Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M, Nature Protocols 2020
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Category: Announcement
Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M
Andrés M. Cardozo Gizzi, Sergio M. Espinola, Julian Gurgo, Christophe Houbron, Jean-Bernard Fiche, Diego I. Cattoni, Marcelo Nollmann
Simultaneous observation of 3D chromatin organization and transcription at the single cell level and with high spatial resolution may hold the key to unveil the mechanisms regulating embryonic development, cell differentiation and even disease. We have recently developed Hi-M, a technology that allows for the sequential labelling, 3D imaging and localization of multiple genomic DNA loci together with RNA expression in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe re-hybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos into microfluidics chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 days including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 days to complete all rounds of labelling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells, or organization of chromatin within complex tissues.
ATP-driven separation of liquid phase condensates in bacteria
B. Guilhas, J.C. Walter, J. Rech, G. David, N.-O. Walliser, J. Palmeri, C., Mathieu-Demaziere, A. Parmeggiani, J.Y. Bouet, A. Le Gall1, M. Nollmann
Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.
Guilhas et al. revealed that the bacterial DNA segregation apparatus behaves as a non-canonical phase separation system. This apparatus employs an ATP-powered motor that splits nanometer-sized condensates and localizes them robustly within the nucleoid to ensure faithful transmission of genetic material.
The ability to communicate effectively with each other is one of the strongest predictors for our chances to get ahead in life. In their latest publication in Science Advances, scientists and engineers from IGF-Montpellier (CNRS, INSERM, Univ. Montpellier), IPAM platform (BioCampus Montpellier, France-Bioimaging Montpellier Node) and ARO-Israel demonstrated that this also holds true for GnRH neurons.
In humans and all vertebrates, species survival depends on a critical step during embryonic development: the migration of a small subset of GnRH neurons (about 2,000 in humans and less than 100 in fish) from the nose to the brain where they join the hypothalamus to control reproduction. Their latest results unveiled that GnRH neurons make a pause at the nose-brain frontier where they function as an inter-hemispheric network that is isolated from the rest of the brain. Only neurons that integrate into the network and are able to communicate with their neighbors will finally cross the barrier and make their way into the brain, towards their hypothalamic destination.
In other words, these GnRH neurons, that are critical for species persistence, face the same challenges like other immigrants: they must learn to communicate effectively if they are to integrate into their new world.
In this study, in vivo 2-photon microscopy was a key tool for:
Long term imaging with minimal bleaching and phototoxicity
Upright configuration enabling dorsal imaging of the fish in its natural position
Long-distance water-immersion objectives allowing imaging of deep tissue structures without sacrificing image quality
Fast calcium imaging
Imaging of red GECI using the higher wavelengths
Precise cell ablation
Photoactivation of ChR2 while monitoring Ca in the red channel
A graphical model illustrating the migration of a single GnRH neuron (marked by black border) from the nasal placode into the zebrafish brain.
L’appel à candidature pour le prix « Pierre Favard » 2021 de la Société Française des Microscopiesest ouvert.
Ce prix récompense depuis 1989 des travaux de thèse réalisés dans une université française dans le domaine de la microscopie électronique, photonique, en champ proche, ou de la sonde atomique. Un prix est attribué en Sciences du Vivant et un autre en Sciences de la Matière. Ils sont décernés tous les deux ans. Pour cette édition, ils couronneront des travaux de thèse soutenus entre le 1er mars 2019 et le 28 février 2021. Chaque lauréat(e) est invité(e) à donner une conférence à l’issue de la remise du prix, lors de la réunion biennale de la Société qui est prévue à Reims du 5 au 9 Juillet 2021. Les prix (1000 € chacun) sont sponsorisés par des compagnies.
La date limite de dépôt des candidatures est fixée au 21 mars 2021.
1. Les candidats doivent fournir un exemplaire papier et une version « pdf » de leurs travaux de thèse, un curriculum vitae d’une page maximum et le fichier modèle ci-dessous dûment complété. Le dossier (papier et électronique) devra être envoyé au président de jury de leur discipline avec copie électronique au secrétariat de la SFμ (sfmu@sfmu.fr) et à la présidente (catherine.venien-bryan@upmc.fr).
2. Les candidats doivent être membre de la Société à la date de dépôt de leur dossier. 3. La Société Française des Microscopies prend en charge l’inscription au congrès de Reims, l’hébergement des lauréats (à hauteur de 90€/nuit) et rembourse le billet aller-retour en 2e classe (train) ou classe économique (pour la voie aérienne), sur présentation des factures originales et d’un ordre de mission sans frais.
Les deux présidents de jury nommés par le Conseil d’administration de la SFμ sont mandatés pour composer un jury de plusieurs personnes. Chaque jury est chargé de sélectionner et de classer les trois meilleurs candidats. Le Conseil de la SFμ, sur proposition des jurys et sur rapport des présidents, désigne les deux lauréats.
Registration for France BioImaging Annual Meeting is now open!
France BioImaging is pleased to invite you to participate to France BioImaging6th Annual Meeting. For this edition, the meeting will be organized as atwo-half days virtual meeting (from 9:00 AM to 1:00 PM) on February 4th & 5th, 2021.
This event, open to all members of the bioimaging community, aims to provide a platform to discuss pivotal subject matters in our field.
The 2021 program of the France BioImaging Annual meeting is built around two pillars:
February 4th: “Building and operating an integrated and open infrastructure for bioimaging“
February 5th: “Latest and future developments in biological imaging“
A l’occasion de la MorningTech Photonique & Santé organisée par Photonics Bretagne et Biotech Santé Bretagne le 2 février 2021 de 9h30 à 12h00, Marc Tramier, coordinateur du Nœud Bretagne-Loire de France BioImaging présentera “lestechnologies photoniques des plateformes de Biogenouest au service de l’imagerie pré-clinique“.
Imaging of proteins, cells, and tissues is critical to understanding health and disease. On December 2nd, 2020, the Chan Zuckerberg Initiative (CZI) announced nearly $32 million in funding to support biomedical imaging researchers, technology development, and the BioImaging North America international network of bioimaging facilities and communities. CZI also opened a new Request for Applications (RFA) aimed at supporting technology development that will allow researchers to see the inner workings of cells, including proteins, at near-atomic resolution to better understand what causes disease and how to develop treatments.
Frontiers of Imaging: Visual Proteomics
The Frontiers of Imaging initiative, part of CZI’s broader Imaging program, supports the development of disruptive imaging technologies that connect biological scales across organs, cells, and proteins, allowing researchers to directly visualize biological processes at the necessary resolution and context to obtain a mechanistic understanding of health and disease. As part of the Frontiers initiative, the new Visual Proteomics Imaging RFA supports technology development that will allow researchers to see the inner workings of cells, including proteins, at near-atomic resolution. CZI invites scientists to apply for this 2 1/2-year grant opportunity to support the development of hardware, software, and methods. Examples of research themes could include hardware and software development to enhance contrast and resolution for electron tomography, high-resolution correlated light and electron microscopy (CLEM), and FIB-SEM; sample preparation improvements for electron tomography; and development of software or new computational techniques and algorithms for identifying protein molecules inside cells and segmenting sub-cellular structures.
The Visual Proteomics Imaging RFA accept applications until February 17, 2021 at 5 p.m. Pacific Time.
For administrative and programmatic inquiries, technical assistance, or other questions pertaining to this RFA, please contact: sciencegrants@chanzuckerberg.com.
Congratulations to Emmanuel Beaurepaire (CNRS Research Director from the Laboratory for Optics and Biosciences CNRS-INSERM-Polytechnique),PI of the ERC Synergy Grant project “HOPE”, and to Laurent Groc (CNRS Research Director ; Interdisciplinary Institute for Neuroscience), coordinator of theERC Synergy Grant project “ENSEMBLE“, Laurent Cognet (CNRS Research Director ; Laboratoire photonique numérique et nanosciences) and U. Valentin Nägerl (Professor at University of Bordeaux ; CNRS Research Director ; Interdisciplinary Institute for Neuroscience), both PIs of theERC Synergy Grant project “ENSEMBLE“.
These grants, each worth around 10 million euro over six years, are designed to enable groups of 2 to 4 scientists to tackle some of the world’s most challenging research problems, spanning several scientific disciplines.
The ERC Synergy Grant scheme is part of the EU’s research and innovation programme, Horizon 2020.
ERC Synergy Grant project “HOPE”
“Reverse engineering the assembly of the hippocampal scaffold with novel optical and transgenic strategies”
Emmanuel Beaurepaire, Directeur de recherche CNRS au Laboratoire d’optique et biosciences – LOB (CNRS/École polytechnique/INSERM),
Rosa Cossart, Directrice de recherche CNRS (Unité INSERM, Aix-Marseille Univ.)
Jean Livet, chercheur INSERM à l’Institut de la vision (CNRS, INSERM, Sorbonne Univ.)
At the heart of our brain, a structure plays a key role in memory, and more particularly in the acquisition and maintenance of our memories: the hippocampus. Classically considered as a “cognitive GPS” for space and time, it is also the seat of our episodic memory.
Over the last decade, the neural circuits of the hippocampus have been better described, in particular by the team of Rosa Cossart, director of the Institut de neurobiologie de la méditerranée (Inmed), but the nature, origin and remodeling of these circuits during development and pathologies remain to be understood.
On the other hand, genetic engineering techniques for staining neurons, developed by Jean Livet, Inserm research director at the Institut de la vision, coupled with multi-photon microscopy developed by the team of Emmanuel Beaurepaire, CNRS research director at the Laboratoire d’optique et biosciences – LOB (illustration below / read the 2019 press release in French), have demonstrated their ability to accurately map the complex architecture of neuronal circuits and their evolution during development.
By combining these exceptional multidisciplinary advances, HOPE aims to answer three interdependent questions:
Is the architecture of the adult seahorse carried by specific circuits?
Are the circuits of the hippocampus pre-wired or shaped by experience?
How does this structure reorganize itself in pathological conditions?
HOPE aims to shed new light on the function of the hippocampus and the role of its neuronal circuits through the design of a new, non-invasive and universal method to monitor the growth and construction of brain circuits located deep in the brain, from their neurogenesis to adulthood, under normal and pathological conditions.
“Structure and functions of the brain extracellular space“
Laurent Groc (Research Director CNRS ; Interdisciplinary Institute for Neuroscience),
Erwan Bézard (Research Director INSERM; Institute of Neurodegenerative Disorders),
Laurent Cognet (Research Director CNRS ; Laboratoire photonique numérique et nanosciences)
U. Valentin Nägerl (Professor at University of Bordeaux ; Research Director CNRS ; Interdisciplinary Institute for Neuroscience)
The ENSEMBLE project aims at underpinning the molecular mechanisms of physiological and pathological brain function. This ambitious and innovative endeavor is based on our ability to develop new approaches in high-resolution microscopy at the service of a new conceptual framework in brain cell communication.
Credit: U.V. Nägerl
This project has roots in the international leadership of the Bordeaux communityin the fields of microscopy, nanophotonics, fundamental and translational neuroscience. The opportunity that is offered to these 4 investigators to break a frontier knowledge was permitted by the continuous support of local institutional actors. The installation of Prof. Valentin Nägerl’s laboratory in 2009 with a “Chaire Accueil” from the Regional Council of Aquitaine, the support of LabEx BRAIN, the Laphia Cluster and the IdEx of the University of Bordeaux provided the ground to build elementary blocks necessary for the challenging adventure of the ERC Synergy project (10 million euros, 6 years).
We are very pleased to announce that FBI Bretagne-Loire Node application to become a Euro BioImaging facility has been evaluated as highly recommended by the EuBI Scientific Advisory Board (SAB) and ratified by the EuBI Board on November 30th, 2020.
The Bretagne Loire Node became a node of the national infrastructure France BioImaging in November 2019 and applied to become a EuBI facility during the last EuBI Call for Nodes (June 2020).
The Bretagne Loire Node brings together four cellular imaging and histology facilities, two in Rennes (MRic and H2P2) and two in Nantes (MicroPIcell and APEX). These facilities have complementary expertise for live imaging and pathological anatomy. The added value of the Bretagne Loire Node is to be able to offer a continuum between biological imaging and medical imaging, through the development of a new line of services as well as methodological and technological transfer to users of microscopy technologies for preclinical studies.
As part of the Euro-BioImaging research infrastructure, the services provided by these facilities will be open to all scientists, regardless of their discipline or affiliation.
The PICsL-FBI microscopy core facility is located on two sites: Centre d’Immunologie de Marseille Luminy (CIML) and Institut de Biologie du Développement de Marseille (IBDM).The PICsL-FBI facility of the CIML called ImagImm (Imaging Immunity) via its microscopy resources – from the molecule to whole organisms – is dedicated to help its users deciphering cellular mechanisms in the fields of immunology.
Major research implications of the ImagImm facility:
In collaboration with Tomasz Trombik (Faculty of Biotechnology, University of Wroclaw – Wroclaw, Poland), Sophie Brustlein (Institut de Convergences Centuri, AMU,CNRS – Marseille, France) and Nicolas Bertaux (Institut Fresnel, AMU, Centrale Marseille, CNRS – Marseille, France), Sébastien Mailfert and Didier Marguet published the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized1. This publication is following the technology transfer made in 2018: the svFCS developed by Didier Marguet’s lab was duplicated by Sébastien Mailfert and Sophie Brustlein and built from scratch in 7 days on site, in Poland.
Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. The published protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment is provided and how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. This fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.
Figure 1:Schematic view of excitation and emission optical paths of the svFCS setup and pictures of the setup. The svFCS setup contains four modules: (1) the output of a fibered 488 nm laser is collimated, (2) a combination of a half-wave plate and polarizing beamsplitter sets the optical power, (3) the laser beam focused on the sample after traveling through a tube-lens free motorized microscope, and (4) the fluorescence is detected through a confocal-like detection path onto an avalanche photodiode coupled to a single photon counting module, which delivers a signal to a hardware correlator. Simplicity gives the system its sensitivity, robustness, and ease of use.
SAPHIR : a Shiny application to analyze tissue section images
In collaboration with Hugues Lelouard (CIML, Inserm, CNRS, AMU) and Elodie Germani, Mathieu Fallet published a powerful method for both basic and medical research to study cell populations in tissues using immunofluorescence. Image acquisitions performed by confocal microscopy notably allow excellent lateral resolution and more than 10 parameter measurement when using spectral or multiplexed imaging. Analysis of such complex images can be very challenging and easily lead to bias and misinterpretation. They developed the Shiny Analytical Plot of Histological Images Results (SAPHIR), an R shiny application for histo-cytometry using scatterplot representation of data extracted by segmentation. It offers many features, such as filtering of spurious data points, selection of cell subsets on scatterplot, visualization of scatterplot selections back into the image, statistics of selected data and data annotation. This application allows to quickly characterize labeled cells, from their phenotype to their number and location in the tissue, as well as their interaction with other cells.
Figure 2: Flow chart of tissue image analysis from image acquisition and segmentation (left side) to extract data analysis with SAPHIR (right side)
Wound healing in C. elegans
In collaboration with Nathalie Pujol and Jonathan Ewbank (CIML, Inserm, CNRS, AMU), Mathieu Fallet and Sébastien Mailfert participated in the project on the immune response by showing that wounding provokes a reorganization of plasma membrane subdomains3. The skin protects animals from infection and physical damage. In Caenorhabditis elegans, wounding the epidermis triggers an immune reaction and a repair response, but it is not clear how these are coordinated. Previous work implicated the microtubule cytoskeleton in the maintenance of epidermal integrity (Chuang et al., 2016). Taffoni et al. show the reorganization of the plasma membrane subdomains by a simple wounding system. This is followed by recruitment of the microtubule plus end-binding protein EB1/EBP-2 around the wound and actin ring formation, dependent on ARP2/3 branched actin polymerization. They show that microtubule dynamics are required for the recruitment and closure of the actin ring, and for the trafficking of the key signaling protein SLC6/SNF-12 toward the injury site. Without SNF-12 recruitment, there is an abrogation of the immune response. These results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of innate immunity.
Figure 3: Time line of events
References:
Mailfert, S., Wojtowicz, K., Brustlein, S., Blaszczak, E., Bertaux, N., Łukaszewicz, M., Marguet, D., Trombik, T. Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells, JoVE, 165, 1-19 (2020).
Germani, E., Lelouard, H., Fallet, M. SAPHIR: a Shiny application to analyze tissue section images, F1000Research, Faculty of 1000, 9, 1276-1285 (2020).
Taffoni, C., Omi, S., Huber, C., Mailfert, S., Fallet, M., Rupprecht, J-F,. Ewbank, J., Pujol., N. Microtubule plus-end dynamics link wound repair to the innate immune response, eLIFE, 9, e45047 (2020)
With his team members, Patrick Lemaire is studying the embryonic development of a small marine invertebrate, the sea squirt Phallusia mammillata, chosen for the simplicity and transparency of its embryos. His latest work has combined microscopy, image analysis and mathematical modeling approaches to describe, cell by cell, the embryogenesis of this animal and to analyze the role of communication between cells.
COMULIS is an EU-funded COST Action that aims at fueling urgently needed collaborations in the field of correlated multimodal imaging (CMI), promoting and disseminating its benefits through showcase pipelines, and paving the way for its technological advancement and implementation as a versatile tool in biological and preclinical research. CMI combines two or more modalities to gather holistic information about the same specimen. It creates a composite view of the sample with multidimensional information about its macro, meso- and microscopic structure, dynamics, function and chemical composition. Since no single technique can reveal all these details, CMI is the only way to understand biomedical processes mechanistically.
In order to encourage correlated multi-modal imaging projects, COMULIS Short Term Scientific Missions (STSMs) provides travel grants to individuals wishing to explore new imaging techniques. The grants are presented in the form of a lump sum of up to 3,500 Euros (depending on the duration of the mission), to cover travel and subsistence. COMULIS COST accepts applications on a continuous basis from Early Career Investigators and Experienced Imaging Scientists who would like to travel internationally to collaborate with a Host facility on a Correlated Multi-modal imaging project. There is a rapid review process and around 10 grants are awarded every year.
In addition, COMULIS STSM can provide funding for core facility staff to learn a new imaging technique or work with new software tools to bring the expertise back to their own facility.
Applications can be submitted any time & will be reviewed at the end of each month.
