Abstract
A complex and still not comprehensively resolved panel of transmembrane proteins regulates the outgrowth and the subsequent morphological and functional development of neuronal processes. In order to gain a more detailed description of these events at the molecular level, we have developed a cell surface biotinylation assay to isolate, detect, and quantify neuronal membrane proteins. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr1, a poorly described cell adhesion molecule. We demonstrated that Negr1 controls the development of neurite arborization in vitro and in vivo. Given the tight correlation existing among synaptic cell adhesion molecules, neuron maturation, and a number of neurological disorders, our assay results are a useful tool that can be used to support the understanding of the molecular bases of physiological and pathological brain function.
Highlights
Genetic analysis indicates that 20% to 30% of the total open reading frame encodes for integral membrane proteins [1]
Cell Surface Biotinylation Assay Allows the Enrichment of Membrane Proteins—Our cell surface biotinylation assay exploits the incubation of living neuronal cultures with nonpermeable biotin moieties to tag protein domains at the extracellular level
Transmission electron microscopy investigation of P2 fractions obtained from DIV6 and DIV16 cultures confirmed that both samples contained a subcellular structure recognizable as the synaptosome
Summary
Francesca Pischedda‡, Joanna Szczurkowska§, Maria Daniela Cirnaru‡, Florian Giesert¶, Elena Vezzoliʈ, Marius Ueffing**‡‡, Carlo Sala‡, Maura Francoliniʈ, Stefanie M. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr, a poorly described cell adhesion molecule. Membrane proteins play an important role in the establishment of functional neuronal circuitries during development This process initially entails the growth, guidance, and stabilization of neuronal processes (axons and dendrites) in a timely, ordered manner involving cell surface molecules that sense the extracellular surroundings and activate signaling cascades [2]. Our assay utilized affinity purification on streptavidin resin of biotinylated membrane proteins extracted from a crude synaptosomal preparation We combined this cell surface biotinylation assay with MS/MS analysis and label-free quantification to investigate protein patterns characterizing immature and mature neuronal cultures. We focused on Negr, and we demonstrated that it
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