Atomic force microscopy and force differentiation assay study on biophysical properties of Aplysia neuronal growth cones associated with adhesion-mediated guidance

Neuronal growth cones are motile structures located at the end of neurites and translate extracellular guidance information into directional movements toward target cells. Despite their important role in the development and regeneration of the nervous system, relatively little quantitative information is available regarding their physical attributes, including three-dimensional structures and mechanical properties of distinct growth cone regions, as well as the spatial, temporal and mechanical parameters of the molecular players involved in functional interactions with the environment and with each other. Here, unique abilities of atomic force microscopy in high-resolution topography imaging and viscoelastic property measurements are explored, on length scales from nanometers to microns, with specific application to the large growth cones of Aplysia bag cell neuronal cultured in vitro. The heights of the peripheral (P) domain, transition (T) zone, and central (C) domain in live growth cones were calculated from contact mode AFM images and averaged to be 183 ± 33, 690 ± 274, and 1322 ± 164 nm, respectively. These findings are consistent with data derived from dynamic mode images of live and contact mode images of fixed growth cones. Nanoindentation measurements revealed that the elastic moduli of the P-domain and T-zone ruffling region ranged between 10-40 and 7-23 kPa, respectively. These regions are significantly stiffer than the C domain, which has a typical range of 3-7 kPa. High-resolution images of the P domain suggest that the higher elastic moduli result from a dense meshwork of actin filaments in lamellipodia and from actin bundles in filopodia. The increased mechanical stiffness of the P and T domains is likely important to support and transduce tension that develops during growth cone steering. We also proposed an innovative method using magnetic tweezer technique to characterize the bond lifetime-force behavior of homophilic apCAM interaction, which plays a key role in adhesion-mediated growth cone steering events. A strong and a weak slip bond were detected with an effective bond length that is characteristic of short-range, stiff intermolecular interactions. The quantitative biomechanical information acquired from these experiments provide direct insights into how adhesion receptors regulate neuronal growth cone motility and guidance, which are important elements to a better understanding of the developmental and regenerative processes of the nervous system