Periodic Reporting for period 2 - R2-TENSION (In-situ spatiotemporal imaging of membrane hydration, electrostatics, tension and curvature to understand cell response to osmotic shocks and cell migration.)
Reporting period: 2023-05-01 to 2024-10-31
Lipid membranes compartmentalize and protect cells from the environment, and selectively permit transport. This functionality derives from unique membrane properties: 5 nm thick, yet fluid, deformable, resistant to stress and chemically complex. The physicochemical properties of membranes are expected to have a major impact on cell life, but the tools to measure the relevant multiscale and dynamic parameters in-vitro and, more importantly, in-vivo membranes are lacking. An essential property of membranes, their hydration and charge state, both needed for membrane integrity and playing a vital role in cell survival is not understood beyond the level of continuum theory. To enable quantitative physics, and physical chemistry for biology Roux and Roke, R2, will join their expertise on molecular biology & biophysics and physics & interfacial chemistry & optics to create tools to measure membrane water and ion fluxes, and image 3D fields of electrostatic free energy, membrane tension and curvature. We will understand how molecular factors, such as the influence of the aqueous phase and interfacial electrostatics, are coupled to tension and curvature under dynamic conditions such as osmosis in artificial and cellular membranes. Obtaining the first temporally resolved, 3D maps of hydration, free energy, tension and curvature at the nanoscale in migrating cells and cells experiencing osmotic shocks we will quantify membrane physical parameters in two processes essential for cell survival, for which currently no data is available: Osmotic shock response and cell migration. Osmotic shock response plays an important role in infections, kidney and intestine function. Cell migration is essential to many cell processes, for example the spreading of cancer, wound repair and the immunological response, as well as food search in unicellular organisms.
ROKE
• A high throughput 2P-FLIM microscope has been constructed.
• A new SFS instrument is under construction.
• Using the microscope we have taken SH images of several samples, testing water imaging, resonant SH fluorophore imaging and 2PF imaging. The images were compared in terms of quality to a modified version of an earlier microscope. Output: High throughput wide field SH imaging of giant unilamellar vesicles. M. Eremchev, D. Roesel, P.-M. Dansette, A. Michailovas, and S. Roke, Biointerphases 18, 2023; doi: 10.1116/6.0002640
• Measurements of water using high-throughput wide-field nonresonant second har-monic (SH) microscopy of membrane water shows that membrane potential fluctua-tions are universally found in lipid bilayer systems. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These tran-sient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through giant unilamellar vesicle (GUV) membranes. First test were also performed with living cells.
Output: 1 ) Passive transport of Ca2+ ions through lipid bilayers imaged by wide-field second harmonic microscopy, M. Eremchev, D. Roesel, C. S. Poojari, A. Roux, J. S. Hub and S. Roke, Biophys. J. (2023), 122(4):624-631
2) Ion induced transient potential fluctuations facilitate pore formation and cation transport through lipid membranes, D. Roesel, M. Eremchev, C. S. Poojari, J. S. Hub, and S. Roke, J. Am. Chem. Soc. (2022), 144, 51, 23352–23357.
ROUX
Over the last few months, students’ in-depth studies focussed on two critical systems for the project. They investigated gradients of flipper lifetime in cells and artificial lipid membranes, finding that these gradients correspond to membrane tension gradi-ents. Additionally, they developed an assay for oscillatory osmotic shocks, discover-ing that the coupling between volume and membrane tension weakens at high fre-quencies. Furthermore, they collected extensive data for a tutorial article on using flipper probes to measure membrane tension, which is currently under submission. A publication based on these findings is also in preparation. Output: 1) Garcia J-M., Guillamat P., Tomba C., Houzet L., Mehidi A., Aumeier C., Colom A., Roux A., Maintenance of membrane tension gradients in migrating and resting cells. Publication July 2024
2) Roffay, C., García-Arcos, J.M. Chapuis, P., López-Andarias, J., Schneider, F., Colom, A., Tomba, C., Meglio, I.D. Dunsig, V., Matile, S., et al. (2023). Technical insights into fluorescence lifetime microscopy of mechanosensitive Flipper probes. bioRxiv, 2022.2009.2028.509885.
• A high throughput 2P-FLIM microscope has been constructed.
• A new SFS instrument is under construction.
• Using the microscope we have taken SH images of several samples, testing water imaging, resonant SH fluorophore imaging and 2PF imaging. The images were compared in terms of quality to a modified version of an earlier microscope. Output: High throughput wide field SH imaging of giant unilamellar vesicles. M. Eremchev, D. Roesel, P.-M. Dansette, A. Michailovas, and S. Roke, Biointerphases 18, 2023; doi: 10.1116/6.0002640
• Measurements of water using high-throughput wide-field nonresonant second har-monic (SH) microscopy of membrane water shows that membrane potential fluctua-tions are universally found in lipid bilayer systems. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These tran-sient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through giant unilamellar vesicle (GUV) membranes. First test were also performed with living cells.
Output: 1 ) Passive transport of Ca2+ ions through lipid bilayers imaged by wide-field second harmonic microscopy, M. Eremchev, D. Roesel, C. S. Poojari, A. Roux, J. S. Hub and S. Roke, Biophys. J. (2023), 122(4):624-631
2) Ion induced transient potential fluctuations facilitate pore formation and cation transport through lipid membranes, D. Roesel, M. Eremchev, C. S. Poojari, J. S. Hub, and S. Roke, J. Am. Chem. Soc. (2022), 144, 51, 23352–23357.
ROUX
Over the last few months, students’ in-depth studies focussed on two critical systems for the project. They investigated gradients of flipper lifetime in cells and artificial lipid membranes, finding that these gradients correspond to membrane tension gradi-ents. Additionally, they developed an assay for oscillatory osmotic shocks, discover-ing that the coupling between volume and membrane tension weakens at high fre-quencies. Furthermore, they collected extensive data for a tutorial article on using flipper probes to measure membrane tension, which is currently under submission. A publication based on these findings is also in preparation. Output: 1) Garcia J-M., Guillamat P., Tomba C., Houzet L., Mehidi A., Aumeier C., Colom A., Roux A., Maintenance of membrane tension gradients in migrating and resting cells. Publication July 2024
2) Roffay, C., García-Arcos, J.M. Chapuis, P., López-Andarias, J., Schneider, F., Colom, A., Tomba, C., Meglio, I.D. Dunsig, V., Matile, S., et al. (2023). Technical insights into fluorescence lifetime microscopy of mechanosensitive Flipper probes. bioRxiv, 2022.2009.2028.509885.
For the SFS instrument, the improvement will significantly advance scientific capabilities in terms of surface spectroscopy.
The construction of the microscope is a high-level technological breakthrough that enables a whole new research area. This is very far advanced beyond the state of the art and our lab is the only one world-wide where such experiments can be performed.
The wide-field microscope enables the SH imaging of water at the interface of membranes and giant unilammelar vesicles in particular. This will result in new biophysical quantification and understanding of membrane properties, energetics, and in particular how the electric double layer dynamics of the hydrated membrane couples to processes such as passive ion transport, ion channel operation, and osmosis. Already the woek about passive ion transport are transforming the way in which the community thinks about the functionality of membranes.
The construction of the microscope is a high-level technological breakthrough that enables a whole new research area. This is very far advanced beyond the state of the art and our lab is the only one world-wide where such experiments can be performed.
The wide-field microscope enables the SH imaging of water at the interface of membranes and giant unilammelar vesicles in particular. This will result in new biophysical quantification and understanding of membrane properties, energetics, and in particular how the electric double layer dynamics of the hydrated membrane couples to processes such as passive ion transport, ion channel operation, and osmosis. Already the woek about passive ion transport are transforming the way in which the community thinks about the functionality of membranes.