Confocal Fluorescence Anisotropy and FRAP Imaging of α-Synuclein Amyloid Aggregates in Living Cells

We assessed the intracellular association states of the Parkinson's disease related protein α-synuclein (AS) in living cells by transfection with a functional recombinant mutant protein (AS-C4) bearing a tetracysteine tag binding the fluorogenic biarsenical ligands FlAsH and ReAsH, The aggregation states of AS-C4 were assessed by in situ microscopy of molecular translational mobility with FRAP (fluorescence recovery after photobleaching) and of local molecular density with confocal fluorescence anisotropy (CFA). FRAP recovery was quantitative and rapid in regions of free protein, whereas AS in larger aggregates was>80% immobile. A small 16% recovery characterized by an apparent diffusion constant of 0.03–0.04 µm2/s was attributed to the dynamics of smaller, associated forms of AS-C4 and the exchange of mobile species with the larger immobile aggregates. By CFA, the larger aggregates exhibited high brightness and very low anisotropy, consistent with homoFRET between closely packed AS, for which a Förster distance (Ro) of 5.3 nm was calculated. Other bright regions had high anisotropy values, close to that of monomeric AS, and indicative of membrane-associated protein with both low mobility and low degree of association. The anisotropy-fluorescence intensity correlations also revealed regions of free protein or of small aggregates, undetectable by conventional fluorescence imaging alone. The combined strategy (FRAP+CFA) provides a highly sensitive means for elucidating both the dynamics and structural features of protein aggregates and other intracellular complexes in living cells, and can be extended to other amyloid systems and to drug screening protocols

High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy

Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of more than 1 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01-1 µm. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells. © The Author(s). Published by IOP Publishing Ltd

Cryptoporic and isocryptoporic acids from the fungal cultures of Polyporus arcularius and P. ciliatus

In a chemical study of several fungal cultures of Polyporus, a methyl ester of cryptoporic H was isolated from P. ciliatus, together with cryptoporic acid H and 5-hydroxymethylfuran-3-carboxylic acid. Furthermore, two additional compounds, named isocryptoporic acids H and I, were isolated from P. arcularius. These isocryptoporic acids are isomers of the cryptoporic acids with drimenol instead of albicanol as the terpenoid fragment; their structural elucidation was determined by application of spectroscopic methods.Fil: Cabrera, Gabriela Myriam. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Roberti, Maria Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Wright, Jorge E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Prog.hongos Que Interv.en Degrad.biológica (p); ArgentinaFil: Seldes, Alicia M.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentin

Confocal fluorescence anisotropy (CFA) microscopy of AS-C4-FlAsH aggregates in SH-SY5Y cells.

<p>(A) CFA image (<i>I_f,</i>) and (B) associated fluorescence anisotropy (<i>r</i>) image. (C) 2D-histogram <i>I_f</i> vs. <i>r</i>. The histogram values were sectioned using threshold values for <i>I_f</i> and <i>r</i>, and the resulting (<i>I_f</i>, <i>r</i>) pairs were backmapped on a pixel-by-pixel basis onto the fluorescence intensity image (red pixels). Four distinct groups were defined: (i) pixels with high <i>I_f</i> and low <i>r</i> (top left), (ii) with high <i>I</i>_f and <i>r</i> (top right), (iii) low <i>I</i>_f and <i>r</i> (bottom left), and (iv) low <i>I</i>_f and high <i>r</i> (bottom right). The colored scale bar represents frequency (number of pixels). The insets in the images in groups (i) and (ii) show an example of an aggregate that displays high <i>I</i>_f and both low and high <i>r</i>, reflecting different dynamics of AS-C4 within its structure.</p

FRAP microscopy of fluorescently labeled AS-C4 aggregates in SH-SY5Y cells.

<p>(A) Cells were transiently transfected to express the biarsenical-binding AS-C4 version of AS, and ReAsH labeling allowed the identification of aggregated and non-aggregated regions with the protein, as opposed to non-transfected control samples that only exhibit a low background staining signal (the transmission image was included for a proper observation of the location of the cell). For a typical FRAP experiment performed on one cell, the regions of interest (<i>ROI</i>s, diameter ∼4 µm) before (B) and immediately after (C) photobleaching, are shown (colored circles): <i>ROI</i> with aggregates (yellow), <i>ROI</i> with no apparent aggregated AS-C4 (green), scanning-photobleaching control <i>ROI</i> (blue), and background <i>ROI</i> (white). (D) Normalized fluorescence recovery for the corresponding ROIs and their fits according to a simple monoexponential model. Fluorescence intensity values before (<i>I_i</i>) and after (<i>I₀</i>) photobleaching, and at the end of the experiment (<i>I_∞</i>), are shown. The green arrow indicates the region of fast recovery for non-aggregated protein. The inset images correspond to the <i>ROI</i> with aggregated protein before photobleaching (<i>t</i> = 0 s), immediately after photobleaching (<i>t</i> = 17 s) and post-bleaching at <i>t</i> = 60, 90 and 120 s. Data shown as means ± standard errors (sample set <i>n</i> = 5).</p

Correlative live and super-resolution imaging reveals the dynamic structure of replication domains

International audienceChromosome organization in higher eukaryotes controls gene expression, DNA replication, and DNA repair. Genome mapping has revealed the functional units of chromatin at the submegabase scale as self-interacting regions called topologically associating domains (TADs) and showed they correspond to replication domains (RDs). A quantitative structural and dynamic description of RD behavior in the nucleus is, however, missing because visualization of dynamic subdiffraction-sized RDs remains challenging. Using fluorescence labeling of RDs combined with correlative live and super-resolution microscopy in situ, we determined biophysical parameters to characterize the internal organization, spacing, and mechanical coupling of RDs. We found that RDs are typically 150 nm in size and contain four co-replicating regions spaced 60 nm apart. Spatially neighboring RDs are spaced 300 nm apart and connected by highly flexible linker regions that couple their motion only <550 nm. Our pipeline allows a robust quantitative characterization of chromosome structure in situ and provides important biophysical parameters to understand general principles of chromatin organization

Engineered HaloTag variants for fluorescence lifetime multiplexing

HaloTag variants offer distinct brightness and fluorescence lifetimes compared with HaloTag7 when labeled with rhodamines. These variants were used for multiplexed imaging with a single fluorophore and to create lifetime-based cell cycle indicators.Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.LI

Imaging nanometer-sized α-synuclein aggregates by superresolution fluorescence localization microscopy

The morphological features of alpha-synuclein (AS) amyloid aggregation in vitro and in cells were elucidated at the nanoscale by far-field subdiffraction fluorescence localization microscopy. Labeling AS with rhodamine spiroamide probes allowed us to image AS fibrillar structures by fluorescence stochastic nanoscopy with an enhanced resolution at least 10-fold higher than that achieved with conventional, diffraction-limited techniques. The implementation of dual-color detection, combined with atomic force microscopy, revealed the propagation of individual fibrils in vitro. In cells, labeled protein appeared as amyloid aggregates of spheroidal morphology and subdiffraction sizes compatible with in vitro supramolecular intermediates perceived independently by atomic force microscopy and cryo-electron tomography. We estimated the number of monomeric protein units present in these minute structures. This approach is ideally suited for the investigation of the molecular mechanisms of amyloid formation both in vitro and in the cellular milieu.Fil: Roberti, Maria Julia. Max Planck Institute For Biophysical Chemistry (karl Friedrich Bonhoeffer Institute); . Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Fölling, Jonas. Institut Max Planck Fuer Gesellschaft; AlemaniaFil: Celej, Maria Soledad. Institut Max Planck Fuer Gesellschaft; AlemaniaFil: Bossi, Mariano Luis. Institut Max Planck Fuer Gesellschaft; AlemaniaFil: Jovin, Thomas M.. Institut Max Planck Fuer Gesellschaft; AlemaniaFil: Jares, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentin