The signaling network that controls neurite growth is much more complex than researchers thought, Fusco et al. reveal.
Neurites, the extensions that sprout from neuronal cell bodies, are highly dynamic. Within a few hours, a neurite can go through initiation, elongation, branching, and retraction phases. Within a few minutes, filopodia at the tips of neurites can change position. Neurites contain more than 200 proteins that might affect or be affected by Rho GTPases. Rac1 and Cdc42 GTPases spur neurite growth, whereas RhoA causes them to shrink. However, previous studies focused on neurites’ final lengths rather than tracking their changes over time.
Fusco et al. used siRNAs to deplete 219 proteins that might interact with Rho GTPases. The researchers captured even subtle alterations in neurite dynamics by photographing a large number of cells every 12 minutes for nearly 20 hours. To make sense of this vast amount of data, the team created a computer vision system, NeuriteTracker, that registers changes in the nuclei, cell bodies, and neurites of neurons from frame to frame and translates them into geometric features. Statistical analyses can then determine which features change upon different siRNA perturbations.
Because RhoA triggers neurite retraction, the researchers expected that knocking down GTPase activating proteins (GAPs) that inhibit RhoA would lead to short neurites. Although depleting one GAP, ARHGAP5, produced short neurites by promoting shrinkage, depleting another GAP, DLC1, caused neurites to elongate. The two GAPs may control different functions of RhoA, indicating that researchers need to systematically analyze Rho GTPases and their interacting proteins to tease out the circuits that manage neurite growth.