A microtubule-based, dynein-dependent force induces local cell protrusions: Implications for neurite initiation.

A key event in neurite initiation is the accumulation of microtubule bundles at the neuron periphery. We hypothesized that such bundled microtubules may generate a force at the plasma membrane that facilitates neurite initiation. To test this idea we observed the behavior of microtubule bundles that were induced by the microtubule-associated protein MAP2c. Endogenous MAP2c contributes to neurite initiation in primary neurons, and exogeneous MAP2c is sufficient to induce neurites in Neuro-2a cells. We performed nocodazol washout experiments in primary neurons, Neuro-2a cells and COS-7 cells to investigate the underlying mechanism. During nocodazol washout, small microtubule bundles formed rapidly in the cytoplasm and immediately began to move toward the cell periphery in a unidirectional manner. In neurons and Neuro-2a cells, neurite-like processes extended within minutes and concurrently accumulated bundles of repolymerized microtubules. Speckle microscopy in COS-7 cells indicated that bundle movement was due to transport, not treadmilling. At the periphery bundles remained under a unidirectional force and induced local cell protrusions that were further enhanced by suppression of Rho kinase activity. Surprisingly, this bundle motility was independent of classical actin- or microtubule-based tracks. It was, however, reversed by function-blocking antibodies against dynein. Suppression of dynein expression in primary neurons by RNA interference severely inhibited the generation of new neurites, but not the elongation of existing neurites formed prior to dynein knockdown. Together, these cell biological data suggest that neuronal microtubule-associated proteins induce microtubule bundles that are pushed outward by dynein and locally override inward contraction to initiate neurite-like cell protrusions. A similar force-generating mechanism might participate in spontaneous initiation of neurites in developing neurons.