Spatiotemporal Corona virus binding dynamics and infection mechanism investigated with 100 Hz ROCS microscopy and thermal fluctuation analysis
Virology
Final Report Abstract
In this project we investigated how SARS-CoV-2 virus-like particles approach living cells by diffusion, attempt to bind to them and are then taken up under favorable conditions. Infection with viruses and particles with similar properties consists of several successive steps, with diffusion contact and attachment being the first crucial processes before the particle enters the cell. Due to the high dynamics of the particles, which are only 0.1 µm in size, the observation and in-depth investigation of these first interaction steps before cell entry has not been possible with conventional measurement technology until now. The aim of this research project was to decipher the dynamic, discontinuous binding processes of viruses, which are determined by thermal fluctuations in position and orientation. We have partially succeeded in this, although the statistics of the individual processes are not yet good enough. First of all, we have succeeded in recording the binding processes, which are based on an initially unspecific interaction with the cell glycocalyx or cell protrusions, using a novel label-free super-resolution microscopy method. The imaging is based on rotating, coherently scattered light from a blue laser (ROCS), with which we record thousands of images at 100 Hz image acquisition rate without loss of image quality. The technique, which we developed in a simplified form around 10 years ago, allows us to observe both small, fast-moving particles and dynamic cells with fast filopodia or microvilli by means of coherent contrast formation on the camera. By automatically tracking and analyzing thermal position fluctuations of the particles, we were able to determine the temporal changes in the binding strength of about 10 particles per cell. Using various, partly new measurement and analysis methods, we were able to quantify different particles in their cell binding behavior, including 100 nm glass or polystyrene beads, beads with SARS-CoV-2 spike proteins on their surface, weakly scattering SARS-CoV-2 virus envelope particles without RNA and pseudotyped VSV-SARS-CoV-2 S-ΔG-mCherry. As targets we used A549 lung epithelial cells, J774 macrophages and ACE-2 receptor-rich Caco2 intestinal epithelial cells. We were only occasionally able to record the multi-step binding process of the particles through their characteristic fluctuation movement, as well as the uptake process through a temporal contrast change in the particle image. A major challenge was to bring the tiny particles between the glass surface and the (basal) cell surface in order to visualize their dynamics or uptake in TIR mode with both ROCS and fluorescence. At least on the dorsal side, which is more accessible to the particles, we were able to see them through the lamellipodia of the cell without labeling and with good quality. The binding behavior with ACE-2 receptor blockers with spike protein-coated particles could not be carried out. Overall, however, the project can be considered a success, as a number of technological obstacles were overcome, new illumination modes were developed and sometimes unique image sequences were recorded, providing insights into virus binding dynamics for the first time. This knowledge, including microscopy methods, can be used for a variety of other research projects.
Publications
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100 Hz ROCS microscopy correlated with fluorescence reveals cellular dynamics on different spatiotemporal scales. Nature Communications, 13(1).
Jünger, Felix; Ruh, Dominic; Strobel, Dominik; Michiels, Rebecca; Huber, Dominik; Brandel, Annette; Madl, Josef; Gavrilov, Alina; Mihlan, Michael; Daller, Caterina Cora; Rog-Zielinska, Eva A.; Römer, Winfried; Lämmermann, Tim & Rohrbach, Alexander
