Cells are complex machines that can process information from their environment and adapt their behaviour to adverse conditions. In this classical description, the role of the intracellular space, and more specifically the cytoplasm, which is densely crowded, is often neglected. Yet, it is now well documented that variations in cytoplasmic density have an impact on cell signalling and cell growth. From the physical point of view, an increase in cytoplasmic density can provoke colloidal phase transitions, drastic decrease of diffusion rates of proteins and, as a result, cell signalling arrest. Remarkably, such observations were made across various species (bacteria, yeast and mammalian cells). This suggests that the crowding properties of cells and their impact on cell functions may represent a core physical feature of living cells. Yet, molecular crowding is usually not considered in the mechanistic description of signalling pathways. It is also neglected as a physical driver of evolution for cell size and growth rate. In addition, it is unknown how molecular crowding is regulated and how this regulation relates to that of cell growth and cell size control. Here, we set out to quantitatively study the physics of the cell interior and to shed light on the relationships between cell density, cell growth and cell dynamics. The key novelty of our project is to combine phase quantitative imaging with fluorescence and volume measurements to extract physical parameters of the cell interior while monitoring the cell growth rate and response to stress. This unprecedented combination of measurements will give a physical description of the impact of molecular crowding on cell dynamics and growth rate. This is the fundamental question, at the frontier of physics and biology, that we want to address. We anticipate that demonstrating the importance of molecular crowding can lead to major advances in our understanding of cell dynamics and cell growth rate.

The mature nervous system is an intricate network in which precise connectivity between neurons is critical for the functioning of the system. A key step in the assembly of neuronal circuits lies in the accurate navigation of developing axons towards their correct targets. Imprecise or ectopic connections during development can lead to major neurological disorders ranging from brain malformation to psychiatric disorders. While guidance signals that wire the brain have mostly been identified, our current knoweldge of the molecular mechanims that modulate axon responsiveness to guidance molecules is far from being complete. In this context, precise coupling between microtubules (MTs) and filamentous actin (F-actin) emerged as a key process in growth cone adaptive mechanical behavior. However, the molecular players that link guidance receptors to both MTs and F-actin and coordinate their remodeling to drive directed axon outgrowth remain poorly characterized. We recently uncovered the MT-depolymerizing enzyme Fidgetin-like 1 (Fignl1) as a key player in zebrafish neuronal circuit wiring. Our preliminary cellular, in vivo and proteomic data positioned Fignl1 at the crossroads between MT, F-actin cytoskeletons and the Slit/Robo repellent guidance pathway. Notably, we showed that Fignl1 directly binds the Robo effector and actin-binding protein Myosin 9b. These results suggest that Fignl1 might be a key integrator of Slit repellent signals modulating and synchronizing MT and actin dynamics to steer growing axons. We will here use a unique combination of biological systems – based on the complementary expertise of the consortium (in vitro cell-free systems, ex-vivo culture of retinal explants and in vivo genetic approaches in zebrafish and mouse embryos) – coupled with cutting edge imaging technologies to dissect the role of Fignl1 in Slit-induced axon repulsion at molecular, cellular and physiological scales. Our project will be divided in two major goals focusing on: (I) Investigating the role of Fignl1 in Slit-mediated actin remodeling and (II) Deciphering how Fignl1 modulates the MT/actin cross talk to drive axon repulsion in response to Slit. Each issue will be addressed following the same experimental plan. We will first use a set of in vitro cell-free assays combined with biochemical methods or live TIRF imaging to investigate how recombinant Fignl1 impacts actin dynamics, structural organization and coupling with MTs. We will next assay how Fignl1 depletion influences Slit-induced F-actin/MT remodelling and interplay (live TIR imaging) as well as axon retraction (DIC imaging) ex-vivo in mouse retinal explants. We will next (iii) conduct rescue and/or dominant-negative experiments (i.e. blocking Fignl1/F-actin, or /Myo9b binding) to dissect the contribution of Fignl1 interactions with F-actin, Myo9b or MTs to these phenotypes. Finally, we will use the same genetic approaches in vivo in zebrafish (live imaging) and mouse embryos (in utero electroporation combined with light sheet imaging) to assess the relevance of these molecular interactions in retinal axon pathfinding at the optic chiasm (i.e. guidance choice point) and their subsequent integration in their main brain targets. Our present proposal addresses major conceptual and technological issues in the cell biology field of neuronal connectivity: (i) the crosstalk between two major cytoskeleton elements (MTs/F-actin) - so far mainly studied as isolated networks –, (ii) the regulation of this crosstalk by guidance signals – which remains an obscure part of axon guidance – and (iii) the development of genetically-encoded tools to manipulate MT/actin crosstalk in vivo in navigating axons of lived embryos. This project will unravel novel key players and critical molecular interactions in the MT/actin crosstalk underlying neuronal connectivity, which should ultimately shed new light in the aetiology of neurodevelopmental disorders.

Genome editing mediated by CRISPR-Cas9 has shown great promise for the treatment of retinal dystrophies (RD). Currently, adeno-associated viruses are the most widely used vectors for retinal gene therapies but their small packaging capacity and permanent transgene expression makes them suboptimal for CRISPR-Cas delivery. Transient delivery of Cas9 protein and its guide RNA as ribonucleoprotein (RNP) complexes have been reported in the retinal pigment epithelium (RPE) and into the inner ear cells in vivo. Members of our consortium investigated transient delivery of either Cas9 mRNA or Cas9 RNP into the retinal pigment epithelium (RPE) and photoreceptors as these are the target cells for most prevalent inherited retinal degenerations. Cas9 mRNA complexed with different lipid or peptide vectors led to low rates of indels at the target sequence in vivo and mostly in the RPE. Major changes to the delivery system are needed to increase the efficiency of gene editing, and finally, safety and cost of the therapeutic approach need to be taken into account when designing such vector systems. Our objectives are to address some of these challenges in this project by developing a novel polymer based non-viral carrier for in vivo targeted delivery of CRISPR-based RNP complexes; and to assess the efficacy and safety of our novel delivery approach in a mouse model of retinal dystrophy.

Fluorescence has become an essential observable in Biology and Medicine. The discrimination of a fluorescent label usually relies on optimizing its brightness and its spectral properties. Despite its widespread use, this approach still suffers from important limitations. First, extraction of a fluorescent signal is challenging in light-scattering and autofluorescent samples. Second, spectral deconvolution of overlapping absorption and emission bands can only discriminate a few labels, which strongly limits the discriminative power of emerging genetic engineering strategies, and falls short from the several tens needed for advanced bioimaging and highly multiplexed diagnostic assays. Our consortium of chemists, physicists, and biologists introduces the HIGHLIGHT concept (PHase-sensItive imaGing of reversibly pHotoswitchable Labels after modulatIon of activatinG ligHT) to achieve chromatic aberration-free highly multiplexed fluorescence imaging with only single and dual wavelength channels in emission and excitation. HIGHLIGHT aims at expanding the discriminative dimensions of fluorophore sets much beyond spectral and concentration information such as classically implemented in multicolor labeling approaches. In HIGHLIGHT, label discrimination will not necessitate anymore singular spectroscopic signatures, sophisticated reading-out instruments, or delicate data processing for signal unmixing. In contrast, it shifts towards designing reactive schemes and observables to selectively promote and retrieve the response of a targeted label. HIGHLIGHT exploits reversibly photoswitchable fluorescent proteins (RSFPs) as labels. Increasingly exploited in super-resolution microscopy and dynamic contrast, they are not only fluorescent but as well engaged in rich photocycles. The HIGHLIGHT protocols exploit their specific fluorescence responses to light modulation under well-designed conditions, which provides several dimensions of dynamic contrast to overcome the limitations encountered with spectral discrimination; These responses will serve as readouts either alone or combined using statistical machine learning strategies, which will enable us to perform real time multiplexed imaging of more than ten spectrally similar fluorescent labels and discriminate more than one hundred hues created by mixing these labels in variable amounts and cell territories. As a proof of principle, we propose to challenge HIGHLIGHT in two types of contexts where the paucity of spectrally distinct fluorescent markers has until now been a major hindrance: the analysis of the lineage of retinal cell subtypes and that of their connectivity. In this project, we will namely (i) design and implement a suite of transgenic tools enabling to express varied combinations of 6-12 RSFPs within a population of cells; (ii) design HIGHLIGHT protocols for wide-field and scanning microscopies as well as relevant barcoding strategies to discriminate different cells; (iii) evaluate the photoswitching properties of several tens of RSFPs with one- and two-photon excitation under various environments; (iv) validate HIGHLIGHT for its implementation in a commercial confocal microscope and in state-of-the art Single Plane Illumination scanning Microscopes to push forward acquisition depth and speed; and eventually (v) perform multiplexed clonal analysis in the vertebrate retina, and single-neuron tracing and analysis of axonal convergence. Eventually, the tools and protocols introduced in this project will have near-universal applicability in Biology for multiplexed fluorescence-based observations within biological samples.

Retinopathy of prematurity (ROP) is the leading cause of blindness in children. This disease is characterized by inflammatory processes and abnormalities in vascular development of the retina. Preliminary results from our laboratories and published data show that specific lipids, such as plasmalogens and endocannabinoids, can regulate inflammatory processes as well as the development of blood vessels in different tissues. The EndoROP project brings together specialists in lipid metabolisms, inflammation and angiogenesis in the retina. It will characterize in details the place of plasmalogens and endocannabinoids in the regulation of retinal vascular development in ROP by: 1) understanding the plasmalogen-dependent molecular mechanisms by which endocannabinoids regulate retinal vascular development in physiologic conditions (Work-Package 1) ; 2) deciphering the cellular and molecular mechanisms involving plasmalogens and endocannabinoids in the pathologic conditions of ROP (Work-Package 2), and 3) identifying new therapeutic targets and develop new therapeutic approaches to prevent or to limit the retinal vascular abnormalities observed in ROP (Work-Package 3). Altogether, our project will document the molecular mechanisms by which plasmalogens regulate the endocannabinoid system in the retina and offer new therapeutic strategies to prevent ROP in premature infants.