The objectives of the ProteOrigami project are to design, produce and characterize self-assembling supramolecular Protein Origami and to use this new platform to precisely organize optical nanomaterials in space. This experimental project gathers four partners with complementary expertise in protein engineering (I2BC, Orsay) , biophysical characterization (IPR, Rennes), electron microscopy and optical nanomaterials design and characterization of the hybrid protein-inorganic architectures (CEMES and CBI, Toulouse). Naturals proteins spontaneously form sophisticated architectures with essential biological functions. However designing self-assembling artificial proteins remains highly challenging and predominantly aimed at biological and medical applications. ProteOrigami will use extremely stable artificial alphaRep proteins, created by I2BC, as elementary units. These proteins have a modular structure that facilitates new designs and are efficiently mass-produced by standard molecular biology methods. We propose a new concept of protein "brick & staple" whereby stable and soluble alphaRep brick proteins are assembled and ordered in a predefined 3D geometry by specific alphaRep staple proteins. The precise molecular recognition between brick and staple is engineered by directed evolution and drives the 3D supramolecular origami assembly. Preliminary results obtained by the four partners clearly demonstrate that this original and potentially general approach works efficiently. A first work package is devoted to the design of a range of new origami building blocks in order to control the origami structure and morphology. Bricks with different sizes or altered geometries will be used to tune the origami shape and its helical periodicity. Stop modules will be designed to control the origami size. Orthogonal bricks and staples pairs will be generated to induce a sequentially ordered protein organization. Finally, multimeric proteins will be built as branching nodes to established 2D/3D origami networks. All origami architectures will be characterized by complementary structural methods (SAXS, TEM, cryoEM). The second work package translates the precise spatial organization of proteins in origamis into the spatial ordering of functional optically active moieties ('cargo') and studies their structural and optical properties . Since the different elements (brick, staple, stop modules) are separately produced, they can be labelled by single and selective coupling reactions with a component to be placed in a predefined position. The regioselective origami decoration will be used to position a single or a spatially ordered ensemble of cargos such as fluorophores, emitting or plasmonic nanoparticles. We anticipate these hybrid nanoconstruction to exhibit synergistic properties when regularly ordered at the nanoscale (superradiance, lasing, coupled plasmon modes,…) or when brought into the near-field (i.e. within 1-10 nm distance) of each other (Purcell effect, FRET, plasmon-mediated energy transfer). Fluorescence, scattering and spectroscopic properties will be explored on bulk and individual objects. A last work package exploits a new approach of alphaRep-templated growth of (111)-faceted plasmonic gold nanocrystals recently established by two ProteOrigami partners. Adding this interacting functionality to the origami bricks is possible as the self-assembly leaves their main affinity surface available. The objective is to grow the plasmonic structure inside or on one side of the origami while programming the position(s) of the emitter(s) at a predefined distance from the gold surface that maximizes the coupling between the emitting dipole and the plasmonic near-field modal landscape. In such a configuration, the protein origami will be the first reported strategy to allow a programmable relative placement of plasmonic and emitting nanostructures on a scalable template. The structural and nanooptical properties will be studied.

The major challenge of cell and developmental biology is to propose a multiscale model able to describe the mechanism through which macromolecules act on the cell, and in cascade on the tissues and organ functions. One of the key goals in this expanding field is to decipher how stress induced by mechanical forces or DNA damage induces cancer. Presently, the multiscale analysis is done by resorting to different tools adapted to imaging at the molecular (nanometer scale), cell (micron scale) or tissue (mm scale) levels. A microscope able to observe the ballet of proteins inside live biological tissues over large fields of view (the dream of all set-ups in one) would drastically increase the available information and open new doors in integrative biology. Widefield fluorescence microscopy is the most widespread tool for getting real time images of specific protein distribution in live specimen over large volumes of observation (hundreds of thousands of microns cube). Unfortunately, its resolution, about 300 nm transversally and 1000 nm axially at best, is not sufficient for an accurate study of the macromolecule organisation and dynamics within living tissues. Super-resolution fluorescence microscopes using saturation or pointillism yield images with a resolution below 50 nm but their toxicity, the time required for the data acquisition and processing restrict their use to small observation volumes and slow temporal dynamics. Structured Illumination Microscopy (SIM) is the best compromise between resolution (about 100 nm transverse and 300 nm axial) and practical implementation on live samples. Yet, as it requires the perfect knowledge of the illuminations, it cannot be used deep inside distorting samples such as biological tissues and its experimental implementation is cumbersome. Recently, we have proposed a technique that gathers the resolution of periodic SIM and the ease of use and field of view of standard fluorescence microscopy. Random Illumination Microscopy (RIM) reconstructs a Super-Resolved (SR) image of the sample from multiple low-resolution frames obtained under different speckle illuminations. It is based on a mathematical analysis, showing that a two-fold resolution gain, can be obtained from the second order statistics of the speckle images. Speckles being insensitive to scattering, distortions and aberrations, RIM is expected to succeed in cases where SIM fails. In the last two years, we have implemented a two-dimensional (2D) version of RIM in which the sample is viewed as a slice limited to the focal plane. This simplified approach yielded remarkable results with 120 nm transverse and 300 nm axial resolutions together with an SR-image rate about 1-5 Hz. This achievement positions 2D-RIM as one of the best super-resolved techniques for live imaging, in particular deep inside biological tissues where aberrations and scattering are redhibitory for SIM. However, it is obvious that 2D-RIM does not exploit the full capacity of RIM and a significant amelioration of the temporal and axial resolutions could be obtained by taking advantage of the structuration of the speckles and observation point spread function along the optical axis. In this project, we propose to extend RIM principle to the three dimensions with an appropriate mathematical analysis and data processing coupled to an up-graded instrumentation. Our objective is to provide images over large field of views with 100 nm transverse and 200 nm axial resolutions and reach frame rate about 10-30 Hz. 3D-RIM will be tested on two key biological issues for which all existing super-resolution microscopes are inoperative: the multiscale interaction of cells with the surrounding tissue during apoptosis, which requires high spatial resolution and large fields of view and the dynamic chromatin loop extrusion during DNA repair which requires high spatiotemporal resolution.

The gut epithelium is single layer of cells and a physical barrier towards the harsh luminal environment. It is also the fastest renewing tissue in the body – the turnover is maintained by cell proliferation in the crypts, migration of differentiated cells along the villi, and cell loss by extrusion. In addition to these intrinsic dynamics, the tissue is constantly mechanically challenged by extrinsic factors, such as muscle contractions. Epithelial cell junctions enable tissue cohesion and collective cell migration – they mechanically link cells, integrating forces across the tissue, and modulating cell behaviour. However, the small intestinal epithelium comprises six different cell types with distinct roles, morphologies and turnover rates. The predominant cell type – enterocytes (absorptive cells) – are columnar with polygonal apical shape, and were shown to turnover fast. The other, rarer cell types, have round apical shapes and secretory roles, and many were shown to renew more slowly than enterocytes. How cell-cell junctions are maintained in a tissue with a diversity of cell types with such distinct morphology and turnover rates is unknown. Here we focus on goblet cells, which have an extremely bulky, round-shaped body, due to mucus granules. Main aims of GOBLET are to understand the impact of goblet cells on epithelial integrity, and to reveal goblet cell dynamics in gut homeostasis. To this end we propose an interdisciplinary and collaborative project, using complementary model systems such as transgenic mice, ex vivo tissue and organoid models, combined with advanced imaging systems and biophysical modelling, to understand the impact of cell heterogeneity in the gut homeostasis.

Despite their high price, super-resolved fluorescence microscopes, either scanning or structured illumination microscopes (SIM), used in biology imaging platforms, often show degraded performances due to sample induced optical aberrations. We have recently developed an easy to use techniquethat provides the same resolution as SIM while being robust to aberrations . The Random Illumination Microscope (RIM) is based on the use of random dynamic illumination and statistical data processing. RIM has demonstrated its ability to image at high resolution samples that were inaccessible to current super-resolution techniques [Mangeat2021]. The objective of this project is to realize a fibered dynamic random illumination module that can be adapted to all microscopes. By accompanying this module with an adapted data processing algorithm, super-resolved microscopy will become accessible to all biology laboratories at a lower cost (less than 20 000 euros). This project is a partnership between the Laboratoire de biologie Intégrative, the Fresnel Institute and the French SME Oxxius specialized in the realization of laser sources in health biology.

Modularity refers to a pattern of connectivity in which elements are grouped into highly connected subsets, modules or building blocks. It is an important property in biology, as it helps a system to "save its current state" while allowing further evolution. Developmental modules are often represented by their physical location and spatial extent in the organism, and they contain informations about the genetic specification of modules. Thus, the properties attributed to modules are: autonomy, discrete organization defined by the expression of specific genes, and occupation of specific physical territories. In vertebrates, the functional and architectural organization of the olfactory system is conserved throughout evolution. The olfactory organ is composed of different types of olfactory sensory neurons, the OSNs, each capable of detecting specific odorant molecules, organized with a specific shape and position, and each expressing a specific set of genes. The hypothesis of the ZOORRO project is that each type of OSNs could be defined by specific modules at key stages of the embryonic development of the olfactory organ. This project aims at breaking down the formation of the olfactory sensory organ into modules: a genetic module and a morphometric-behavioral module. A multidisciplinary consortium of three teams will use a combination of qualitative and quantitative imaging on the zebrafish embryo (Julie Batut, CBI-MCD Toulouse), data science, machine learning and image analysis (Christian Rouvière, CBI Toulouse) together with mathematical models (David Sanchez, INSA Toulouse) to identify the two modules and assemble them to generate a single-cell transcriptomic atlas dynamically linked to cellular behavior and capable of predicting the architecture of a complex biological system: herein, the olfactory sensory organ.