The Level of Substrate Deformation and not Traction Force Regulates Adhesion-Mediated Neuronal Growth Cone Advance

Abstract

Although pulling forces have been observed in axonal growth for several decades, their exact roles are not fully understood. Here, we quantified retrograde traction forces in neuronal growth cones as they develop over time in response to an adhesion substrate using two different experimental approaches. In the first approach, we used force-calibrated glass microneedles coated with ligands for the Aplysia cell adhesion molecule apCAM to guide the advance of Aplysia Californica growth cones. The traction force exerted by the growth cone was measured by monitoring the microneedle deflection using an optical microscope. In the second approach, we developed a novel method for measuring traction forces using an atomic force microscope (AFM) with a static cantilever that contained a ligand-coated microbead and was monitored in real time. Both approaches showed that Aplysia growth cones can develop maximum traction forces up to 100 nN, which is an order of magnitude higher than previously reported for other experimental methods or growth cones. Moreover, our results suggest that the traction force is directly correlated to the stiffness of the microneedle, which is consistent with a reinforcement mechanism supported by other research. Contrary to our expectation, the level of force does not predict whether the growth cone will advance towards the adhesion site or not, but the level of microneedle deflection does. In cases of adhesion-mediated growth cone advance, the mean deflection was 1.07 ± 0.09 μm. By contrast, the mean deflection was significantly lower (0.49 ± 0.04 μm) when the growth cones did not advance in response to the adhesion substrate. In summary, our results provide novel insights into significance of substrate deformation as opposed to traction force for the regulation of adhesion-mediated directional growth cone advance.