title: Substrate Deformation Predicts Neuronal Growth Cone Advance.
abstract: Although pulling forces have been observed in axonal growth for several decades, their underlying mechanisms, absolute magnitudes, and exact roles are not well understood. In this study, using two different experimental approaches, we quantified retrograde traction force in Aplysia californica neuronal growth cones as they develop over time in response to a new adhesion substrate. In the first approach, we developed a novel method, to our knowledge, for measuring traction forces using an atomic force microscope (AFM) with a cantilever that was modified with an Aplysia cell adhesion molecule (apCAM)-coated microbead. In the second approach, we used force-calibrated glass microneedles coated with apCAM ligands to guide growth cone advance. The traction force exerted by the growth cone was measured by monitoring the microneedle deflection using an optical microscope. Both approaches showed that Aplysia growth cones can develop traction forces in the 10(0)-10(2) nN range during adhesion-mediated advance. Moreover, our results suggest that the level of traction force is directly correlated to the stiffness of the microneedle, which is consistent with a reinforcement mechanism previously observed in other cell types. Interestingly, the absolute level of traction force did not correlate with growth cone advance toward the adhesion site, but the amount of microneedle deflection did. In cases of adhesion-mediated growth cone advance, the mean needle deflection was 1.05 ± 0.07 μm. By contrast, the mean deflection was significantly lower (0.48 ± 0.06 μm) when the growth cones did not advance. Our data support a hypothesis that adhesion complexes, which can undergo micron-scale elastic deformation, regulate the coupling between the retrogradely flowing actin cytoskeleton and apCAM substrates, stimulating growth cone advance if sufficiently abundant.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana; Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana.
authors.forename: Ahmad I M
authors.initials: AI
authors.lastname: Athamneh
authors.affiliation: Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana; School of Mechanical Engineering, Purdue University, West Lafayette, Indiana.
authors.forename: Alexander X
authors.initials: AX
authors.lastname: Cartagena-Rivera
authors.affiliation: Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana; School of Mechanical Engineering, Purdue University, West Lafayette, Indiana.
authors.forename: Arvind
authors.initials: A
authors.lastname: Raman
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana; Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana; Bindley Bioscience Center, Purdue University, West Lafayette, Indiana. Electronic address: dsuter@purdue.edu.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Suter
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001048:Aplysia; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000737:chemistry; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D004548:Elasticity; D005898:Glass; D020439:Growth Cones / Q000502:physiology*; D018625:Microscopy, Atomic Force; D008863:Microspheres; D009474:Neurons / Q000502:physiology*; D061848:Optical Imaging
chemical_list: D000199:Actins; D015815:Cell Adhesion Molecules