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Cells in tissues are tightly packed and undergo strong deformations, as a result of internal dynamics but also external forces applied by neighboring cells. Cytoskeleton organization is strongly linked with cell shape dynamics and is known to be mechanosensitive. The cytoskeleton is also an important biochemical transducer. In particular, the cortical actin skeleton located beneath the plasma membrane possesses a highly dynamic nature that is thought to affect ion channels activity, nuclear envelope, gene expression.
It comprises polydispersed actin filaments undergoing continuous turnover and organized in a dense network with mesh-sizes of 10–15 nm, and an estimated thickness of 100–500 nm1,2,3. Attempts to dissect the organization of the actin cortex have mostly relied on diffraction-limited fluorescence imaging, whose spatial resolution is insufficient to reveal its structure at the nanoscale.
Recently, important advances have been made possible thanks to super-resolution microscopy, or nanoscopy4, which have shown how these techniques can be used to visualize and measure cortical mesh networks at the nanoscale,
This study was performed in the laboratory of Mathieu Piel (Systems Biology of Cell Polarity and Cell Division, Institut Curie, Paris) by Larisa Venkova in collaboration with Abbelight, and i focused on the actin cortex reorganization following acute cell deformation. Round cells were confined between two horizontal plates by using a cell confiner previously developed in the laboratory of Mathieu Piel5, which allows a precise control of the height of confinement. Round cells become flat and cylindrical upon deformation, but they do not die, and the contact area with the substrate increases as confinement height decreases. These deformations induce a rapid and strong reorganization of the actin cortex that was followed by super-resolution microscopy on cells fixed at the time of confinement.
These observations will allow to define short timescale the actomyosin responses to global cell compressive deformation of large amplitudes, a regime that has not been explored so far.
Abbelight features the highest 3D resolution (15x15x15 nm) in single molecule imaging, and thanks to the implementation of DONALD6 technology it is insensitive to axial drift and provides a reference-free axial position. These features were used to dissect the sub-diffraction cortical mesh of actin filaments in non-adherent cells acutely compressed between two glass plates at defined heights. In A, the cortical actin of a non-compressed HeLa cell (20 mm of confinement height); the cortical mesh is interspersed with thicker filament bundles; in B, a profile view in confocal microscopy (membrane bound GFP), and a schema of the confinement strategy. In C, axial distribution of the cortical actin, showing an average thickness of 350 nm, and a density peak around 220 nm above the coverslip.
In D and F, the cortical actin of HeLa cells compressed, respectively, at 10 mm and 5 mm; several blebs filled with a finer and denser actin mesh appear under compression. In E, the axial distribution of the actin at different confinement heights, showing a shift of axial density closer to the plasma membrane (coverslip). In G and H, representation of the compression scheme, with lateral view of the cells. In I, the cell contact area with the substrate (coverslip) increases with decreasing confinement heights, as measured in live cells with RICM and fixed cells with nanoscopy.
Adherent HeLa cells where gently detached via Calcium chelation, and the rounded cells compressed using a cell confiner (described in 5), in glass-bottom 6 well plates (1.5, Mattek). To preserve cortex ultrastructure, the cells were fixed at the time of confinement with glutaraldehyde. Cortical actin filaments were stained with phalloidin-AF647. For STORM imaging the cells were incubated in Abbelight SMART-Kit buffer
3D imaging was performed on an Olympus Ix83 microscope with a 100X 1.49NA objective, equipped with Abbelight SAFe360 that implements DONALD technology. Based on ratiometric measures of supercritical angle fluorescence emissions, DONALD allows absolute axial localization and 10 nm precision of localization near the coverslip. and imaged using a 632nm laser for ~30000-60000 frames at 20 FPS.
Single molecule planar and axial localization, image reconstruction and drift correction were performed in real time with Abbelight NEO software. NEO software was also used for 3D visualization and analysis of single molecule data, that is the spatial coordinates of each detected molecule.
References
1.A. G. Clark, K. Dierkes, E. K. Paluch, Biophys. J. 105, 570–580 (2013).
2.M. Fritzsche, C. Erlenkämper, E. Moeendarbary, G. Charras, K. Kruse, Sci. Adv. 2 (2016), doi:10.1126/sciadv.1501337.
3.M. Fritzsche et al., Nat. Commun. 8, 14347 (2017).
4.M. P. Clausen, H. Colin-York, F. Schneider, C. Eggeling, M. Fritzsche, J. Phys. D. Appl. Phys. 50, 064002 (2017).
5.M. Le Berre, E. Zlotek-Zlotkiewicz, D. Bonazzi, F. Lautenschlaeger, M. Piel, in Methods in Cell Biology (Academic Press Inc., 2014), vol. 121, pp. 213–229.
6.N. Bourg et al., Nat. Photonics. 9, 587–593 (2015).
Samples and images provided by:
Sample preparation, imaging and analysis by Larisa Venkova, PhD, Institut Curiie, Paris
The data was presented at the 2018 Biophysical Society meeting.