Membrane curvature participates in a wide range of cellular processes, and acts as a hotspot for protein interactions and intracellular signalling. Curvature also occurs at the interface between cells and nanotopography of biomaterials and biomedical devices, which could influence the performance of tissue engineering scaffolds and implantable devices. Precisely manipulating membrane curvature is thus of great interest in probing intracellular activities involved with curved membranes. Here we present a detailed protocol to design, fabricate, and characterize nanoscale structures for manipulating membrane curvature and probing curvature-induced phenomena in live cells. This protocol first describes a detailed procedure for the design and fabrication of nanoscale structures using electron-beam lithography. Then, the protocol describes how to use these nanostructures to manipulate local membrane curvature and probe intracellular protein responses. Finally, the protocol describes a procedure to characterize the nanostructure-cell membrane interface using focused ion beam and scanning electron microscopy.
The dynamic interface between the cellular membrane and 3D nanostructures determines biological processes and guides the design of novel biomedical devices. Despite the fact that recent advancements in the fabrication of artificial biointerfaces have yielded an enhanced understanding of this interface, there remain open questions on how the cellular membrane reacts and behaves in the presence of sharp objects on the nanoscale. Here we provide a multifaceted characterization of the cellular membrane's mechanical stability when closely interacting with high-aspect-ratio 3D vertical nanostructures, providing strong evidence that vertical nanostructures spontaneously penetrate the cellular membrane to form a steady intracellular coupling only in rare cases and under specific conditions. The cell membrane is able to conform tightly over the majority of structures with various shapes while maintaining its integrity.
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