Spatially Selective Dissection of Signal Transduction in Neurons Grown on Netrin‑1 Printed Nanoarrays via Segmented Fluorescence Fluctuation Analysis

Axonal growth cones extend during neural development in response to precise distributions of extracellular cues. Deleted in colorectal cancer (DCC), a receptor for the chemotropic guidance cue netrin-1, directs F-actin reorganization, and is essential for mammalian neural development. To elucidate how the extracellular distribution of netrin-1 influences the distribution of DCC and F-actin within axonal growth cones, we patterned nanoarrays of substrate bound netrin-1 using lift-off nanocontact printing. The distribution of DCC and F-actin in embryonic rat cortical neuron growth cones was then imaged using total internal reflection fluorescence (TIRF) microscopy. Fluorescence fluctuation analysis via image cross-correlation spectroscopy (ICCS) was applied to extract the molecular density and aggregation state of DCC and F-actin, identifying the fraction of DCC and F-actin colocalizing with the patterned netrin-1 substrate. ICCS measurement of spatially segmented images based on the substrate nanodot patterns revealed distinct molecular distributions of F-actin and DCC in regions directly overlying the nanodots compared to over the reference surface surrounding the nanodots. Quantifiable variations between the populations of DCC and F-actin on and off the nanodots reveal specific responses to the printed protein substrate. We report that nanodots of substrate-bound netrin-1 locally recruit and aggregate DCC and direct F-actin organization. These effects were blocked by tetanus toxin, consistent with netrin-1 locally recruiting DCC to the plasma membrane via a VAMP2-dependent mechanism. Our findings demonstrate the utility of segmented ICCS image analysis, combined with precisely patterned immobilized ligands, to reveal local receptor distribution and signaling within specialized subcellular compartments.