Single Layer Graphene (SLG) has emerged as a critically important nanomaterial due to its unique optical and electrical properties and has become a potential candidate for biomedical applications, biosensors, and tissue engineering. Due to its intrinsic 2D nature, SLG is an ideal surface for the development of large-area biosensors and, due to its biocompatibility, can be easily exploited as a substrate for cell growth. The cellular response to SLG has been addressed in different studies with high cellular affinity for graphene often detected. Still, little is known about the molecular mechanism that drives/regulates the cellular adhesion and migration on SLG and SLG-coated interfaces with respect to other substrates. Within this scenario, we used quantitative super-resolution microscopy based on single-molecule localization to study the molecular distribution of adhesion proteins at the nanoscale level in cells growing on SLG and glass. In order to reveal the molecular mechanisms underlying the higher affinity of biological samples on SLG, we exploited stochastic optical reconstruction microscopy (STORM) imaging and cluster analysis, quantifying the super-resolution localization of the adhesion protein vinculin in neurons and clearly highlighting substrate-related correlations. Additionally, a comparison with an epithelial cell line (Chinese Hamster Ovary) revealed a cell dependent mechanism of interaction with SLG.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.