High‐resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy

Abstract

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three-dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force-distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F-actin bundles are stiffer than surrounding F-actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions.

Identification of functional marker proteins in the mammalian growth cone

“…The growth cone is comprised of morphologically and functionally distinct regions referred to as the central (C) and peripheral (P) regions (31). The C region is enriched in vesicles and microtubules and is probably involved in membrane expansion for axonal growth.…”

Kinetic-structural analysis of neuronal growth cone veil motility

“…Further, the dendritic pathway biomarker, Arp2/3, was localized to the leading edge of protruding veils by both immunostaining and by expression of tagged Arp2/3 (both Myc and GFP). It is not clear why this staining pattern was not seen in the study of Strasser et al (Strasser et al, 2004), but one possibility is that their wide field imaging missed the edge localization because of the greatly differing optical thickness of the neuronal shaft versus the lamellipodium [see Grzywa et al (Grzywa et al, 2006) and comparison of TIRF and widefield imaging of growth cones in supplementary material Fig. S3A].…”

Probing nanomechanical properties from biomolecules to living cells

“…The cholesterol-rich domains, or rafts, are denoted by arrows, while variations in Young's modulus are color-coded. The scanning size is 2 μm (reproduced with the permission of C. Roduit) [75], neural growth cones [26], focal adhesions [22], and lamellipods [55,79].…”