Photosensitive or phototransformable fluorescent molecules able of photoactivation, photoswitching or photoconverison are powerful tools in bioimaging to unambiguously track labeled biomolecules over large spatiotemporal scales. In addition to this feature these molecules have led to significant advances in biophotonics due to their ability to be used in super resolution imaging (PALM and STORM). Since the discovery of paGFP in 2002, phototransformable fluorescent proteins are predominant in the field of bioimaing. Although this approach is robust and powerful it is not universal as it is limited to proteins and not straightforward, as it requires a transfection step thus leading to heterogeneous samples and toxic effects. Conversely, molecular probes are characterized by their ease of use; they homogenously stain the cells and can be used in tissue imaging. Moreover their accessible chemical modifications offer more possibilities for improvement compared to proteins. Although photoactivatable fluorophores have drawn a notable attention, they only reveal their fluorescence upon activation thus mainly finding their use in super resolution imaging. Similarly, photoswitchable fluorophores generally switch from non-emissive state to a fluorescent form and require UV irradiation, which is phototoxic. Surprisingly, dual-color photoswitchable fluorophores (DCPSF) able to switch from a bright color to another, and thus that can be advantageously detected prior to conversion, were only poorly developed. While complex FRET pair system that can be separated by photocleavage have been proposed, the chemical development of single molecular DCPSF remains extremely rare. Indeed in most studies, dual color switching properties were evaluated on existing commercially available fluorophores like AlexaFluor 647 and cyanines thus not allowing an extensive comprehension in the development of dual-color photoswitchable fluorophores. This project aims at developing bright and multicolor small Dual-Color Photoswitchable Fluorophores (DCPSFs) able to switch from a color to another by connecting conjugated photoactivatable moieties to the p-system of fluorescent dyes. This project is based on promising preliminary results where organelle-specific (mitochondria and plasma membrane) DCPSFs were synthesized and successfully used in photoconversion and super resolution imaging. We herein propose to develop DCPSF bearing a clickable moiety allowing post functionalization in order to target not only proteins (by SNAP or Halo tag) but also specific organelles (mitochondria, nucleus, reticulum, etc) using identified targeting moieties to provide universal tools in cellular imaging. This project also aims at understanding the mechanism leading to the photoswitching by establishing a structure/photophysical-properties relationship involving advanced photophysical caracterizations. Once fully characterized (brightness, photostability, reversibility, etc), the probes will be evaluated in cells using conventional microscopy and then used in bioimaging with advanced microscopy techniques including tracking, FRAP and super resolution. The project will provide an extensive comprehension in the development of molecular DCPSF and the developed probes will be of wide use and interest in the field of bioimaging.

Single molecule imaging of biomolecules in living cells using fluorescence microscopy has become of key importance for understanding biological processes at the molecular level. Following directly their dynamics requires localizing single emitters inside cells with a spatial and temporal resolution at the level of the molecular events. The achievable resolution depends strongly on the fluorescent markers used for labeling. The brighter the emitter the higher its imaging speed and the better its localization in space. Furthermore, fluorescence intermittency of the labels due to blinking or photoactivation allows resolving emitters at distances below the diffraction limit using, e.g., direct stochastic optical reconstruction microscopy (dSTORM). This makes, however, tracking of the emitters more cumbersome. The limited brightness of organic fluorophores and fluorescent proteins can be overcome by using fluorescent nanoparticles (NPs). Fluorescent polymer NPs are currently attracting increasing interest due to their versatility, biocompatibility and their potential to overcome the limitations of quantum dots in terms of brightness and control of blinking. We recently showed that encapsulating a charged dye with a bulky counterion in polymer NPs led to increased brightness and a strong cooperativity of the dyes resulting in whole NP blinking. The main objective of this project is to design the smallest possible ultrabright fluorescent polymer NPs with controlled blinking required for resolving and tracking single molecules with superresolution and their adaptation to the intracellular environment. The most versatile and straightforward approach to fluorescent polymer NPs is loading with organic fluorophores. In these systems the first challenge is to achieve extreme brightness as the dyes usually undergo aggregation self-quenching at the high concentrations needed for high brightness. A second challenge is that the on-off-switching of the entire NP necessary for superresolution imaging requires a collective behavior of hundreds of fluorophores. In this project we will use encapsulation of salts of rhodamine B derivatives with bulky hydrophobic counterions in polymer NPs to create ultrabright NPs with controlled blinking. For this we will engineer the organization of dyes within the NPs by varying hydrophobicity and glass transition of the polymer, hydrophobicity of the dye salt, and assembly conditions. When using NPs as labels two further aspects have to be considered, in order to obtain optimum resolution and perturb as little as possible the observed system. First, their size should be of the order of the proteins they label. Second, aggregation and nonspecific interactions with intracellular proteins and cellular components have to be avoided. In this project we will introduce multiple charged and zwitterionic groups in polymers. Nanoprecipitation will lead to very small NPs with the thinnest possible noninteracting shells. Studying the interactions of these NPs inside cells and optimization of their surface chemistry will enable us to obtain <10 nm intracellular stealth NPs. We will validate our NPs for intracellular tracking by directly tracking cytoplasmic dynein, a molecular motor, for the first time in mammalian cells – a challenge due to the speed and resolution needed. Individual dynein motors will be labeled with our small stealth NPs. For this, NPs bearing benzyl-guanine groups will be introduced in cells expressing dynein-SNAP-tag. The NPs will be imaged using videomicroscopy and 3D-dSTORM. Adjusting the blinking of these ultrabright NPs will make it possible to track single dyneins in living cells, resolve individual dyneins at sites of high concentration, and thus gain information on their colocalization and cooperativity. The NPs developed here will, due to their size, surface properties, brightness, and controlled blinking, open the way to unprecedented single molecule superresolution tracking in living cells.

Efficient light energy collection, transport and transfer to a functional « acceptor » is a fundamental process at the origin of natural and artificial photosynthesis, photocatalysis, (organic) photovoltaic energy conversion, as well as single molecule detection e.g. in bio-medical sciences. The nature and function of the acceptor is very different in all these processes: while the photosynthetic reaction center uses this energy to perform a multistep electron transfer, a fluorophore reemits light after collecting the energy from the antenna. The latter is of key importance for amplifying fluorescence signal, which enables detection of single molecules at low excitation power and designing ultrasensitive biosensing assays. On the other hand, the function of the so-called “antennas” for collection and transport is fundamentally the same in all cases: to harvest excitation light and efficiently transfer to the acceptor. Currently the field of optical nanoantennas is dominated by plasmonics, which employs collective excitation of the conduction electrons in metallic nanoparticles or surfaces. It was successfully used to transport excitation energy along nanostructured metallic surfaces or locally enhance the antenna-acceptor coupling and transfer efficiency. It has been widely applied for single molecule detection for biosensing, and also finds applications in enhancing solar cell harvesting efficiency. However, achieving high amplification efficiency from plasmonic nanoantenna requires very precise control of positioning of functional acceptor in the hot spots. Thus, in case of single-molecule detection, the fluorophore should be placed between two metallic beads using sophisticated approaches. We recently achieved a breakthrough in the field of single molecule detection with the demonstration of single-molecule detection under ambient light illumination using organic nanoparticles (ONPs) as giant light-harvesting nanoantennas. An extremely efficient excitation energy transfer is achieved with cationic rhodamine dyes encapsulated in a poly(methyl methacrylate) (PMMA) matrix using bulky fluorinated counterions to avoid p-p stacking and self-quenching. With 60 nm-sized biocompatible ONPs, we achieved a 1000-fold amplification of the effective brightness of a single energy acceptor (Cy5) located within the nanoparticle. Remarkably, this antenna effect is 3-fold higher than that of the best plasmonic nanoantennas. In parallel, we also observed that a large number of rhodamine dyes (>500), encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) 30 nm ONPs, behaves as a single emitter displaying ON-OFF blinking with nearly 100% contrast, further supporting the presence of an extremely fast energy transport/delocalization over the entire nanoparticle. The blinking occurs because a single energy trap is able to efficiently quench the emission from the dye ensemble as it was already demonstrated for conjugated polymers. However, this quenching process limits the length, over which the energy can be transported and limits the use of these systems in organic photonics. Here, we propose to design synthetic light-harvesting nanomaterials (LHN’s) made of chromophore-doped organic nanoparticles (0D) and nanowires (1D). We will apply these LHN’s to single molecule detection under ambient light excitation via Förster resonance energy transfer to single acceptor dyes. This application will also serve as a benchmark to demonstrate the performances of our LHN’s for light collection, transport and transfer to any kind of acceptor paving the route for numerous applications.

Numerous cell mechanisms and pathways rely on dynamic interactions of RNAs or DNAs with proteins that induce local and transient changes in their secondary and tertiary structures. Though structural methods, such as X-ray diffraction, NMR or electron microscopy provide invaluable information on the structure of the protein/nucleic acid complexes, they are less suited for monitoring the dynamics of these interactions, especially in diluted solutions. Due to their exquisite sensitivity, fluorescence-based techniques are highly potent for this purpose. However, in the case of nucleic acids, these techniques suffer from the fact that natural nucleobases are almost not fluorescent, so that labeling with external and generally bulky probes is requested. A breakthrough has been recently achieved with the introduction of thienoguanosine (thG), a truly faithful emissive and responsive surrogate for G, which actually reproduces the structural context and dynamics of the parent native nucleoside. In addition, this fluorescent G analogue remains well fluorescent when incorporated in ODNs, and exhibits environment sensitive fluorescence properties. thG is expected to transform nucleic acid biophysics by allowing for the first time to selectively and faithfully monitor the conformations and dynamics of a given G residue in a nucleic acid sequence. To further extend the applications of this outstanding nucleoside analogue and provide for the first time relevant information on the local and transient modifications of nucleic acids at the single molecule level, the objective of the SMFLUONA project is to implement and apply thG-based single molecule experiments. To this end, one of the main challenges is to increase thG brightness and photostability. This will be achieved by using the surface plasmon resonances of Al and Mg nanoparticles (NPs) that match with thG absorption (300-400 nm), but require a precise control of the distance between the dye and the metallic surface. This control will be achieved by synthesizing Al and Mg NPs of controlled size and surface, and then grafting them at a 1:1 stoichiometry with thG-labeled ODNs of appropriate length or by accurately positioning them together with thG-labeled ODNs on DNA origami platforms. To validate and apply this thG-based single molecule approach, we will monitor the base flipping steps of the DNA methylation replication by the tandem formed by the DNA methyltransferase 1 (DNMT1) and its guide, the UHRF1 protein (Ubiquitin-like, containing PHD and RING finger domains). DNA methylation of cytosines in CpG sites at very precise positions on the genome provides key epigenetic marks that allow a cell to express a well-defined number of genes which determine the cell identity. When the cells multiply, they must transmit this information by faithfully copying the methylation marks. Therefore, thG-based single molecule experiments should provide for the first time a complete picture of the SRA- and DNMT1-induced base flipping processes, as well as their dependence on the CpG context, DNA length and key protein residues. This thG-based single molecule fluorescence approach will help solving a large range of biological questions involving local and transient structural nucleic acid changes. Moreover, the deciphering of the molecular mechanism of the base flipping steps in DNA methylation replication by the UHRF1/DNMT1 tandem should provide new clues on its possible blockage and thus, lead to major therapeutic applications in pathologies, such as cancers and neurodegenerative diseases, where the methylation profile is modified. This interdisciplinary project will be performed by two highly complementary partners with expertise in fluorescence, advanced fluorescence techniques and UHRF1/DNMT1 (Partner 1, Y. Mély, U. Strasbourg) and metal-induced fluorescence enhancement and synthesis of Al NPs (Partner 2, J. Plain, UTT Troyes).

The arterial wall is the target of plasma-borne components like LDL and proteolytic enzymes known to be involved in the evolution of the atherothrombotic disease. Vascular cells must be able to protect themselves from proteolytic injuries by producing antiproteases. Serine protease inhibitors (serpins) form a large family of structurally related proteins present in the plasma or in tissues and play a central role in the regulation of protease activity. Among them, serpinE2 or protease nexin-1 (PN-1) which is produced by most cell types, including vascular and inflammatory cells, is often found overexpressed at sites of tissue injury. PN-1 emerged as an important actor in the regulation of tissue proteolytic degradation since it is a powerful inhibitor of several serine proteases including thrombin, plasminogen activators (uPA, tPA) and plasmin. Many of these target serine proteases are known to be involved in thrombus formation and degradation, in matrix degradation and in cell loss. We have indeed demonstrated that PN-1 constitutes a key factor in the responses of vessels to injury, via its antithrombotic and antifibrinolytic properties. The present project is aimed at demonstrating the protective role of PN-1 in vascular tissue, in particular the arterial wall and the “neo-tissue” that is the thrombus. We want to demonstrate that the presence of PN-1 in either the arterial wall or the thrombus represents a mechanism of tissue defense against aggression by blood-borne proteases. PN-1 avidly binds glycosaminoglycans (GAGs) such as heparan sulfates which potentiate its activity, target it to the pericellular space, and impede its diffusion, proposing PN-1 as a model of tissue serpin, able to bind serine proteases and form complexes which can be, in situ, removed and degraded by endocytosis via the LRP1 scavenger receptor. In tissue, the pericellular activated serine proteases bind to serpin, forming complexes that are cleared by LRP1. This mechanism of protease-antiprotease clearance and its consequences have not yet been extensively explored in human arterial wall in vivo and particularly in VSMCs. The potential close relationships between PN-1/protease complexes & LRP1 in VSMCs underline a possible function of these two proteins in the control of protease activities in the vascular wall. We hypothesize that PN-1/protease complexes and LRP1 interact in VSMCs to eliminate deleterious proteases, participating in maintaining the homeostatic function of the vascular wall, and that this physiological clearance function of VSMCs could be overwhelmed in vascular pathology. When thrombosis affects the arterial cerebral bed, it is the most frequent cause of stroke. Interestingly, PN-1 is not only present in platelet, but has also been identified in the central nervous system as a regulator of thrombin effects on nervous cells. We demonstrated that platelet PN-1 present in blood clot contributes to clot resistance to fibrinolysis by its ability to inhibit plasminergic enzymes. However this latter property does not seem to be the only reason of such an effect of PN-1. Indeed, we observed a direct influence of PN-1 on blood clot structure and retraction. Our objective will consist to decipher at the molecular level by which mechanism PN-1 interferes on clot structure and retraction. Moreover, the characterization of PN-1 impact in blood clots opens new therapeutic possibilities for the thrombolytic treatment of stroke. Only few teams in the world are working on PN-1 and LRP1 in physiology and pathology. The Inserm U698 is leader in the field of PN-1 in the vascular system and has provided clear-cut evidence for a relevant role of PN-1 in vascular biology. In parallel, the team of Philippe Boucher is leader in the field of LRP1 functions in VSMC. The novelty of our project is to highlight new aspects of PN-1-dependent processes in vascular wall physiological protection and the role of PN-1 in the thrombus resistance to proteolysis.