Axons of the corpus callosum (CC), the white matter tract that connects the left and right hemispheres of the brain, receive instruction from a number of chemoattractant and chemorepulsant cues during their initial navigation towards and across the midline. While it has long been known that the CC is malformed in the absence of Myristoylated alanine-rich C-kinase substrate (MARCKS), evidence for a direct role of MARCKS in axon navigation has been lacking. Here, we show that MARCKS is necessary for Netrin-1 (NTN1) signaling through the DCC receptor, which is critical for axon guidance decisions. Marcks null (Marcks) neurons fail to respond to exogenous NTN1 and are deficient in markers of DCC activation. Without MARCKS, the subcellular distributions of two critical mediators of NTN1-DCC signaling, the tyrosine kinases PTK2 and SRC, are disrupted. Together, this work establishes a novel role for MARCKS in axon dynamics and highlights the necessity of MARCKS as an organizer of DCC signaling at the membrane.

Through the process of neuronal differentiation, newly born neurons change from simple, spherical cells to complex, sprawling cells with many highly branched processes. One of the first stages in this process is neurite initiation, wherein cytoskeletal modifications facilitate membrane protrusion and extension from the cell body. Hundreds of actin modulators and microtubule-binding proteins are known to be involved in this process, but relatively little is known about how upstream regulators bring these complex networks together at discrete locations to produce neurites. Here, we show that Myristoylated alanine-rich C kinase substrate (MARCKS) participates in this process. Marcks−/− cortical neurons extend fewer neurites and have less complex neurite arborization patterns. We use an in vitro proteomics screen to identify MARCKS interactors in developing neurites and characterize an interaction between MARCKS and a CDC42-centered network. While the presence of MARCKS does not affect whole brain levels of activated or total CDC42, we propose that MARCKS is uniquely positioned to regulate CDC42 localization and interactions within specialized cellular compartments, such as nascent neurites.