Membrane-based nano-mechanobiology: Role of mechanical forces in remodelling the spatiotemporal nanoarchitecture of the plasma membrane

Periodic Reporting for period 3 - NANO-MEMEC (Membrane-based nano-mechanobiology: Role of mechanical forces in remodelling the spatiotemporal nanoarchitecture of the plasma membrane)

Periodo di rendicontazione: 2021-12-01 al 2023-05-31

Cells communicate with other cells and their surrounding environment through a myriad of receptors, proteins and lipids that are all located at the cell surface. Specific receptors recognize their ligands (on the cell outside) and upon binding to them, they rely this information to the cell interior by initiating signalling cascades that ultimate result in a appropriate cell response. Research over the last twenty years has evidenced that aside from receptor expression at the cell surface, their spatial organization within the plane of the membrane plays a crucial role of initiating cellular function. Importantly, this organization occurs at multiple spatial scales, starting from the nanometer scale and moreover it is not fixed in time, i.e. receptors and proteins aggregate and disassemble dynamically in a highly complex manner. All these studies have provided unique insights on receptor spatiotemporal organization and cell function but have been performed so far in the absence of any mechanical stimuli. In contrast, cells in our body are exposed to different types of mechanical stimuli. For instance, during their life, cells of the immune system experience a large range of mechanical stimuli, from shear stress in blood and lymph, irregular topographical cues of extracellular matrix fibres, and changes in cell contractility and tension during extravasation. Components of the cell machinery such as the actin cytoskeleton, the extracellular matrix, and even the cell surface and cell nucleus will feel and sense these mechanical cues. How do mechanical stimuli affect the spatiotemporal organization of receptors on the cell surface?; how these changes transduce to the cell interior to ultimately modulate cellular response? These are the two major questions that we would like to address in NANO-MEMEC. Since the entire process of mechanical sensing and transduction (also known as mechanobiology) starts at the molecular and nanoscale levels, a mechanistic understanding at the nanoscale is vital to further progress in the field. This in turns crucially depends on our availability to visualize, probe and quantify these processes at the relevant spatiotemporal scales. The overall objective of NANO-MEMEC is therefore to provide quantitative and mechanistic understanding on the role of mechanical stimuli and biochemical coupling in the spatiotemporal organization of receptor nano-assemblies at the cell membrane. We intend to directly visualize, probe and quantify these processes at the relevant spatiotemporal scales with single molecule detection sensitivity. We hope that NANO-MEMEC will open new frontiers of research by establishing membrane-based nano-mechanobiology as a novel mechanism that crucially contributes to signal transduction and cellular response. Although NANO-MEMEC has a fundamental character, the impact for Society could be large: receptor organization is important for proper cell function, and any mis-regulation of this organization will result in disease and different pathologies including cancer, neurological disorders and auto-immune diseases amongst others. Insight on how receptors organize and respond in the presence of mechanical stimuli will allow us to identify what, when and how it goes wrong to hopefully trigger the development of more targeted therapies to correct for these mis-functions.

NANO-MEMEC is structured along three main objectives: Objective 1: to dissect mechanical and biochemical coupling of mechanosensing at the cell membrane ascertaining how forces alter membrane physical properties (tension, fluidity), the cortical actin cytoskeleton and the glycocalyx matrix, affecting the nanoscale organization and molecular conformation of integrin receptors. Objective 2: to visualize and quantify the coordinated spatiotemporal recruitment of integrin-associated signaling proteins in response to force and associated to mechanotransduction, dissecting how these events couple back to remodel the cell membrane. Objective 3: to determine how changes in spatiotemporal remodeling of integrin receptor nanoplatforms and interactions with their ligands propagate through the intracellular machinery to impact on cell response, from adhesion to migration of immune cells. In this reporting period we have mainly concentrated on objectives 1 and objectives 3 and started to perform preliminary experiments along objective 2. In particular, we have developed a new high-density single molecule-based approach to map how receptors at the cell surface dynamically explore the 2D space over multiple spatiotemporal scales. By using the prototypical receptor CD44, known to directly interact with the cortical actin cytoskeleton and the extracellular matrix, we have been able to discriminate the role of actin and the glycocalyx matrix orchestrating the organization of many cell surface receptors (Mol. Bio. Cell 2020). Our results indicate that the glycocalyx matrix strengthen interactions between receptors, while the cortical cytoskeleton imposes dynamic fences that constrain and relax the diffusion and interaction between multiple partners of the cell membrane. Interestingly, actin remodeling occurs at multiple temporal scales and our work conciliates the different dynamical scales that have been observed in separate experiments by us and other groups around the world. In addition, as part of objective 1, we have exploited our photonic antennas configuration to investigate the role of extra-cellular glycans on the patterning of the lipid bilayer composing the cell surface. Our work reveals that glycans have a profound impact modulating the physico-chemical properties of the lipid bilayer itself, synergizing with cholesterol to alter the local fluidity and mechanical properties of the membrane at the nanoscale (J. Phys. Chem. Lett. 2021). In relation to objective 3, we have provided evidence that prolonged physiological shear-forces promote the formation of ICAM-1 nanoclusters on endothelial cells and cause actin-dependent polarization of ICAM-1 against the flow direction. Importantly, we determined for the first time, to our knowledge, that this shear-force induced ICAM-1 nanoclustering is sufficient to alter the migration of T cells under flow, inducing a more migratory T cell profile and accelerating their migration (Biophys. J. 2021). Regarding objective 2, we are currently investigating the nanoscale organization of different integrin receptors and their main molecular adaptors (paxillin, talin and vinculin) both on fibroblasts and T cells. So far, our unpublished data reveals that: a) integrins and their molecular adaptors organize as segregated nanoclusters inside adhesion structures forming dynamic nano-hubs of activity; b) interactions between integrins and their molecular adaptors (in particular talin and vinculin) are highly dynamic so that at a given moment of time, only a few integrins are actively engaged with their partners; c) integrin receptors are highly sensitive to mechanical force, with a modest shear stress application being sufficient to induce conformation changes of integrins that promote their activation.

So far, our research has produced interesting results beyond the current state-of-art. In terms of technology development, we have implemented a new methodology based on high-density single particle tracking together with a dedicated software algorithm capable to dynamically mapping how molecules diffuse in their environment at multiple spatiotemporal scales (manuscript in preparation). Within the context of NANO-MEMEC we have applied this methodology for identifying the role of the actin cytoskeleton and the glycocalyx matrix. Nevertheless, this methodology is equally powerful to reveal compartmentalization of many other cell organelles such as the cell nucleus or Golgi complex. We hope that this methodology will be adapted by many other researchers in the field. We have also implemented a multiplexed approach for simultaneous readout of hundreds of photonic antennas in a single measurement (in preparation). This approach will allow us to record with unprecedented high throughput the dynamics of individual molecules on the cell surface. We have determined the role of extracellular glycans on the organization of the lipid bilayer composing the cell membrane. It has been known that glycans interact with receptors on the cell surface, but their role on the lipid bilayer itself was unclear. Our results now reveal that glycans synergize with cholesterol to modulate the local fluidity and mechanical properties of the bilayer, impacting on the organization of membrane components. We have identified for the first time, to our knowledge, the role of mechanical forces on the nanoscale organization of ICAM-1 expressed on endothelial cells (ECs). Although the role of mechanical forces on the activation of the integrin LFA-1 is well-established, the impact of forces on its major ligand ICAM-1, has received less attention. Using a parallel-plate flow-chamber combined with confocal and super-resolution microscopy, we showed that prolonged shear-flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear-forces caused actin re-arrangements and promoted actin-dependent ICAM-1 nanoclustering prior to LFA-1 engagement. T-cells adhered to mechanically pre-stimulated ECs or nanoclustered ICAM-1 substrates, developed a pro-migratory phenotype, migrated faster and exhibited shorter-lived interactions with ECs than when adhered to non-mechanically stimulated ECs, or to monomeric ICAM-1 substrates. Together, our results indicate that shear-forces increase ICAM-1/LFA-1 bonds due to ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. This mechanism might thus be important for firm leukocyte adhesion and migration during inflammation. Our data also underscores the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation. Our current research indicates that integrin receptors and their molecular adaptors organize in segregated nano-hubs that dynamically interact with each other to engage in adhesion and/or mechanical sensing. Moreover, single molecule experiments reveal a profound effect on the activation of integrins in the presence of flow. We expect to elucidate in the next period how forces contribute to this dynamic exchange of activity and ultimately determining the consequences for cell adhesion and migration.

Multi-color super-resolution imaging of adhesion molecules on human fibroblasts

Reconstructed hd-SPT maps for (left) simulations of random motion & (right) experiments on CD44

Periodic Reporting for period 3 - NANO-MEMEC (Membrane-based nano-mechanobiology: Role of mechanical forces in remodelling the spatiotemporal nanoarchitecture of the plasma membrane)

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