Sylvain Trepout

Nanobody-functionalized PEG-b-PCL polymersomes and their targeting study.

We prepared and characterized polymersomes functionalized with nanobodies (VHHs) on the basis of biocompatible, biodegradable and FDA-approved poly(ethylene glycol)-block-poly(ϵ-caprolactone) (PEG-b-PCL). Fluorescein isothiocyanate (FITC) and N-beta-maleimidopropyl-oxysuccinimide ester were allowed reacting with H2N-PEG-b-PCL to produce FITC and maleimide (Mal) functionalized copolymers, Mal-PEG-b-PCL and FITC-PEG-b-PCL. A mixture of MeO-PEG-b-PCL, Mal-PEG-b-PCL and FITC-PEG-b-PCL was used to prepare polymersomes by thin film hydration and nanoprecipitation methods. Morphological studies by cryogenic transmission electron microscopy (Cryo-TEM) showed that the nanoparticles exhibited predominantly vesicular structures (polymersomes). Their mean diameters measured by dynamic light scattering were around 150 nm and the zeta-potentials around -1 mV at pH 7.4. The nanoparticles were functionalized with either anti-HER2 (VHH1) or anti-GFP (VHH2) nanobodies using maleimide-cysteine chemistry. Their particle size and zeta-potential increased slightly after nanobody-functionalization. The specific binding of VHH-functionalized polymersomes and control nanoparticles towards HER2 positive breast cancer cells was analyzed by flow cytometry and confocal microscopy. The collected results represent the first report which experimentally demonstrates that VHH1-functionalized PEO-b-PCL polymersomes can target specifically breast cancer cells expressing HER2 receptors. The detailed morphological and cell-binding studies described herein pave the way for future in vivo studies to evaluate the feasibility to use such nanoparticles for targeted drug delivery.

Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase

Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.

New insight into the structure and function of Hfq C-terminus.

Accumulating evidence indicates that RNA metabolism components assemble into supramolecular cellular structures to mediate functional compartmentalization within the cytoplasmic membrane of the bacterial cell. This cellular compartmentalization could play important roles in the processes of RNA degradation and maturation. These components include Hfq, the RNA chaperone protein, which is involved in the post-transcriptional control of protein synthesis mainly by the virtue of its interactions with several small regulatory ncRNAs (sRNA). The Escherichia coli Hfq is structurally organized into two domains. An N-terminal domain that folds as strongly bent β-sheets within individual protomers to assemble into a typical toroidal hexameric ring. A C-terminal flexible domain that encompasses approximately one-third of the protein seems intrinsically unstructured. RNA-binding function of Hfq mainly lies within its N-terminal core, whereas the function of the flexible domain remains controversial and largely unknown. In the present study, we demonstrate that the Hfq-C-terminal region (CTR) has an intrinsic property to self-assemble into long amyloid-like fibrillar structures in vitro. We show that normal localization of Hfq within membrane-associated coiled structures in vivo requires this C-terminal domain. This finding establishes for the first time a function for the hitherto puzzling CTR, with a plausible central role in RNA transactions.

Scanning transmission electron microscopy through-focal tilt-series on biological specimens

Since scanning transmission electron microscopy can produce high signal-to-noise ratio bright-field images of thick (≥500 nm) specimens, this tool is emerging as the method of choice to study thick biological samples via tomographic approaches. However, in a convergent-beam configuration, the depth of field is limited because only a thin portion of the specimen (from a few nanometres to tens of nanometres depending on the convergence angle) can be imaged in focus. A method known as through-focal imaging enables recovery of the full depth of information by combining images acquired at different levels of focus. In this work, we compare tomographic reconstruction with the through-focal tilt-series approach (a multifocal series of images per tilt angle) with reconstruction with the classic tilt-series acquisition scheme (one single-focus image per tilt angle). We visualised the base of the flagellum in the protist Trypanosoma brucei via an acquisition and image-processing method tailored to obtain quantitative and qualitative descriptors of reconstruction volumes. Reconstructions using through-focal imaging contained more contrast and more details for thick (≥500 nm) biological samples.

Overview of chemical imaging methods to address biological questions

Chemical imaging offers extensive possibilities for better understanding of biological systems by allowing the identification of chemical components at the tissue, cellular, and subcellular levels. In this review, we introduce modern methods for chemical imaging that can be applied to biological samples. This work is mainly addressed to the biological sciences community and includes the bases of different technologies, some examples of its application, as well as an introduction to approaches on combining multimodal data.

Bacterial kinesin light chain (Bklc) links the Btub cytoskeleton to membranes

Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today’s eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules.

Nonparametric Denoising Methods Based on Contourlet Transform with Sharp Frequency Localization: Application to Low Exposure Time Electron Microscopy Images

Image denoising is a very important step in cryo-transmission electron microscopy (cryo-TEM) and the energy filtering TEM images before the 3D tomography reconstruction, as it addresses the problem of high noise in these images, that leads to a loss of the contained information. High noise levels contribute in particular to difficulties in the alignment required for 3D tomography reconstruction. This paper investigates the denoising of TEM images that are acquired with a very low exposure time, with the primary objectives of enhancing the quality of these low-exposure time TEM images and improving the alignment process. We propose denoising structures to combine multiple noisy copies of the TEM images. The structures are based on Bayesian estimation in the transform domains instead of the spatial domain to build a novel feature preserving image denoising structures; namely: wavelet domain, the contourlet transform domain and the contourlet transform with sharp frequency localization. Numerical image denoising experiments demonstrate the performance of the Bayesian approach in the contourlet transform domain in terms of improving the signal to noise ratio (SNR) and recovering fine details that may be hidden in the data. The SNR and the visual quality of the denoised images are considerably enhanced using these denoising structures that combine multiple noisy copies. The proposed methods also enable a reduction in the exposure time.

Compressed sensing for dose reduction in STEM tomography

We designed a complete acquisition-reconstruction framework to reduce the radiation dosage in 3D scanning transmission electron microscopy (STEM). Projection measurements are acquired by randomly scanning a subset of pixels at every tilt-view (i.e., random-beam STEM or “RB-STEM”). High-quality images are then recovered from the randomly downsampled measurements through a regularized tomographic reconstruction framework. By fulfilling the compressed sensing requirements, the proposed approach improves the reconstruction of heavily-downsampled RB-STEM measurements over the current state-of-the-art technique. This development opens new perspectives in the search for methods permitting lower-dose 3D STEM imaging of electron-sensitive samples without degrading the quality of the reconstructed volume. A Matlab code implementing the proposed reconstruction algorithm has been made available online.

Bidirectional intraflagellar transport is restricted to two sets of microtubule doublets in the trypanosome flagellum

Intraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. In this study, we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Trypanosoma brucei. Focused ion beam scanning electron microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3–4 and 7–8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occurs on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3–4 s while remaining on the same side of the axoneme.

Fluorescent Polymersomes with Aggregation-Induced Emission

Fluorescent polymersomes are interesting systems for cell/tissue imaging and in vivo study of drug distribution and delivery. We report on bright fluorescent polymersomes with aggregation-induced emission self-assembled by a series of tetraphenylethylene (TPE)-containing amphiphilic biodegradable block copolymers, where the hydrophilic block is a polyethylene glycol and hydrophobic block is a TPE-substituted trimethylenecarbonate polymer P(TPE-TMC). Their self-assemblies in water were prepared by nanoprecipitation using dioxane or tetrahydrofuran as co-solvent, and the self-assembling processes were studied in detail by cryo-electron microscopy, dynamic light scattering, and spectrofluorometer. The polymersomes are formed via the closure of bilayer lamellae self-assembled first by amphiphilic block copolymers. The polymersome membrane affords a nanosize bright fluorescent system with self-assembly induced emission in the thickness scale of 10-15 nm. The control of the whole size of polymersome is achieved by the choice of co-solvent for self-assembling and by the design of a suitable hydrophilic/hydrophobic ratio of block copolymers. These polymersomes can be potentially used as a stable fluorescent tool to monitor the transportation and distribution of drugs and bioconjugates in living cells.