Spatial mapping of the neurite and soma proteomes reveals a functional Cdc42/Rac regulatory network

Neurite extension and growth cone navigation are guided by extracellular cues that control cytoskeletal rearrangements. However, understanding the complex signaling mechanisms that mediate neuritogenesis has been limited by the inability to biochemically separate the neurite and soma for spatial proteomic and bioinformatic analyses. Here, we apply global proteome profiling in combination with a neurite purification methodology for comparative analysis of the soma and neurite proteomes of neuroblastoma cells. The spatial relationship of 4,855 proteins were mapped, revealing networks of signaling proteins that control integrins, the actin cytoskeleton, and axonal guidance in the extending neurite. Bioinformatics and functional analyses revealed a spatially compartmentalized Rac/Cdc42 signaling network that operates in conjunction with multiple guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) to control neurite formation. Interestingly, RNA interference experiments revealed that the different GEFs and GAPs regulate specialized functions during neurite formation, including neurite growth and retraction kinetics, cytoskeletal organization, and cell polarity. Our findings provide insight into the spatial organization of signaling networks that enable neuritogenesis and provide a comprehensive system-wide profile of proteins that mediate this process, including those that control Rac and Cdc42 signaling.