Periodic Reporting for period 4 - NANO-MEMEC (Membrane-based nano-mechanobiology: Role of mechanical forces in remodelling the spatiotemporal nanoarchitecture of the plasma membrane)
Reporting period: 2023-06-01 to 2025-05-31
Cells communicate with other cells and their surrounding environment through multiple receptors, proteins and lipids located at the cell surface. Specific receptors recognise their ligands and upon binding to them, they rely this information to the cell interior by initiating signalling cascades that ultimate result in appropriate cell responses. Research over the last two decades has evidenced that aside from receptor expression, their spatial organisation within the plane of the membrane plays a crucial role for initiating cellular function. Importantly, this organisation occurs at multiple spatial scales, starting at the nanometre scale and being highly dynamic. These studies have provided unique insights on receptor spatiotemporal organisation and cell function but have been mostly performed in the absence of mechanical signals. Yet, cells in our body are exposed to different types of mechanical stimuli: shear stress in blood and lymph, irregular topographical cues of extracellular matrix fibres, changes in cell contractility and tension, etc. Components of the cell machinery such as the actin cytoskeleton, the glycocalyx matrix, and the lipid bilayer itself, sense these mechanical cues. How do mechanical stimuli affect the spatiotemporal organisation 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 addressed in NANO-MEMEC. Since the process of mechanical sensing and transduction starts at the molecular and nanoscale levels, a mechanistic understanding at the nanoscale is vital to further progress in the field. The overall objective of NANO-MEMEC has been thus to provide quantitative and mechanistic understanding on the role of mechanical stimuli and biochemical coupling in the spatiotemporal organisation of receptor nano-assemblies at the cell membrane. Our ambition has been to visualise, probe and quantify these processes at the relevant spatiotemporal scales with single molecule detection sensitivity within the whole complexity of the living cell. By doing so, 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. NANO-MEMEC poses fundamental questions to the field of cell membrane mechanobiology, but with an impact for society in the long run: receptor organisation is important for proper cell function, and any mis-regulation of this organisation will result in diseases including cancer, neurological disorders and auto-immune pathologies amongst others. Insight on how receptors organise 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 was structured along three main objectives: Objective 1: to dissect mechanical and biochemical coupling of mechanosensing at the cell membrane; Objective 2: to visualise and quantify the coordinated spatiotemporal recruitment of integrin-associated signalling proteins in response to force and associated to mechanotransduction; Objective 3: to determine how changes in spatiotemporal remodelling of integrin receptor nanoplatforms and interactions with their ligands propagate through the intracellular machinery to impact on cell response. During the project, we developed and extensively applied new methodologies based on high-density multi-colour single molecule imaging and/or spectroscopy at the nanoscale fully compatible with force application. In relation to objective 1, we investigated the role of the glycocalyx matrix (in particular galectins), membrane tension, lipid packing and the immediate cortical cytoskeleton. From these, the actin cytoskeleton and its interaction with the proximal membrane had the most profound effects on the organisation of transmembrane receptors, coordinating their dynamics at multiple temporal scales, modulating the interactions with other receptors and ultimately impacting in receptor function. Regarding objective 2, we investigated how mechanical forces impact the nano- and meso-scale organisation and activation of integrin mechanosensitive receptors and the recruitment of signalling adaptor molecules in the process of mechanotransduction. We found that while integrin nanoclusters are force independent, their organisation inside focal adhesions is highly regulated by force loading on integrins, underscoring the importance of spatial compartmentalisation on cell adhesion. Regarding objective 3, we discovered that prolonged shear-forces induces actin-dependent ICAM-1 nanoclustering on the surface of endothelial cells, increasing in turn adhesion and migration capacity of leukocytes in the presence of forces. In a similar and highly orchestrated manner, the integrin LFA-1 expressed in leukocytes, and receptor for ICAM-1, becomes further activated in the presence of lateral mechanical forces. This overall force-induced spatial re-arrangement of receptors and their cognate ligands guarantees successful leukocyte migration and extravasation during the adhesion cascade of immune cells. We have disseminated our results at more than 70 international conferences, workshops and topical meetings and have produced so far a total of 19 publications in journals of high impact in the field (several papers are still in preparation or revision). The PI has also been invited as keynote or plenary speaker at multiple conferences in the field and has been invited to write several specialised reviews in the field.
The importance of mechanobiology regulating a broad range of biological processes has been known for decades. Moreover, a range of mechanosensitive molecules had been already identified and their responses studied at the single molecule level but mostly under in-vitro conditions, isolated from the full complexity of the living cell. Yet, prior to NANO-MEMEC, our knowledge on how mechanical forces affect individual multi-molecular interactions and membrane spatiotemporal organisation of mechanosensitive platforms was quite limited. Likewise, it remained unknown how physical and biochemical inputs couple and integrate to initiate mechanical sensing and signal transduction.
NANO-MEMEC has created a palette of exclusive biophysical tools that combine super-resolution microscopy and single molecule imaging together with different ways to exert mechanical stimuli fully compatible with these highly challenging techniques. By doing so, we have discovered that mechanical forces play a crucial role in re-modelling the cortical actin cytoskeleton and therefore impacting directly on the organisation of multiple receptors on the plasma membrane, even for those receptors that do not directly interact with actin. We have identified different temporal dynamics associated to receptor interactions with the cortical actin and have directly visualised how interactions between multiple receptors on the plasma membrane are ultimately coordinated by the actin machinery, with a major impact in receptor function. Finally, our project demonstrated that mechanical forces play a major role regulating the spatial re-arrangement of receptors and their cognate ligands to guarantee successful leukocyte adhesion and migration, a process that is crucial for the extravasation of immune cells.
NANO-MEMEC has created a palette of exclusive biophysical tools that combine super-resolution microscopy and single molecule imaging together with different ways to exert mechanical stimuli fully compatible with these highly challenging techniques. By doing so, we have discovered that mechanical forces play a crucial role in re-modelling the cortical actin cytoskeleton and therefore impacting directly on the organisation of multiple receptors on the plasma membrane, even for those receptors that do not directly interact with actin. We have identified different temporal dynamics associated to receptor interactions with the cortical actin and have directly visualised how interactions between multiple receptors on the plasma membrane are ultimately coordinated by the actin machinery, with a major impact in receptor function. Finally, our project demonstrated that mechanical forces play a major role regulating the spatial re-arrangement of receptors and their cognate ligands to guarantee successful leukocyte adhesion and migration, a process that is crucial for the extravasation of immune cells.