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.

Membrane contact sites (MCSs) enable specific lipid exchange between organelles. Our recent results on the archetypical OSBP/VAP complex suggest that the architecture and dynamics of MCS are influenced by intrinsically disordered regions (IDRs). These overlooked structural attributes enable formation of MCS of adjustable thickness and reduce protein crowding. These effects are likely to be general given the abundance of IDRs in MCS proteins. We posit that IDRs guarantee the lateral and/or vertical flexibility of proteins. We will dissect the link between these characteristics and the function, dynamics and organization of MCSs, by using a multidisciplinary and multi-scale approach involving quantitative and super-resolution cell imaging, in vitro reconstitution of membrane systems, structural analysis by cryo-electron tomography, and the development of innovative pharmacological tools. This project will offer new perspectives on the efficiency, plasticity and specificity of MCSs.

Mammalian cells can adopt a large variety of shapes often associated with a specific internal organisation called cell polarity, which is instrumental for cell migration and tissue morphogenesis. The capacity of cells to control their shape and polarity relies in part on a thin layer of actin filaments associated to the plasma membrane, the actin cortex. We will use novel tools that we developed to measure locally and simultaneously, in live cells, the parameters defining the physical state of the actin cortex (its thickness, stiffness, viscosity and molecular tension), while controlling its polarity using optogenetics. This study will be first performed on single cells in a controlled micro-environment, then in the context of a polarised epithelial monolayer. With this project, we propose to understand how, in addition to molecular components, mechanical properties of the cell cortex can also be polarised, and how this polarity contributes to single cell and tissue morphogenesis.

Fast progress in genomics and cell biology is uncovering the complexity of the mechanisms underlying diseases and aging. The application of this new understanding in health, however, raises stronger and stronger technological challenges. This slows down the current pace of progress, and leads to an explosion of costs in pharmaceutical research, and to increasing difficulties to choose the right treatment among a constantly increasing choice of possible drugs and therapeutic options. This diversification let to the concept of “precision” or “personalized” medicine, aiming at selecting the best treatment for each patient using a more and more detailed characterization of his/her specific pathology, at the molecular and cellular level. The DROMOS project aims at helping the development of more powerful, more specific and less costly diagnosis and treatments, by combining two innovative technological fields. The first is “organoids”: it consists in growing, from cells from patients or stem cells, 3D cellular aggregates reproducing the structure of tissues. On can then use them to test drugs, in a more powerful and less expensive way than with previously used animal models. The second technological field is microfluidics: microfluidic systems can be seen as “microprocessors” able to manipulate minute fluid volume, as conventional microprocessors manipulate information. Microfluidic devices allow to considerably increase the number of tests feasible with a sample, and to accelerate testing. Unfortunately, so far the wide transfer of these technologies to daily life was hindered by their cost and their complexity. The project will overcome this limitation using a new technology “Free Flow Textile Microfluidics”, allowing the mass-production of integrated and low-cost microfluidic systems, by textile technologies. These systems use a combination of innovations by the partners, which allow the integration of complex fluidic architectures and functionalities within the textile “chip”. A preliminary study has validated an original approach, in which hundreds of organoids can be cultivated within droplets prepared in the textile chip, and tested individually against various drug combinations. This approach will find applications e.g. in pharmaceutical research or in precision medicine. The project will develop in parallel the textile microfluidic chips and the instrument allowing their operation and the implementation of assays. This will be followed by a biological and clinical validation, and finally by the development of a commercial instrument, to transfer as fast as possible the project’s results to patients and the society. Validation will focus on a specific problem: the screening of drugs against pediatric cancers. The textile microfluidic technology is very generic, however, and it will be applicable to a number of other biomedical problems, such as the diagnosis of infectious diseases, regenerative medicine or patient’s follow-up by “intelligent” clothing. This interdisciplinary project will involve close collaboration between: the microfluidics laboratory MMBM, affiliated to Curie Institute, CNRS and IPGG (Pierre-Gilles de Gennes Institute, first European institute fully dedicated to microfluidics); the GEMTEX laboratory of ENSAIT (National Superior School of Textile Arts and Industries), a European leader in textile innovation; the Clinical and fundamental research laboratory U830 Institut Curie/INSERM, who will develop biological and clinical aspects and testing; and finally the biomedical startup INOREVIA, who will develop the pre-industrial prototype, and prepare industrialization and commercialization of the technology after its validation within the project.

We aim to understand how the primitive streak (PS) is patterned in the mouse embryo. This transient structure plays a crucial role in the organization of the body plan. Classical embryology has established that cells emerging from it at different levels and times adopt different cell identities, but how this is achieved is still not understood. The BMP, WNT and NODAL signaling pathways are known to be involved, but altering precisely the level and duration of these signals to examine how it impacts cell-fate decisions is challenging in the mouse embryo. However, the development of an embryonic stem-cell-based in vitro model system recapitulating key aspects of gastrulation, and resulting in the formation of 2D gastruloids (2Dgas), has allowed us to investigate its underlying mechanisms. The results we have obtained so far are consistent with those previously obtained in the embryo, but are at odds with current theoretical models of PS patterning. In particular, we found that in addition to promoting proximal PS cell fates in m2Dgas, BMP actively suppresses distal ones. We propose to address two fundamental questions: First, what are the BMP, WNT and NODAL signaling requirements for the correct allocation of cell-fates within the PS? Second, what are the molecular mechanisms underlying the patterning of the PS, and in particular, how is BMP signaling controlling the expression of NODAL and WNT signaling targets? To carry out these investigations we recently designed a microfluidic chip in which pluripotent cells can be exposed to defined gradients of molecules for defined length of time. This allows us to explore in vitro the signaling parameters that ensure the patterning of a complete PS and the formation of all its derivatives. In combination with imaging, genome editing, single cell transcriptomics and epigenomics approaches, our in vitro models will give us the means to dissect the respective roles played by the BMP and NODAL signaling pathways in PS patterning. The results of our experiments will we processed and analyzed using a machine learning-based program, designed by one of us, to infer causality. These analyses will infer possible regulatory interactions playing critical roles in the PS cell identity decision process. The validity of these inferences will tested both in vitro and in vivo. We expect that the completion of our project will lead to advances in our understanding of the signaling landscape that shapes the formation and patterning of the PS. We should also finally understand how, within the PS, BMP signaling redirects the action of NODAL and WNT signaling, and affects the process that underlies cell-fate decisions. Finally, we should make some significant advance in our understanding of how the grammar of TF binding sites at an enhancer translates into a specific pattern of activity. Completing PATTERNING will be an opportunity to identify general principles that will apply to other biological contexts, and may have applications in healthcare or bioengineering.