Abstract
During brain development, Uncoordinated locomotion 5 (UNC5) receptors control axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into cholesterol-enriched membrane raft microdomains is crucial for the efficient transduction of the recognized signals. However, whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we combine the use of confocal microscopy, live-cell FRAP analysis and single-particle tracking PALM to characterize the distribution of UNC5 receptors into raft microdomains, revealing differences in their membrane mobility properties. Using pharmacological and genetic approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibits the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that the specific localization has functional consequences for the axonal chemorepulsion against Netrin-1.
Highlights
The establishment of proper neuronal connections relies on the ability of axons to locate and reach their target during neural development
To investigate the membrane distribution and dynamics of Uncoordinated locomotion 5 (UNC5) receptors involved in the repulsion associated with the recognition of Netrin-1 ligand, we fused the fluorescent protein YFP to the C-terminus of all four members of the UNC5(A–D) family of proteins
UNC5 receptors lead external granular layer (EGL) axons away from the source of Netrin-1 [13]; little is known about the importance of their localization in lipid rafts during cerebellar Netrin-1-dependent growth cone chemorepulsive events
Summary
The establishment of proper neuronal connections relies on the ability of axons to locate and reach their target during neural development. ◂Fig. 1 Distribution of YFP-tagged UNC5 receptors expressed in HEK-293AD cells. Representative confocal images of HEK-293AD cells expressing a UNC5A-YFP, c UNC5B-YFP, e UNC5C-YFP and g UNC5D-YFP receptors. I-l Confocal images from HEK-293AD cells expressing YFP-tagged UNC5A–D receptors. M Quantification of the colocalization observed between the UNC5 receptor channel and WGA channel (range 0–1). UNC5(A–D) are single-spanning transmembrane proteins whose extracellular region consists of two immunoglobulin domains (Ig) that bind Netrin-1 and two type I thrombospondin domains (TSP) [9]. A ZU-5 domain, a DCC-binding (DB) motif and a death domain (DD) motif comprise the cytoplasmic region [10]
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