Multimodal Imaging Center (US43-UMS2016)


Year of publication 2020

Adrià Sogues, Mariano Martinez, Quentin Gaday, Mathilde Ben Assaya, Martin Graña, Alexis Voegele, Michael VanNieuwenhze, Patrick England, Ahmed Haouz, Alexandre Chenal, Sylvain Trépout, Rosario Duran, Anne Marie Wehenkel, Pedro M Alzari (2020 Apr 4)

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum.

Nature communications : 1641 : DOI : 10.1038/s41467-020-15490-8 Learn more

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

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Emilie Mathieu, Anne-Sophie Bernard, H Y Vincent Ching, Andrea Somogyi, Kadda Medjoubi, Jennifer Rodon Fores, Hélène C Bertrand, Amandine Vincent, Sylvain Trépout, Jean-Luc Guerquin-Kern, Andreas Scheitler, Ivana Ivanović-Burmazović, Philippe Seksik, Nicolas Delsuc, Clotilde Policar (2020 Feb 6)

Anti-inflammatory activity of superoxide dismutase mimics functionalized with cell-penetrating peptides.

Dalton transactions (Cambridge, England : 2003) : 49 : 2323-2330 : DOI : 10.1039/c9dt04619d Learn more

A superoxide dismutase mimic (Mn1) was functionalized with three positively charged-peptides: RRRRRRRRR (Mn1-R9), RRWWWRRWRR (Mn1-RW9) or F-r-F-K (Mn1-MPP). Characterization of the physico-chemical properties of the complexes show that they share similar binding affinity for Mn, apparent reduction potential and intrinsic superoxide dismutase activity. However, their accumulation in cells is different (Mn1-R9 < Mn1-MPP < Mn1-RW9 < Mn1), as well as their subcellular distribution. In addition, the three functionalized-complexes display a better anti-inflammatory activity than Mn1 when assayed at 10 μM. This improvement is due to a combination of an anti-inflammatory effect of the peptidyl moiety itself, and of the SOD mimic for Mn1-RW9 and Mn1-MPP. In contrast, the enhanced anti-inflammatory activity of Mn1-R9 is solely due to the SOD mimic.

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Year of publication 2019

Tao X., Chen H., Trepout S., Cen J., Ling J., Li M.H. (2019 Oct 15)

Polymersomes with Aggregation-Induced Emission Based on Amphiphilic Block Copolypeptoids

Chem. Commun. : 55 : -13530-13533 : DOI : 10.1039/C9CC07501A Learn more

Biocompatible polymersomes are prepared from amphiphilic block copolypeptoids with aggregation-induced emission, where the hydrophobic block P(TPE-NAG) is a tetraphenylethylene (TPE)-modified poly(N-allylglycine) and the hydrophilic block is polysarcosine. These nanoparticles are non-cytotoxic and show strong fluorescence emission in aqueous solution.

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Sylvain Trépout (2019 Jul 19)

Tomographic Collection of Block-Based Sparse STEM Images: Practical Implementation and Impact on the Quality of the 3D Reconstructed Volume.

Materials (Basel, Switzerland) : DOI : E2281 Learn more

The reduction of the electron dose in electron tomography of biological samples is of high significance to diminish radiation damages. Simulations have shown that sparse data collection can perform efficient electron dose reduction. Frameworks based on compressive-sensing or inpainting algorithms have been proposed to accurately reconstruct missing information in sparse data. The present work proposes a practical implementation to perform tomographic collection of block-based sparse images in scanning transmission electron microscopy. The method has been applied on sections of chemically-fixed and resin-embedded cells. There are 3D reconstructions obtained from various amounts of downsampling, which are compared and eventually the limits of electron dose reduction using this method are explored.

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David Partouche, Jérémie Mathurin, Antoine Malabirade, Sergio Marco, Christophe Sandt, Véronique Arluison, Ariane Deniset-Besseau, Sylvain Trépout (2019 Apr 1)

Correlative infrared nanospectroscopy and transmission electron microscopy to investigate nanometric amyloid fibrils: prospects and challenges.

Journal of microscopy : 274 : 23-31 : DOI : 10.1111/jmi.12779 Learn more

Propagation of structural information through conformational changes in host-encoded amyloid proteins is at the root of many neurodegenerative disorders. Although important breakthroughs have been made in the field, fundamental issues like the 3D-structures of the fibrils involved in some of those disorders are still to be elucidated. To better characterise those nanometric fibrils, a broad range of techniques is currently available. Nevertheless none of them is able to perform direct chemical characterisation of single protein fibrils. In this work, we propose to investigate the structure of the C-terminal region of a bacterial protein called Hfq as a model amyloidogenic protein, using a correlative approach. The complementary techniques used are transmission electron microscopy and a newly developed infrared nanospectroscopy technique called AFM-IR. We introduce and discuss the strategy that we have implemented as well as the protocol, challenges and difficulties encountered during this study to characterise amyloid assemblies at the nearly single-molecule level. LAY DESCRIPTION: Propagation of structural information through conformational changes in amyloid proteins is at the root of many neurodegenerative disorders. Amyloids are nanostructures originating from the aggregation of multiple copies of peptide or protein monomers that eventually form fibrils. Often described as being the cause for the development of various diseases, amyloid fibrils are of major significance in the public health domain. While important breakthroughs have been made in the field, fundamental issues like the 3D-structures of the fibrils implied in some of those disorders are still to be elucidated. To better characterise these fibrils, a broad range of techniques is currently available for the detection and visualisation of amyloid nanostructures. Nevertheless none of them is able to perform direct chemical characterisation of single protein fibrils. In this work, we propose to investigate the structure of model amyloidogenic fibrils using a correlative approach. The complementary techniques used are transmission electron microscopy and a newly developed infrared nanospectroscopy technique called AFM-IR that allows chemical characterisation at the nanometric scale. The strategy, protocol, challenges and difficulties encountered in this approach are introduced and discussed herein.

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Year of publication 2018

Ptissam Bergam, Johannes M Reisecker, Zsófia Rakvács, Nóra Kucsma, Graça Raposo, Gergely Szakacs, Guillaume van Niel (2018 Jun 26)

ABCB6 Resides in Melanosomes and Regulates Early Steps of Melanogenesis Required for PMEL Amyloid Matrix Formation.

Journal of molecular biology : 3802-3818 : DOI : S0022-2836(18)30662-4 Learn more

Genetically inheritable pigmentation defects provide a unique opportunity to reveal the function of proteins contributing to melanogenesis. Dyschromatosis universalis hereditaria (DUH) is a rare pigmentary genodermatosis associated with mutations in the ABCB6 gene. Here we use optical and electron microscopy imaging combined with biochemical tools to investigate the localization and function of ABCB6 in pigment cells. We show that ABCB6 localizes to the membrane of early melanosomes and lysosomes of the human melanocytic cell line MNT-1. Depletion of ABCB6 by siRNA impaired PMEL amyloidogenesis in early melanosomes and induced aberrant accumulation of multilamellar aggregates in pigmented melanosomes. PMEL fibril formation and normal maturation of pigmented melanosomes could be restored by the overexpression of wild-type ABCB6 but not by variants containing an inactivating catalytic mutation (K629M) or the G579E DUH mutation. In line with the impairment of PMEL matrix formation in the absence of ABCB6, morphological analysis of the retinal pigment epithelium of ABCB6 knockout mice revealed a significant decrease of melanosome numbers. Our study extends the localization of ABCB6 to melanosomes, suggesting a potential link between the function of ABCB6 and the etiology of DUH to amyloid formation in pigment cells.

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Year of publication 2017

Sylvain Trépout, Anne-Marie Tassin, Sergio Marco, Philippe Bastin (2017 Dec 18)

STEM tomography analysis of the trypanosome transition zone.

Journal of structural biology : 51-60 : DOI : S1047-8477(17)30228-9 Learn more

The protist Trypanosoma brucei is an emerging model for the study of cilia and flagella. Here, we used scanning transmission electron microscopy (STEM) tomography to describe the structure of the trypanosome transition zone (TZ). At the base of the TZ, nine transition fibres irradiate from the B microtubule of each doublet towards the membrane. The TZ adopts a 9 + 0 structure throughout its length of ∼300 nm and its lumen contains an electron-dense structure. The proximal portion of the TZ has an invariant length of 150 nm and is characterised by a collarette surrounding the membrane and the presence of electron-dense material between the membrane and the doublets. The distal portion exhibits more length variation (from 55 to 235 nm) and contains typical Y-links. STEM analysis revealed a more complex organisation of the Y-links compared to what was reported by conventional transmission electron microscopy. Observation of the very early phase of flagellum assembly demonstrated that the proximal portion and the collarette are assembled early during construction. The presence of the flagella connector that maintains the tip of the new flagellum to the side of the old was confirmed and additional filamentous structures making contact with the membrane of the flagellar pocket were also detected. The structure and potential functions of the TZ in trypanosomes are discussed, as well as its mode of assembly.

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Sylvain Trépout, Anne Marie Wehenkel (2017 Sep 5)

Bacterial Tubulins: A Eukaryotic-Like Microtubule Cytoskeleton.

Trends in microbiology : 782-784 : DOI : S0966-842X(17)30194-4 Learn more

Ever since their discovery, bacterial tubulins, found in several Prosthecobacter species, have raised curiosity as they are closely related to eukaryotic tubulin. Deng and colleagues now present new evidence for the functional homology of the two cytoskeletal systems where in vitro reconstituted Btub-microtubules display eukaryote-like biochemical and dynamic properties.

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Ilse Hurbain, Maryse Romao, Ptissam Bergam, Xavier Heiligenstein, Graça Raposo (2017 May 1)

Analyzing Lysosome-Related Organelles by Electron Microscopy.

Methods in molecular biology (Clifton, N.J.) : 43-71 : DOI : 10.1007/978-1-4939-6934-0_4 Learn more

Intracellular organelles have a particular morphological signature that can only be appreciated by ultrastructural analysis at the electron microscopy level. Optical imaging and associated methodologies allow to explore organelle localization and their dynamics at the cellular level. Deciphering the biogenesis and functions of lysosomes and lysosome-related organelles (LROs) and their dysfunctions requires their visualization and detailed characterization at high resolution by electron microscopy. Here, we provide detailed protocols for studying LROs by transmission electron microscopy. While conventional electron microscopy and its recent improvements is the method of choice to investigate organelle morphology, immunoelectron microscopy allows to localize organelle components and description of their molecular make up qualitatively and quantitatively.

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Year of publication 2014

Guillaume van Niel, Ptissam Bergam, Aurelie Di Cicco, Ilse Hurbain, Alessandra Lo Cicero, Florent Dingli, Roberta Palmulli, Cecile Fort, Marie Claude Potier, Leon J Schurgers, Damarys Loew, Daniel Levy, Graça Raposo (2014 Nov 13)

Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells.

Cell reports : 43-51 : DOI : 10.1016/j.celrep.2015.08.057 Learn more

Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

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