Periodic Reporting for period 4 - MechaDynA (Multi-scale mechanics of dynamic leukocyte adhesion)
Okres sprawozdawczy: 2023-03-01 do 2024-08-31
One of the first steps in immune response involves the slowdown of white blood cells flowing with the blood stream for then firmly adhere to the vascular wall and crawl to reach the site of injury or inflammation. During this process, the blood flow and active force generation by the cells result in important mechanical forces that deform the cells and break individual adhesion linkages between them. These forces are generated at different time scales, from very short to very long lapses of time. Thus, knowing the forces involved during the different steps is essential to better understand this process. However, measurement of forces on cells at the small length scales of the cell and covering all the range of time scales requires development of new nanotools: instruments working at the nanometre scale. The aim of this project was to develop new nanotools to understand the physics behind white blood cells activity over the widest temporal range, from sub-microsecond to minutes and hours. The outcomes have established these novel nanotools applicable to understand relevant biological processes such as cell-cell and virus-cell binding. The results provided fundamental understanding of the binding mechanisms behind the adhesion of cells and viruses and the regulation of cell mechanics. This may help the rational desing of drugs and vaccines and to the discovery of novel biomakers of disease based on cell and tissue mechanics.
A team of experts in the fields of biophysics, engineering, nanotechnology and computational biology was formed. The team developed two new nanotools to probe adhesion and mechanics on living cells: 1) high-speed atomic force microscopy (HS-AFM) coupled to fast confocal fluorescence microscopy that gives access to force measurements at the shortest timescales (sub-microsecond), and 2) acoustic force spectroscopy (AFS) coupled to reflection interference contrast microscopy (RICM) that gives access to biomechanical processes at long timescales (hours). The new nanotools were calibrated on model systems, and adapted and improved to work on living cells. We developed dedicated control software for the two instruments and to synchronize them with the coupled optical microscopes. We developed associated data analysis software that allows fast and robust extraction of the mechanical parameters from experimental data. We applied the system to living cells revealing the mechanics and adhesion of white blood cells and viruses. We found that white blood cells become stiffer and stickier under conditions under inflammatory conditions. The technology developed allowed us to study the first step of SARS-CoV-2 virus infection, by deciphering the mechanisms of the spike protein binding to its receptor. The AFS development allowed us to probe the interaction between receptor and ligands at ultraslow loading rates. Combined with our HS-AFM system and simulations, we cover an unprecedented total range expanding 15 orders of magnitude in time. This opens the door to unexplored biophysical regimes.
We have published our results through various works in peer reviewed journals (Karageorgi et al. 2020; Ilić et al. 2022; Martins et al. 2022; Junior et al. 2023; Eroles, et al. 2023; Wang et al. 2023; Mesbah et al. 2024; Saha et al. 2024). Two methodological works on AFM calibration methods, necessary for robust and quantitative measurements (Sumbul et al. 2020; Rodriguez-Ramos and Rico, 2021). Five review articles on HS-AFM, AFM instrumnetation, cell mechanics, and mechanical biomarkers (Valotteau et al. 2019; Casuso et al 2020; Lacaria et al. 2023; Casuso et al. 2023; Eroles and Rico 2023). We have disseminated the developed software as open-source trhough repositories for being used and improved by the research community (Alonso et al. 2023).
We have published our results through various works in peer reviewed journals (Karageorgi et al. 2020; Ilić et al. 2022; Martins et al. 2022; Junior et al. 2023; Eroles, et al. 2023; Wang et al. 2023; Mesbah et al. 2024; Saha et al. 2024). Two methodological works on AFM calibration methods, necessary for robust and quantitative measurements (Sumbul et al. 2020; Rodriguez-Ramos and Rico, 2021). Five review articles on HS-AFM, AFM instrumnetation, cell mechanics, and mechanical biomarkers (Valotteau et al. 2019; Casuso et al 2020; Lacaria et al. 2023; Casuso et al. 2023; Eroles and Rico 2023). We have disseminated the developed software as open-source trhough repositories for being used and improved by the research community (Alonso et al. 2023).
This project has established two new nanotools to study the mechanics of living cells at unprecedented timescales. The tools allow exploring fundamental biophysical processes that occur at very short and very long timescales, so far unexplored. The development of the nanotools may lead to patents and will help the development of an open-source instrument for the emerging field of mechanobiology.
The application of the nanotools to study other biological and physical systems, such as virus-cell binding and polymers. The results provide information from a novel perspective on essential steps during immune response, important for better diagnosis, prognosis and treatment of disease. It may also allow the detection of a novel kind of biomarkers of diseases based on the mechanics of cells and tissues. In the context of the COVID-19 pandemic, we were able to apply our tools to explore the binding mechanisms of the spike protein of SARS-CoV-2 from a novel perspective.
All the developed software is publicly available in open-source. This will allow other researchers to use it, improve it and adapt it to their needs.
The application of the nanotools to study other biological and physical systems, such as virus-cell binding and polymers. The results provide information from a novel perspective on essential steps during immune response, important for better diagnosis, prognosis and treatment of disease. It may also allow the detection of a novel kind of biomarkers of diseases based on the mechanics of cells and tissues. In the context of the COVID-19 pandemic, we were able to apply our tools to explore the binding mechanisms of the spike protein of SARS-CoV-2 from a novel perspective.
All the developed software is publicly available in open-source. This will allow other researchers to use it, improve it and adapt it to their needs.