title: Neuron Subpopulations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment.
abstract: Correct wiring of the nervous system requires guidance cues, diffusible or substrate-bound proteins that steer elongating axons to their target tissues. Netrin-1, the best characterized member of the Netrins family of guidance molecules, is known to induce axon turning and modulate axon elongation rate; however, the factors regulating the axonal response to Netrin-1 are not fully understood. Using microfluidics, we treated fluidically isolated axons of mouse primary cortical neurons with Netrin-1 and characterized axon elongation rates, as well as the membrane localization of deleted in colorectal cancer (DCC), a well-established receptor of Netrin-1. The capacity to stimulate and observe a large number of individual axons allowed us to conduct distribution analyses, through which we identified two distinct neuron subpopulations based on different elongation behavior and different DCC membrane dynamics. Netrin-1 reduced the elongation rates in both subpopulations, where the effect was more pronounced in the slow growing subpopulation. Both the source of Ca(2+) influx and the basal cytosolic Ca(2+) levels regulated the effect of Netrin-1, for example, Ca(2+) efflux from the endoplasmic reticulum due to the activation of Ryanodine channels blocked Netrin-1-induced axon slowdown. Netrin-1 treatment resulted in a rapid membrane insertion of DCC, followed by a gradual internalization. DCC membrane dynamics were different in the central regions of the growth cones compared to filopodia and axon shafts, highlighting the temporal and spatial heterogeneity in the signaling events downstream of Netrin-1. Cumulatively, these results demonstrate the power of microfluidic compartmentalization and distribution analysis in describing the complex axonal Netrin-1 response.
authors.affiliation: Bionanoscience Group, School of Chemistry and Chemical Biology, University College Dublin , Belfield, Dublin 4, Ireland.
authors.forename: Agata
authors.initials: A
authors.lastname: Blasiak
authors.affiliation: Bionanoscience Group, School of Chemistry and Chemical Biology, University College Dublin , Belfield, Dublin 4, Ireland.
authors.forename: Gil U
authors.initials: GU
authors.lastname: Lee
authors.affiliation: Bionanoscience Group, School of Chemistry and Chemical Biology, University College Dublin , Belfield, Dublin 4, Ireland.
authors.forename: Devrim
authors.initials: D
authors.lastname: Kilinc
mesh_terms: D000818:Animals; D001369:Axons / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D049108:Cell Enlargement; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D000075389:DCC Receptor; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D046210:Microfluidic Analytical Techniques; D009414:Nerve Growth Factors / Q000008:administration & dosage / Q000378:metabolism*; D000075388:Netrin-1; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D011554:Pseudopodia / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000008:administration & dosage / Q000378:metabolism*
chemical_list: D000075389:DCC Receptor; C501910:Dcc protein, mouse; D009414:Nerve Growth Factors; C000617353:Ntn1 protein, mouse; D011956:Receptors, Cell Surface; D019837:Ryanodine Receptor Calcium Release Channel; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; D002118:Calcium
keywords: Microfluidics; UNC-5; filopodia; growth cones; receptor dynamics