroblasts the primary function of which under normal conditions is to maintain the tissue’s extracellular matrix. In response to hypertension these fibroblasts can convert to myofibroblasts (MFBs). This phenotype, which plays an important role in wound healing, has properties of both normal fibroblasts and smooth muscle cells. MFBs continue to secrete collagen but also are larger and have increased contractility. The collagen production and increased contractility of the mFBs stiffens the heart muscle, impairing its ability to relax and leading to a “dystolic dysfunction” that can lead to heart failure and death. Moreover, MFBs can interrupt the orderly spread of electrical excitation that is required for normal heart contraction and can lead to reentrant arrhythmias. Also, in response to a myocardial infarction the MFBs replace dead CMs, generating an infarct scar that helps to maintain the integrity of the heart wall but that also impairs the electromechanical function of the muscle. It is therefore important to understand how MFBs affect heart muscle function.
Engineered heart tissues (EHTs) that model important aspects of heart muscle function provide a promising approach to this subject. EHTs can be assembled from embryonic CMs (and also embryonic stem cells) and MFBs embedded in a collagen matrix (Eschenhagen, Fink et al. 1997). The cells form a connected network of myofibrils that undergo periodic twitch contraction. Using methods that we have developed to measure the mechanical properties and twitch contractile force of EHTs, we have shown that they display characteristic features of heart muscle (Asnes, Marquez et al. 2006). Our current aim is to develop an experimental system in which we can construct EHTs with specified proportions of CMs and MFBs either randomly intermixed or with the MFBs organized into defined patterns, e.g., MFB “islands” that mimic infarct scars. We will then test the effects of the MFBs on the electromechanical function of the EHTs via measurements both of the spread of excitation along the EHT using potential sensitive fluorescent dyes and of the stiffness and the twitch contractions of the EHTs. We have also developed computational models that include a Hodgkin-Huxley-type account of excitation, excitation-contraction coupling, and the mechanical properties of the cells, matrix and the tissue overall.
REF: Asnes, C. F., J. P. Marquez, et al. (2006). "Reconstitution of the Frank-Starling mechanism in engineered heart tissues." Biophys J 91(5): 1800-10.
Eschenhagen, T., C. Fink, et al. (1997). "Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system." Faseb J 11(8): 683-94.
Yves Engelborghs (Speaker)
Protein diffusion and interactions in vitro and in the living cell as studied by fluorescence correlation spectroscopy.
Yves Engelborghs, University of Leuven, Biomolecular Dynamics, Celestijnenlaan 200 G, box 2403, B3001 Leuven, Belgium
The purpose of this study is to understand the diffusion behaviour of proteins in the living cell. The technique used is Fluorescence Correlation Spectroscopy (FCS) applied to GFP-fusion proteins. A number of potential FCS artefacts in living cells have been identified (1). It will be described here how these can be minimized. We have applied FCS to study the diffusion of several proteins in the living cell and compared this with diffusion in lysates. In almost all cases the diffusion of GFP in the cell can be described as simple single component diffusion. For most protein-GFP fusion products the behaviour in the cell is more complex. This is the case for the PPAR receptor and biliverdine reductase (2), HIV-integrase and LEDGF (3). The diffusion can most often be described, equally well by two components as by anomalous diffusion. The application of site directed mutagenesis allows to interfere with the specific or non-specific interactions of the fusion proteins. Generally there seems to be a correlation between the anomaly parameter and the size of the protein.
1. Enderlein, J., Gregor, I., Patra, D., and Fitter, J. (2004) Curr. Pharm. Biotechnol. 5, 155-161.
2. Tudor, C., Feige, J.N. et al. (2007) J.B.C. 282, 4417-4426.
3. Hendrix J, Gijsbers R, De Rijck J, Voet A, Hotta J, McNeely M, Hofkens J, Debyser Z, Engelborghs Y. (2011) Nucleic Acids Res. 39, 1310-25
Laura C. Estrada (Speaker)
The gold enhancement nano-imaging method: a 3D super-resolution technique for imaging of filamentous actin by directed motion of gold nanoparticles.
Laura Estrada and Enrico Gratton, Laboratory for Fluorescence Dynamics, University of California, Irvine, CA
More than 100 years ago, Ernst Abbe established that diffraction of light prevents optical-based microscopes from having spatial resolution beyond a value comparable to the illumination wavelength. However, since the mid-90s, several super-resolution techniques have emerged. Stimulated Emission Depletion (STED), Saturated Structured-Illumination Microscopy (SSIM), Photo-Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) are some of the imaging techniques now available. In this contribution, I will present a new optical super-resolution method based on a nanoparticle (NP) tracking/pushing principle. Although the image of a single NP is diffraction-limited, the precision of determining its position scales with the inverse of the square root of the number of collected photons. Using the gold enhancement nano-imaging (GENI) method, we can push single gold NPs along biological fibers by using a two-photon microscope and the localized surface plasmon at the NP. Gold NPs binds to collagen and actin but it can gently slide over the fiber without detaching. While the particle is sliding, we determine its 3D location with nanometer precision by a 3D tracking algorithm. The recorded trace follows the 3D structure of the underlying fiber without the need of staining. In this talk, I will show the latest examples that illustrate the capabilities of the GENI method.
New methods for FLIM and FCS for confocal laser scanning microscopy.
S. Fore (*), B. Krämer, F. Koberling, M. König, V. Buschmann, M. Wahl, S. Orthaus, U. Ortmann, R. Erdmann. (*) PicoQuant Photonics North America Inc., 61 Union Street, Westfield, MA 01085; PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany, info@picoquant.com
Confocal laser scanning microscopes (CLSM) are an essential tool in biological and biomedical research. Their functionality can be further enhanced by adding sensitive time-resolved data acquisition capabilities, enabling Fluorescence Lifetime Imaging (FLIM), lifetime based Förster Resonance Energy Transfer (FRET) and Fluorescence (Lifetime) Correlation Spectroscopy (F(L)CS) down to the single molecule level. By using the non-descanned (NDD) internal detectors of the laser scanning microscope, also NDD FLIM acquisition becomes easy and powerful. Based on a recently added network interface, the FLIM and FCS data acquisition can now be directly accessed from the CLSM computer. This unique integration enables a seamless work flow.
Förster Resonance Energy Transfer (FRET) studies provide a very powerful tool for a broad range of biological applications since this technique enables to measure intra- and intermolecular distances down to several nanometres. Other than intensity-based FRET measurements, FLIM can further reveal sub-populations; thus, allowing to determine the fraction of free donors compared to associated donor molecules within a complex. The result of such an analysis yields not only the FRET efficiency distribution of FLIM-FRET images, but also the fraction and distribution of complete to incomplete FRET complexes. In addition, so-called lifetime sensitive sensors allow the monitoring of environmental conditions such as pH and ion concentration.
Fluorescence lifetime imaging for cell biology, drug discovery and label-free diagnosis.
Paul French, Imperial College London, UK
This talk will describe fluorescence lifetime imaging and metrology applied across the scales in single point measurements, micrcoscopes ranging from STED-based super resolution to automated multiwell plate readers, endoscopes and tomographic imaging systems. Our aim is to translate assays of biomolecular interactions from solution phase and cell cultures to in vivo physiological contexts in disease models (murine and zebrafish). We are also applying FLIM to label-free diagnosis using autofluorescence to readout changes in metabolites and tissue matrix components and aim to correlate this with, e.g. FLIM FRET readouts of signalling networks in disease models.
Analysis of the uptake and localization of Botulinum neurotoxin serotype A in neuronal cells using raster imaging correlation spectroscopy.
Patton E. Garay1, Justin A. Ross2, Dudley J. Williams1, Lance E. Steward1, Roger Aoki1, and Ester Fernández-Salas1 , 1Department of Biological Sciences, Allergan Inc., Irvine, CA; 2Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawai`i at Manoa University of Hawaii, Honolulu, HI
Botulinum neurotoxin serotype A (BoNT/A) is an effective therapeutic for a variety of muscle hyperactivity disorders, hyperhidrosis, and pain. BoNTs are 150 kDa proteins consisting of three 50 kDa domains: a receptor binding domain (HC), a translocation domain (HN), and a metallo-protease domain (light chain, LC). Once inside the cell, the LC is translocated to the cytoplasm and cleaves one of three SNARE proteins depending on the neurotoxin’s serotype (A-G). The LCs of serotypes A and E cleave SNAP25 at a different amino acid sequences, however, serotype A has a longer duration of action than E. Previously, we reported that a chimera of GFP with LC/A (GFP-LCA) localizes to the membrane while GFP-LCE localizes to the cytoplasm (Fernández-Salas et al. PNAS 2004). To complement these studies, Raster Image Correlation Spectroscopy (RICS) was used to determine the diffusion rates of GFP (23 µm2/min), GFP-LC/A (0.1 µm2/min) and GFP-LCE (6 µm2/min). This data supports earlier evidence that the long duration of serotype A maybe due to the association of the LC/A to a protein complex in the plasma membrane that protects it from proteolytic degradation.
In order to gain entry into a neuron, the HC binds a cellular receptor complex composed of proteins and gangliosides. Members of the synaptic vesicle glycoprotein family (SV2) have been identified as cellular receptors for BoNT/A. They are reported to mediate BoNT/A uptake by facilitating toxin binding and internalization during vesicle recycling. Previously, we indentified Fibroblast Growth Factor Receptor Type 3 (FGFR3) as a high affinity component of the receptor complex for BoNT/A on neuronal cells. The interaction between FGFR3 and recombinant HC/A (rHC/A) was analyzed using Cross Correlation RICS (ccRICS). Differentiated PC-12 and SH-SY5Y cells expressing a fluorescent FGFR3-Halo tag chimera protein were treated with rHC/A labeled with Alexa-Fluor 633, and imaged at 3, 6, 9, and 24 hours post treatment. At 3 hours post treatment, the labeled rHC/A was present mainly at the cellular periphery and localized to structures containing the ganglioside GT1b. After 6 hours, rHC/A and FGFR3 colocalized in PC-12 cells. The ccRICS analysis demonstrated that these proteins traffic together in PC-12 and SH-SY5Y cells after 6 hours and 9 hours treatment, respectively. Furthermore, the spatial correlation data also indicate that these fluorescent proteins are moving directionally in the cell and are not freely diffusing. These data support a model where FGFR3 is part of a high-affinity receptor complex for BoNT/A on the cell surface and suggest that the FGFR3-BoNT/A interaction may play a role in trafficking the toxin through neuronal cells.
Following single molecules in cells and tissues by correlation spectroscopy.
Enrico Gratton, Laboratory for Fluorescence Dynamics, University of California, Irvine, CA
Methods in Fluorescence Correlation Spectroscopy (FCS) allow us to detect and characterize the behavior of single molecules directly in cells and in tissue. However the “tracking” of single molecules is a much more difficult operation. Single particle tracking (SPT) in 2D and in 3D is a well-established method in biophysics. The application of SPT requires bright and isolated particles so that the trace of the particle can be recognize in successive image frames. By combining the sensitivity of FCS to single molecules and methods for spatial cross-correlations we show that we can produce a characteristic signature through an analysis method that allow us to determine specific paths that molecules follow in complex environments such as the cell interior and specifically the nucleus. In this talk we present the statistical basis of the pair correlation function approach and the application of this method to the flow of molecules in the nucleus of cells. We show that the chromatin structure has peculiar properties in directing the motion of molecules along the chromatin structure but at the same time regulating the crossing of molecules in and out of the close chromatin environment. This regulation produces regions of connected flow which could have a role in regulating gene expression.
Further experience with the pair correlation approach is leading us to the study of molecular flow between cells and flow in the extracellular matrix. The development of time and space cross-correlation methods is opening new possibilities and applications for fluctuation spectroscopy in cells.
Enrico Gratton (Sponsor talk)
The SimFCS program for data acquisition and analysis.
Enrico Gratton, Laboratory for Fluorescence Dynamics, University of California, Irvine, CA
SimFCS is a comprehensive platform for data fluorescence data analysis, simulation and data acquisition. SimFCS, contains drivers for data acquisition, algorithms for data reduction and methods for data display and export. Different technologies work together to provide new kinds of information, which is not available using each one independently. The challenge is both technical: combine technologies to achieve new functions; and scientific: plan experiments that could make use of the combined new capabilities. By designing a software platform that is agile and can access information from different modalities of data acquisition, we can join different conceptual measurements in the same platform. For example, while performing tracking experiments, we can also acquire and analyze the data for the diffusion of molecules in the proximity of the particle being tracked, or detect binding. As another example, since data are collected and analyzed by the same software, we can rapidly manipulate the data set to appear as a “RICS” experiment and then analyze and fit the data for models of diffusion. This conceptual integration allows us to combine experimental modalities in a way that would be impossible if we have independent programs for each technology. Another example is the collection of data for the FLIM/Phasor analysis that can, at the same time, be used for the RICS and N&B analysis. Another example is to perform FLIM analysis while acquiring data for the pair correlation fluctuation experiment. The result of this integration that is achieved by the SimFCS program is a powerful new microscope software tool that can reveal molecular interactions at the basis of such fundamental processes as cell signaling, cancer metastasis and stem cell differentiation.
Zygmunt (Karol) Gryczynski (Invited talk)
Novel probes for ultrasensitive diagnostics.
Tadeusz W. Zerda, Badri P. Maliwal, Rafal Fudala, Sangram Rout, Ignacy Gryczynski, Eric Simanek, Julian Borejdo, Ryan Rich, Irina Akopowa, and Zygmunt Gryczynski
In this presentation we will discuss emerging applications of new diamond fluorescent nanoparticles (NDs). Fluorescent nanodiamonds (NDs) are new emitting nanoparticles with point defects embedded in the diamond crystal lattice resulting in exceptional luminescent properties - perfect almost 100% quantum yield and infinite photostability. We demonstrate that diamond particle (can be as small as 4-5 nm in diameter) can be decorated with many (10-100) organic dyes suitable as energy donors to nitrogen-vacancy pair and may easily exceed extinction coefficient of 1,000,000. In addition diamond material is extremely heard and has one of the highest refractive index (2.32) what enable novel nanophotonics and plasmonics applications. We present novel applications of these particles for molecular binding (like DNA hybridization) within the evanescent field.
Probing cellular protein complexes using single-molecule pull-down.
Ankur Jain, Ruijie Liu, Biswarathan Ramani, Edwin Arauz, Yuji Ishitsuka, Kaushik Ragunathan, Jeehae Park, Jie Chen, Yang K. Xiang & Taekjip Ha
Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.
Peeking and poking at GTPase circuits in living cells.
Klaus M. Hahn, Thurman Distinguished Professor of Pharmacology, Departments of Pharmacology and Medicinal Chemistry, University of North Carolina, Chapel Hill
This talk will provide an overview of new approaches to study and manipulate signaling in living cells and animals. Topics will include genetically encoded proteins activated or inactivated by light, multiplexed imaging of biosensors using red shifted fluorescent proteins, and novel methods to confer drug sensitivity on kinases. These tools are being used to understand the dynamics of GTPase/adhesion signaling in cell migration, specifically the role of RhoG and Src family kinases in cell polarization.
Alejandro P. Heuck (Invited talk)
Insights into the membrane-interaction and assembly of the Pseudomonas aeruginosa Type III secretion translocators PopD and PopB.
Fabian B. Romano, Kyle C. Rossi, Alejandro P. Heuck.
Pathogenic Gram-negative bacteria can secrete and deliver proteins directly into the cytoplasm of an adjacent eukaryotic host cell. A conserved and efficient delivery mechanism, called the type III secretion (T3S) system, acts as a syringe that injects these protein effectors across the eukaryotic plasma membrane. In Pseudomonas aeruginosa PopB and PopD are essential proteins required for effectors translocation. Substantial genetic and biochemical data support a model with PopB and PopD inserting into the target membrane and forming an oligomeric translocon pore, however virtually nothing is known about the structure of this transmembrane complex.
Our primary goal is to determine the arrangement of PopD in membrane assembled translocons. How do we established an efficient in vitro system to study the T3S translocon? What factors govern the interaction and the insertion of the translocators into membranes? Does PopB influence the PopD/membrane interaction? Using membrane binding assays, site-directed fluorescence labeling, and multiple fluorescence approaches we have analyzed the interaction of PopD with model membranes and the pore formation activity of the translocator in the presence and absence of PopB.
Despite that both proteins can individually form pores in membranes, we found that PopD oligomerizes with PopB and forms pores with an inner diameter of approximately 30Å. Our data support a model that consists in the binding of the denatured translocators to the membrane, oligomerization, and formation of discrete transmembrane pores.
Alejandro P. Heuck (Poster)
Determining cholesterol levels in cell membranes using Perfringolysin O derivatives.
Benjamin B. Johnson1, Paul Moe1, Juan Anguita2, Bernardo Trigatti3, and Alejandro P. Heuck1. 1Dept. Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003, USA. 2Dept. Veterinary and Animal Sciences, University of Massachusetts, Amherst. 3Dept. Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada
Cholesterol is an essential component of mammalian membranes and important for regulating the structure and function of cell membranes. Changes in cholesterol content affect the formation of arterial plaques, virus entry to cells, receptor organization, and many other physiological and pathological events. Measuring cholesterol trafficking and distribution is essential to understand how cells regulate cholesterol.
Perfringolysin O (PFO) is a cytolysin secreted by Clostridium perfringens that requires cholesterol in the target cell membrane for binding. Once bound to the membrane PFO oligomerizes into ring-shaped pre-pore complexes, that ultimately insert and form large transmembrane β-barrels. The specificity of PFO for high concentrations of cholesterol makes the toxin a potential tool for the detection of cholesterol distribution and trafficking.
To better understand the distribution of cholesterol in cell membranes under different physiological conditions we have endeavored to adapt PFO into a molecular probe capable of binding differentially to membranes with high, and low levels of cholesterol. To this end we have taken a non-lytic derivative of PFO and introduced several point mutations in the membrane-interacting domain. These mutations altered the threshold for the amount of cholesterol required in the membrane to trigger binding.
The cholesterol-dependent binding of each PFO derivative was tested on liposomal membranes containing different amounts of cholesterol. Three derivatives that showed a different cholesterol-binding threshold were selected and labeled with a fluorescent probe. The binding of the PFO probes to macrophage plasma membranes was evaluated using flow cytometry and fluorescence microscopy. The amount of cholesterol on macrophage membranes was modified with b-methyl-cyclodextrin/cholesterol mixtures to evaluate the response of the fluorescent probes to the cholesterol content of the plasma membrane. Our data showed that the generated PFO derivatives differentially respond to the total cholesterol content in macrophage membranes. Interestingly, the binding analysis suggested that the maximum amount of “free” cholesterol tolerated by macrophages in the plasma membrane is lower than the one present in liposomes with POPC/cholesterol 6:4 ratio.
Biosensor FRET detection by the phasor approach to fluorescence lifetime imaging microscopy (FLIM).
Elizabeth Hinde, Michelle A. Digman, Christopher Welch, Klaus Hahn, and Enrico Gratton. Laboratory for Fluorescence Dynamics, University of California, Irvine, CA
FRET based biosensors report on the concentration of a ligand by switching between a high FRET (HF) and a low FRET (LF) state. In principle this change of state can be quantified using either an intensity based ratiometric method of analysis or by monitoring the change in donor lifetime. In practice this measurement is difficult because changes in the concentration of the LF and HF states are often small, and a significant portion of the biosensors present are not optically active due to photobleaching or protein synthesis malfunction. Furthermore in every pixel of a FRET image we have a different fractional contribution of fluorescence not derived from the biosensor (i.e. cell auto-fluorescence) as well instrumental fluorescence background. Clearly determination of the concentrations of the multiple fluorescent components at any one pixel in a FRET image cannot be accomplished using only two emission wavelengths. It is however possible to determine the presence of several fluorescence lifetime components by measuring the fluorescence decay at a single pixel. We present here the phasor approach to fluorescence lifetime imaging (FLIM) as a method for quantitative determination of a biosensor FRET signal. We demonstrate this capacity on a series of dual and single chain GTPase biosensors, performing concomitant normalized FRET ratio analysis on the acquired FLIM data by the generalized polarization (GP) approach. By testing biosensors constructed from different designs, we place different demands on the two detection methods tested and elucidate subtle differences in the ability of each to quantitatively image the concentration of the ligand as a function of time and space.
What cell shape oscillations can tell us about cortical Actin-microtubule interactions and amoeboid migration.
Ken Jacobson. Univ. North Carolina, Dept. Cell and Development Biology, Chapel Hill, NC
Many cellular responses to environmental stimuli involve large-scale changes in cell morphology. For example signaling molecules, such as hormones or growth factors, can induce cell differentiation, proliferation, or migration. These global changes in cell shape are highly coordinated and require dynamic regulation of the actin cytoskeleton. Therefore understanding how the actin and microtubule cytoskeleton and associated regulatory proteins function as an integrated system is a central challenge for cell biology. The morphological oscillations that occur during cell spreading are ideally suited for performing a systems-level investigation into the biochemical and biomechanical mechanisms that drive changes in cell shape. In the broader context, these oscillations constitute a mechanochemical prototype of how signaling networks regulate cytoskeletally driven mechanical behavior that in turn feeds back to modulate the signaling network. Importantly, fluorescently labeled cytoskeletal proteins and fluorescent biosensors allow dynamic structural features of the actin-based cortex and spatiotemporal activity of signaling molecules and visualized. I will discuss what we know about the dynamic structural changes in the actin cortex during oscillations, how Rho proteins regulate these oscillations, how this model may be related to amoeboid migration and emerging theoretical models for this phenotype.
Nicholas James (Invited talk)
Application of the phasor method to in vitro studies.
Nicholas James
The phasor method is a graphical, model-less approach in which the raw data from lifetime measurements (either time or frequency domain) can be converted to a single point on an x-y plot. Although originally developed in 1984 for the graphical demonstration of differences in the frequency domain parameters τmod and τphase, and applied soon after that for background subtraction in the frequency domain, this method has gained in popularity in lifetime studies during the last decade. Specifically, the phasor approach has been increasingly applied to fluorescence lifetime imaging microscopy (FLIM) to provide instantaneous visualization of lifetime properties in cells. FRET studies in vivo have been particularly amenable to the phasor approach. The phasor approach has also recently been shown to be useful for analyzing in vitro systems. A particularly interesting application is the analysis of protein systems in which the complex decay of intrinsic tryptophan fluorescence is monitored. In this talk, we will provide a general overview of the theory of phasor analysis and its application to diverse in vitro systems including protein unfolding, quenching studies and multiple fluorophore systems.
David M. Jameson (Speaker)
Misregulation of Dynamin 2 associated with centronuclear myopathy.
David M. Jameson, University of Hawaii, Cell & Molecular Biology, Honolulu, HI
Dynamin 2 is a ubiquitously expressed GTPase of approximately 100 kDa that polymerizes around the necks of vesiculating membranes, thereby promoting their constriction and scission. Numerous mutations have been generated in Dynamin 2 that impair its ability to self-assemble or to bind and/or hydrolyze GTP. Expression of these mutants in cells generally results in severe inhibition of endocytosis. We have expressed, purified, and characterized Dynamin 2 mutants associated with Centronuclear Myopathy (CNM) and Charcot-Marie-Tooth (CMT) diseases. The GTPase activities of the CNM mutants are as high, or higher, than that of wild-type Dynamin 2. The CNM mutants, however, form polymers in vitro that are more resistant to disassembly by salt or GTP than wild-type. This defect may have functional implications, because GTPase-coupled disassembly of dynamin polymers appears to be necessary not only for recycling of dynamin subunits to new sites of membrane budding, but also for completion of the membrane scission process. We have studied the assembly properties of these Dynamin 2 mutants both in vitro and in living cells, using spectroscopic methods including FCS, and shall describe some of these studies in this talk.
David M. Jameson (Opening talk)
Fundamental contributions of Gregorio Weber to fluorescence spectroscopy and protein research.
David M. Jameson, University of Hawaii, Cell & Molecular Biology, Honolulu, HI
Gregorio Weber is acknowledged to be the person responsible for many of the more important theoretical and experimental developments in modern fluorescence spectroscopy. In addition to these achievements he also made fundamental contributions to protein research. This lecture will describe and discuss many of his fundamental discoveries and insights in both fluorescence spectroscopy and protein chemistry. In addition to these seminal contributions, Gregorio Weber also trained and inspired generations of spectroscopists and biophysicists who went on to make important contributions to their fields, including both basic research as well as the commercialization of fluorescence methodologies and their extension into the clinical and biomedical disciplines. In addition to outlining Gregorio Weber’s scientific career and contributions I will also endeavor to illuminate his outstanding personal characteristics.
Fluorescent binding studies of Phosphofructokinase from Bacillus stearothermophilus using a tryptophan-shifted mutant.
Amy M. Knutson, Gregory D. Reinhart. Texas A&M Univ., College Station, TX
Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is an allosteric, homotetrameric enzyme containing one tryptophan per subunit. Unlike the homologous PFK from E. coli (EcPFK), the fluorescence of the native tryptophan is unresponsive to ligand binding. This study utilizes a tryptophan-shifted mutant, in which W179 has been mutated to phenylalanine, and F240 has been mutated to tryptophan. The variant is functionally similar to wild-type, however a decrease in the fluorescence of ~6.5% is associated with substrate, fructose 6-phosphate (Fru-6-P), binding and a decrease of ~16% is associated with inhibitor, phosphoenol pyruvate (PEP), binding. Dissociation constants of 1.9±0.3μM for Fru-6-P and 107±13μM for PEP were thereby determined. This dissociation constant for PEP is in good agreement with that determined by kinetic assays (128±5μM). Due to the absence of known ATP antagonism, the dissociation constant for Fru-6-P in the absence of MgATP is lower than with steady-state kinetic assays determined at saturating MgATP (36±1μM). The coupling between PEP and Fru-6-P has also been determined and increases with temperature as observed with steady-state kinetic assays using wild-type BsPFK. Like wild-type PFK, the coupling free energy results from compensating enthalpy and entropy components. The sign of the coupling free energy is opposite that of the enthalpy and is therefore determined by the larger absolute value of the entropy term. This is opposite the thermodynamic basis of the allosteric response in EcPFK, where the sign is established by the enthalpy component. The functional and thermodynamic similarity of the mutant enzyme to wild-type, together with the added ability to follow ligand binding with fluorescence, make it a good candidate for further studies probing a deeper understanding of the thermodynamics involved in PFK allosterism. Funding provided by the following: NIH-GM33261, NIH-CBI, and the Welch Foundation.
Jay R. Knutson (Invited talk)
Routes to multicolor, low-power nanoscopy.
Tilman Rosales1, Gary L. Griffiths2 and Jay R. Knutson1,
1NHLBI Laboratory of Molecular Biophysics and the 2NHLBI IIPDC, National Institutes of Health, Bethesda, Maryland 20892-1412
The recent advent of PALM(and varietals) and STED nanoscopy have opened the door to fluorescence imaging at the nanoscale within living cells. The former technique, much like the pontillist style of painting, requires long times (minutes) to develop enough detail, while the latter has video nanoscopy potential but subjects the living tissue to large (and damaging) power levels. The latter problem is due to the reduced cross section for stimulating emission found on the red tail of fluorophores. We have therefore developed synthetic schemes to increase the deactivation cross section an order of magnitude or more.
To test this strategy, we have constructed a STED nanoscope based on picosecond Ti: Sapphire and PPCTA (MIRA/MIRA-OPO) source lasers. A portion of the Ti:Sapphire 783nm pump beam is diverted and formed into a “donut” profile deactivation beam with a spiral staircase phase mask (center wavelength: 796nm) from RPI photonics. Excitation is at 542nm (doubled OPO output). Scanning and remanent emission collection is accomplished with an ISS Alba driving a MadCity piezo stage on a Zeiss Axiovert microscope body. The temporal coincidence of the excitation and deactivation beams is tested via cross-correlation mixing.
The first such dye (a Rhodamine 6G-like dye, amine-reactive version) has been conjugated to sub-resolution (20nm) beads and we have preliminary evidence of its efficacy: an apparent FWHM of 53nm using only 50mW of deactivating power. Fluorescein-like dyes and those with even higher cross sections are being synthesized.
Don C. Lamb (Invited talk)
The pH dependence of actin polymerization and the dynamics of the mitochondria HSP70 chaperon: molecular insights into the dynamics of proteins using ultrasensitive fluorescence methods.
Martin Sikor; Nikolaus Naredi-Rainer, Dejana Mokranjac, Alvaro H. Crevenna, Don C. Lamb
Actin polymerization drives cellular motility. One interesting aspect of actin polymerization that has not been investigated in detail is the pH dependence. Using FCS, we have investigated the critical concentration of actin needed for polymerization as a function of pH. We have also investigated the size and frequency of the bursts resulting from polymerization using a burst search algorithm and PCH. The observed pH sensitivity can be explained from the pH-dependent net charge of actin on protein-protein interactions.
Recently, we have investigated the conformation of Mitochondrial Heat Shock Protein 70 (mtHsp70), a protein involved in the import of proteins into mitochondria and in protein folding in the mitochondrial matrix, using spFRET (Mapa et al, Mol. Cell 2010). To study the dynamics of conformational transitions observed in the chaperon, we performed spFRET TIRF experiments on molecules immobilized within lipid vesicles. The FRET efficiencies and thus conformations of the states occupied in the presence of ADP were found to be different than in the presence of AMP-PNP or substrate peptide. MtHsp70 displayed fast interdomain dynamics with rates of ~1.5s-1 while the dynamics of the substrate binding lid are slower by a factor of ~4. We measured the dynamics as a function of ADP concentration. The dynamics of the lid as well as separation of the domains were independent of ADP concentration whereas the rate of domain closing increased with increasing ADP concentration. This suggests that the closed state is an ADP bound state while the open state is a nucleotide free state and the dynamics of the lid is independent of the domain dynamics for mtHsp70. However, the nucleotide bound conformation of mtHsp70 was found to influence the affinity of the chaperone with its cochaperone, TIM44.
Modulation tracking of single microvilli: imaging protein distribution and dynamics.
Luca Lanzano, Michelle Digman, Hector Giral*, Moshe Levi* and Enrico Gratton. Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA; *Department of Medicine, University of Colorado Denver, Aurora, CO
Brush border microvilli (MV) are specialized cell protrusions that provide an increase in the surface area for the absorption of nutrients. In the kidney proximal tubule, reabsorption of inorganic phosphate (Pi) occurs via the sodium gradient-dependent Pi (NaPi) cotransport proteins localized in the MV membrane. The proper localization and regulation of the NaPi transporters on the MV are related to a set of specific protein-protein interactions. These include interaction with the PDZ proteins, a family of proteins generally acting as scaffolders of subcellular structures, and mediating the interaction with the actin-based MV cytoskeleton.
MV are mobile structures which are microns long with a lateral size of the order of 100nm, and can be difficult to study with conventional imaging. We image MV in live cultured proximal tubule cells using the Modulation Tracking (MT) method that, instead of scanning following a predetermined pattern, uses a feedback approach to adapt the scan to the shape and movement of the object. We describe the principles of this method and observe the distribution and dynamics of different proteins within the MV.
Work supported in part by NIH RO1 DK066029-01A2 (ML, EG), NIH-P41-RRO3155 (EG and LL) and P50-GM076516 (EG and LL) and NIH-U54 GM064346 Cell Migration Consortium (MD and EG).
Characterization of cytosolic Phosphatidylinositol 4-kinase II using fluorescence fluctuation spectroscopy.
Jinhui Li1, Barbara Barylko2, Joseph Albanesi2, Joachim Mueller1, Yan Chen1. Physics Department1, University of Minnesota, Minneapolis, MN 55455; Pharmacology Department2, University of Texas Southwestern Medical Center, Dallas, Texas, 75239
Mammalian cells express two classes of phosphatidylinositol 4-kinase (PI4K), designated Types II and III. These enzymes phosphorylate phosphoinositol to generate PI4P. A number of studies indicate that these enzymes are important for Golgi trafficking and early as well as late stages of endocytosis. There are two Type II kinases : PI4KIIalpha, an integral membrane protein involved in membrane trafficking; and PI4KIIbeta, distributed evenly between membranes and cytosol and believed to participate in phosphoinositide signaling. In this study, we focus on PI4KIIbeta. Using fluorescence fluctuation spectroscopy, we found that PI4KIIbeta exists as two distinct species in the cytosol. One species exists as a soluble monomer, while the other species is associated with cytosolic vesicles. We further identified that PI4KIIbeta on vesicles co-moves with clathrin light chain using dual-color fluorescence fluctuation spectroscopy with heterospecies partition analysis. This analysis allows us to isolate the co-moving species in the presence of the strong background contribution from the monomeric PI4K. Our results demonstrate that brightness analysis is a powerful tool to characterize cellular cytosolic vesicles that are otherwise difficult to characterize by other techniques. This work is supported by the National Institutes of Health (R01 GM64589).
Non-linear optical probes of membrane potential.
Ping Yan, Corey Acker, Les Loew. U. Conn. Health Center, Farmington, CT
The chemistry and the physics of voltage sensitive dyes should be understood and appreciated as a prerequisite for their optimal application as probes of cellular physiology. The physical and chemical properties of the dyes can be tuned to optimize delivery to and staining of the cells in different experimental preparations. High resolution microscope imaging, including confocal and non-linear optical microscopy, are being used in conjunction with the voltage sensitive dyes to map electrical activity in both space and time; these optical approaches will be briefly surveyed. I will highlight some of our recently developed dyes which are optimized for non-linear optical microscopy. Using these dyes we have been able to record backpropagating action potentials in individual dendritic spines within a brain slice. (supported by a Human Frontier Science Program grant and by USPHS NIH grants R01 EB001963 and P41 RR13186)
Porous inorganic silica host for living cell encapsulation.
Alexander M. Macmillan1, Dylan M. Owen2, Katharina Gaus2, David J. S. Birch3 & Jan Karolin3. 1Biomedical Imaging Facility, University of New South Wales, Sydney, Australia; 2Centre for Vascular Research, University of New South Wales, Sydney, Australia; 3Centre for Molecular Nanometrology, University of Strathclyde, Glasgow, United Kingdom
The encapsulation of proteins in silica sol-gels has attracted a lot of attention since the first demonstration of the technique by Braun et al in 1990[1]. However, the technique of encapsulating live cells is still in its relative infancy. Here we report on the application of silica sol-gels to the encapsulation of Jurkat T cells with minimal perturbation to the cell membrane. During the gelation process of silica sol-gels, hydrated pores are formed around individual cells, matching their size whilst connecting to the network by smaller pores of a few nanometers diameter. The latter acts as conduits for analytes of interest while excluding potentially interfering macromolecules. The accessibility of Jurkat T cells when encapsulated and immobilized in the silica sol-gel is demonstrated by the relatively free diffusion of 7-ketocholesterol (7-kc) through the porous framework causing a disruption of the cell membrane[2]. The time required between addition of 7-kc to the gel surface and the first indication of cell membrane ordered phase perturbation was 60 seconds. The improved bioencapsulation of biological samples that we demonstrate could provide a generic method for immunoprotection of transplanted cells whilst, as demonstrated here, still retaining accessibility to drugs of interest. Furthermore, the improved biocompatibility coupled with the porous nature of sol-gels could have applications to tissue engineering based on scaffolds integrated with biological cells[3].
REFERENCES
1. Braun, S., et al., BIOCHEMICALLY ACTIVE SOL-GEL GLASSES - THE TRAPPING OF ENZYMES. Materials Letters, 1990. 10(1-2): p. 1-5.
2. Rentero, C., et al., Functional Implications of Plasma Membrane Condensation for T Cell Activation. Plos One, 2008. 3(5).
3. Hollister, S.J., Porous scaffold design for tissue engineering. Nature Materials, 2005. 4(7): p. 518-524.
Moving towards a technical specification for fluorescence excitation-emission mapping and absorbance analysis of colored dissolved organic matter.
Karen Gall, Jim Mattheis, Adam Gilmore, Fluorescence Group Horiba Jobin Yvon, Inc., 3880 Park Ave., Edison, NJ 08820
Colored dissolved organic matter (CDOM) measurements with fluorescence and absorbance are important for evaluating a wide variety natural and industrial water sources. However, uncertainties and ambiguities continue to be propagated regarding interpretation of CDOM spectral data due to the variety of instruments, sampling chemistry conditions and types of analysis algorithms. Recent efforts have focused on standardization and interlaboratory comparisons of CDOM samples with respect to preparation, spectroscopic evaluation and mathematical analysis. This study deals with correlating absorbance and fluorescence data measured with the same sample to minimize interlaboratory variation. The theoretical significance of true simultaneous acquisition of the corrected (NIST Traceable) absorbance spectrum and fluorescence excitation spectral profile and excitation emission map is discussed as a means to provide the least ambiguous spectral data. Key issues considered are the variations introduced by ‘serial’ acquisitions of absorbance and fluorescence data. Variation can be caused by the different light-exposure history (especially UV) in the instruments, dissolved oxygen content associated with temperature changes and oxidation kinetics of the CDOM and in many cases concentration- and pH-related changes associated with diluting and pH buffering of the CDOM sample, respectively. Concentration changes in CDOM can be associated with optical anomalies including self-quenching and -absorption which systematically alter the fluorescence spectrum. Clearly, monitoring the absorbance and fluorescence simultaneously would deal with the above sampling variations and facilitate correcting the absorbance based fluorescence anomalies. The proposed method(s) described will be discussed in view of their potential to serve as the basis for an international technical specification in terms of the optical instrument and sampling conditions for CDOM analysis and reporting.
EGFR clustering away from Cholesterol- or Sphingolipid-enriched microdomains revealed by PALM/dSTORM.
Hideaki Mizuno1,2, Mitsuhiro Abe3, Peter Dedecker2, Asami Makino3, Yoshiko Ohno-Iwashita4, Johan Hofkens2, Toshihide Kobayashi3, and Atsushi Miyawaki1. 1Cell Function and Dynamics, Brain Science Institute, RIKEN; 2Department of Chemistry, Katholieke Universiteit Leuven; 33Lipid Biology Laboratory, RIKEN Advanced Science Institute; 4Faculty of Pharmacy, Iwaki Meisei University
Lipids in biomembranes such as cholesterol and sphingomyelin form raft-like microdomains. These microdomains are thought to serve as platforms for signal transduction and molecular trafficking, but it is difficult to elucidate their detailed structure since their size is smaller than the resolution of light microscopy. We employed superresolution microscopy to circumvent this limitation. To this end, we designed dedicated probes for cholesterol- and sphingomyelin-enriched microdomains. PALM imaging revealed two types of cholesterol-enriched microdomains, line-shaped with widths of around 150 nm and round-shaped with the average radius of 118 nm. All sphingomyelin-enriched microdomains were round-shaped with an average radius of 124 nm. Both the cholesterol- and sphingomyelin-enriched microdomains vanished by the depletion of cholesterol. The sphingomyelin-enriched microdomains also vanished by the depletion of sphingomyelin whereas the cholesterol-enriched microdomains were unaffected. We conclude that cholesterol- and sphingomyelin-enriched domains occupy different regions on the plasma membrane. EGF receptors clustered on the plasma membrane with no apparent change upon short stimulation with EGF. The clusters of EGF receptors were on different regions from cholesterol- or sphingomyelin-enriched microdomains and unaffected by depletion of cholesterol and sphingomyelin. The clusters were gathered and endocytosed upon prolonged stimulation with EGF. The endocytosis required membrane cholesterol but not sphingomyelin.
BODIPY phosphatidylinositol probes incorporation into giant unilamellar vesicles membrane.
I.L. Salvemini, J. Reid and P. Moens
The use of Giant Unilamellar Vesicles (GUVs) composed of fluorescently labelled lipid analogues has become an increasing popular model to study both structural and complex biophysical properties of bilayers. However, there is a common assumption that the number of probes incorporated into the membrane of the GUVs is proportional to the mole fraction (%) of these lipid molecules in the original solvent solution. To test this assumption, a commercial confocal laser scanning microscope (Nikon C1) was used to obtain single point fluorescence correlation spectroscopy (FCS) data.
We measured the diffusion coefficient and number of molecules incorporated into the membrane of the GUVs for several BODIPY labelled lipid i.e. BODIPY TMR-phosphatidylinositol (4,5) bisphosphate, BODIPY TR- phosphatidylinositol (4,5) bisphosphate and BODIPY (530/550) hexadecanoyl-sn-glycero-3-phosphocholine. We investigated the effect of various mole fraction of these lipids and compared the results with (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate [DiIC18].
We show that the number of DiIC18 molecules incorporated into the membrane of the GUVs (formed by the electroformation method) is in agreement with the expected number of molecules calculated from the mole fraction of the organic stock solution. However, we find that the actual proportion of β-BODIPY-HPC, TR-PI(4,5)P2, and TMR-PI(4,5)P2 incorporated into the bilayer is significantly less than the proportion of these lipids in the organic solvent stock solution.
Recent developments in brightness analysis.
Joachim Mueller. School of Physics and Astronomy, Institute for Molecular Virology, Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455
Association of fluorescently labeled proteins into complexes can be detected by the increased fluorescence intensity emitted by the complex. This increase in brightness is suitable for the characterization of homo-protein interactions using a single fluorescent color. Characterization of hetero-protein interactions requires dual-color brightness analysis, where each of the two protein species carries a differently colored label. Although the theoretical concept of brightness analysis is well understood, quantitative applications inside the complex environment of the cells remain challenging despite significant progress over the last few years. Here I will focus on quantitative characterization of protein stoichiometry and binding curves inside cells. The heterogeneous mixture of brightness species typically found in cells provides a substantial roadblock for the quantitative characterization of protein binding. To overcome this and other challenges encountered in cells requires new fluorescence fluctuation methods. I will discuss recent advances in addressing these challenges.
Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity.
Helen Hwang, Hajin Kim and Sua Myong. University of Illinois, Department of Bioengineering, Urbana, IL 61801
Single-molecule FRET has been widely used for monitoring protein–nucleic acids interactions. Direct visualization of the interactions, however, often requires a site-specific labeling of the protein, which can be circuitous and inefficient. In addition, FRET is insensitive to distance changes in the 0–3-nm range. Here, we report a systematic calibration of a single molecule fluorescence assay termed protein induced fluorescence enhancement. This method circumvents protein labeling and displays a marked distance dependence below the 4-nm distance range. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein. Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. We demonstrate the general applicability and distance sensitivity using (a) a finely spaced DNA ladder carrying a restriction site for BamHI, (b) RNA translocation by DExH enzyme RIG‐I, and (c) filament dynamics of RecA on single-stranded DNA. The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.
Structural and mechanical bases for coordinated activities of dyneins of Chlamydomonas flagella.
Kazuhiro Oiwa
Coordinated beating and bend propagation of cilia and flagella are generated by active sliding of the peripheral doublet microtubules, driven by ensembles of various types of dyneins. These dyneins are localized in projections called dynein arms, which extend periodically from the side-wall of each peripheral doublets. Axonemal dyneins show large-scale integrated behavior that is responsible for the beating of flagella. To understand the basis underlying the coordinated behavior of dyneins, we have precisely measured and characterized spatial arrangement of axonemal components and their mechanical properties. Intense X-ray beams generated by the third-generation synchrotron radiation facilities (Spring-8, BL45XU) have made it feasible to analyze axonemal structure by means of small-angle X-ray diffraction. With using this X-ray, we have precisely measured the axial repeat of the outer dynein arms of Chlamydomonas flagella to be 24.7± 0.7nm, which corresponds to length of three tubulin dimers (8.25 nm × 3 =24.8nm)[1]. Further, we carried out electron cryo-tomography on a whole axoneme and showed the three-dimensional arrangement of dynein arms in situ [2-4]. The tomograms showed that an outer-dynein arm is composed of three plates corresponding to the head rings of dynein heavy chains. The tomograms further showed the presence of three inter-dynein arm connections between the outer arms: two outer arm-outer arm linkages and one outer arm-inner arm linkage. These structural features are probably the structural bases of the coordination.
For mechanical analysis, we have characterized the mechanical properties underlying the coordination among multiple dynein molecules with using in vitro motility assay and advanced fluorescence microscopy [5,6]. Now, we are developing a new technique, in which single molecule fluorescence microscopy is combined with DNA scaffold technique. DNA scaffold permits studies on coordination of multiple motors by manipulating the space between motors with nanometer precisions and stiffness of the linkage between them. We have manipulated the number and spacing of motors at specific sites on a single DNA scaffold and first constructed complexes that engage teams of either kinesin-1 or kinesin-14. The travel distances of both kinesins were greatly extended when the number of molecules was increased, whereas the velocities were only slightly affected. We then linked together two opposing kinesin motors, kinesin-1 and kinesin-14, by a single molecular scaffold, which leads to a tug-of-war on microtubules. In the tug-of-war, reversals of direction and periodical, step-like movements were frequently observed. We are now trying to extend this method to dyneins. The combination of quantitative in vitro and in silico experiments using this simplified system will provide fundamental knowledge of coordination mechanism of dyneins.
REFERENCES:
(1)Oiwa, K., Kamimura, S., & Iwamoto, H. Meth. Cell Biol. 91, 89-109. (2009).
(2)Ishikawa, T. Sakakibara, H., & Oiwa, K. J. Mol. Biol. 368, 1249-1258. (2007).
(3)Bui, K. H., et al., J. Cell Biol. 183, 923-932. (2008).
(4)Movassagh, T., et al. Nat Struct Mol Biol 17, 761-767. (2010).
(5)Kotani, N., et al. Biophys. J. 93, 886-894. (2007).
(6)Toba, S. et al. Mol Biol Cell 22, 342-353. (2011).
Membrane organisation and dynamics studied by correlated FCS and Laurdan spectroscopy and super-resolution imaging.
D M Owen, A Magenau, D Williamson, J Rossy and K Gaus. UNSW, Centre for Vascular Research, Sydney, Australia
The efficiency of signalling networks depends on the rate of interactions (collisions) between biomolecules. This is in turn determined by their rate and mode of diffusion and the degree of segregation/clustering. Here, we examine the diffusion and clustering of two proteins involved in the activation of T cells; the tyrosine kinase Lck and the scaffold protein LAT. Both are membrane-associated and are more-specifically thought to be targeted to high-order membrane lipid domains. We study the diffusion and clustering of these proteins using a combination of quantitative fluorescence techniques: Fluorescence correlation spectroscopy (FCS) combined with simultaneous membrane-lipid-order analysis using Laurdan spectral-ratiometric measurements and super-resolution PALM imaging combined with quantitative statistical cluster analysis based on Ripley’s K-functions.
The combination of membrane protein diffusion and quantitative order analysis allows the correlation between membrane lipid order and protein diffusion to be determined for the first time. In live HeLa cells, we find a significant negative correlation between membrane order and the protein diffusion coefficient (high order = slow diffusion). This correlation is ablated when using non-raft targeted mutants or by disruption of the ordered-phase using 7-ketocholesterol or the cytoskeleton using Latrunculin-B. In terms of clustering, we find that these proteins are relatively evenly distributed in the plasma membrane of resting cells but that the removal of ordered-phase targeting, high membrane order or the actin cytoskeleton all increase protein clustering levels.
Collectively, the data therefore suggest that high-order lipid microdomains work synergistically with the actin cytoskeleton (possibly by actin-anchored proteins acting as nucleation sites for larger, more stable lipid domains) to retard membrane protein diffusion and this in turn regulates the formation of large-scale protein clusters in the plasma membrane.
Using immunofluorescence to determine treatment depth of a novel antibacterial agent.
M. Pastagia, V.A. Fischetti. The Rockefeller University, Dept. of Microbiology, New York, NY 10065
Staphylococcus aureus is a major human pathogen responsible for a number of serious and sometimes fatal infections. One of its reservoirs on the human body is the skin, which is known to be a source of invasive infection. The potential for an engineered staphylococcal-specific phage lysin (ClyS) to be used for topical decolonization is presented. Staphylococci in mouse tissue were visualized by immunofluorescence using the purified binding domain of ClyS labeled with Alexa fluor 647. Alexa fluor-labeled ClyS was incubated with 0.6mm mouse skin sections. This technique was used on control, placebo, and ClyS treated mouse skin sections. Staphylococcal burden was determined by measuring the depth from the epidermis to dermis where immunofluorescent staphylococci could be found (treatment depth). Treatment depth was significantly higher in mice treated with ClyS than in placebo treated mice.
Rajesh Ramachandran (Invited talk)
Allosteric conformational coupling in Dynamin.
Rajesh Ramachandran, Case Western Reserve University School of Medicine, Cleveland, OH
The polymerizing GTPase dynamin catalyzes the scission of deeply invaginated coated pits at the plasma membrane utilizing domain-specific protein-membrane and protein-protein interactions. Multiple lines of evidence now indicate that a structural and functional coupling between its membrane-inserting pleckstrin homology (PH) domain and the distal GTPase domain underlies this function. However, the molecular mechanism(s) by which this putative conformational coupling is achieved remains ill-defined. Through site-specific fluorescence labeling of its PH and GTPase effector (GED) domains, and the real-time detection of domain-specific protein-membrane and protein-protein interactions, the requirement for membrane insertion-coupled GED-zippering in dynamin function is revealed. A membrane insertion-defective dynamin 1 mutant, Dyn1 I533A, uncouples intermolecular GED-GED interactions from PH domain-membrane insertion and fails to elicit assembly-dependent GTPase activity. Thus, stable PH domain-membrane interactions in dynamin allosterically trigger intermolecular GED-zippering to effect assembly-dependent GTPase activation.
Gregory D. Reinhart (Speaker)
Illuminating the dynamic changes associated with heterotropic allosteric communication in Phosphofructokinase from Bacillus stearothermophilus.
Gregory D. Reinhart and Stephanie Perez. Department of Biochemistry and Biophysics, Texas A&M University and Texas, AgriLife Research, College Station, TX 77843-2128
Phosphofructokinase from Bacillus stearothermophilus (BsPFK) undergoes dynamic changes in its otherwise rigid structure upon binding of ligands, fructose 6-phosphate (F6P) in the active sites, and allosteric inhibitor phosphoenolpyruvate (PEP) in the allosteric sites. PFK can exist in four distinct ligation states: Apo BsPFK, BsPFK-F6P, PEP-BsPFK, and PEP-BsPFK-F6P; however, the biophysical properties of each ligation state are poorly understood particularly with respect to their dynamic character. Since the four active sites and four allosteric sites in the homotetramer of BsPFK are involved in an intricate network of homotropic and heterotropic interactions, a hybrid strategy has been developed to isolate and subsequently characterize the allosteric contribution of an individual heterotropic interaction. For this study, the hybrid approach is simultaneously used to isolate and utilize a single intrinsic fluorophore to report the local side-chain dynamics upon ligand binding to the active and allosteric sites specific to the isolated individual heterotropic interaction.
Tryptophan was strategically positioned by constructing conservative tryptophan-shift mutant enzyme forms in which one of the sixteen native phenylalanine and tyrosine residues in each subunit was substituted with tryptophan and the native tryptophan was changed to tyrosine. Eight of these targets have been characterized so far. Steady-state anisotropy measurements reveal little change in response to ligand binding of four targeted regions in the monomer. For the remaining four tryptophan-shift mutants, time-resolved experiments indicated that changes in rotational correlation times of the ligation states were significant, above 1 nsec, when compared to their unligated forms. Of these significant dynamic changes, however, only two regions were found likely to be involved in the allosteric inhibition of the enzyme. These results may suggest a molecular basis for the observation that the nature of allosteric communication in BsPFK is entropically driven. Funding is provided by NIH grant GM33216 and Welch Foundation grant A1543.
Gregory D. Reinhart (Poster)
Effect of hydrostatic pressure on the allosteric couplings in a tryptophan-shifted variant of Phosphofructokinase from Bacillus stearothermophilus.
Amy M. Knutson and Gregory D. Reinhart. Department of Biochemistry and Biophysics, Texas A&M University and Texas, AgriLife Research, College Station, TX 77843-2128
Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is an allosteric, homotetrameric enzyme containing one tryptophan per subunit. Unlike the homologous PFK from E. coli (EcPFK), the fluorescence of the native tryptophan is largely unresponsive to ligand binding. This study utilizes a tryptophan-shifted mutant, in which W179 has been mutated to phenylalanine, and F240 has been mutated to tryptophan. The variant is functionally similar to wild-type, however a decrease in the fluorescence of ~6.5% is associated with substrate, fructose 6-phosphate (Fru-6-P), binding and a decrease of ~20% is associated with inhibitor, phosphoenol pyruvate (PEP), binding. Dissociation constants of 1.9 ± 0.3μM for Fru-6-P and 107 ± 13μM for PEP were thereby determined. This dissociation constant for PEP is in good agreement with that determined by kinetic assays (128 ± 5μM). Due to ATP antagonism, the dissociation constant for Fru-6-P in the absence of MgATP is lower than with steady-state kinetic assays determined at saturating MgATP (36 ± 1μM). The coupling between PEP and Fru-6-P has also been determined and increases with temperature as observed with steady-state kinetic assays using wild-type BsPFK. Like wild-type PFK, the coupling free energy results from compensating enthalpy and entropy components. The sign of the coupling free energy is opposite that of the enthalpy and is therefore determined by the larger absolute value of the entropy term. This is opposite of the thermodynamic basis of the allosteric response in EcPFK, where the sign is established by the enthalpy component.
The functional and thermodynamic similarity of the mutant enzyme to wild-type, together with the added ability to follow ligand binding with fluorescence, make it a good candidate for further fluorescence studies. Fluorescence intensity of the variant BsPFK increases by ~15% linearly as a function of pressure from 0 to 2 kbar. Pressure induced changes to the fluorescence intensity in this pressure range are completely reversible and the enzyme maintains 100% of its activity. Current studies focus on determining how the thermodynamic components of the coupling free energy change as a function of pressure with the goal of better understanding the thermodynamic forces involved in the allosteric effect. Funding provided by the following: NIH-GM33261, NIH-CBI Training Grant, and Welch Foundation grant A1543.
Advanced fluorescence correlation techniques to study membrane dynamics.
Jonas Ries and Petra Schwille
Fluorescence Correlation Spectroscopy (FCS) is a powerful tool to measure important physical quantities such as concentrations, diffusion coefficients, diffusion modes or binding parameters, both in solution and in membranes. However, it can suffer from severe artifacts, especially in non-ideal systems. Here we develop several novel implementations of FCS which overcome these limitations and facilitate accurate and quantitative determination of dynamic parameters in membranes.
Scanning FCS with a scan path perpendicular to the membrane plane allows to correct for instabilities permitting long measurement times necessary to study slow diffusion. It can easily be extended to measure calibration-free diffusion coefficients with two-focus scanning FCS and to quantify binding with dual color scanning FCS. Spectral crosstalk can be avoided effectively by using alternating excitation. Using this method we were able to perform measurements in systems previously not accessible with FCS, such as membranes of living zebrafish embryos, where we could determine binding affinities of the morphogen Fgf8 to its receptors in situ.
Line-scan FCS with a scan path in the membrane plane uses the parallel acquisition along the line to increase the statistical accuracy and decrease the measurement times. Knowledge of the scan speed serves as an internal calibration, enabling accurate diffusion and concentration measurements within seconds, hardly affected by photobleaching. Both realizations of scanning FCS can be easily implemented with commercial laser scanning microscopes.
Often, a fluorescence background around the membrane cannot be avoided. The high surface selectivity needed in this case can be achieved efficiently by using a novel objective for FCS, the supercritical angle objective, which produces a very flat and laterally confined detection volume.
The novel FCS techniques developed here will have a high impact on the use of FCS to address key questions in biological systems, previously inaccessible by other methods.
Transfection alters mobility of transcription factors in the nucleus.
Tilman Rosales1, Zuqin Nie2, Jay R. Knutson1 and David Levens2. 1Laboratory of Molecular Biophysics, Optical Spectroscopy Section, NHLBI, NIH; 2Laboratory of Pathology, NCI, NIH
C-myc is the classic product of a proto-oncogene. It is carefully regulated and usually expressed at very low levels (few nM) in the cell. It has been demonstrated to regulate expression of many genes and controls cell proliferation. We properly expressed c-myc-eGFP as knock-in from its native locus in primary murine fibroblasts and NIH 3T3 cells and observed its diffusion within the nucleus using Fluorescence Correlation Spectroscopy. When increasing the total concentration of c-myc-eGFP with transient transfection(s), total c-myc levels increase steadily and the ‘mobile’ pool rapidly climbs toward 90% of the total c-myc-eGFP (at about 100nM), while the ‘immobile’ component concentration remains near endogenous levels, thus decreasing from 50% to only a few percent of total signal. The importance of recognizing the small, easily saturated endogenous pool of binding sites in regulation will be discussed.
We also studied the accumulation of eGFP-c-myc over time after serum stimulation. A peak at 2-3 hrs after serum stimulation was observed for the cells without the proteasome inhibitor MG132 (with an increase of about 30% from the final steady state level of about 8 +/-1.3 nM after 8hrs). For those with MG132, we see further increase of the eGFP-c-myc concentration, with a peak at about 3 hrs and increasing again later.
We also demonstrate the relationship of c-myc with Max protein by knockdown with RNAi and inhibitors. We found an increase of the mobile population at low expression levels of 20%.
Measuring biophysical properties of antibodies coated on microparticles.
Qiaoqiao Ruan, Sylvia C. Saldana, Jane Beebe and Sergey Y. Tetin; Diagnostics Research, Abbott Diagnostics Division, Abbott Park, IL 60064
Antibody coated microparticles are used in numerous immunoassays and it is important to understand and control their biophysical properties. Anti-NGAL (Neutrophil Gelatinase-Associated Lipocalin) antibody coated microparticles are used as the model system. The binding affinities and binding kinetics of antibodies on microparticles are different from the solution phase. We have performed detailed studies of properties of antibodies coated on microparticles using flow-cytometery and fluorescence microscopy and found that the total concentration and the fraction of active antibodies on microparticles are greatly affected by coating conditions. We also did not find preferences in antibody orientation under different coating conditions when probed with fluorescently labeled anti-Fab or anti-Fc specific antibody reagents.
Super-resolution single-molecule FRET sheds light on biomolecular structure, dynamics and interactions.
Claus Seidel, Suren Felekyan, Stanislav Kalinin, Thomas Peulen, Simon Sindbert. Heinrich-Heine-University, Duesseldorf, Germany
Although static structures are known for many biomolecules, the functions of biomolecules are governed in space and time ultimately by their dynamic character. Using a confocal fluorescence microscope the newly developed multi-parameter fluorescence detection (MFD) enables us to simultaneously collect all fluorescence information such as intensity, lifetime, anisotropy in several spectral ranges) from picoseconds to seconds and to circumvent the classical pitfalls of the FRET method in ensemble measurements [1]. In addition, we developed single-molecule (sm) super-resolution (SR)-FRET as a quantitative procedure for the proper consideration of the position distribution of the dye and of linker and a very high accuracy of FRET based structure determination [4]. To achieve super-resolution (RMSD < 0.2 nm) in FRET experiments with dyes flexibly linked to biomolecules, it is indispensible considering three effects to achieve correct positioning in our newly developed rigorous FRET position system (FPS): (1) analysis of shot noise broadening by photon distribution analysis (PDA) [8], (2) distinct dye brightnesses [7], and (3) all possible dye positions. We applied FPS to proteins and RNA [2, 3]. These novel FRET-based detection and analysis methodologies allowed us to resolve several structural subpopulations with Ångström resolution und derived FRET based structural models for several species from the same measurement. SmFRET studies show the presence of multiple conformational states, which are related to their biochemical function. Moreover I will present novel perspectives of MFD to study protein-protein interactions in live cells [5, 6].
1. Sisamakis, et al. Methods in Enzymology 475 Chapter 18, 455-514 (2010).
2. Gansen, et al. Proc. Natl. Acad. Sci. USA. 106, 15308-13 (2009).
3. Woźniak, et al. Proc. Natl. Acad. Sci. USA. 105, 18773-42 (2008);
4. Sindbert, et al. J. Am. Chem. Soc. 133, 2463-2480 (2011).
5. Kudryavtsev, et al. Anal. Bioanal. Chem. 387, 71-82 (2007.
6. Weidtkamp-Peters, et al. Photobiol. Sci., 8, 470-480 (2009).
7. Kalinin, et al. J. Phys. Chem. B. 114, 6197–6206 (2010).
8. Kalinin, et al. J. Phys. Chem. B. 114, 7983–7995 (2010).
Characterization of microparticle coating by direct fluorescent labeling using green fluorescent protein.
Joseph P. Skinner, Stefan Hershberger and Sergey Y. Tetin,
Diagnostics Research, Abbott, Abbott Park, IL
Magnetic microparticles coated with specific receptors provide an excellent means for allowing manipulation of proteins and peptides in solution by providing a solid surface to which target molecules bind. Moving microparticles to and from different solutions for processes such as purification or concentration measurement is provided by simple magnetic manipulation. Binding of molecules to the microparticle depends upon coating procedures and homogeneity of receptor molecules coated on the surface. Here we characterize particles by directly coating them with green fluorescent protein and applying fluorescence imaging. Direct labeling with fluorescent protein uses common procedures for microparticle coating and enables imaging without the need for secondary fluorescent labeling. Using two-photon laser scanning microscopy we can construct 3D images which enable qualitative surface characterization of individual microparticles. Application of TIRF microscopy reveals variations of protein concentrations on the surface of a number of microparticles present in a single image. By applying image correlation spectroscopy to these images, we can study the size of variations on the particles in a quantitative manner.
Aleksandr Smirnov (Poster)
Maximizing speed and depth of multiphoton microscopy with aspherical reflective surfaces.
A. V. Smirnov1, C. A. Combs2, B. M. Lucotte3, D. Chess3, D. B. McGavern4, J. L. Schroeder3, S. S. Kang4, J. R. Knutson1, and R. S. Balaban3. 1NHLBI Laboratory of Molecular Biophysics; 2NHLBI Light Microscopy Facility; 3NHLBI Laboratory of Cardiac Energetics; 4NINDS Viral Immunology and Intravital Imaging Unit, National Institutes of Health, Bethesda, MD 20892-1412
Two-photon (2p) excitation of fluorophores is a key event to image formation in microscopy with ultra-short laser pulses. Imaging relies on both the 2p-absorption volume being minimal and emission being non-directional. Thus, schemes that either optimize effectiveness of available excitation photons or maximally collect all available emitted photons become useful.
To the latter end, for in vivo applications we developed a version of Total Emission Detection (TED) built to obtain images inside living tissue without direct physical contact. The device employs a parabolic reflector and is mounted on a periscope (LSM Tech) to facilitate approach to live animals. Observation of blood vessels tagged with DsRed-actin ~ 100 um deep within an exposed mouse brain provide over 2.5X more light in this epi-TED device (compared to light collected by the 20X water 0.95NA dipping objective alone).
Optical elements of a microscope (including relay lenses in epi-TED) as well as the sample itself can be sources of chromatic and spherical aberrations that disperse the sharp point-spread-function needed for multiphoton imaging. To further push limits of speed, depth and signal-to-noise ratio (SNR) we have employed an open-loop controlled continuous-membrane deformable mirror (DM) and a combination of wavefront sensing/reconstruction and image analysis algorithms on a commercial multiphoton laser scanning microscope. We demonstrate that compensation of aberrations by DM provides sharper and brighter images at large depths in tissue phantoms.
Thus, epi-TED alone allows increased scanning speed with the same SNR (to more than twice the normal rate). Alternatively, to minimize photodamage one could reduce laser power by 60%. At depths >50 um, adaptive optics with wavefront sensing should become advantageous to compensate for sample-induced optical aberrations.
High throughput FLIM/PLIM based on temporal focusing wide-field multiphoton microscopy.
Heejin Choi, Dimitris Tzeranis, Jae Won Cha, Philippe Clemenceau, Sander De Jong, Peter T. C. So, MIT, Cambridge, MA
This presentation focus on high throughput fluorescence and phosphorescence lifetime resolved imaging in 3D based on temporal focus wide-field multiphoton excitation. Lifetime resolved imaging is performed based on frequency domain heterodyne detection with a gated camera. This approach offers signal-to-noise ratio advantage over state-of-the-art method by at least two-orders of magnitude.
Fluorescence study of membrane rafts formation using Cholera toxin protein.
Martin Štefl, Jana Humpolíčková, Marek Cebecauer, Martin Hof. J. Heyrovsky Institute of Physical Chemistry; Prague, CR
Characterization of lateral compartmentalization in cellular membranes is crucial for better understanding of their function. Currently, the model of the plasma membrane suggests the existence of more rigid domains (frequently called lipid rafts or ordered phase in model systems) surrounded by fluid membrane (disordered phase in model membranes). The mechanism of lipid rafts formation has always been a key question for membrane biologists and biophysicists. Two processes of rafts formation are discussed in literature so far: a) lipid rafts are directly formed in single step by accumulating of raft lipids into the rafts, or b) pre-existing nanodomains are always present in the membrane, and merge into the raft-like structures when sufficient number of these domains is reached or by the external factor (e.g. cholera toxin subunit B, CTxB) [1, 2]. Since the size of pre-existing nanodomains is below the light microscope resolution (< 200 nm), visualisation of such domains is challenging. Moreover, because of relatively high rigidity of these domains, a limited number of fluorescent dyes are available for their imaging (poor partitioning of the probes for ordered membranes). Thus, cholera toxin B subunit, due to its specific and high affinity interaction with GM1 ganglioside enriched in ordered phase, is often used as an indirect lipid raft marker.
Here, I am presenting the primary data supporting the existence of microscopically unresolvable highly ordered nanodomains. The presented data were aquired using the model membrane system, giant unilamellar vesicles (GUVs), with varying lipid compositions. Using diffusion times determined by z-scan FCS approach [3], we were able to demonstrate the CTxB clustering at nanoscopic levels and, therefore, creation of small, rigid nanodomains in microscopically homogenous disordered phase (herein mimicking the main membrane). The lipid clustering in prevalently disordered membranes has been also confirmed by Förster Resonance Energy Transfer and phasor plot analysis of time-resolved data [4]. All our observations indicate that membrane lipid rafts are formed from pre-existing nanoscopic structures.
[1] A.T.Hammond,F.A.Heberle,T.Baumgart,D.Holowka,B.Baird ,G WFeigenson. Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes. Proc Natl Acad Sci U S A 102 (2005) 6320-6325;
[2] T.S.van Zanten,J.Gómez,C.Manzo,A.Cambi,J.Buceta,R.Reigada ,M FGarcia-Parajo. Direct mapping of nanoscale compositional connectivity on intact cell membranes. Proc Natl Acad Sci U S A 107 (2010) 15437-15442;
[3] A.Benda,M.Benes,V.Marecek,A.Lhotsky,W.T.Hermens ,MHof. How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. Langmuir 19 (2003) 4120-4126;
[4] M.Stefl,N.G.James,J.A.Ross ,D MJameson. Applications of phasors to in vitro time-resolved fluorescence measurements. Anal Biochem 410 (2011) 62-69.
Chiara Stringari (Speaker)
In vivo metabolic mapping of stem cells and cancer cells by phasor FLIM.
Chiara Stringari1 Peter Donovan2,3 and Enrico Gratton1. 1Laboratory of Fluorescence Dynamics, Biomedical Engineering Department, University of California, Irvine; 2 Departments of Developmental & Cell Biology, University of California, Irvine; 3Department of Biological Chemistry and Sue and Bill Gross Stem Cell Research Center, University of California, Irvine
Here we present a novel label-free method for deriving metabolic maps of cells and living tissues in vivo. We use the phasor approach to fluorescence lifetime imaging and intrinsic biochemical fluorescence biomarkers such as NADH, flavins, retinoids and porphyrin. Metabolic signatures of living tissues are obtained by calculating the phasor fingerprint of cells and by mapping the relative concentration of FAD, free and bound NADH. Phasor fingerprints of embryonic and adult stem cells cluster according to their differentiation state, both in vitro and in a living tissue such as the C. elegans germ line. This method can detect small changes in metabolic signatures of colon cancer cells with different phenotypes and identify highly metabolically active cells that correspond to the stem cells at the base of living crypts from colon and small intestine. Phasor FLIM provides a label-free, fit-free and sensitive method to identify different metabolic states of cells and to classify stem cells, normal differentiated cells and cancer cells. This method is a promising non-invasive optical tool for monitoring metabolic pathways during differentiation and carcinogenesis and for high throughput screening.
Sergey Y. Tetin (Speaker)
A brief overview of the history and new trends in immunoassay design.
Sergey Y. Tetin, Qiaoqiao Ruan, Joseph P. Skinner, Sylvia Saldana. Diagnostics Research, Abbott Diagnostics Division, Abbott Park, IL 60064
In this short presentation we will review the history of the detection techniques used in immunoassays and experimental immunoassay design. Starting as radioimmunoassays (RIA) in the early 1960s this technology has evolved to the vast research field of modern biotechnology. The work of G. Weber on theory and applications of biological fluorescence, extrinsic fluorescent probes and ligand binding moved the field toward using non-radioactive materials and was realized in automated immunoassay instrumentation. Consequent introduction of chemiluminescent detection brought the technology to picomolar levels of sensitivity. Currently, we are replacing empirical reagent optimization with biophysical tools and computations of the required reagent characteristics. We are also implementing modern fluctuation techniques and imaging microscopy as sensitive and information-rich new detection technologies.
Winfried Wiegraebe (Invited talk)
Tools for genome wide high-content FCS-screening (HCS-FCS) in S. Cerevisiae.
Christopher Wood1, Joseph Huff1, 2, Will Marshall1, Elden Yu1, Jay Unruh1, Brian Slaughter1, and Winfried Wiegraebe1. 1Stowers Institute for Medical Research, Kansas City, MO, 64110; 1presently at Carl Zeiss Microimaging, Thornwood, NY, 10594
We developed tools to do automated auto- and cross-correlation FCS measurements on thousands of individual, living cells. We combined the data with the images we took for each cell we measured and we used this system to investigate the local concentrations and diffusion properties proteome-wide in the budding yeast S. cerevisiae using auto-correlation. For this screen we took over 200,500 measurements on more than 50,000 individual cells for 4082 clones of eGFP tagged ORFs. We extended the set-up to do interaction studies based on cross-correlation.
In addition to the data acquisition, the automatic data analysis turned out to be a major challenge. We developed algorithms based on machine learning to classify the images of the measured cells to include only measurements that were taken on healthy, non-moving, and in-focus cells. We developed a statistical approach to exclude erroneous data, such as cells moving during measurement, aggregates drifting through the confocal volume, and bleaching of immobile dyes. In addition, to allow for automation, this approach had the advantage of eliminating any human bias. We fit different fluctuation models to the data and selected the most appropriate one using Akaike’s ‘An Information Criterion’ (AIC). We are implementing mixed-effects models to fit correlation curves measured for the same protein at multiple cells.
To minimize spectral cross-talk in cross-correlation measurements, we switched between the two excitation lasers with a frequency of about 10 kHz. For data evaluation we extracted the actual frequency and phase from the raw data using a Fourier transformation.
Measuring synaptic receptor distributions and oligomerization using spatial intensity distribution analysis.
A. G. Godin1, S. Costantino1, L.E. Lorenzo3, J. L. Swift2, M. Sergeev1, A. Ribeiro-da-Silva3, Y. De Koninck4, P. W. Wiseman1,2
McGill University Departments of Physics1, Chemistry2 and Pharmacology3 ; Laval University Department of Cellular Neurobiology4
The ability to measure protein interactions in subcellular compartments is key to understanding cell signalling mechanisms, but quantitative analysis of these interactions in situ has remained a major challenge. We present a fluorescence imaging based analysis technique called spatial intensity distribution analysis (SpIDA) (Godin et al., PNAS 2011). SpIDA allows for quantitative measurement of fluorescent particle densities and oligomerization states within individual images acquired with conventional confocal laser-scanning microscopy. The method, an analog imaging extension of the photon counting histogram approach, is based on fitting intensity histograms calculated from images with super-Poissonian distributions to obtain density maps of fluorescent molecules and their quantal brightness values. Importantly, because distributions are acquired in space, SpIDA can be applied, not only to analysis of live cells, but also to that of chemically fixed tissue.
We will show the limits of the technique using computer-based simulations and control experiments on cellular distributions of receptors with known subunit composition (heterodimeric GABAB receptors, monomer-dimer mixtures EGFRs). SpIDA is applicable to sampling within small areas (6 µm2) with complex morphology and reveals the presence of monomers and oligomers with dye labelling of one type of fluorophore. Using SpIDA, we also show, for the first time, that the substance P receptor (NK-1r) almost exclusively forms homodimers on the plasma membrane and is primarily monomeric in the cytoplasm of adult spinal dorsal horn neurons. Triggering receptor internalization caused a measurable decrease in homodimer density on the membrane surface (from 3 +/- 2 to 21+/- 1 µm-2). Independent EM ultrastructural immunocytochemical analysis confirmed the differential distribution of NK-1r monomers and homodimers in distinct subcellular compartments. Finally we will show that SpIDA measurements of epidermal growth factor receptor (EGFR)-GFP dimerization in transfected CHO-k1 cells can be measured following direct activation with EGF ligand.
Bin Wu (Speaker - Weber Prize Winner)
Develop fluorescence fluctuation spectroscopy to quantify molecular interaction in living cells.
Bin Wu and Joachim D Müller
Macromolecules, such as protein, RNA and DNA, are the basic structural and functional units of cells. Quantitative information about the affinity and stoichiometry between these macromolecules are crucial for building physical models of cellular processes. Fluorescence fluctuation spectroscopy (FFS) is a promising experimental technique for addressing such interactions directly in living cells. However, the signal / noise ratio of traditional FFS analysis is insufficient to resolve the heterogeneous mixtures found in the cell environment. We established time-integrated fluorescence cumulant analysis (TIFCA), an exact FFS theory of factorial cumulant of photon counts for arbitrary sampling time. We demonstrate that TIFCA increases the signal / noise ratio of FFS data significantly. To quantify hetero molecular interactions, we introduced hetero-species partition (HSP) analysis for the interaction scheme D + nA = DAn. HSP directly identifies the hetero-interacting species from the sample mixture and determines the binding curve and stoichiometry in the cellular environment. Recently, we have generalized HSP to protein-mRNA interaction system. To demonstrate the application of FFS, we studied the nuclear receptor Retinoid X Receptor (RXR) and the coactivator Transcription Intermediate Factor 2 (TIF-2) and we identify the canonical interaction stoichiometry between them. Furthermore, we applied FFS to probe the regulation mechanism of Zipcode Binding Protein 1 (ZBP1) dependent β-actin mRNA metabolism.
Femtosecond time resolved fluorescence spectroscopy in proteins.
Jianhua Xu, Arianna Biesso and Jay R. Knutson. Optical Spectroscopy Section, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1412
Fluorescence decay and quenching of Tryptophan (Trp) were performed in a variety of proteins, including Thioredoxins, Cyanovirin, GB1 and Interleukin-1β using an upconversion spectrophotofluorometer combined with a time correlated single photon counting apparatus to span the ~200fs to 20ns time scale. All reveal multiexponential behavior. Besides a ubiquitous “bulk water” relaxation (~2 ps), a 50 ps fluorescence decay was found in single-Trp thioredoxin which corresponded to a component we had found previously in two-Trp Anabaena and E. coli thioredoxins. The sub-100ps component is consistently found (in all but one of these proteins) with positive amplitudes even at longer wavelengths (e.g., 390nm). The exception is GB1, a very small protein which Toptygin and Brand previously found carried a negative preexponential term near 390nm. The positive-definite amplitude DAS (decay-associated spectra) we see in most proteins are indicative of ultrafast quenching processes. Candidate mechanisms include ET to nearby acceptors and/or direct collisional quenching. This comports with our prior observation that ultrafast quenching, not desorbing water, dominates the time-resolved emission spectra (TRES) of monellin.
Surface-enhanced spectroscopy of dipeptides in Silver sol solution.
Sanjun Zhang1, Haifeng Pan1, Jing Zhou1, Rongrong Liang1, Zhandong Tao1, Jianhua Xu1,2, Jay Knutson2. 1State Key Lab. of Precision Spectroscopy, East China Normal University
3663 Zhongshan North Road, Shanghai 200062, China
2Optical Spectroscopy Section, Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1412
Surface-enhanced phenomena in Raman scattering (SERS), fluorescent emission, and nonlinear optical harmonic generation have been studied for decades. Raman spectra of tryptophan and tryptophan dipeptides of the form Trp-X and X-Trp, where X is another aminoacyl residue, have been investigated in silver sol solutions with a Jobin Yvon T64000 Raman system using the 514 nm line of an argon ion laser as the exciting source. The most prominent features in the SER spectra of all compounds are the strongly enhanced peaks at about 930 and 1370 cm-1 due to the C-COO- stretching and COO- symmetric stretching vibrational modes, respectively. Furthermore, steady state fluorescence and time-resolved emission/excitation spectra of dipeptide water solutions with or without silver nano-particles were obtained using a wide-band tunable ultraviolet upconversion spectrophotofluorometer with a temporal cross-correlation of less than 80fs (FWHM).
Martin vandeVen (Sponsor talk)
Digital frequency domain for fast FLIM measurements and phasor analysis: applications to FRET pairs.
S.-C. Liao1, M. vandeVen1,2, A. Periasamy3, E. Gratton4 and B. Barbieri1. 1ISS; 1602 Newton Dr., Champaign, IL 61822; 2Biomedical Research Institute, Hasselt University, B-3590 Diepenbeek, Belgium; 3Biology Department, University of Virginia, Charlottesville, VA 22904; 4Laboratory for Fluorescence Dynamics, Biomedical Engineering Department, University of California, Irvine, CA 92697
Fluorescence Lifetime Imaging (FLIM) in combination with a variety of fluorescence techniques is becoming an important tool in cell biology for the study of dynamic interactions of molecular species, for example protein-protein interactions, and for the intracellular mapping of ion concentration and pH imaging. We present FLIM data obtained with a new method called “Digital Frequency Domain” (DFD). It offers superior sensitivity, which results in a factor of four reduction in the time required for image acquisition as compared to the traditional approach using time-correlated single photon counting (TCSPC). This improves capturing images of motile samples, eg. live cells.
We report the measurement of Förster Resonance Energy Transfer (FRET) efficiency by using FLIM on mouse pituitary GHFT1 cells labeled with Cerulean (donor) and Venus (acceptor) fluorescent proteins. Several genetic constructs were developed tethering these fluorescent proteins via different amino acid linkers. A standard ISS FastFLIM unit was attached to an ISS Alba confocal FLIM microscope using one-photon excitation. The energy transfer efficiency was measured as a function of the FRET pair separation distance. Data analysis was achieved by using the phasor plot approach. Values for the FRET efficiencies could rapidly be obtained, which allowed for an immediate visualization of the location of FRET processes.
