| Autor: |
Gopal, Angelica A., Ricoult, Sebastien G., Harris, Stephanie N., Juncker, David, Kennedy, Timothy E., Wiseman, Paul W. |
| Zdroj: |
ACS Nano; August 2017, Vol. 11 Issue: 8 p8131-8143, 13p |
| Abstrakt: |
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 viaimage 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 viaa 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. |
| Databáze: |
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