Macrophages play key roles in tissue regeneration and inflammation at steady-state, two processes that are essential to maintain tissue homeostasis and, if dis-regulated, can lead to pathology. We have recently discovered a peculiar population of macrophages in the distal colon that is needed to locally maintain the survival of epithelial cells in homeostasis. These macrophages are localized in the stroma, but insert “balloon-like protrusions (BLPs)” through the basement membrane in between epithelial cells. BLPs sample the fluids that are absorbed through the epithelium and instruct epithelial cells to stop absorption if fluids are overloaded with fungal toxins. We here propose a project aiming to identify the key players involved in the tripartite interaction between macrophages, epithelial cells and fungi (Aim 1), unraveling the underlying molecular and cellular mechanisms (Aim 2) and evaluating whether and how failure in this “ménage à trois” can lead to colon pathology (Aim 3).

Cells can sense and respond to external forces and mechanotransduction events appear to be critical for most cellular functions. While mechanotransduction has been extensively studied at the plasma membrane and at the nucleus, the effect of forces on other intracellular organelles is still not clear. Our project will study mechanotransduction at the Golgi apparatus, a central organelle for intracellular transport pathways. We will focus on the role of the small G protein CDC42, a Golgi-localized protein involved in polarized membrane trafficking, which has recently been suggested to be involved in mechanosensing in the secretory pathway. We have three objectives: 1) quantifying the impact of external constraints on Golgi mechanics and tension; 2) measuring the effects of CDC42 activity on force transduction at the Golgi apparatus; and 3) investigating the role of CDC42 in the mechanosensitivity of the secretory pathway. To achieve these objectives, we will use a combination of biophysics and cell biology approaches. A range of mechanical cues will be applied for instance by intracellular optical tweezers, by osmotic shocks, or by plating cells on substrates of defined rigidities. In parallel, Golgi tension will be monitored with recently developed biosensors. The role of CDC42 in mechanotransduction at the Golgi apparatus will be investigated by modulating CDC42 expression and activity. Conversely, we will study the putative effects of mechanical constraints on CDC42 activity and Golgi tension. We hope to better decipher how the Golgi apparatus can act as a tension sensor and regulate, via CDC42, the mechanosensitivity of post-Golgi trafficking.

Proximity between proteins plays an essential and ubiquitous role in many biological processes. Molecular tools enabling to control and observe the proximity of proteins are thus essential for studying the functional role of physical distance between two proteins. In this context, chemically induced proximity (CIP) technologies have been transformative for studying the role of protein proximity in cellular and physiological mechanisms. Based on the genetic fusion of proteins to dimerization domains that interact in a specific manner in presence of a small chemical inducer of proximity, CIP technologies enable to modulate protein interactions and thus biological processes with high temporal control. In this project we propose to develop a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities for real-time monitoring of proximity. The developed technology will rely on genetic fusion to small dimerizing domains that interact together in presence of fluorogenic inducers of proximity that fluoresce upon formation of the ternary assembly, allowing real-time monitoring of chemically induced proximity. This technology should be game-changing as it will open a wide range of possibilities to study the role of protein proximity in biology and control biological processes for biological/medical applications. We will demonstrate our ability to regulate and track biochemical and cellular processes through various mechanisms including regulated localization/compartmentalization of proteins regulated transport of organelles. In addition, we will show that the intrinsic imaging/sensing capabilities of the system provide unprecedented opportunities to design sensors of cell signaling and cell physiology, and image and control endogenous proteins.

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.

Biomembranes are ubiquitous lipid structures that delimit the cell surface and organelles and operate as platforms for a multitude of biomolecular processes. Tension is a major property of membranes. It contributes significantly to the regulation of membrane deformation and topological alteration during fusion and fission events. Therefore, membrane tension probably regulates all membrane dynamics in cells to some extent. Unfortunately, limited data are available on intracellular membrane tension and its regulation due to lack of established tools to measure it. This precludes an understanding of major regulatory mechanisms of organelle dynamics. The objective of this proposal is to comprehensively reveal the molecular mechanisms regulating intracellular membrane tension during autophagy, a conserved autodigestive pathway that is characterized by major endomembrane deformations. Particularly, we will focus on membrane tension regulation by actin-dependent motors of the myosin family and their control of membrane dynamics during autophagy. Our hypothesis is that unconventional myosins, Myo1B and Myo1C, known to regulate autophagy, potentially helped by Myo7A found at lysosomes, exert their distinct functions through control of lysosomal membrane tension. Here we will perform 3D resolved measurements of lysosomal membrane tension through the development of fast and high resolved FLIM microscopy in cells during autophagy (WP1). We will combine this approach with mechanistic studies using giant unilamellar vesicles (GUVs)- and organelle-based in vitro reconstitution assays (WP2) and structural analysis of the motors (WP3) to provide unprecedented detail on how cytoskeletal motors regulate membrane tension. We believe that our multi-scale study will allow to fully grasp the molecular mechanisms of intracellular membrane tension control by unconventional myosins 1 and 7A during autophagy.