Cell-Shaping Micropatterns for Quantitative Super-Resolution Microscopy Imaging of Membrane Mechanosensing Proteins

摘要

Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells. This approach, based on fibronectin microcontact printing on hydrophobic organosilane monolayers, allows evanescent wave and light-sheet microscopy of cells whilst fulfilling the stringent optical demands of point reconstruction optical microscopy. By imposing steady-state mechanical cues on cells grown in these micropatterns, we reveal nanoscale remodeling in the dynamics and the structural organizations of the nuclear envelope mechanotransducing protein emerin and of the plasma membrane mechanosensing protein caveolin-1 using single particle tracking photoactivated localization microscopy and direct stochastic optical reconstruction microscopy imaging. In addition to allowing quantitative biophysical studies of mechanoresponsive membrane proteins, this approach provides an easy means to probe mechanical regulations in cellular membranes with high optical resolution and nanometer precision.