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.

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.

Small interfering RNAs (siRNAs) are specific and effective molecules for gene silencing, but fail to enter cells unassisted due to their negative charges and their high molecular weight/size. The conjugation of siRNA to ligands targeting cell surface receptors is a promising approach to silence genes associated with various pathologies. However, besides the external cell barrier, to reach the cytosol where the siRNA associates with the RISC (RNA-induced silencing complex) machinery, the siRNA has to escape from intracellular compartments (ICC). However, despite encouraging perspectives, RNA therapeutic applications are hindered by a low rate of ICC escape. To enhance the cytosolic delivery of siRNAs, one strategy is based on multi-ligand conjugates. Its efficacy has been proven with recently marketed drugs composed of three N-acetylgalactosamine ligands conjugated to siRNA. This bioconjugate has led the way for the use of siRNA conjugates in therapy. However, these conjugates are not transposable to pathologies other than liver diseases. Moreover, mechanisms of escape across ICC remain unknown, the underlying mechanisms relating affinity/avidity/specificity to siRNA cytosolic delivery remain to be elucidated, and active targeting strategies for organs/cells other than liver/hepatocytes, remain to be explored. Our project aims at deciphering and optimizing the delivery of siRNAs mediated by aptamers. Aptamers are nucleic acid molecules which bind to their targets with high affinity and selectivity. When the target is a cell-surface biomarker, aptamers have huge potential as specific cell-targeting ligands. If the receptor is internalized, aptamers can drive conjugated siRNA inside cells. Moreover, their chemical synthesis allows for a variety of modulable constructs. Our project aims to provide strategies based on multivalent/multispecific nucleic acid aptamers as innovative active targeting tools to enhance selective siRNA delivery to targeted cells. We will develop multifunctional molecules, in which the aptamer(s) is/are the ‘cell-targeting part(s)’ and the siRNA is the ‘therapeutic part’. These aptamers conjugated to siRNA are named AsiC for aptamer-siRNA conjugates. This project, based on one siRNA and three aptamers targeting three different cell surface receptors, aims to 1) synthesize conjugates composed of 1-3 aptamers conjugated to siRNA, 2) characterize their interaction kinetics, and 3) follow their intracellular trafficking, to in fine provide insights to improve the delivery of siRNA by active targeting.

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.

Human pathogens such as enveloped viruses enter and exit host cells by interacting with the cell membrane. In addition, they acquire their lipid envelop from the host cell. Consequently, the characteristics of the viral lipid membrane is of high importance for virus infectivity. There are numerous reported evidences of the manipulation of the host cells’ lipid metabolism and dynamics by viruses during their assembly or cell entry. Using live cell super-resolution dynamic microscopy, we have previously shown how a retrovirus sorts specific lipids during assembly at the plasma membrane of the infected cell to create a favorable environment. However, very little is known about how other viruses, like the recent pandemic SARS-CoV2, assemble at and acquire lipids from the host cells’ and more complex inner membranes, like the endoplasmic reticulum-Golgi Intermediate compartment (ERGIC). There are several key questions pending. Is the lipid composition of viral envelop defined by that of ERGIC, site of virus assembly, and/or by the viral structural proteins? How do these specific lipids control the mobility of viral envelop proteins and thus infectivity? How do these lipids affect viral particle endocytosis or fusion during viral entry? In this project, we will use dynamic super-resolution microscopy in combination with novel functional membrane probes and fluorescent SARS-CoV-2 particles from model systems up to wild-type viruses to decipher the role of lipids in virus assembly and entry. Apart from cholesterol, no other lipid has been identified to play a role in virus-host cell fusion or assembly of SARS-CoV-2. In this project, we will first develop new fluorescent tools for observing host-lipid interactions during virus infection, in particular with the structural viral proteins N/M and S, which includes new generation of fluorescent SARS-CoV-2 virus-like particles, fluorescent molecular probes for ER/ERGIC/Golgi, and adapted advanced fluorescence microscopy tools. Then, we will (i) identify virus particle lipids using lipidomic, (ii) sense lipid organization and interactions with the viral structural proteins (nucleocapsid N and membrane protein M and the viral envelop Spike S protein) by specially designed fluorescent proteins and environment-sensitive probes that specifically label the inner cell membrane compartments, (iii) monitor changes in lipid dynamics inside living cells while tracking the viral structural proteins, both at few/single molecule level thanks to super-resolution microscopy, and (iv) customize virus-like particles to decipher the interplay with identified lipids and the viral envelop S proteins during recognition to the receptor and during entry by fusion or endocytosis. Our project will address these issues thanks to our strong complementary expertise in virology, lipidic membranes, super-resolution dynamic microscopy and innovative fluorescent probes. Expected outcomes will shed the light on molecular manipulation of inner cell lipids by viruses during their assembly and how this facilitates their entry into new host cells.