Angewandte Chemie International Edition, 2022

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity

Vladimir Khayenko,Clemens Schulte,Sara L. Reis,Dr. Orly Avraham,Dr. Cataldo Schietroma,Rafael Worschech,Noah F. Nordblom,Sonja Kachler,Dr. Carmen Villmann,Dr. Katrin G. Heinze,Dr. Andreas Schlosser,Dr. Ora Schueler-Furman,Dr. Philip Tovote,Dr. Christian G. Specht,Dr. Hans M. Maric
Article reference https://doi.org/10.1002/anie.202202078
Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding “Sylite”—a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.
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BioRxiv 2022

Escherichia coli membrane microdomain SPFH protein HflC interacts with YajC and contributes to aminoglycoside and oxidative stress tolerance

Aimee K. Wessel, Yutaka Yoshii, Alexander Reder, Rym Boudjemaa, Magdalena Szczesna, Jean-Michel Betton, Joaquin Bernal-Bayard, Christophe Beloin, Daniel Lopez, Uwe Völker, Jean-Marc Ghigo
Article reference https://doi.org/10.1101/2022.07.25.501378
Here we studied the determinants of the native, chromosomal QmcA and HflC cell localization using a domain swap analysis and fluorescent and super-resolution microscopy. We showed that full QmcA and HflC protein is required for achieving their native inner-membrane localization and that impairing the synthesis of cardiolipin and isoprenoid lipids known to associate with FMMs alters QmcA and HflC localization pattern. Finally, using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress. This phenotype is exclusively due to the absence of HflKC and a cross-linking and mass spectrometry analysis showed that YajC, a SecDF translocon accessory protein, interacts with HflC and also contributes to E. coli stress tolerance. Our study therefore provides insights into the function and interactions associated with SPFH proteins in E. coli FMMs.
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Nature Communications, 2021

Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields

Adrien Mau, Karoline Friedl, Christophe Leterrier, Nicolas Bourg & Sandrine Lévêque-Fort
Article reference https://doi.org/10.1038/s41467-021-23405-4
Non-uniform illumination limits quantitative analyses offluorescence imaging techniques. Inparticular, single molecule localization microscopy (SMLM) relies on high irradiances, butconventional Gaussian-shaped laser illumination restricts the usablefield of view to around40μm×40μm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), aversatile illumination technique that generates uniform and adaptable illumination. ASTER isalso highly compatible with optical sectioning techniques such as total internal reflectionfluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonablelaser power overfields-of-view up to 200μm × 200μm. We demonstrate that ASTERimproves clustering analysis and nanoscopic size measurements by imaging nanorulers,microtubules and clathrin-coated pits in COS-7 cells, andβ2-spectrin in neurons. ASTER’ssharp and quantitative illumination paves the way for high-throughput quantification ofbiological structures and processes in classical and super-resolutionfluorescencemicroscopies.
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Progress in Neurobiology,2021

Mutual functional dependence of cyclase-associated protein 1 (CAP1) and cofilin1 in neuronal actin dynamics and growth cone function

Felix Schneider, Thuy-An Duong, Isabell Metz, Jannik Winkelmeier, Christian A. Hübner, Ulrike Endesfelder, Marco B. Rust
Article reference https://doi.org/10.1016/j.pneurobio.2021.102050.
Abstract: Neuron connectivity depends on growth cones that navigate axons through the developing brain. Growth cones protrude and retract actin-rich structures to sense guidance cues. These cues control local actin dynamics and steer growth cones towards attractants and away from repellents, thereby directing axon outgrowth. Hence, actin binding proteins (ABPs) moved into the focus as critical regulators of neuron connectivity. We found cyclase-associated protein 1 (CAP1), an ABP with unknown brain function, abundant in growth cones. Super-resolution microscopy and live cell imaging combined with pharmacological approaches on hippocampal neurons from gene-targeted mice revealed a crucial role for CAP1 in actin dynamics that is critical for growth cone morphology and function. Growth cone defects in CAP1 knockout (KO) neurons compromised neuron differentiation and was associated with impaired neuron connectivity in CAP1-KO brains. Mechanistically, by rescue experiments in double KO neurons lacking CAP1 and the key actin regulator cofilin1, we demonstrated that CAP1 was essential for cofilin1 function in growth cone actin dynamics and morphology and vice versa. Together, we identified CAP1 as a novel actin regulator in growth cones that was relevant for neuron connectivity, and we demonstrated functional interdependence of CAP1 and cofilin1 in neuronal actin dynamics and growth cone function.
Keywords: Actin dynamics; Growth cone; Axon outgrowth; Srv2; Cyclase-associated protein; Cofilin
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Nature Photonics 2021

Nanometric axial localization of single fluorescent molecules with modulated excitation

Pierre Jouchet, Clément Cabriel, Nicolas Bourg, Marion Bardou, Christian Poüs, Emmanuel Fort & Sandrine Lévêque-Fort
Article reference doi: https://doi.org/10.1101/865865
Distance measurements are commonly performed by phase detection based on a lock-in strategy. Super-resolution fluorescence microscopy is still striving to perform axial localization but through entirely different strategies. Here we show that an illumination modulation approach can achieve nanometric axial localization precision without compromising the acquisition time, emitter density or lateral localization precision. The excitation pattern is obtained by shifting tilted interference fringes. The molecular localizations are performed by measuring the relative phase between each fluorophore response and the reference modulated excitation pattern. We designed a fast demodulation scheme compatible with the short emission duration of single emitters. This modulated localization microscopy offers a typical axial localization precision of 6.8 nm over the entire field of view and the axial capture range. Furthermore, the interfering pattern being robust to optical aberrations, a nearly uniform axial localization precision enables imaging of biological samples by up to several micrometres in depth.
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eLife Sciences, 2021

Full assembly of HIV-1 particles requires assistance of themembrane curvature factor IRSp53

Kaushik Inamdar, Feng-Ching Tsai, Aurore de Poret, Rayane Dibsy, John Manzi, Peggy Merida, Remi Muller, PekkaLappalainen, Philippe Roingeard, Johnson Mak, Patricia Bassereau, Cyril Favard, and Delphine Muriaux
Article reference https://doi.org/10.7554/eLife.67321
During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. siRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single-molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.
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Methods Volume 193, September 2021, Pages 107-115

Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework

Koen J.A.Martens, Abbas Jabermoradi, Suyeon Yang, Johannes Hohlbein
Article reference https://doi.org/10.1016/j.ymeth.2020.07.010
In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub-pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies.
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BioOptics 2021

STORM made easy for high-throughput research

Valentina Caorsi, Håkan Karlsson
Article reference
Single-molecule localization microscopy (SMLM) methods (PALM,1,2 PAINT,3 and STORM4,5) have become important tools to access biological information at the nanoscale. These techniques can resolve structures with a quasi-isotropic 3D localization precision of ~10 nm.6 However, SMLM is reliant on fluorophore photoswitching characteristics,7 which depend on the wavelength and significant chromatic aberration at the nanoscale. These challenges are hampering straightforward, multicolor imaging with SMLM. So far, the multicolor information has been accessed by sequential acquisitions at different excitation wavelengths, which on the other hand induce uncontrollable drifts between the different color recordings.

But now, researchers have demonstrated a novel solution where only a single laser is used to excite multiple fluorophores in the same spectral range (far-red) coupled to a spectral demixing strategy that overcomes these limits and permits 3D multicolor nanoscopy with increased simplicity, speed, and reliability of output data.

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BioRxiv 2021

Concentration of intraflagellar transport proteins at the ciliary baseis required for proper train injection

Jamin Jung, Julien Santi-Rocca, Cécile Fort, Jean-Yves Tinevez, Cataldo Schietroma and Philippe Bastin
Article reference doi: https://doi.org/10.1101/2021.08.02.454739
Construction of cilia and flagella relies on Intraflagellar Transport (IFT). Although IFT proteins can be found in multiple locations in the cell, transport has only been reported along the axoneme. Here, we reveal that IFT concentration at the base of the flagellum of Trypanosoma brucei is required for proper assembly of IFT trains. Using live cell imaging at high resolution and direct optical nanoscopy with axially localized detection (DONALD) of fixed trypanosomes, we demonstrate that IFT proteins are localised around the 9 doublet microtubules of the proximal portion of the transition zone, just on top of the transition fibres. Super-resolution microscopy and photobleaching studies reveal that knockdown of the RP2 transition fibre protein results in reduced IFT protein concentration and turnover at the base of the flagellum. This in turn is accompanied by a 4- to 8-fold drop in the frequency of IFT train injection. We propose that the flagellum base provides a unique environment to assemble IFT trains.
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BioRxiv 2021

Visualization of bacterial type 3 secretion system components down to the molecular level by MINFLUX nanoscopy

Alexander Carsten, Maren Rudolph, Tobias Weihs, Roman Schmidt, Christian A. Wurm, Andreas Diepold, Antonio Virgilio Failla, Manuel Wolters, Martin Aepfelbacher
Article reference doi: https://doi.org/10.1101/2021.09.27.461991
Type 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.
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Nature Communications 2021

Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

Thomas Orré, Adrien Joly, Zeynep Karatas, Birgit Kastberger, Clément Cabriel, Ralph T. Böttcher, Sandrine Lévêque-Fort, Jean-Baptiste Sibarita, Reinhard Fässler, Bernhard Wehrle-Haller, Olivier Rossier & Grégory Giannone
Article reference Nature Communications volume 12, Article number: 3104 (2021)
Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.
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eLife Sciences 2021

Nanoscale architecture and coordination of actin cores within the sealing zone of human osteoclasts

Marion Portes, Thomas Mangeat, Natacha Escallier, Ophélie Dufrancais, Brigitte Raynaud-Messina, Christophe Thibault, Isabelle Maridonneau-Parini, Christel Vérollet, Renaud Poincloux
Article reference https://doi.org/10.7554/eLife.75610
Osteoclasts are unique in their capacity to degrade bone tissue. To achieve this process, osteoclasts form a specific structure called the sealing zone, which creates a close contact with bone and confines the release of protons and hydrolases for bone degradation. The sealing zone is composed of actin structures called podosomes nested in a dense actin network. The organization of these actin structures inside the sealing zone at the nano scale is still unknown. Here, we combine cutting-edge microscopy methods to reveal the nanoscale architecture and dynamics of the sealing zone formed by human osteoclasts on bone surface. Random illumination microscopy allowed the identification and live imaging of densely packed actin cores within the sealing zone. A cross-correlation analysis of the fluctuations of actin content at these cores indicates that they are locally synchronized. Further examination shows that the sealing zone is composed of groups of synchronized cores linked by α-actinin1 positive filaments, and encircled by adhesion complexes. Thus, we propose that the confinement of bone degradation mediators is achieved through the coordination of islets of actin cores and not by the global coordination of all podosomal subunits forming the sealing zone.

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BioRxiv 2021

Dynamin-2 controls complement receptor 3- mediated phagocytosis completion and closure

Anna Mularski, Ryszard Wimmer, Floriane Arbaretaz, Gabriel Le Goff1, and Florence Niedergang
Article reference doi: https://doi.org/10.1101/2021.02.14.431164
Phagocytosis is the mechanism of the internalization of large particles, microorganisms and cellular debris. The complement pathway represents one of the first mechanisms of defense against infection and the complement receptor 3 (CR3), which is highly expressed on macrophages, is a major receptor for many pathogens and debris. Key to dissecting the mechanisms by which CR3-mediated phagocytosis occurs, is understanding how the complex actin binding protein machinery and associated regulators interact with actin during phagocytosis, from triggering of receptor, through to phagosome formation and closure. However, how CR3-mediated phagosome completion and closure are orchestrated is not known. Here, we reveal that dynamin-2 is recruited concomitantly with polymerised actin at the site of the nascent phagosomes and accumulates until membrane scission. Inhibition of dynamin activity leads to stalled phagocytic cups and a decrease in the amount of F-actin at the site of phagocytosis. Acute inhibition of dynamin activity in living phagocytosing cells established that dynamin-2 plays a critical role in the effective scission of the CR3-phagosome from the plasma membrane. Thus, dynamin-2 has two distinct roles in CR3-mediated phagocytosis, in the assembly of the F-actin phagocytic cup and during phagosome scission.
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Nature Materials 2020

Stress fibres are embedded in a contractile cortical network

Timothée Vignaud, Calina Copos, Christophe Leterrier, Mauricio Toro-Nahuelpan, Qingzong Tseng, Julia Mahamid, Laurent Blanchoin, Alex Mogilner, Manuel Théry, and Laetitia Kurzawa
Article reference doi: 10.1038/s41563-020-00825-z
Contractile actomyosin networks are responsible for the production of intracellular forces. There is increasing evidence that bundles of actin filaments form interconnected and interconvertible structures with the rest of the network. In this study, we explored the mechanical impact of these interconnections on the production and distribution of traction forces throughout the cell. By using a combination of hydrogel micropatterning, traction-force microscopy and laser photoablation, we measured the relaxation of traction forces in response to local photoablations. Our experimental results and modeling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignement and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.
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Journal of Cell Science 2020

Myosin 1b flattens and prunes branched actin filaments

Julien Pernier, Antoine Morchain, Valentina Caorsi, Aurélie Bertin, Hugo Bousquet, Patricia Bassereau , Evelyne Coudrier
Article reference J Cell Sci (2020) 133 (18): jcs247403. doi:10.1242/jcs.247403
Motile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins, including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Here, by performing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy, we show that this molecular motor flattens (reduces the branch angle) in the Arp2/3-dependent actin branches, resulting in them breaking, and reduces the probability of new branches forming. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and dynamics of actin networks in different cellular regions.
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Frontiers in Physics 2020

Ionizing Radiation Effects onHs 578Bst Microtubules

L. Bruni, M. Manghi, E. Gioscio, V. Caorsi, F. M. Rizzi and S. Croci
Article reference https://doi.org/10.3389/fphy.2020.579081
Microtubules are one of the three components of the cell cytoskeleton. They are hollow wires, with a diameter of about 25 nm, formed by 13 laterally associated protofilaments composed by dimers of α and β-tubulin. Microtubules are dynamic structures which undergo constant modifications by shrinking and elongating in a phenomenon called treadmilling. Microtubules intervene in various fundamental aspects of the biology of a cell. They contribute to determine the shape of a cell, play a role in the cell movement, and in the intracellular transport of organelles during motion and mitotic chromosome segregation. Despite the relevance of the processes mediated by microtubules most studies on the effects of ionizing radiations focus their attention on the damages delivered to DNA. In this paper we attempt to assess the effects borne by IRs to the microtubules network as a biological target. In this study we irradiated Hs 578Bst cells (a no-cancer, no-immortalized, human breast epithelial cell line) with an 8 Gy single dose of either X-rays or protons. After the irradiated cells fixation, the microtubules were imaged by means of stochastic optical reconstruction microscopy to characterize the network disruption. In our results, Microtubules fibers integrity appears to not have been significantly affected at the administered dose of protons and X-rays, nonetheless we observed differences in the MT network distribution and fiber curvatures.
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mBio2020

The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori

Alejandro Tejada-Arranz, Eloïse Galtier, Lamya El Mortaji, Evelyne Turlin, Dmitry Ershov, Hilde De Reuse
Article reference DOI: https://doi.org/10.1128/mBio.01173-20
Posttranscriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation, and some were reported to be compartmentalized inside these organelleless cells. The minimal RNA degradosome of the important gastric pathogen Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and superresolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes nor by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into nonpolar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote focus formation in H. pylori. Finally, RNase J-GFP (RNase J-green fluorescent protein) molecules and foci in cells were quantified by three-dimensional (3D) single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity.
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BioRxiv, 2020

Quantitative dSTORM super-resolution microscopy localizes Aurora kinase A/AURKA in the mitochondrial matrix

Béatrice Durel, Charles Kervrann, Giulia Bertolin
Article reference https://doi.org/10.1101/2020.11.20.390948
Mitochondria are dynamic organelles playing essential metabolic and signaling functions in cells. Their ultrastructure has largely been investigated with electron microscopy (EM) techniques, which provided a wide range of information on how mitochondria acquire a tissue-specific shape, how they change during development, and how they are altered in disease conditions. However, quantifying protein-protein proximities using EM is extremely challenging. Superresolution microscopy techniques as direct stochastic optical reconstruction microscopy (dSTORM) now provide a fluorescent-based alternative to EM with a higher quantitative throughput. Recently, super-resolution microscopy approaches including dSTORM led to valuable advances in our knowledge of mitochondrial ultrastructure, and in linking it with new insights in organelle functions. Nevertheless, dSTORM is currently used to image integral mitochondrial proteins only, and there is little or no information on proteins transiently present at this compartment. The cancer-related Aurora kinase A/AURKA is a protein localized at various subcellular locations, including mitochondria. After performing dSTORM, we here use the Geo-coPositioning System (GcoPS) image analysis method to quantify the degree of colocalization of AURKA with compartment-specific mitochondrial markers. We show that two-color dSTORM provides sufficient spatial resolution to visualize AURKA in the mitochondrial matrix. We conclude by demonstrating that optimizing fixation procedures is a key step to follow AURKA in the matrix. In this light, we show that a methanol-based fixation leads to a better detection of the matrix pool of AURKA than an aldehyde-based fixation. Our results indicate that dSTORM coupled to GcoPS colocalization analysis is a suitable approach to explore the compartmentalization of non-integral mitochondrial proteins as AURKA, in a qualitative and quantitative manner. This method also opens up the possibility of analyzing the proximity between AURKA and its multiple mitochondrial partners with exquisite spatial resolution, thereby allowing novel insights into the mitochondrial functions controlled by AURKA.
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Nature Communications 2019

Combining 3D single molecule localization strategies for reproducible bioimaging

Clément Cabriel, Nicolas Bourg, Pierre Jouchet, Guillaume Dupuis, Christophe Leterrier, Aurélie Baron, Marie-Ange Badet-Denisot, Boris Vauzeilles, Emmanuel Fort & Sandrine Lévêque-Fort
Article reference https://doi.org/10.1038/s41467-019-09901-8
Here, we present a 3D localization-based super-resolution technique providing a slowly varying localization precision over a 1 μm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently to provide a weakly anisotropic 3D resolution over the imaging range. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several-μm range by direct merging of multiple acquisitions at different depths.
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Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration 2018

Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions

Philippe Codron, Julien Cassereau, Patrick Vourc’h, Charlotte Veyrat-Durebex, Hélène Blasco, Selma Kane, Vincent Procaccio, Franck Letournel, Christophe Verny, Guy Lenaers, Pascal Reynier & Arnaud Chevrollier
Article reference https://doi.org/10.1080/21678421.2018.1431787
Objective: Sporadic amyotrophic lateral sclerosis (sALS) is a fatal neurodegenerative disorder affecting upper and lower motor neurons. In view of the heterogeneous presentation of the disease, one of the current challenges is to identify diagnostic and prognostic markers in order to diagnose sALS at early stage and to stratify patients in trials. In this study, we sought to identify cytological hallmarks of sALS in patient-derived fibroblasts with the aim of finding new clinical-related markers of the disease. Methods: Primary fibroblasts were prospectively collected from patients affected with classical, rapid, and slow forms of sALS. TDP-43 localization, cytoskeleton distribution, mitochondrial network architecture, and stress granules formation were analyzed using 3D fluorescence microscopy and new super-resolution imaging. Intracellular reactive oxygen species (ROS) production was assessed using live imaging techniques. Results: Six sALS patients (two classical, two rapid, and two slow) and four age-matched controls were included. No difference in fibroblasts cell growth, morphology, and distribution was noticed. The analysis of TDP-43 did not reveal any mislocalization nor aggregation of the protein. The cytoskeleton was harmoniously distributed among the cells, without any inclusion noticed, and no difference was observed regarding the mitochondrial network architecture. Basal ROS production and response to induced stress were similar among patient and control fibroblasts. Conclusions: ALS cytological lesions are absent in patient-derived fibroblasts and thus cannot contribute as diagnostic nor prognostic markers of the disease.
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Nature Communications 2018

Combining 3D single molecule localization strategies for reproducible bioimaging

Clément Cabriel, Nicolas Bourg, Pierre Jouchet, Guillaume Dupuis, Christophe Leterrier, Aurélie Baron, Marie-Ange Badet-Denisot, Boris Vauzeilles, Emmanuel Fort, Sandrine Lévêque-Fort
Article reference https://doi.org/10.1038/s41467-019-09901-8
We developed a 3D localization-based super-resolution technique providing an almost isotropic 3D resolution over a 1 μm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several μm range by direct merging of multiple acquisitions at different depths.
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Antimicrobial Agents and Chemotherapy 2018

Failure of daptomycin to kill Staphylococcus aureus: impact of bacterial membrane fatty acid composition

Rym Boudjemaa, Clément Cabriel, Florence Dubois-Brissonnet, Nicolas Bourg, Guillaume Dupuis, Alexandra Gruss, Sandrine Lévêque-Fort, Romain Briandet, Marie-Pierre Fontaine-Aupart, Karine Steenkeste
Article reference https://doi.org/10.1128/AAC.00023-18
Daptomycin is a last-resort membrane-targeting lipopeptide approved for the treatment of drug-resistant staphylococcal infections such as bacteraemia and implant-related infections. Although cases of resistance to this antibiotic are rare, increasing clinical, in vitro and animal studies report treatment failure, notably against Staphylococcus aureus. The aim of this study was to identify the features of daptomycin and its target bacteria that lead to daptomycin treatment failure. We show that daptomycin bactericidal activity against S. aureus to significantly varies with the growth state and strain according to membrane fatty acid composition. Daptomycin efficacy as an antibiotic relies on its ability to oligomerize within membranes and form pores that subsequently lead to cell death. Our findings ascertain that daptomycin interacts with tolerant bacteria and reaches its membrane target, regardless of its functionality. However, the final step of pore formation does not occur in cells that are daptomycin-tolerant, strongly suggesting its incapacity to oligomerize. Importantly, membrane fatty acid contents correlated with poor daptomycin bactericidal activity, which could be manipulated by fatty acid addition. In conclusion, daptomycin failure to treat S. aureus is not due to a lack of antibiotic-target interaction but is driven by its capacity to form pores, which depends on membrane composition. Manipulation of membrane fluidity to restore S. aureus daptomycin bactericidal activity in vivocould open the way to novel strategies of antibiotic treatment.
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Nature Methods 2018

Quantitative mapping and minimization of super-resolution optical imaging artifacts

Siân Culley, David Albrecht, Caron Jacobs, Pedro Matos Pereira, Christophe Leterrier, Jason Mercer & Ricardo Henriques
Article reference https://doi.org/10.1038/nmeth.4605
Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.
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ArXiv 2018

Multicolor localization microscopy by deep learning

Eran Hershko, Lucien E. Weiss, Tomer Michaeli, Yoav Shechtman
Article reference arXiv:1807.01637
Deep learning has become an extremely effective tool for image classification and image restoration problems. Here, we apply deep learning to microscopy, and demonstrate how neural networks can exploit the chromatic dependence of the point-spread function to classify the colors of single emitters imaged on a grayscale camera. While existing single-molecule methods for spectral classification require additional optical elements in the emission path, e.g. spectral filters, prisms, or phase masks, our neural net correctly identifies static as well as mobile emitters with high efficiency using a standard, unmodified single-channel configuration. Furthermore, we demonstrate how deep learning can be used to design phase-modulating elements that, when implemented into the imaging path, result in further improved color differentiation between species.
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Science Advances 2018

TADs are 3D structural units of higher-order chromosome organization in Drosophila

Quentin Szabo, Daniel Jost, Jia-Ming Chang, Diego I. Cattoni, Giorgio L. Papadopoulos, Boyan Bonev, Tom Sexton, Julian Gurgo, Caroline Jacquier, Marcelo Nollmann, Frédéric Bantignies and Giacomo Cavalli
Article reference DOI: 10.1126/sciadv.aar8082
Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, we provide an integrative view of chromatin folding in Drosophila. We observed that repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.
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Neuron 2017

Localized Myosin II Activity Regulates Assembly and Plasticity of the Axon Initial Segment

Stephen L. Berger, Alejandra Leo-Macias, Stephanie Yuen, Latika Khatri, Sylvia Pfennig, Yanqing Zhang, Esperanza Agullo-Pascual, Ghislaine Caillol, Min-Sheng Zhu, Eli Rothenberg, Carmen V. Melendez-Vasquez, Mario Delmar, Christophe Leterrier, and James L. Salzer
Article reference https://doi.org/10.1016/j.neuron.2017.12.039
The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity.
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Scientific Reports 2017

Nuclear pore complex plasticity during developmental process as revealed by super-resolution microscopy

Julien Sellés, May Penrad-Mobayed, Cyndélia Guillaume, Alica Fuger, Loïc Auvray, Orestis Faklaris & Fabien Montel
Article reference https://doi.org/10.1038/s41598-017-15433-2
Nuclear Pore Complex (NPC) is of paramount importance for cellular processes since it is the unique gateway for molecular exchange through the nucleus. Unraveling the modifications of the NPC structure in response to physiological cues, also called nuclear pore plasticity, is key to the understanding of the selectivity of this molecular machinery. As a step towards this goal, we use the optical super-resolution microscopy method called direct Stochastic Optical Reconstruction Microscopy (dSTORM), to analyze oocyte development impact on the internal structure and large-scale organization of the NPC. Staining of the FG-Nups proteins and the gp210 proteins allowed us to pinpoint a decrease of the global diameter by measuring the mean diameter of the central channel and the luminal ring of the NPC via autocorrelation image processing. Moreover, by using an angular and radial density function we show that development of the Xenopus laevis oocyte is correlated with a progressive decrease of the density of NPC and an ordering on a square lattice.
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ACS Nano 2017

Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring

Anaïs Bouissou, Amsha Proag, Nicolas Bourg, Karine Pingris, Clément Cabriel, Stéphanie Balor, Thomas Mangeat, Christophe Thibault, Christophe Vieu, Guillaume Dupuis, Emmanuel Fort, Sandrine Lévêque-Fort, Isabelle Maridonneau-Parini, and Renaud Poincloux
Article reference https://doi.org/10.1021/acsnano.7b00622
Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.
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Optics Letters 2017

Aberration-accounting calibration for 3D single-molecule localization microscopy

Clément Cabriel, Nicolas Bourg, Guillaume Dupuis, and Sandrine Lévêque-Fort
Article reference https://doi.org/10.1364/OL.43.000174
We propose a straightforward sample-based technique to calibrate the axial detection in 3D single-molecule localization microscopy. Using microspheres coated with fluorescent molecules, the calibration curves of point spread function-shaping or intensity-based measurements can be obtained over the imaging depth range. This experimental method takes into account the effect of the spherical aberration without requiring computational correction. We demonstrate its efficiency for astigmatic imaging in a 1.2 μm range above the coverslip.
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Nature Photonics 2015

Direct optical nanoscopy with axially localized detection

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort & S. Lévêque-Fort
Article reference https://doi.org/10.1038/nphoton.2015.132
Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Here, we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called supercritical-angle fluorescence can be captured using a high-numerical-aperture objective and used to determine the axial position of the fluorophore with nanometre precision. We introduce a new technique for three-dimensional nanoscopy that combines direct stochastic optical reconstruction microscopy (dSTORM) with dedicated detection of supercritical-angle fluorescence emission. We demonstrate that our approach of direct optical nanoscopy with axially localized detection (DONALD) typically yields an isotropic three-dimensional localization precision of 20 nm within an axial range of ∼150 nm above the coverslip.
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Nature Communications 2015

Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy

Pierre Bon, Nicolas Bourg, Sandrine Lécart, Serge Monneret, Emmanuel Fort, Jérôme Wenger & Sandrine Lévêque-Fort
Article reference DOI: 10.1038/ncomms8764
Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging
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