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ACS Nano. 2017 Aug 22;11(8):8131-8143. doi: 10.1021/acsnano.7b03004. Epub 2017 Jul 20.

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

Author information

1
Department of Chemistry, ‡Department of Neurology and Neurosurgery, Montreal Neurological Institute, §Department of Biomedical Engineering, Genome Quebec Innovation Centre, and ∥Department of Physics, McGill University , Montreal, Quebec H3A 0G4 Canada.

Abstract

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.

KEYWORDS:

chemotropic guidance; deleted in colorectal cancer; fluorescence fluctuation analysis; image correlation spectroscopy; nanopatterned cell substrates; receptor aggregation

PMID:
28679208
DOI:
10.1021/acsnano.7b03004

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