AFM study of F-actin on chemically modified surfaces

Surfaces that modulate the behaviour of surface-immobilised biomolecules have been of critical interest in the last decades, with applications in biomedical microdevices. Recently, the advances in nanofabrication also allowed the contemplation of a leap-through in the form of the design and fabrication of biomimetic surfaces that replicate the molecular surfaces of large biomolecular structures such as cytoskeleton aggregates. The process of formation of these long-range ordered nanostructures have enormous biological interest, but increasingly they are good examples of ‘fabrication’ processes leading to large nanostructured areas with the design embedded in their smaller components. To this end, we report here an atomic force microscopy (AFM) study of the high order self assembly of F-actin on mica. Actin is one of the principal structural proteins in eukaryotic cells and is an ideal biopolymer for investigations of new modes of higher order self-assembly. G-actin monomer can be polymerized into long right-handed double helical filaments (F-actin) whose formation is induced by Mg2+, K+, Na+, and ATP. F-actin can be considered as a semiflexible and highly charged polyelectrolyte, with diameter DA ~ 8 nm and persistence length of 10 μm [1]. Atomic force microscopy (AFM) is a useful tool for elucidating the topography of biomolecules-covered surfaces, including proteins, and mica is commonly used as a substrate for AFM imaging at molecular resolution due to its atomically-flat surface. In our previous [2] and present work, we visualised different morphologies of assemblies of actin filaments adsorbed on mica and silicon surfaces with different geometries and physical-chemical properties. However, it is notoriously difficult to immobilize negatively charged samples as actin (theoretical pI 5.23) because the electrostatic repulsion between the negatively charged mica surface and the actin assembly results is a weak immobilisation of the filaments, thus rendering the AFM scanning very difficult and prone to artefacts. On silicon surfaces large, flat and long F-actin fibre bundles were visualised, which could indicate a substrate repulsion that favours the assembly of fibres in the bulk and/or the vicinity of the surface, followed by deposition of pre-‘fabricated’ F-actin rafts on the silicon surface. To counter-act the technological problem described above, mica was chemically treated with Mg2+ to increase the affinity towards F-actin. Interestingly, this treatment rendered the mica surface more hydrophobic. On this treated surface we could visualise with great reproducibility F-actin patterns by both contact and tapping mode AFM approaches (Figures 1 and 2). The most interesting aspect was the capability of fabrication ordered patterns formed by F-actin filaments, through the balanced interplay between F-actin self-assembly forces and forces applied by the AFM tip in a contact mode. More specifically, increasing the force applied by the AFM tip we could observe the shift from the visualisation of individual actin filaments (Figure 3, “e” surface) to parallel actin filaments ‘rafts’ (Figure 1 and Figure 3, surfaces “a” and “c”). In ‘visualisation-only’ contact mode, the average height F-actin filaments above the mica surface was 5.5 nm (Figure 2), consistent with the theoretical value. The spacing between rows was found to be a function of the applied tip force, as can be seen in Figure 2 where three different regions were scanned at forces ranging from 7 to 20 nN. Further increasing the force applied by the AFM tip to 50 nN we could clean the mica surface and ‘dragged’ the filaments, still in an ordered manner, i.e., in a direction perpendicular to their axis (Figure 3, transition from surface “d” to surface “b”). Future work will study the parallel characterization of nanostructured solid surfaces which can modulate, either enhancing or retarding, the oligomerisation and fibrillization of actin filaments, w...