A 3D-matrigel/microbead assay for the visualization of mechanical tractive forces at the neurite-substrate interface...

Mechanical properties of neuronal processes contribute to neuronal function, to resistance of fiber tracts against mechanical trauma, and to morphological changes during development and neurodegeneration. Conventional in vitro cell culture systems on inflexible substrates do not allow for the visualization of changing mechanical stress between neurites and their substrate. To solve this problem, we adapted a three-dimensional gel matrix assay to visualize mechanical traction forces at the neurite-substrate interface. We chose matrigel as substrate because in this matrix various types of neurons initially adapt a bipolar morphology while migrating, similar to migrating neurons in vivo. To visualize emerging traction forces between neurites and their substrate, microbeads were embedded into the matrix as visible landmarks. We first analyzed mechanical distortion of matrigel by stepwise movements of a glass pipette tip under control of a micromanipulator to ensure reproducibility of induced bead displacement. The assay was then used to study the effect of the microtubule disrupting drug nocodazole on neuronal processes. By monitoring displacement of matrigel-embedded microbeads, we visualized here for the first time emerging mechanical traction forces between the leading process and the substrate during nocodazole-induced soma translocation. We did not observe bead displacement by processes of aged neurons.