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.

DOI https://doi.org/10.1038/s41596-019-0269-9

Contact: Marcelo Nolmann marcnol@gmail.com

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.

DOI: https://doi.org/10.1016/j.molcel.2020.06.034

Contact: Marcelo Nolmann marcnol@gmail.com

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.

M. Golan, J. Boulanger-Weill, A. Pinot, P. Fontanaud, A. Faucherre, D. S. Gajbhiye, L. Hollander-Cohen, T. Fiordelisio-Coll, A. O. Martin, P. Mollard, Synaptic communication mediates the assembly of a self-organizing circuit that controls reproduction. Sci. Adv. 7, eabc8475 (2021). doi: 10.1126/sciadv.abc8475

Contact: Patrice Mollard, IGF, Montpellier patrice.mollard@igf.cnrs.fr

During embryonic development, cells take on increasingly precise roles in the body as they divide. Be they skin cells, muscle cells or neurons, the different cell types that make up the embryo emerge gradually from a very fine orchestration of their positions and identities, coordinated by the signals they exchange with each other. Like us, the cells need to “talk” to each other to make decisions.

Screaming or whispering: the embryonic cell dilemma

In vertebrate embryos, cells have a very dynamic behaviour. They move around, exchange their neighbours or migrate over long distances. The signals they exchange therefore need to have a long range, which could be characterized as “shouting”. The study of the embryonic development of a sea squirt, a small marine animal with optically transparent embryos, has enabled scientists from several teams at CNRS and INRIA in France, in collaboration with a team from the European Molecular Biology Laboratory (EMBL, Germany), to capture and describe in detail a more discreet mode of cell communication.

The scientists recorded the development of live embryos every two minutes with a new-generation « light-sheet » microscope. They then created software to automatically detect each cell and analyze its position, shape and neighbours up to an advanced stage of development. This work revealed an unusually reproducible mode of development, in which the same cell can be found in the same position across all embryos and where cells move very little in relation to each other. The authors of the study then annotated the films thus made with information on the cell type and the molecular signals emitted by each cell. Using mathematical modelling to integrate the quantitative description of the embryonic geometry with these annotations, their work suggest that cells communicate with very short-range signals. Moreover, the interpretation of these signals is modulated by the area of the contacts between cells. Unlike vertebrates, the cells of ascidian embryos thus have a static and fixed behaviour and the range of their “whispered” signals is very small.

Top: embryonic development of an ascidian from egg to tadpole. The part framed in white is the part of embryogenesis that we have imaged and then segmented (below, segmented cells coloured according to their cell fate). The lower part of the figure illustrates that the light green cells “whisper” instructions to their immediate neighbours by short-range signals.

This study indicates that the dynamics of cell movement varies greatly between animals and that these different modalities could be strongly related to the range of signals that the cells exchange with each other. By extending the repertoire of cellular communication mechanisms, this work opens new perspectives on the understanding of self-organization strategies of living forms.

Article: L. Guignard*, U.-M. Fiuza*, B. Leggio, J. Laussu, E. Faure, G. Michelin, K. Biasuz, L. Hufnagel, G. Malandain, C. Godin#, P. Lemaire# (2020) Contact-area dependent cell communications and the morphological invariance of ascidian embryogenesis (Science, July 10 2020 issue, https://science.sciencemag.org/content/369/6500/eaar5663)

Guignard-et-al.-2020-Contact-area–dependent-cell-communication-and-the-morphological invariance of ascidian embryogenesisDownload

2 recent publications using the laser irradiation and photoablation systems available on the MRic facility from the Bretagne-Loire Node are presented here:

  • Esmangart de Bournonville and Le Borgne (IGDR) characterized the assembly and interactions of tricellular junction components in Drosophila epithelial cytokinesis using laser ablation on a SP5 confocal. Their article entitled “Interplay between Anakonda, Gliotactin, and M6 for Tricellular Junction Assembly and Anchoring of Septate Junctions in Drosophila Epithelium” was published last august in Current Biology (https://doi.org/10.1016/j.cub.2020.07.090).
  • Rebecca Smith, post-doc in Sébastien Huet’s team (IGDR) in collaboration with Szilvia Juhász from Gyula Timinszky’s team (Szeged, Hungary) used laser irradiation to study chromatin remodeling following DNA damage. Their paper entitled “The chromatin remodeler ALC1 underlies resistance to PARP inhibitor treatment” has just been accepted in Science Advances.