9 research outputs found

    Sonic hedgehog multimerization: A self-organizing event driven by post-translational modifications?

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    <p>Sonic hedgehog (Shh) is a morphogen active during vertebrate development and tissue homeostasis in adulthood. Dysregulation of the Shh signalling pathway is known to incite carcinogenesis. Due to the highly lipophilic nature of this protein imparted by two post-translational modifications, Shh’s method of transit through the aqueous extracellular milieu has been a long-standing conundrum, prompting the proposition of numerous hypotheses to explain the manner of its displacement from the surface of the producing cell. Detection of high molecular-weight complexes of Shh in the intercellular environment has indicated that the protein achieves this by accumulating into multimeric structures prior to release from producing cells. The mechanism of assembly of the multimers, however, has hitherto remained mysterious and contentious. Here, with the aid of high-resolution optical imaging and post-translational modification mutants of Shh, we show that the C-terminal cholesterol and the N-terminal palmitate adducts contribute to the assembly of large multimers and regulate their shape. Moreover, we show that small Shh multimers are produced in the absence of any lipid modifications. Based on an assessment of the distribution of various dimensional characteristics of individual Shh clusters, in parallel with deductions about the kinetics of release of the protein from the producing cells, we conclude that multimerization is driven by self-assembly underpinned by the law of mass action. We speculate that the lipid modifications augment the size of the multimolecular complexes through prolonging their association with the exoplasmic membrane.</p

    Experimental Proof of Concept of Nanoparticle-Assisted STED

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    We imaged core–shell nanoparticles, consisting of a dye-doped silica core covered with a layer of gold, with a stimulated emission depletion, fluorescence lifetime imaging (STED-FLIM) microscope. Because of the field enhancement provided by the localized surface plasmon resonance of the gold shell, we demonstrate a reduction of the STED depletion power required to obtain resolution improvement by a factor of 4. This validates the concept of nanoparticle-assisted STED (NP-STED), where hybrid dye-plasmonic nanoparticles are used as labels for STED in order to decrease the depletion powers required for subwavelength imaging

    Analysis of DNA Binding and Nucleotide Flipping Kinetics Using Two-Color Two-Photon Fluorescence Lifetime Imaging Microscopy

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    Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the “binding” and “base flipping” steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations

    Multispectral fluorescence intensity and FLIM images acquired from normal skin and BCCs.

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    <p>(a–i) Fluorescence intensity and false color FLIM images from a single field of view acquired at a depth of 110 µm with all spectral channels taken near a dermal papilla from normal skin. (j–n) FLIM images taken from the green channel only of different depths within a sample of normal skin. (o–u) FLIM images taken from the green channel illustrating visual architectural features seen in BCC using MPT. (v) FLIM image taken from the blue channel of a BCC. (w,x) paired FLIM images taken from the green and blue channels respectively of a BCC nest. KEY SG-Stratum Granulosum, SS- Stratum Spinosum, BCL-Basal Cell Layer, DP-Dermal Papilla. Scale bar 25 µm.</p

    Exemplar segmented fluorescence intensity images, fitted fluorescence decay and fluorophore emission spectra.

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    <p>(a) Total fluorescence intensity image, same image with (b) manually and (c) automatically defined cellular regions of interest overlayed. (d) Top – exemplar fluorescence decay from one region of interest (black), biexponential fit to data (green) and instrument response function (blue). (d) Bottom – normalized residuals. (e) The emission spectra from endogenous fluorophores plotted in relation to the four spectral detection channels.</p
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